PHOTOPHYSICS OF SOME ORGANIC BICHROMOPHORIC MOLECULES AND THEIR MODULATION IN DIFFERENT ORGANIZED MEDIA

Transcription

1 PHOTOPHYSICS OF SOME ORGANIC BICHROMOPHORIC MOLECULES AND THEIR MODULATION IN DIFFERENT ORGANIZED MEDIA THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (SCIENCE) OF JADAVPUR UNIVERSITY BY DIBAKAR SAHOO DEPARTMENT OF SPECTROSCOPY INDIAN ASSOCIATION FOR THE CULTIVATION OF SCIENCE JADAVPUR, KOLKATA INDIA

2 PHOTOPHYSICS OF SOME ORGANIC BICHROMOPHORIC MOLECULES AND THEIR MODULATION IN DIFFERENT ORGANIZED MEDIA

3 CHAPTER ONE Introduction

4 CHAPTER TWO Experimental: Apparatus, Methods, Materials and Theoretical Approaches

5 Chapter THREE Dye-surfactant interaction: modulation of photophysics of an ionic styryl dye

6 Chapter FOUR Spectra and Dynamics of an ionic Styryl dye in reverse micelles

7 Chapter FIVE Orientational dynamics of a charge transfer complex in cyclodextrin cavity as receptor

8 Chapter Six On the Spectral Behavior of an Ionic Styryl Dye: Effect of Micelle- Poly ethyleneblock-polyethylene glycol Diblock Copolymer Assembly

9 Chapter Seven A quest for binding site in Serum Albumin in a model biological system with an ionic Styryl dye

10 Chapter Eight Spectroscopic study on the mode of binding of 2-(4- (dimethylamino) styryl)-1- methylpyridinium iodide with calf thymus DNA

11 Appendix I SYNOPSIS OF THE RESULTS

12 LIST OF PUBLICATIONS Appendix II

13 INDIAN ASSOCIATION FOR THE CULTIVATION OF SCIENCE 2A & B Raja S. C. Mullick Road, Jadavpur, Kolkata , INDIA Phone: (Extn. 250), Fax: , spsc@iacs.res.in Professor S. Chakravorti, Ph.D, FAScT Department of Spectroscopy, Head, CSS October 23, 2009 This is to certify that the thesis entitled Photophysics of some organic bichromophoric molecules and their modulation in different organized media submitted by Sri Dibakar Sahoo who got his name registered on 31/07/2008 for the award of Ph. D. (Science) degree of Jadavpur University, is absolutely based upon her own work under the supervision of Professor Sankar Chakravorti and that neither this thesis nor any part of it has been submitted for any degree / diploma or any other academic award anywhere before. (S. Chakravorti) Signature of sole supervisor & Date with Official seal

14 Preface The present dissertation entitled Photophysics of some organic bichromophoric molecules and their modulation in different organized media is submitted to fulfill the requirements for the degree of Doctor of Philosophy (Science) of Jadavpur University. The study has been done for the investigations on the photophysical characteristics of organic bichromophoric molecule under various environmental conditions by analyzing their steady state and timeresolved spectroscopic method. Some theoretical calculations have been done to compare with experimental findings. All the studies have been done under the supervision of Professor Sankar Chakravorti in the Department of spectroscopy, Indian Association for the Cultivation of Science, Jadavpur, Kolkata , India. The thesis contains eight chapters and two appendices. Chapter 1 contains a brief review of the existing knowledge to the present work and outline of the purpose and problems studied in the present investigation. Second chapter contains a description of the experimental techniques, materials, methods and theoretical calculation used. Chapter 3-8 contain photochemical and photophysical properties of 2(4-(dimethylamino) styryl)-1-methylpyridinium iodide (DASPMI) in different media. Most of the results incorporated in this thesis have been published in referred international scientific journals, a list of which is appeared at the end of this dissertation. (Dibakar Sahoo) Date: Department of Spectroscopy Indian Association for the Cultivation of Science Jadavpur, Kolkata India

15 Acknowledgement I humbly express my deep sense of gratitude to my supervisor Prof. Sankar Chakravorti for his extremely helpful guidance and constant encouragement throughout the course of my research work. I have learned a lot from him not only about research but also many things which helped me to be a better human being. It was really a great experience working under the guidance of Prof. Sankar Chakravori. I would like to express my sincere thanks to Prof. Prasanta Kumar Mukherjee, Prof Abhijit Das, Prof Manika Mukherjee, Prof. S. C. Roy and Prof. R. Venkteswaran of IACS for valuable discussion, encouragement and technical supports. I would like to thank to Dr. Papia Chowdhury, Dr. Subhasis Panja and Tirthada for their suggestions and useful advices. I fail to utter my heartfelt thanks with appropriate words to Prof. Anjan Kumar Dasgupta, Mr. Hirak Kumar Patra, Mr. Santiswarup Singha, of Ballyguange Science College for their constant co-operation and kind support. I would be failing in my duty if I do not mention the name of my dear friend Late Tapan Sarkar without his active cooperation till last breath I would not have ventured into the biological world. I am thankful to all faculties of my department for providing me a healthy atmosphere. In last three years many people have helped and encouraged me to continue with the present work. I am grateful to all of them. It is not possible to acknowledge everyone by name. Still I wish to thank Prosenjit, Anamika, Manasda, Karanda, Bhaskar for their cooperation and support. I acknowledge my indebted to all nonteaching staff of my department for their constant support. I am grateful to Indian Association for the Cultivation of Science for providing the financial support. I would like to take this opportunity to express my respect and love to my parents. From the very beginning of my life they instilled me a desire for dreaming about scientific research. They provided every type of opportunities and supports that helped me to step forward on this way. I express my love to my sweet sister who is always with me. I also express my respect to my grand father and love to all my near and dear ones for their contribution that helped me to touch my dream of my life. Finally I yield to gratify my wife Paulami for her cooperation, encouragement and mental support in the present work without which it would not have been possible for me to devote all my times to the present study. ----The Author

16 DEDICATED TO MY PARENTS Any success of my life is due their inspiration and sacrifices

17 Contents 1. Introduction 1.1 Preliminary remarks Interaction between light and organic molecules Singlet and triplet states Physical characteristics of excited state Deactivation routs for excited molecule Radiative transitions Nature of radiationless process Environmental effect on electronic spectra Solvent effects Acidity effects Different complex in excited state Excimer formation Exciplex formation Hydrogen bond formation Excited state charge transfer complex Environmental effects on excited state complex Homogeneous environments Microheterogenious environments The motivation and aim of the thesis Outline of the problems presented in the thesis 41 Bibliography Experimental: Apparatus, Methods, Materials and Theoretical Approaches 2.1 Experimental techniques Absorption Spectra Excitation spectra Fluorescence spectra Time-resolved spectroscopy.61

18 2.1.5 Scanning electron microscopy (SEM) and field emission SEM ( FESEM) Circular dichroism (CD) Dynamic light scattering Methods of calculations Polarization spectra Quantum yield Purification of samples Chemicals and solvents used throughout the work Computational methods Gaussian programs Molecular modeling studies..78 Bibliography Dye-surfactant interaction: modulation of photophysics of an ionic styryl dye 3.1 Introduction Results and discussion Steady state emission and absorption results Effect of inorganic salt Time resolved emission Conclusion Bibliography Spectra and Dynamics of an ionic Styryl dye in reverse micelles 4.1 Introduction Results and discussion Absorption and emission of DASPMI in n-heptane/aot and benzene/bhdc reveres micelle Time resolved emission in AOT/n-heptane and BHDC/benzene Emission of DASPMI in n-heptane/aot/water and benzene/bhdc/water Steady state fluorescence anisotropy Micropolarity around the fluorophore

19 4.2.6 Microviscosity around the fluorophore Metal induced fluorescence quenching Time resolved studies of DASPMI in n-heptane/aot/water and benzene/bhdc/water Pico-second anisotropy decay Conclusion.123 Bibliography Orientational dynamics of a charge transfer complex in cyclodextrin cavity as receptor 5.1 Introduction Results and discussion Spectroscopic and photophysical properties of complexes Pico-second time resolved emission and anisotropy decay Conclusion Bibliography On the Spectral Behavior of an Ionic Styryl Dye: Effect of Micelle- Polyethyleneblock-Polyethylene glycol Diblock Copolymer Assembly 6.1 Introduction Results and discussion Steady state absorption and emission spectra Time resolved emission studies Time resolved fluorescence anisotropy measurement Conclusion Bibliography A quest for binding site in Serum Albumin in a model biological system with an ionic Styryl dye 7.1 Introduction Results and discussion Conclusion Bibliography.178

20 8. Spectroscopic study on the mode of binding of 2-(4-(dimethylamino) styryl)-1- methylpyridinium iodide with calf thymus DNA 8.1 Introduction Results and discussion Absorption study Steady state fluorescence spectra Fluorescence quenching study Effects of ionic strength Equilibrium binding titration CD studies Steady state anisotropy Fluorescence lifetime Molecular modeling of DASPMI-DNA interaction Wobbling motion Conclusion 194 Bibliography 195 Apendix I: Synopsis of the results 197 Apendix II: Paper published in international journals...201

21 Introduction Preliminary Remarks: A visible or ultraviolet absorption peak is caused by the promotion of an electron in one orbital (usually a ground state orbital) to a higher orbital [1]. Normally the amount of energy necessary to make this transition depends mostly on the nature of the two orbitals involved and much less on the rest of the molecule. A group that causes absorption is called a chromphore. Among the different chromophores the aromatic bichromophore type molecules have attracted special attention. All these bichromophoric molecules contain aromatic donor and aromatic acceptor. These types of molecules energetically and kinetically favor typical excited state geometry, which is very much different from the Franck-Condon excited state geometry. The probability or rate of absorption of light is given by Beer-Lambert Law which states that Fraction light absorption is proportional to the concentration (C) and thickness (dl) of the absorbing system i. e., di = α ν C dl (1.1) I where α ν is the proportionality constant. The integrated form of the above equation is where I 0 log = ε ν C l = optical density (1.2) I ε ν = α ν /2.303 is called the decadic molar extinction coefficient and is a function of frequency/wavelength. 1.2 Interaction between light and organic molecules: The energy level at which a molecule exists is referred to as its state. Under normal conditions, molecules exist in their most stable states. We refer to that most stable level as the ground state. The laws of quantum mechanics tell us that molecules do not increase their energy levels gradually. A molecule goes directly from one state to another without going through

22 Introduction 2 intermediates. We refer to these "quantum leaps" as transitions. The transition from the ground state (written as S o ) to the first excited state (S 1 ) requires some form of energy input. When a photon passes through the organic molecule the molecule may absorb the photon. The energy of the absorbed photon is used to energize an electron and causes it to jump to higher energy orbital that is energetically unstable relative to the ground state and is said to be excited state. In order to induce the formation of an electronically excited state, a molecule must absorb a photon of radiation with energy that is equal to the difference between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the ground state: hυ = E = E E ( LUMO) ( HOMO) (1.3) The energy of this electronic excited state may be defined as the energy difference between the vibrational levels of quantum number zero of the two states. Two different electronic orbital configurations may be produced by absorption of the photon. In one state the electron spins are paired (anti-parallel) and in other state the electron spins are unpaired (parallel). The state with paired spins has no spin magnetic moment, but the state with unpaired spins possesses a net spin magnetic moment. A state with paired spin remains a single state in presence of a laboratory magnetic field and is termed as singlet state. A state with unpaired spins interacts with a laboratory magnetic field and splits into three quantized states and is termed as triplet state Singlet and Triplet States: A triplet state has a lower energy than the corresponding singlet state because of the repulsive nature of the spin-spin interaction between electrons of the same spin (in accord with Hund's rule, which states that the most stable arrangement of electrons in atoms (or molecules) is that with maximum multiplicity). The magnitude of the difference in energy varies according to the degree of spatial interaction (overlap) between the orbitals involved. For orbitals which occupy

23 Introduction 3 substantially different regions of space (as in (n, π*) states of carbonyl compounds) orbital overlap is small and the singlet-triplet energy difference (splitting) is relatively small. For orbitals which occupy similar regions of space (as in (π,π*) states of alkenes) the difference is much larger. The excited states initially produced by absorption of a photon are almost always singlet states, designated by S 1, S 2,..., S n, the increasing order of subscripts representing the states with increasing energy. This is because practically all molecules have a singlet ground state, denoted by S 0 and the selection rules for absorption strongly favor conservation of spin during the absorption process. Since the energy needed to raise an electron from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) is usually much larger than kt, almost all molecules are in their electronic ground state at room temperature. Singlet triplet absorption bands in the absorption spectra of some compounds can be observed with sensitive spectrophotometers, and these bands can often be enhanced by the presence of a paramagnetic species such as molecular oxygen, magnetic fields, and heavy atoms but they are in general very much weaker than singlet singlet bands. (1) Spin-forbidden Transitions: Transitions in which the spin of electron changes are not allowed, because a change from one spin to the opposite involves a change in angular momentum and such a change would violate the law of conservation of angular momentum. Therefore, singlet-triplet and triplet-singlet transitions are forbidden, whereas singlet-singlet and triplet-triplet transitions are allowed. (2) Symmetry-Forbidden transitions: Among the transitions in this class are those in which a molecule has a center of symmetry. In such cases, a gerade (g) gerade (g) or ungerade (u) ungerade (u) transition is forbidden, while the g u or u g transition is allowed.

24 Introduction The physical characteristics of excited state: Mainly the four characteristics, viz., energy, lifetime, quantum yield and polarization are associated with molecular emission. a) Energy: For polyatomic molecules the excess vibrational energy gained in a vibration coupled electronic transition loses their energy to the surrounding within a very short lifetime ~ sec. This energy associated with the emission process is the easiest of the characteristic qualities to determine. Then the molecule reaches zero vibrational level of the first excited state (S 1 ) and exist there for about 10-8 sec. After that normally it returns to the ground state. b) Lifetime: The lifetime is defined as the time taken for the radiation intensity to decay to e 1 of its initial value [2]. It is observed that the fluorescence intensity decays after the withdrawal of the source of excitation according to a first order rate equation, which is 0 t I = I e τ (1.4) where I 0 is some initial intensity, I is the intensity at a later time t and τ is a constant known as the mean lifetime of the excited state. When the time t over which the decay is measured is equal to τ, the intensity reduces to 1 e of its initial value. The lifetime τ of the excited state is related to the radiative transfer rate as 1 τ = (1.5) 0 k F where k F is the rate constants for fluorescence emission. The intrinsic or natural lifetime for phosphorescence emission is also defined in the same way as

25 Introduction 5 1 τ = (1.6) k P where k p is the rate constant for phosphorescence. c) Quantum yield: The quantum yield of an emission is defined as the number of quanta emitted per exciting quanta absorbed. This is one of the most difficult characteristic parameter to measure [3-7]. The quantum yield of fluorescence emission is Number of fluorescence quanta emitted φ F = Number of quanta absorbed by a singlet excited state And the quantum yield of phosphorescence is defined as φ p = Number of phosphorescence quanta emitted Number of quanta absorbed by a singlet excited The general relation between quantum yield and observed lifetime τ Q is related as τ Q ϕ = (1.7) τ ϕ 0 d) Polarization: With the knowledge of polarization characteristics of absorption bands it is possible to assign the nature of the state from which the emission occurs. The molecule, whose absorption transition moments are parallel with the electric vector of the light, will be excited most but less preferred orientations will contribute less. The degree of polarization is defined as I I I = + I + 2 3cos ( θ ) 1 2 cos ( θ ) 3 (1.8) where I and I are the intensities of the observed parallel and perpendicular component, θ is the angle between emission and absorption transition moments.

26 Introduction 6 e) Fluorescence anisotropy: Fluorescence anisotropy assays the rotational diffusion of a molecule between the exciting and emitted (fluorescent) photons. This decorrelation can measure the "tumbling time" of the molecule as a whole, or of a part of the molecule relative to the whole. From the rotational diffusion constants, one can estimate the rough shape of a macromolecule. Fluorescence anisotropy is a method for measuring the binding interaction between two molecules, and can be used to measure the binding constant or the disassociation constant for the interaction. The basic idea is that a fluorophore excited by polarized light will also emit polarized light. However, if a molecule is moving, it will tend to "scramble" the polarization of the light by radiating at a different direction from the incident light. The "scrambling" effect is greatest with fluorophores freely tumbling in solution and decreases with decreased rates of tumbling. The steady state fluorescence anisotropy (r) was calculated using the relation given below r= (I VV -GI VH )/(I VV +2GI VH ), where G is the correction factor for detector sensitivity to the polarizer direction of emission and I VV and I VH represents the vertically and horizontally polarized emission intensity obtained on excitation with vertically polarized light. f) Time-resolved emission spectra: Time resolved emission spectra (TRES) is frequently used to study the excited state dynamics and kinetics of fluorescence molecules in solution. The standard interpretation of TRES assumes a prior knowledge of the number of fluorescent species in the ground state (usually a single species). This assumption may fail if the fluorophore is present in a complex environment such as a microheterogeneous media. Time-resolved area normalized emission spectra (TRANES) is a step forward and, with this analysis, it is possible to determine the number of species in the sample that contribute to the observed fluorescence emission. In this way, TRANES, which is a modified version of TRES, is obtained without assumption of ground or excited-state kinetics. TRANES gives fluorescence spectra that are

27 Introduction 7 analogous to the absorption spectra of transient species. In particular, an isoemissive wavelength in TRANES has the same significance as an isosbestic wavelength in absorption spectra. 1.4 Deactivation routes for excited molecule: A molecule, photochemically promoted to an excited state, does not stay there for long. Excitations to S 2 and higher singlet states take place, but in liquids and solids these higher states usually drop very rapidly to the lowest singlet S 1 state (about to sec) by giving up the energy to the environment. This process is called an energy cascade. In a similar manner, molecules at different vibrational levels of S 1 cascade down to the lowest vibrational level of S 1. Therefore, in most cases, the lowest vibrational level of the S 1 state is the only important excited singlet state. This state can undergo various physical and chemical processes. The physical processes are shown in the modified diagrams (Fig. 1.1). Figure 1.1: A Jablonski diagram showing excitation and deactivation routes. FLUOR: fluorescence (~ _ 10-6 s); PHOS: Phosphorescence (~ 10-6_ 10-3 s); VT: vibrational cascade (~ s); ic: internal conversion (~10-10 ); isc: internal system crossing (~ 10-6 s).

28 Introduction 8 According to Kasha [8], for most photophysical processes we need to consider only the lowest excited singlet state (S 1 ) or the lowest triplet state (T 1 ) as likely candidates for the initiation of a process. The various deactivation routes of an excited molecule may be broadly classified into two categories, one is the radiative transitions and the other is non-radiative transitions. Radiative transitions routes are fluorescence, phosphorescence and radiative energy transfer (trivial process). On the other hand, possible routes open to non-radiative transitions are (i) Internal conversion (IC), (ii) Intersystem crossing (ISC), (iii) Non-radiative energy transfer, (iv) Photoinduced electron transfer (ET) and (v) Excited state proton transfer (ESPT) Radiative Transitions: Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon. Luminescence is divided into two types, depending upon the nature of the ground and excited states. Substances which display significant fluorescence generally possess delocalized electrons formally present in conjugated double bonds. The emission of radiation is also governed by Franck-Condon principle. (a) Fluorescence : On photoexcitation a molecule is excited from a vibrational level in the electronic ground state to one of the many vibrational levels in the excited electronic state. This excited state is usually the first excited singlet state. A molecule in a high vibrational level of the excited state will quickly fall to the lowest vibrational level of this state by losing energy to other molecules through collision. The molecule will also partition the excess energy to other possible modes of vibration and rotation. Fluorescence occurs when the molecule returns to the electronic ground state, from the excited singlet state, by emission of a photon (Figure 1.2). Such transitions are quantum mechanically allowed and the emissive rates are typically near 10 8 sec - 1. These high emissive rates result in fluorescence lifetimes near 10-8 s. or ns.

29 Introduction 9 Figure 1.2 : Mechanism of fluorescence according to Frank-Condon principle (b) Phosphorescence: The phenomenon of phosphorescence involves an intersystem crossing, or transition, from the singlet to the triplet state. The transition from the ground state to the triplet excited state is a forbidden (highly improbable) transition. Internal conversion from the singlet to the triplet (electronic spin reversal) is more probable since the energy of the lowest vibrational level of triplet state is lower than that of singlet excited state (Figure 1.3). Molecules in triplet state can then return to the ground state directly. Transition times of 10-4 to 10 sec are observed in phosphorescence. Hence a characteristic feature of phosphorescence is an afterglow, that is, emission that continues after the exciting source is removed. 1.3 : Mechanism of phosphorescence

30 Introduction Nature of radiationless processes: Radiationless transition between two electronic states may be represented as the presence of the point of intersection of potential energy surfaces. A crossing point is the point of equal energy for both the curves. The irreversible radiationless transfer of energy involves two steps: (a) the horizontal transfer of energy at the isoenergetic point from the zero-point level of higher electronic energy state to the high vibrational level of the lower electronic state and (b) the rapid loss of excess vibrational energy after transfer. The radiationless transitions are governed by three factors. They are (1) Density of state ρ E which is defined as the number of vibrational levels per unit energy interval at the energy of the initial value. (2) Energy gap E between the interacting electronic states (3) Vibronic overlap or Franck-Condon factor. The major nonradiative deactivation routes from excited molecules are (i) internal conversion and (ii) intersystem crossing (iii) Fluorescence resonance energy transfer (FRET). (i) Internal Conversion (IC): It is a radiationless passage between the electronic energy states of same spin multiplicity or between the different vibrational levels of the same electronic state [9,10]. The internal conversion rate constant for S1 S0 is small ( sec -1 ) compared to the rate constant for higher states like S2 S1 etc. ( sec -1 ). However it has been observed that with the increase of energy gap between S 1 and S 0, the internal conversion efficiency decreases [11,12]. Internal conversion between T2 T1 has also been observed for some molecules [13].

31 Introduction 11 (ii) Intersystem Crossing (ISC): It describes the radiationless transition process between states of different spin multiplicity and depends on spin orbit coupling [9]. Due to large energy gaps, transition form S 1 to S 0 is not always probable by radiationless transition mechanism. Under these circumstances the molecule has two alternatives: (i) to return to the ground state by fluorescence emission or (ii) to cross over to the lowest triplet state non-radiatively. Since T 1 S 0 energy differences are normally of the order of 20,000 cm -1 and S 1 T 1 energy differences are commonly of the order of cm -1, the intersystem crossing probability ( S1 T1 ) is considerably greater than the probability of direct transition from the ground state to the triplet state. The intersystem crossing rate [14] for S1 T1 transition is sec -1 and that for T1 S0 transition is sec -1 i.e. it depends on the energy gap between singlet and triplet states. (iii) FRET: Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two chromophoric molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole dipole coupling. This mechanism is termed "Förster resonance energy transfer" or "fluorescence resonance energy transfer". FRET is analogous to near field communication, in that the radius of interaction is much smaller than the wavelength of light emitted. In the near field region, the excited chromophore emits a virtual photon that is instantly absorbed by a receiving chromophore. These virtual photons are undetectable, since their existence violates the conservation of energy and momentum, and hence FRET is known as a radiationless mechanism. The FRET efficiency depends on many parameters that can be grouped as follows:

32 Introduction 12 The distance between the donor and the acceptor The spectral overlap of the donor emission spectrum and the acceptor absorption spectrum. The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment. E depends on the donor-to-acceptor separation distance r with an inverse 6th power law due to the dipole-dipole coupling mechanism: (1.9) with R 0 being the Förster distance of this pair of donor and acceptor, i.e. the distance at which the energy transfer efficiency is 50%. The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation as expressed by the following equation: (1.10) where Q 0 is the fluorescence quantum yield of the donor in the absence of the acceptor, κ 2 is the dipole orientation factor, n is the refractive index of the medium, N A is Avogadro's number, and J is the spectral overlap integral calculated as (1.11)

33 Introduction 13 where f D is the normalized donor emission spectrum, and ε A is the acceptor molar extinction coefficient. κ 2 =2/3 is often assumed. This value is obtained when both dyes are freely rotating and can be considered to be isotropically oriented during the excited state lifetime. If either dye is fixed or not free to rotate, then κ 2 =2/3 will not be a valid assumption. In most cases, however, even modest reorientation of the dyes results in enough orientational averaging that κ 2 = 2/3 does not result in a large error in the estimated energy transfer distance due to the sixth power dependence of R 0 on κ 2. Even when κ 2 is quite different from 2/3 the error can be associated with a shift in R 0 and thus determinations of changes in relative distance for a particular system are still valid. Fluorescent proteins do not reorient on a timescale that is faster than their fluorescence lifetime. In this case κ 2 remains in the limit 0 κ Environmental effect on electronic spectra: Solvent effect: The electronic spectral shift in presence of solvent molecules arises due to the introduction of local field around the solute molecule and also due to alternation in the charge density of the molecule. The shift is different from that in free space. There are two types of solute solvent interactions: (i) universal interaction and (ii) specific interaction. (i) Universal interactions: This long-range effect is due to the collective influence of the solvent as a dielectric medium. It depends on the refractive index n and dielectric constant D of the solvent and includes the following interactions: (a) Bayliss polarization interaction: Bayliss [15] showed that the contribution of this interaction to spectral shift ( ν cm -1 ) in solution relative to gas phase is given by ν polarization = f A ν 3 a 2 n 1 2n (1.12)

34 Introduction 14 where ν and f are the wave number and oscillator strength of solute transition, A is a number factor whose value was calculated to be according to classical or quantum mechanical models respectively. (b) London dispersion interaction: The interaction between two non-polar but polarizable solute and solvent molecules is the dispersion interaction [16] that results from the fluctuation of their instantaneous dipoles. The contribution of this interaction to the solvent spectral shift [17,18] is given by ' 2 α 3 II ( n 1) ν dispersion = 3 ' 2 hca 2( I + I ) ( n + 2) (1.13) where α is the difference between ground and Franck-Condon (FC) excited state polarizabilities, a is the radius of the cavity in solvent where solute molecule lies, h is Planck s constant and I and I are the ionization potentials of the solute and solvent molecules respectively. (c) Solvent stark effect: This small effect arises due to the polarization of nonpolar solute molecule by the permanent dipole moment of the dipolar medium. The contribution of this interaction of the spectral shift is given by [19-21] α ( ) ( ) 3 hca 2 { f n } ν solvent Stark effect = L f D (1.14) where L is a function factor and f(d) and f(n 2 ) are Onsagar polarity functions expressed as 2 2( D 1) 2 2( n 1) f n 2 f( D) = and ( ) = (2D+ 1) (2n + 1) (1.15) (d) Dipole-induced dipole interaction: This is the interaction between the total dipole moment of the solute molecule and the induced dipole moment in the polarizable solvent. The solvatchromic shift due to the dipole-polarizabilty interaction is given by [21]

35 Introduction 15 µ µ 2 2 g e 2 ν induction = f ( n ) (1.16) 3 2hca Utilizing these effects of solvent on the absorption and emission spectra, useful information regarding physical properties like change in dipole moment [22-24] and polarizabilty [25] in the two combining states can be obtained. (ii) Specific interaction: There are low lying unfilled orbitals in the solvent. It is likely to have a strong affinity for electrons. If the solvent molecule accepts electrons from the solute molecule, a charge transfer to solvent complex is said to be formed. Other specific interactions are the formation of hydrogen-bonded complexes and exciplexes. All these are short-range interactions. In general, the electronic charge distribution of (π, π *) state is more extended than that of the ground state due to which the former is more polarizable. Change from a nonpolar to polar solvent increases the solute-solvent interaction in both ground and excited states but the stabilization of the excited states is greater. This produces a red shift in absorption and emission spectra. Due to these reasons, the molecules having more polar excited states give longer wavelength fluorescence in more polar solvents. On the other hand, non-bonding lone pair on hetero-atoms remains hydrogen bonded with the polar and hydrogen-bonding solvents in ground state resulting in greater decrease in ground state energy. The excited (n, π *) state is not much depressed as the promotion of n-electron into π *- orbital reduces this hydrogen bonding forces in excited state. The result is a blue shift of electronic spectra. The solvent in which the absorbing species is dissolved also has as effect on the spectrum Acidity effect on electronic spectra: Addition of acid or base to the solvent can affect the electronic spectra of the solute molecule having dissociable protons or non-bonded electron pairs in the following two ways. If the acidity of the molecule is insufficient to protonate non-bonded electron pairs or to abstract a proton from dissociable group of the solute molecule, the acid or base may

36 Introduction 16 form hydrogen bonds with the basic or acidic group of the solute molecule. The effect of this type of hydrogen bonding is similar to that described in earlier section. If the acidity of the medium is sufficient to protonate a group having lone electron pairs or to abstract a proton from acidic functional group, the effect on electronic spectra are more dramatic but qualitatively similar to the effect produced by hydrogen bonding. Protonation of basic molecule or dissociation of an acidic molecule produces a chemical species whose reactivity and electronic structure is different from that of the original molecule. Protonation of non-bonded pairs on functional groups [26], which are charge transfer acceptor in the excited state (carbonyl and carboxyl group), enhances the acceptor properties of these groups and results in stabilization of the excited state relative to the ground state. Therefore, it produces shifting of intramolecular, electronic, charge transfer spectra to longer wavelength. Protolytic dissociation from functional group which is C-T acceptors in the excited state (carboxyl group) leaves residual negative charge on the functional groups, inhibiting charge acceptance from the aromatic ring and producing a shift to shorter wavelength of electronic spectra. Protonation of lone pairs of functional groups which are C-T donors in the excited state (e. g. NH 2 ) inhibits the donor properties of these groups and results in decrease as well as shifting of the electronic spectra to the shorter wavelength. Deprotonation of these type of groups (e. g. phenolic groups) enhance charge donation because of repulsive effect of the residual negative charge of the dissociated group upon the lone pair electrons and results in shifting of electronic spectra to the longer wavelength.

37 Introduction Different complexes in excited state: Excimer Formation: In some cases, simultaneously with the quenching of normal fluorescence, a new structureless emission band appears on the red side of the monomer fluorescence spectrum with increasing concentration of the solution. This phenomenon was first observed in pyrene solution by Förster [27] and was explained as due to transitory complex formation between the ground and excited state molecules, since the absorption spectrum was not modified by increase in concentration. These short lived excited state dimers are called excimers [28] to differentiate them from electronically excited dimeric ground state species. The excimers dissociate when they return to the ground state by emission, giving rise to a structureless envelope for the emission spectrum. This suggests that the ground state PE surface must be repulsional. Excimer emission [29-41] is often observed from planner aromatic hydrocarbon molecules when the two component molecules are placed in parallel configuration. The condition for possible eximer formation are (i) the two planer molecules should approach within a distance ~0.35 nm of each other. (ii) The concentration should be high enough for interaction to occur within the excited lifetime and (iii) Interaction energy between an excited and a normal molecule should be attractive, such that the excited state enthalpy - H* is greater than the thermal energy RT Exciplex Formation: When two dissimilar molecules collide, attractive tendencies are usually greater, depending on polarity and ability to polarize. This type interaction involves some degree of charge transfer (CT) within the lifetime of the excited fluorescer molecule and the complex formed between two different moieties of a bichromophore or between the excited fluorescer and some added foreign molecule in ground state, is called exciplex [42-49]. So exciplexes are polar entities while excimers have zero dipole moment. Exciplex are generally nonfluorescent, but when the exciplex lifetimes are long, they may degrade by light emission. The exciplex fluorescence may

38 Introduction 18 be quenched by addition molecules with heavy atoms [50] or by suitable electron donor or acceptor [51-56]. There are other exciplex systems in which the importance of CT in the exciplex state is much less [57-67] Hydrogen Bond Formation: The hydrogen bond formation is weakly associative interaction that produces cluster of molecular aggregates, which conform in many respects to the criteria identifying molecules. In N-heteroatom molecules n π * absorptive transitions are usually more affected by H-bond donor properties of the solvents and the ground state is more stabilized than the excited one. The strength of hydrogen bond of a complex in excited state may be different from that in ground state. Generally hydrogen bonded excited state complexes are nonfluorescent [68] but there may be exception [69-72]. The formation of CT complex due to hydrogen bonding and related CT theory has been nicely discussed by Mataga and Ratajczak [73,74]. Excited state hydrogen bond formation often effects quenching of fluorescence of 1 A * without appearance of new bands in the spectrum even if the added quencher is not a π -electron system [73,75,76] evidently because excited state hydrogen bonding affects a very significant increase in the efficiency of internal conversion Excited state charge transfer complex: Separation of electrical charges is one of the important processes which is the basis of numerous present and possible future developments including organic conductors and super conductors as well as the production and storage of electricity. Most important, all of earth s life forms use directly or indirectly the transformation of sunlight into chemical energy which occurs via charge-separation process. The classic examples of natural processes involving charge transfer are photosynthesis in green plants and solar energy conversion to many other forms of chemical energy [77]. Since charge-separation on a molecular level is often involved on the study of model system displaying charge separations essential to understand the mechanisms involved.

39 Introduction 19 An especially powerful tool is provided if such charge separation systems luminous, because internal and environmental influences affects this luminescence in a characteristic way, yielding detailed information on thermodynamics, kinetics and other photophysical and even photochemical properties of such species The concept of charge transfer: The concept of charge transfer refers to excitation into a state that involves a complete transference of an electron from a donor to an acceptor or between them in the excited state of either of the species. This phenomenon imparts an effect on the emission spectra by developing a new structureless band at a longer wavelength region. There are two phenomenologically different ideas concerning the charge transfer. The first one, through space interaction, is applicable when the two chromophoric parts are within van der Waals distance [78] and the other one, through bond interaction, deals with rigid systems when the distance is much larger [79]. In the ground state the donor acceptor (D A) complex possesses no stabilization energy apart from the very small resonance energy due to the ionic structure D + A -. In spectroscopy we generally see two fluorescence band, a normal fluorescence band or the locally excited (LE) band and a second, strongly red-shifted charge transfer (CT) band. Locally excited state of the exciplex, DA*is the origin of LE band, A* being the lowest singlet excited state of A. The ideal systems for the study of the charge transfer processes are electron donor acceptor molecules [80,81]. These molecules not only provide a testing ground for the contemporary theories of CT process, but they are also useful for the study of solvation dynamics [82] N, nonlinear optical properties [83], etc. The CT reactions may be classified as (i) intermolecular charge transfer and (ii) intramolecular charge transfer.

40 Introduction Intermolecular charge transfer: In an intermolecular charge transfer process an electron from a molecule with high charge density (donor) is transferred to a molecule with low charge density (acceptor) leading to the formation of a new complex called charge transfer (CT) complex. In this case the transition occurs from the highest filled molecular orbital of the donor to the lowest empty molecular orbital of the acceptor. (Fig. 1.4)Additional charge transfer is also possible on promotion of an electron to higher unoccupied orbitals of the acceptor. Charge transfer complexes are regarded as important materials due to their ample applications [84-87]. The CT complexes have been reported as important reaction intermediates in many chemical reactions [88,89]. The question of participation of CT complexes in organic synthesis, and in understanding reaction mechanisms has been debated since the classical work interaction of iodine with aromatic hydrocarbons of Benesi and Hildebrand [90]. The review work of Mataga et al. has focused elaborately on the fundamental aspects of charge transfer process and CT complex chemistry [91]. Fig. 1.4: Schematic diagram of charge transfer transition. In a very recent report Shen et al. has shown the coexistence of inter- and intramolecular charge transfer in polymethylphenylsilane/c60 films [92]. Besides, several authors have focused their attention in studying intermolecular charge transfer reaction in micellar and reverse micellar environments [93,94]

41 Introduction Intramolecular charge transfer: Intramolecular charge transfer is a fundamental process and has always been attracting considerable attention as a topic of central importance in photochemistry and biochemistry [95-100]. A charge separation may result from the transfer of an electronic charge upon excitation from a donor (D) site to an acceptor (A) site within a suitable molecule. In such an internal charge transfer (ICT) process, positive and negative charges are localized in two different and separated functional parts of the same molecule. This new charge distribution generally leads to a large increase of the dipole moment, and hence will cause a marked solvatochromic effect and a large Stokes shift. Recently the solvent polarity sensitive organic donor acceptor (D A) compounds have been claiming increasing interest and are being used as the fluorescent probes to study different microenvironments [ ] (Fig 1.5). Among various fluorophores, showing intramolecular charge transfer properties coumarins and aminonaphthalimides are the prominent ones [102,81, ]. Through their extensive studies Berces, Kossanyi, and co-workers discussed on the influence of geometry of the molecule or intramolecular geometrical relaxation Figure 1.5: Excited state schematic diagram of donor-acceptor compound. process on the emitting properties of various ICT probes [ ]. Yuan and Brown studied the photophysical properties of aminonaphthalimides in various media [113,114]. They have also

42 Introduction 22 studied the effect of ph of the medium on the fluorescence properties of aminonaphthalimides and explored possible use of these systems in fluorescence sensing of the transition-metal ions such as Cu 2+ [115] Twisted Intramolecular charge transfer: The discovery of dual fluorescence of the simple donor-acceptor substituted benzene derivative 4 N, N-dimethyaminobenzonitrile (DMABN) by Lippert et. al. [116] and the subsequent model compound studies by Grabowski et. al. [ ] including rigidized and pre-twisted compounds gave birth to the idea of Twisted Intramolecular Charge Transfer (TICT) states. This was the start of an expanding area of physical, physical-organic and organic chemistry connected with mechanistic and kinetic questions of electron transfer, and with a rationalization of the excited state behavior of many dye systems. The number of applications is growing in various fields such as tailor made fluorescence dyes [122,123], sensing of free volume in polymer [ ], fluorescence ph or ion indicator [126,127], fluorescence solar collectors [122] and electron transfer photochemistry [128,129]. In the TICT model the dual fluorescence of DMABN with its normal band (B band) at around 350 nm and its anomalous one (A band, around 450 nm in medium polar solvents) depends on the conformational freedom of the dimethylamino (DMA) group. For the pre-twisted compounds TMABN and CBQ where the nitrogen lone pair is nearly in plane with the benzonitrile skeleton and perpendicular to the π-orbital system, on the other hand, only the anomalous A band is observed. The conclusion regarding DMABN was therefore that a reaction path exists in the excited state leading from the near planar conformation (emitter of the B band) to a photochemical product with an energetic minimum at the perpendicular conformation (emitter of the A band). These two emitters were called the B* state and A* state and were shown to possess a mother- daughter relationship later substantiated by direct kinetic measurements. In many case, the back reaction A* B* also occurs leading to an excited state

43 Introduction 23 equilibrium. The ground state of DMABN is known to possess an energy barrier for the perpendicular conformation (the rotation barrier), therefore emission from the perpendicular excited state minimum occurs to a repulsive potential and is expected to lead a structureless spectra. In recent times large numbers of molecules have been discovered to have TICT emission [ ]. Another important point to emphasize is that the reaction co-ordinate is not simply the intramolecular twisting motion but involves other coordinates too, such as electron transfer, solvent dipolar relaxation and most probably, some rehybridization at the amino nitrogen (umbrella motion)[ ]. For the perpendicular TICT conformation, donor (dialkylamino group) and acceptor (benzonitrile) π-orbitals are orthogonal (zero overlap) and thus decoupled leading to maximum for dipole moment in the excited state (and a minimum in the ground state). These maxima of the dipole moments (near full electron transfer from donor to acceptor) connected with the energetic minimum for the perpendicular conformations are essential ingredients of the so-called minimum overlap rule [117]. For the near planar conformation (B* state), mesomeric interaction between the donor acceptor π-system exists and diminishes the dipole moment (in strong donor acceptor system the difference between the dipole moments of TICT and B* state may be small, however). The reaction process has been summarized in figure 1.6 Figure 1.6 The TICT model involves a twisted product species with charge transfer (A* state) formed through an adiabatic photoreaction from the precursor (B*) state which is often (but not always) of near plane conformation.

44 Introduction 24 Equation (1.17) and (1.18) can be used to predict possible new TICT system [123,146]. Whether or not the energetic minimum of the A*/TICT state is lower than that of the precursor B* state sensitively depends on the electron donor-acceptor properties of the sub system which can be quantified by ionization (or oxidation) potential IP and electron affinity EA (or reduction potential) of donor D and acceptor A. E B * E TICT > 0 (1.17) E = IP( D) EA( A) + C + (1.18) TICT E solv The B* state responds much less to changes in donor and acceptor properties than the TICT state, and equation (1.17) can often easily be fulfilled by increasing donor and/or acceptor strength. In addition to these two factors which deliver the decisive part of the reaction driving force, polar solvent stabilization E solv and the mutual Coulombic attraction C of the linked donor and acceptor radical anion /cation pair also help to preferentially stabilize the TICT state with respect to the precursor B* state. The photophysical properties of the stilbazolium dyes in the light of viscosity and temperature has been described by Rettig et.al. [147] the simultaneous dependence of photophysics on the solvent viscosity and polarity was found for stilbazolium and related pyridine dyes by Fromherz and co-workers [ ]. These authors discuss the results by invoking single bond twisting in the excited state toward a twisted intramolecular charge transfer state (TICT)[ ] as the main cause of this dependencies and also for very short fluorescence lifetime in dilute solution at room temperature. The validity of the TICT- model was approved by synthesis of a model compound [154] where the flexible single bond between the pyridium and aniline group was bridged to obtain a rigid molecule (2-methyl-7- (dimethylamino)-2-azafluorenium).the comparative photophysical study of three positional isomers of dimethylaminostyrylpridinium salt at room temperature and low temperature has been

45 Introduction 25 described by Rettig and his co-workers [155]. Also the photophysical properties of o-,m- and p- (dimethylamino) stilbazolium dyes were investigated by semiempirical quantum chemical calculations[156]. In their work Leinhos et. al. [157] showed that DMABN in toluene undergoes charge transfer reaction in the excited state. The interpretation is supported by the experiments with 4- amino benzonitrile MABN (1H) and ABN (1HH). The fluorescence decay of these molecule were single exponential in toluene, indicated the absence of any excited state reaction. The explanation of their observation was based on the increase in energy of the CT state with respect to the LE state in those molecules, brought about by the replacement of methyl substitutents of amino group by hydrogen. As dual emission and excited state charge transfer occurred with DMABN in toluene, they concluded that 1:1 complex formation involving lone pair electrons of the solvent is not a general mechanism for Intramolecular Charge Transfer (ICT). 1.7 Environmental effect on excited state complex: Works from quite an early period have witnessed the importance of environments on different photo-processes in chemistry as well as in biology [158]. Solvents have their own intrinsic properties due to which they can alter the reaction pathways by changing the energies of the reactants and contributing to the final states of a reaction [159]. The environment around the participating molecule/s in the intramolecular charge transfer reactions takes a major role in controlling the phenomena. The following section will deal with the characteristics of ICT in homogeneous and microheterogeneous environments imparted by micelles, reverse micelles, proteins and cyclodextrins.

46 Introduction Homogeneous environments: The photophysical properties of many fluorophores, especially those containing polar substitutents on the aromatic rings are known to be sensitive to the chemical and physical properties of the solvents. The process of rearrangement of solvent dipoles around an instantaneously created charge or dipole is known as solvation dynamics [81,82, ]. Ultra short laser pulses have been developed to study the sub-picosecond time scale phenomena of solvation dynamics [82, ]. Redistribution of electron density in the excited state results in a change in the dipole moment of the probe, thus creating a nonequilibrium state. The solvent environment cannot follow this sudden change and undergoes a modification in terms of rearrangement around the probe leading to a shift in the fluorescence band of the dye system [164,165]. The positions of the charge transfer absorption bands are different in different solvents. If there are low unfilled orbitals in the solvents, it is likely to have a strong affinity for electrons. If the solvent accepts electron from the solute molecules, a charge transfer to solvent (CTTS) complex is said to be formed [164,165]. The donor capacity of the solute in the ground state and excited state are different. If it is greater in the ground state, then the ground state energy level gets stabilized and blue shift occurs. Greater solute solvent interaction in the excited state compared to the ground state leads to red shift. Mataga et al. [78,90] has interpreted the red shift of indole and its derivatives in terms of generalized dipole dipole interaction between the solute and the solvent in the excited state. Although in most of the cases it has been shown that the dynamics of the ICT process is controlled by the nature of the solvent [83, ], the different experimental ICT rates obtained for many different ICT probe molecules in the same solvent suggest that the overall dynamics depends not only on the solvation but also on the structural aspect of the probe molecules. Dynamics of the ICT process is therefore dependent upon solute as well as the solvent. Recently Isihida et al. [169] studied the dynamics of the intramolecular charge transfer process in ultracold clusters of 9,9-bianthryl with water in

47 Introduction 27 supersonic jet using picoseconds time-resolved spectroscopy. They observed that the LE state is changed to the unrelaxed CT state in a time scale <20 ps. Following this, the unrelaxed CT state relaxes in 50 ps time scale to a new equilibrium state. The dynamics of the ICT process depends strongly on the solvent polarity of the medium. To investigate the effect of solvent polarity on the ICT process a large number of reports have come out [83, ]. An increase in the solvent polarity is expected to stabilize the ICT state. With a rise in the solvent polarity, the quantum yield of the CT emission initially increases and after reaching a maximum decreases with a further rise in polarity while the emission maximum monotonically shifts to the red [ ,175]. To explain this, it was considered that there are two competing processes formation of the ICT state and subsequent non-radiative decay of the ICT state. The rate of formation of the ICT state increases with an increase in the solvent polarity [180,181]. In a highly polar solvent like water, although the formation of CT state is highly favored, the stabilization of the state is so great that, owing to the proximity of the stabilized CT state and the low lying triplet/ground states, the nonradiative decay is facilitated very much, resulting in the lowering of the net fluorescence yield of the CT emission [81,180,181]. The formation time for the ICT state can be determined experimentally, either from the rate of decay of the LE emission or from the rate of formation (growth) of the CT state, the latter one being more dependable [160,182,183]. The solvent dipoles relax around the newly created solvent dipoles at a rate corresponding to longitudinal relaxation time [184,185]. Depending on the relative rates of the molecular relaxation and the solvent relaxation, two distinct cases may arise. When solvation is slower than the molecular relaxation rate, the overall rate is governed by the solvation. This is called the dynamic polarity effect. In this case creation of the ICT state can be considered instantaneous and after the formation, the ICT state gradually loses energy due to solvation. As a consequence the ICT emission exhibits a time dependent or dynamic Stokes shift [ ]. A dynamic Stokes shift is conclusive evidence for dynamic polarity effect. The other extreme is static

48 Introduction 28 polarity effect, when molecular rate is slower than the solvation and hence is rate determining. For DMABN, Hicks et al. observed that the TICT rate is slower than the salvation relaxation time and there is no dynamic Stokes shift [182,183] and the process falls under the purview of static polarity Microheterogeneous environments: The last two decades have witnessed the importance of the organized assemblies on biological and photophysical processes. Reactants accommodated in molecular assemblies like micelles, reverse micelles, cyclodextrins, vesicles, etc., often achieve a greater degree of organization compared to their geometries in homogeneous solution, can mimic reactions in bio-systems and also have potential for energy storage [188]. The interiors of these assemblies are quite different from the bulk solution phase. For aqueous solutions of micelles, cyclodextrins, proteins, the inner core is relatively less polar than the bulk aqueous phase. A reverse picture is found when one deals with reverse micelles. Not only the polarity but also the local viscosity/rigidity within the organized assemblies are appreciably different from the bulk liquid medium; as a result, these can alter a photo-process drastically. In this section, we will discuss the architecture of a few organized assemblies and see how they can modify the photo-processes like ICT Micelles: Surfactants can self-organize under specific environmental conditions in solution to form micelles. The micelle-forming amphiphiles or surfactants essentially fall in two categories, ionic and nonionic. They basically contain non-polar hydrocarbon or polyoxyethylenic chain (called the tail) and an ionic or polar group (called the head). When self-interactions of both surfactants and solvent molecules cannot be compensated by their mutual interactions, the surfactant molecules tend to associate in a regular pattern forming association colloids or micelles [189]. Generally micelle in aqueous solution forms a roughly spherical or globular aggregate but ellipsoid, cylindrical and bi-layer are also possible. The size of micellar aggregates

49 Introduction 29 is normally 1 10 nm and the aggregation number, i.e., the number of surfactant molecules per micelle, ranges from 20 to 200. The core of a micelle is essentially dry and consists of the hydrocarbon chains with polar and/or charged head groups projecting outward into the bulk water. The core is surrounded by a polar shell, which is called the Stern layer for an ionic micelle and palisade layer for a non-ionic micelle (Fig. 1.7). Comprehensive information on the structure of the micelles has recently been obtained through small angle X-ray and neutron scattering studies [ ]. According to these studies, the thickness of the Stern layer is 6 9A for cationic cetyl trimethyl ammonium bromide (CTAB) and anionic sodium dodecyl sulphate (SDS) micelles, whereas the palisade layer is about 20 thick for non-ionic Triton X-100 (TX- 100). Radius of the dry hydrophobic core of TX-100 is about A, and thus overall radius of TX-100 micelles is about 51 A. The overall radius of CTAB and SDS micelles are about 50 and 30 A, respectively. The difference in polarity and viscosity in the different sites of micelles render them as very attractive model media for many important photo-processes including ICT [102,162,192]. Figure 1.7: Schematic diagram of micelle

50 Introduction 30 The surfactant above a certain concentration (critical micellar concentration, CMC) forms aggregates (micelles) with a relatively hydrophobic (water repellent) core and a hydrophilic (water attracting) surface (Figure 1.7). Water is not completely excluded from the inner core and the individual detergent monomer exchange continuously with free monomer in solution [193]. Micelles can be formed from anionic, cationic and neutral surfactants. The ionic micelles are the most common one and the polar or ionic fluorophore behave differently according to their charge (Figure 1.8). The charge of the surfactants has no relevance for neutral molecule. Below the CMC the surfactants is present as monomers and in above the CMC any increase of the surfactant concentration leads to more micelles being formed with no variation of the monomer concentration. This relationship can be expressed as [ M ] = [ Sur] [ CMC] where [M] is the micelle concentration, [Sur] is the surfactant concentration and N is the aggregation number, i.e., the number of surfactant monomers per micelle. The CMC can be determined by variety of methods that are sensitive to physiochemical change in the solution due to micelle formation. One of the most common methods is variation of the relative fluorescence intensity with surfactant concentration. Generally above CMC the fluorescence intensity shows discontinuity. In micellar environment the ICT process is largely inhibited, controversially, due to either the reduction in polarity or enhanced steric constraint, and hence the fluorescence yield, fluorescence lifetime and fluorescence anisotropy of the CT emission increase markedly [102,194,195]. Since the dynamics of the ICT process is sensitive to the polarity of the medium, the microscopic polarity of micellar microenvironment can influence the rate of ICT process [170]. In case of structurally modified 4-aminophalimide derivatives, which is nearly nonfluorescent in water, the emission quantum yield increases nearly 7, 9 and 22 times and the corresponding lifetime 4, 2 and 5 times upon binding with SDS, CTAB and TX-100, respectively N

51 Introduction 31 [196]. For p-toluidinonaphthalene sulphonate, TNS (a TICT probe), which is also nearly nonfluorescent in water, the emission quantum yield and lifetime increases nearly 500 times on binding to non-ionic micelles TX-100 [175]. Such increase in the emission intensity and lifetime arises due to the marked reduction of the non-radiative ICT process inside the less polar micellar microenvironment. Figure 1.8: Approximate representation of anionic and cationic micelle in aqueous solution. The two basic concepts of microheterogeneous systems are compartmentalization and preferential location of substance. By compartmentalization we understand that substance cannot move freely between different parts of the system i.e. a barrier is present. Preferential location refers to the fact that substances are more soluble or selectively binds to a particular part of the microheterogeneous systems. For example a water insoluble 'oily' compound can be incorporated into the micellar core i.e. the partition coefficient will be highly favored towards the micellar phase. Studies in this field are aimed at understanding how the above mentioned concepts can influence physical and chemical processes. The preferential location can accomplish higher reaction efficiency by increasing local reagent concentrations, which is very much important for

52 Introduction 32 reagents that are soluble in different environments. It is reported in literature that the size and hydration of SDS micelle increases significantly with the salt addition [197,198]. The addition of salt causes increased micellar aggregation number and the content of mechanically trapped water in the Stern layer [199,200] along with increase in micellar hydration layer. Though there is an increase of hydration in the Stern Layer but around the ions present a kind of clustering occurs [199,200] which give rise to microviscosity. Further, the properties of the environment can influence the chemical reactivity of the substances Reverse micelles: Reverse micelles are the aggregates of surfactants formed in non-polar solvents, in which the polar head groups of the surfactants point inward while the hydrocarbon chains project outward into the non-polar solvent [201]. The wonderful property held by the reverse micelles is the ability to encapsulate fairly large amount of water molecules to form what is known as microemulsion. Up to 50 water molecules, per molecule of the surfactant, can be incorporated inside the bis (2-ethylhexyl) sulphosuccinate (commonly known as AOT) reverse micelles. Such a surfactant coated nanometer-sized water droplet, dispersed in a non-polar liquid, is called a water pool. The radius of a water pool varies linearly with the water to surfactant mole ratio; W. Reverse micelles afford the opportunity of examining molecules with various states of hydration, simulating situations present in water restricted environments prevailing in supramolecular systems [202]. Such model systems are able to capture a number of essential features of biological membranes though lacking much of their complexities. In particular, reverse AOT micelles have been studied extensively during the past decades [ ]. It is a nontoxic system and can be used in pharmaceutical and medicinal preparations. Unlike most amphiphiles and like many phospholipids, it is a double tailed anionic surfactant, which can be conveniently used for solubilization and emulsification purposes. As one of the several advantages of a reverse AOT micellar system, the size of the water pool can be controlled

53 Introduction 33 precisely at the nanometer level through the molar ratio of water/surfactant. Therefore, size effect on chemical and physical properties in nanometer dimension can be studied by the use of AOT reverse micelles [201]. In heptane the radius of such water pool is about 2W (in Ǻ) where W is the number of water to the surfactant molecules [207,208]. Several non-ionic surfactants have recently been reported to form reverse micelles in pure and mixed hydrocarbon solvents [209,210]. The difference in polarity and viscosity in the different sites of the reverse micellar environments render them as a very attractive model for many important photo-processes like ICT [211]. In AOT microemulsions, presence of the negatively charged head group causes a sharp gradient in ph/poh over the nanometer sized water pool. Menger and Saito reported that the acid base property of p-nitrophenol (PNP) gets substantially modified in AOT microemulsion [211]. On the basis of their experimental observation they concluded that the pka of PNP in the AOT microemulsion is greater than that in bulk water. The dynamics of excited state proton transfer processes in organized assemblies are often quite different from those in ordinary solutions. The sharp variation of local ph on the water pool of the AOT microemulsions affects the intermolecular proton transfer process strongly. Politi et al. studied the excited state deprotonation of a tri-negatively charged probe hydroxypyrene trisulphonate in AOT reverse micelles [212]. They observed that in large water pool the proton transfer process is similar to that in bulk water while it is quite different in small water pool. They concluded that in large water pool, due to electrostatic repulsion from the negatively charged AOT ions, the negatively charged probe remains in the water pools, far from AOT anion and experiences almost bulkwater like environment. But in small water pool, the very different local ph, near the AOT anions, renders the deprotonation/reprotonation behavior quite different from the ordinary aqueous solutions. Marzola and Gratten, studied frequency domain fluorescence spectroscopy on proteins in reverse micelles [213]. Measuring fluorescence and anisotropy decays of the tryptophan residue of human serum albumin (HSA) and liver alcohol dehydrogenase (LADH) in

54 Introduction 34 AOT reverse micelles; they concluded that the rotational correlation times for the internal motion and the overall protein rotation in reverse micelles decreases with an increase in hydration [213]. Karukstis et al. analyzed the emission spectra of another ICT probe 6-propionyl-2- dimethylaminonaphthalene (PRODAN) in microemulsion and showed that PRODAN molecules are distributed at different sites within the microemulsion exhibiting different emission spectra [214]. Inside the water pool of the microemulsion TICT process of several probes including 1- anilino-8-naphthalene sulphonate (ANS), TNS and nile red have been found to be significantly retarded compared to that in bulk water and the lifetimes of the probes increase from picoseconds to several nanoseconds in the water pool [215]. In a recent study Hazra and Sarkar have investigated the relative retardation of the ICT rate and solvation dynamics of coumarin 490 in the water pool of AOT reverse micelle compared to that in normal water [108] using picosecond time-resolved emission spectroscopy. The retardation of ICT is much smaller compared to the 1000-fold decrease in the solvation dynamics in the water pool of the reverse micelle. The retardation of the ICT/TICT process inside the water pool of the microemulsion is ascribed to the lower static polarity of the water pool compared to the bulk water. Through a series of works Sarkar et al. have studied various dynamical aspects of ICT process in both aqueous and reverse micellar environments. They have investigated the relative retardation rate of ICT process and salvation dynamics in aqueous and non-aqueous reverse micellar environments using different coumarin derivatives [101,74,177,203]. One of our recent works has exploited the modification of the photophysics of 3-acetyl-4-oxo-6,7-dihydro-12H-indolo- [2,3- a] quinolizine (AODIQ), to determine the polarity and viscosity of the microenvironment around the probe with a variation of the core size (W) of the reverse micelle [216]. The fluorescence anisotropy is found to decrease sharply with an increase in the W value. The energy transfer process is modulated more dramatically in reverse micellar environments than the micellar environments. The rate constant of energy transfer process increases by more than one

55 Introduction 35 order of magnitude in organized surfactant media. De et al. showed that the energy transfer process is very much favored in micelles and reverse micelles compared with neat water [217]. In the same report by calculating the overlap integral, energy transfer efficiency, rate constant of energy transfer, etc., they established the reverse micellar environment as a better medium for energy transfer compared to the normal micellar media. Within the reverse micelles, smaller water pools facilitate energy transfer due to proximity between the donor and acceptor molecules. Seth et al. have described the role of structures of the donors in the energy transfer processes. Using various 7-amino coumarin dyes as donors and only rhodamine 590 as the acceptor they investigated the energy transfer process in methanol and acetonitrile solubilized non-aqueous reverse micelles using steady-state and time-resolved fluorescence spectroscopy [218]. The detailed investigation reveals that apart from the environmental factors the energy transfer process depends on the structure of the donor Proteins: Among the various available proteins, in the present overview we have focused our attention on serum albumins (human serum albumin and bovine serum albumin), the most widely studied abundant proteins in plasma that are identified as major transport proteins in blood plasma for many compounds like fatty acids, hormones, bilirubin and many important drugs [219]. Moreover, albumins are involved in the maintenance of colloid blood pressure and are implicated in the facilitated transfer of many ligands across organ-circulatory interfaces such as in the liver, intestine, kidney and brain [219]. The three dimensional structure of human serum albumin (HSA) has been resolved [220]. However, for bovine serum albumin (BSA), there are conflicting results so far as the structural aspect is concerned. As a matter of fact both BSA and HSA are similar in sequence and conformation, but differ in the number of tryptophan (Trp) residues. BSA (583 aa) and HSA (585 aa) are characterized by a high homology (80%) and similar conformation, containing 17 disulphide bridges and a series of nine loops, assembled in three

56 Introduction 36 domains (I III), each containing two subdomains, A and B [220,221]. The principal binding sites are located in subdomains IIA and IIIA and it is generally assumed that in BSA and HAS these sites are homologous, although they may differ in affinities [221]. From the determined crystallographic structure of HAS it was proposed that in this protein the single tryptophan residue (Trp-214) is located in IIA binding site, while Lys-199 and His-242 are involved in the protein ligand interaction. In case of BSA, there are two tryptophan residues, Trp-212 and Trp Trp-212 is located in a similar hydrophobic microenvironment as the single Trp-214 in HSA (subdomain IIA) [220,221], whereas Trp-134 is more exposed to solvent and it is localized in the subdomain IA. The interior of the serum albumin protein is undoubtedly less polar than the bulk water phase [222]. The reduced polarity in the proteinous environment changes photoprocesses like ICT remarkably Cyclodextrins: Cyclodextrins (CD) comprises a family of cyclic oligosaccharides of six, seven or eight D- glucopyranose (C 6 H 10 O 5 ) units (the three popular and well known ones are α, β and γ CD with diameters of ~5.7, 8.5 and 9.5 Å respectively) obtained from a starch by enzymatic Figure 1.9 Structure of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin Figure 1.10 Functional structural scheme Figure 1.11 Molecular dimension of cyclodextrins of β-cyclodextrin

57 Introduction 37 degradation (Figure 1.9).In aqueous solutions they form a well-defined hydrophobic cavity and encapsulate molecules of suitable sizes and the resulting supramolecules often exhibit properties drastically different from those of the free guest molecules in aqueous solutions [223]. The relatively hydrophobic interior and hydrophilic exterior of their molecular pockets make them suitable and fascinating hosts for supramolecular chemistry and for studying the spectroscopy and dynamics of several molecular systems. Investigation of cyclodextrins chemistry has been on the increase for several decades. The description of the structure and properties of cyclodextrins and their application has been the subject of several monograms [224,225] and numerous review articles [226,227]. Studies on CD complexes with aromatic molecules using steady state and nanosecond spectroscopy have been reported [228]. These studies are aimed to understand and control the photophysical and photochemical behavior of organic guests (such as fluorescence and phosphorescence enhancement), charge and proton transfer, excimer / exciplex formation, photocleavage and photo-isomerization etc. Most of these reports show the effects of molecular restriction, due to the cavity size of the host and protection of the guest provided by CD cavity and its low polarity relative to that of water on the photophysical and photochemical properties of the encapsulated guest. The α-, β- and γ-cyclodextrins contains six, seven and eight amylose units respectively. For each of them the height of the cavity is ~ 8 Å. Figure 1.10 shows the functional structure of β-cyclodextrin and Figure 1.11 shows the molecular dimensions of cyclodextrins. In aqueous cyclodextrin solutions one encounters three regions of distinctly different polarity, namely, the interior of the cavity, the rim of the cavity and bulk aqueous solution. The interior of the cyclodextrin cavities resembles cyclic ethers (e.g. dioxane) and is relatively non-polar while the rims are moderately polar due to the hydroxyl groups and nearby water molecules.the main interest of studying charge transfer processes (such as TICT process in cyclodextrin solutions) is to find out how the cavity affects the dynamics of the charge transfer

58 Introduction 38 processes, the twisting motion and whether it is due to the reduced polarity of the cavity or due to the restrictions imposed on the twisting motion. It is also interesting to investigate whether the dynamics in the interior of the cavity are different from those at the rims or not Polymers: A polymer is a large molecule (macromolecule) composed of repeating structural units typically connected by covalent chemical bonds. While polymer in popular usage suggests plastic, the term actually refers to a large class of natural and synthetic materials with a variety of properties. A heteropolymer or copolymer is a polymer derived from two (or more) monomeric species, as opposed to a homopolymer where only one monomer is used [229]. Copolymerization refers to methods used to chemically synthesize a copolymer. Since a copolymer consists of at least two types of constitutional units (not structural units), copolymers can be classified based on how these units are arranged along the chain [230]. These include: Alternating copolymers with regular alternating A and B units (A, B are the basic units) Periodic copolymers with A and B units arranged in a repeating sequence (e.g. (A-B-A- B-B-A-A-A-A-B-B-B) n ) Statistical Copolymers are copolymers in which the sequence of monomer residues follows a statistical rule. If the probability of finding a given type monomer residue at a particular point in the chain is equal to the mole fraction of that monomer residue in the chain, then the polymer may be referred to as a truly random copolymer [231]. Block copolymers comprise two or more homopolymer subunits linked by covalent bonds. The union of the homopolymer subunits may require an intermediate nonrepeating subunit, known as a junction block. Block copolymers with two or three distinct blocks are called diblock copolymers and triblock copolymers, respectively.

59 Introduction 39 Block Copolymers: A special kind of copolymer is called a "block copolymer". Block copolymers are made up of blocks of different polymerized monomers [232]. For example, PS-b-PMMA is short for polystyrene-b-poly(methyl methacrylate) and is made by first polymerizing styrene, and then subsequently polymerizing MMA from the reactive end of the polystyrene chains. This polymer is a "diblock copolymer" because it contains two different chemical blocks. You can also make triblocks, tetrablocks, multiblocks, etc. Diblock copolymers are made using living polymerization techniques, such as atom transfer free radical polymerization (ATRP), reversible addition fragmentation chain transfer (RAFT), ring-opening metathesis polymerization (ROMP), and living cationic or living anionic polymerizations. An emerging technique is chain shuttling polymerization. Recent research in block copolymers suggests that they may be useful in creating selfconstructing fabrics with potential utility in semiconductor arrays (for example, computer memory devices) by assembling fine details atop a structured base created using conventional microlithography methods[233] DNA: Calf-thymus DNA (ct-dna) is a polymer. The DNA backbone contains an alternative sugar phosphate sequence. ct-dna has relatively low protein content with a highly polymerized skeleton. DNA posses a unique structure consisting of a hydrophobic core of the hydrogen bonded base pairs surrounded by a hydrophilic sugar-phosphate backbone [234]. DNA consists of the four natural nucleotides adenine (A), guanine (G), cytosine(c) and thymine (T) (ref same). Interaction of DNA with drug molecules, in general, involve three types of binding modes: (1) electrostatic binding between the negatively charged DNA phosphate backbone that is along the external DNA double helix and the cationic or positive end of the polar drug, (2) groove binding

60 Introduction 40 involving hydrogen bonding or van der Waals interaction with the nucleic acid bases in the deep major groove or the shallow minor groove of the DNA helix and finally, (3) intercalative binding where the drug intercalates itself within the nucleic acid base pairs. Groove binding involves docking the thin ribbon-like molecules in the DNA minor groove, in close proximity to the sugar phosphate backbone. 1.8 The motivation and aim of the thesis: Photo-induced charge transfer reactions is so extensive and diverse that it is one of the most interesting topics in current photochemistry both from the point of view of fundamental and applied science. Organic bichromophoric molecules in which the donor and acceptor moiety is connected by a single bond or double bond are of great interest to observe the intramolecular charge transfer state in the excited relaxed most stable and most favorable energy state. A great attention has been drawn to explore the different mechanism of intramolecular charge transfer of several bichromophore as it is the primary function for basic mechanism of biological and chemical energy conversion. All the three isomers of (4-(dimethylamino) styryl)-1-methylpyridinium iodide (DASPMI) possess interesting excited state photophysical properties [235]. One of its isomers selectively stains mitochondria of living cells [236], it has a satisfactory long-term photo stability and has application in polymer science [237]. The application of this molecule also exits in medicinal aspects where it may be used as fluorescence indicator because it exhibits strong charge shift in light excitation [237]. As fluorescence intensity of the probe is a dynamic measure for membrane potential of mitochondria [235], this property may be used to detect the membrane potentials of different biological membrane. These molecules interestingly have the possibility of multiple single-bond and a double-bond twisting involvement towards the nonradiative TICT deactivation. In ortho-isomer (DASPMI) the twisting of double bond and the twisting of

61 Introduction 41 dimethylamino group do not contribute to the TICT state only the rotation of remaining single bonds are important. Different microheterogeneous organized systems such as cyclodextrins and micelles have of great importance in spectroscopy due to their biological implication. The interior of some organized assemblies (cyclodextrins, micelles) is quite non-polar but the bulk media is highly polar. The photophysics and photodynamics of organic bichromophore are greatly influenced due to subsequent non-polar or more polar environment as well as different viscosity and also due to the impediment imposed on the free motion of the molecule by the restricted geometry inside the organized assemblies. Influence of photophysics and photodynamics excited state of bichromophore in different biological environment like protein, DNA helps us to probe the nature of the biological environment. In the dissertation efforts have been made to explore the importance of Twisted Intramolecular Charge Transfer (TICT) state over the Intramolecular Charge Transfer (ICT) process of 2-(4-(dimethylamino) styryl)-1-methylpyridinium iodide (DASPMI), an organic bichromophore in different environments including nano cavities, diblock polymer, protein, DNA and microheterogenous medium. In addition to gaining insight about the excited state of DASPMI in those environments considerable efforts have been made to understand nature of host media and specific interactions with those media. Apart from experimental study some quantum chemical calculation has been aimed at in order to understand the interaction of bichromophore with the environment. 1.9 Outlines of the problems presented in the thesis: The ground and excited state photodynamics of a dye in different restricted environments like micelles, reverse micelles, cyclodextrins, polymers, protein and DNA has been analyzed in this work. The present thesis focuses its attention towards unrevealing the properties of different

62 Introduction 42 organized media through probing the environment sensitive dye. The thesis comprises six chapter ( chapter 3 to chapter 8) including a review on charge transfer reaction in Chapter 1 followed a brief account of the experimental techniques with the details of instrumentation set up and methods of calculations of certain physical parameters in Chapter 2. The methods of compound preparation and purification are also described in Chapter 2. Excited state photophyiscs of 2-(4-(dimethylamino) styryl)-1-methylpyridium iodide (DASPMI) in ionic and nonionic micelles have been reported in Chapter 3. The probing of three micelles have been done with the help of DASPMI using the environmental sensitivity. The necessary shift and change in intensity reveals that DASPMI enters into all three micelles in different positions from the water solution due to active hydrophobic force and electrostatic field. The binding constant and variation of emission intensity reveal that only non-ionic micelle the probe enters in the core region whereas in ionic micelles it is anchored in the interfacial region with different orientations. From the observed data it may be guessed that the donor group (NMe 2 ) of the probe is in Gouy-Chapman layer and the acceptor group resides in the Stern layer for cationic micelle whereas for anionic micelle the reverse phenomena is observed With salt addition increase in aggregation number, mechanically trapped water and the microviscosity modulate emission intensity. Excited state photophysics of 2-(4-(dimethylamino) styryl)-1-methylpyridium iodide (DASPMI) in anionic and cationic reverse micelles (RMs) have been described in Chapter 4. From steady state data and metal quenching effect there was an indication to reside the molecule in the interfacial region near the core keeping acceptor group towards water pool in anionic micelle and for cationic micelle it was away from the core. The partition constant K p in two RMs correlate well with the position of emitting probe DASPMI in RMs interface and it corresponds nicely with the values obtained from anisotropy data. The rotational restriction experienced by DASPMI in anionic reverse micelle decreases with increasing hydration. The in-

63 Introduction 43 situ measurements of micropolarity and microviscosity around the probe with increasing pool size (W) show enhanced micropolarity and a decrease in microviscosity. Chapter 5 deals with photophysics of 2-(4-(dimethylamino) styryl)-1- methylpyridium iodide (DASPMI) in different Cyclodextrin environments. Nice 1:1 inclusion complex with β-cd in the excited state could be found with the dimethylamino group of the molecule sticking out as revealed from steady state and time-resolved emission. The minimum energy of the complex found to be nearly 5 Å length of the molecule with dimethylamino part sticking out in the bulk water and the molecule enters into β-cd at an angle 10 0 as reveled from semiemperical calculations. In γ-cd the o-daspmi molecules remain hydrogen bonded with the rim as 1:1 or 1:2 association and enter the γ-cd cavity after urea breaks the hydrogen bonds. In γ-cd the orientation of o-daspmi is along the axis of the cavity and the long axis of the molecule is perpendicular to the opening of the cavity. The interaction of anionic micelle Sodium dodecyl sulfate (SDS) and amphiphilic block copolymers poly ethylene-b-polyethylene glycol (PE-b-PEG) and the sharp change of excited state charge transfer complex photophysics of 2-(4-(dimethylamino) styryl)-1- methylpyridinium iodide (DASPMI) inside the supramolecular assembly have been addressed in Chapter 6. The dramatic enhancement of emission intensity of DASPMI incorporated inside the nano structure formed by micellar and polymeric chains indicates completely different environment, compared to that in water and micellar system. Huge increase in rotational relaxation time obtained from time-resolved anisotropy decay and the value of order parameter is indicative of much restrictive regime in the self assembly system. The wobbling and translational motion of the probe is also restricted inside micelle-polymer aggregate due to the presence of polymer chains. The translational diffusion coefficient gets drastically reduced due to the aggregation.

64 Introduction 44 Chapter 7 deals with the mode of interaction of 2-(4-(dimethylamino) styryl)-1- methylpyridinium iodide (DASPMI) with Bovine Serum Albumin (BSA) in buffer as well as in reverse micelle (RM) environments. The most important thing of the investigation is that subdomain IIA of BSA is doubly active inside RM compared to that in buffer solution and the activity of subdomain IIIA in RM is halved compared to that in buffer solution. The fluorescence resonance energy transfer (FRET) from BSA to DASPMI in buffer solution is measured to be 27% whereas, for that in reverse micelle it is found to be increased to 36%. The greater FRET efficiency also indicates that probe is located much more nearer to Tryptophan (Trp) of BSA and stronger binding between BSA-DASPMI complex in RM than in buffer. The mode of binding of 2-(4-(dimethylamino) styryl)-1-methylpyridinium iodide (DASPMI) with calf thymus DNA as revealed from different steady state and timeresolved emission spectroscopic measurements has been reported in Chapter 8. Fluorescence enhancement of DASPMI and its quenching by KI and also strong dependence on ionic strength or salt in controlling the binding of DASPMI with DNA by electrostatic interaction confirms groove binding. The value of binding constant from emission and association constant from circular dichroic spectrum also indicates weak binding. Increase in steady state anisotropy and fluorescence lifetime hints at binding with DNA. Visualization of theoretical modeling confirms groove binding as a crescent with a curvature of Ǻ, which is complementary to the natural curvature of the minor of B-DNA.

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78 Experimental Experimental Techniques: Experimental investigations on the photophysical studies in different environments of organic bichromophore, selected for the present research work were carried out by steady state electronic absorption, steady state and time resolved (nano-second order) emission, steady state excitation, steady state and time resolved (second/millisecond) anisotropy and Scanning electron microscopy. The measurements of some relevant parameters like fluorescence quantum yield, degree of polarization of fluorescence emission and excitation have been done by standard procedure. The different techniques used for experimental measurements and the different instruments used throughout the work have been presented briefly in this chapter. The different methods used for solvent purification as well as for purification of different compounds have also been included in the text Absorption Spectra: At ambient temperature (296 K) the steady state electronic absorption spectra of dilute solutions (~ mol dm -3 ) of all the samples, used for all investigations, were recorded using 1 cm path length rectangular quartz cells by means of an absorption spectrophotometer (Shimadzu UV-VIS 2401PC). A schematic diagram of the spectrophotometer is shown in figure-2.1. Light from a suitable lamp is focussed on the slit S 1 and then allowed to fall on a grating (1200 lines/cm.) using mirror M 1 and M 2. The light incident on the grating is dispersed and then dispersed light is successively reflected by mirror M 3 and M 4 to form an image on slit S 2. The monochromatic beam from S 2 is chopped by rotating mirror RM 1 rotating at a speed of 1500 rpm. The reference and sample beams after passing through the respective cells is recombined by another rotating mirror RM 2 and finally allowed to fall on the photomultiplier tube (PM). The rotation of the two mirrors is synchronized by a belt system to make the recombination effective. Components of a typical absorption spectrophotometer are shown in figure 2.2. The measurements were performed with an

79 Experimental 59 optically matched pair of quartz cell, one containing the sample and the other the solvent as reference. Figure - 2.1: Schematic diagram of absorption spectrophotometer Figure 2.2: Components of a typical absorption spectrophotometer Excitation Spectra: In order to supplement the absorption spectral data, particularly to locate lowest excited singlet and triplet states, the technique of excitation spectroscopy [1-5] was employed. The excitation spectra have been recorded with Hitachi fluorescence spectrophotometer (model F-

80 Experimental ) with the emission monochromator set at the maximum of fluorescence intensity and the exciting monochromator is scanned from shorter wavelength values in the vicinity of the emission band of the molecule. The details of the spectrophotometer have been described in the following section. The excitation spectra obtained in this procedure were found to agree fairly well with the corresponding absorption spectra of the compounds, studied in the present research work, these findings confirm the absence of any impurity in the solvents Fluorescence Spectra: Throughout the work all the fluorescence emission spectra are recorded by with a Hitachi fluorescence spectrophotometer (model F-4500) at ambient temperature. For room temperature emission measurements 1cm quartz cell was used while special Dewar in which a long cylindrical quartz tubing (cell) (3 mm in diameter) could be introduced, was used for low temperature (77 K) measurements. The steady state and time resolved (second/millisecond order) phosphorescence emission spectra also were recorded by Hitachi F-4500 fluorescence spectrophotometer. The figure-2.3 depicts the schematic diagram of the Hitachi F-4500 fluorescence spectrophotometer. A 150 W Xenon (Xe) lamp enclosing gas at Figure - 2.3: Schematic diagram of the Hitachi fluorescence spectrophotometer.

81 Experimental 61 high pressure of around 9 atm. was used as the excitation source in Hitachi F-4500 fluorescence spectrophotometer. The beam from the Xe lamp is incident on the excitation monochromator to irradiate sample. Part of this beam is directed to detector by means of a beam splitter. Emitted light from the irradiated sample is incident on the monochromator. The photomultiplier ( nm) detects the emission intensity of particular wavelength selected by the monochromator. The output of the photomultiplier in the fluorescence (or phosphorescence) measurement mode is divided by the output of the monitor detector to obtain data for the purpose of light quantity compensation of the light source. The resolution of the spectrophotometer is 1.0 nm and the response time is 4 ms. The instrument is corrected in order to give the corrected spectra by eliminating instrumental response such as wavelength characteristic of the excitation and emission monochromators and the detector (photomultiplier). The corrected emission and excitation spectra could be measured in the region nm Time-resolved spectroscopy: There are different time-resolved techniques that are used to determine properties like the emission lifetimeτ, or charge transfer rate constants Time-correlated single photon counting: Time correlated single photon counting (TCSPC) spectrometer used for measuring the time resolved fluorescence intensities could be described as follows [6,7]. In this method the sample is excited with a laser pulse, but the detection measures the time difference between the pulse of excitation and the arrival of the first fluorescence photon from the sample. The light intensity must be adjusted so that only a single photon is observed for a large number of excitation pulses. Such a low count rate must be used to ensure that only a single photon arrives for which a photon is counted [8]. If more than a single photon arrives per pulse, then

82 Experimental 62 the time resolved intensity profile is artificially shifted to shorter times, which does not reflect the actual profile. Figure 2.4: Schematic diagram of Time Correlated Single Photon Counting (TCSPC) spectrometer. To determine the time difference between the exciting laser pulse and the arrival of the fluorescence photon a time to amplitude converter (TAC) coupled with a multi channel pulse height analyzer (MCPHA) is used (Figure 2.4). A fraction of the incident laser light pulse to the laser pulses of the other wavelength from the same laser which is not used for the excitation of the sample are fed to a photodiode (or PMT). This photodiode sends a reference signal, which is called as START signal and the START signal starts TAC to generate a voltage ramp that increases linearly with time. The incident laser pulse fed to the sample gives fluorescence and is collected by a lens and focused on to micro channel plate photomultiplier (MCP PMT) after selecting the wavelength of interest by a monochromator. The arrival of fluorescence photon at MCP PMT gives a signal called as STOP signal to TAC, which then stops the increasing voltage ramp. The voltage generated by voltage ramp

83 Experimental 63 between the START and STOP signal is proportional to the time difference between the exciting laser pulse and the arrival of the first photon. If no fluorescence photon is detected within the TAC time range (which can be preset) the voltage ramp is reset and TAC waits for the next START signal from the photodiode. The voltage generated by the voltage ramp between the consecutive START and STOP signals in the TAC is recorded into the MCPHA bins (channels) in a digital manner. Each channel counts the number of times a specific voltage level is obtained and by repeating the cycle many times per second a complete spectrum produced in the memory of the MCPHA. The probability that an emitted photon will be detected within a given time interval, which corresponds to a single channel on the MCA, decreases with increasing time. This decay profile can give very precise estimates of radiative lifetimes. Constant fraction discriminator (CFD) is used for the precise timing of the arrival of the photon. The same procedure for the determination of arrival of fluorescence is repeated until a large number of counts are achieved as a histogram recorded in MCPHA. All the measurements are performed by keeping the excitation polarizer at the vertical position and the emission polarizer at the magic angle (54.7 ) with respect to the excitation polarizer. The fluorescence collected at the magic angle with respect to the excitation polarization is free of any anisotropy components and represents the actual total fluorescence intensity decay. The fluorescence collected in the perpendicular direction with respect to the direction of excitation from the sample by a focusing lens is dispersed in a monochromator. Appropriate cut-off filters are placed just before the focusing lens to prevent any scattered exciting light from entering the monochromator from the sample chamber Fluorescence lifetime measurement: The fluorescence lifetime of a molecule is the average time that the molecule resides in the excited state before photon emission occurs, i.e. the average time that passes between absorption and emission of a photon. The most direct way to determine the fluorescence

84 Experimental 64 lifetime is to excite the sample with a short pulse of light and measure the fluorescence response as a function of time. When a fluorescent sample is excited using a short light pulse, many probes enter the excited state at the same instant. The probes relax at different times t after the excitation pulse and the fluorescence intensity, F(t), decays in time following a first order kinetic law. The simplest form of a fluorescence intensity decay following a short excitation pulse is described by a single exponential function with time constant [8] exp t F0 F t (2.1) Where F 0 is the intensity at time t = 0. The single exponential function cannot describe all decay processes. In general decay processes are described by a sum of exponential decay functions with different amplitudes F i0 and the time constant i F t exp t Fi 0 i (2.2) i when the complete time course is covered by the analysis, from the excitation pulse till the time where all probes are relaxed to the ground state, the average fluorescence lifetime is define as the average of the fluorescence lifetime of each component weighted by its integral intensity [8]: i 2 Fi0 i. (2.3) F i i0 i The integral intensity of an exponential decay function equals t F0exp dt F 0 0. (2.4) Equation (2.1) represents an ideal case where the excitation pulse is infinitely narrow. If the pulse has a finite temporal width, the exponential fluorescence decay of equation (2.1) will be convoluted with the pulse profile function L(t), resulting in the more general expression ' F t F0 L t t dt t ' t ' exp (2.5) 0

85 Experimental 65 when the width of L(t) becomes zero, equation (2.5) transforms into equation (2.5). In a typical experiment both L(t) and F(t) are measured and the lifetime ( ) is recovered numerically, usually by an iterative least square algorithm. If the functions L(t) and F(t) were measured with infinite precision, then there would not be any restrictions on the measurement of short lifetimes with a given pulsed source. In a real experiment the noise component will eventually cause L(t) and F(t) to become indistinguishable from one another when the lifetime becomes significantly shorter than the excitation pulse width Nanosecond single photon counting techniques: Fluorescence lifetime of different organic molecules having lifetime >2 ns were measured by using time correlated single photon counting technique (TCSPC) fluorimeter (Figure 2.4.) constructed from components purchased from Edinburgh Analytical Instruments (EAI). The measurements were made in a conventional L-format arrangement. This instrument closely resembles Edinburgh Instruments model 199, UK. The excitation source consists of an allmetal coaxial N 2 flash lamp with an instrumental response function of about 1.3 ns full-width at half-maximum (FWHM) at repetition rate of 25 khz. A photomultiplier tube (PMT) XP2020Q was used for collecting the photons emitted by the fluorophores. The data were stored in a multichannel analyzer (MCA). A deconvolution fit was used to analyze the fluorescence decay curves over the entire region including the rising edge. The statistical parameter e.g., 2 (DW) values gave an indication of the goodness of fit. is given by definition, 2 and Durbin-Watson 2 2 Yi () FD () i N () i (2.6)

86 Experimental 66 2 Yi () FD () i χ = N σ () i (2.6) where Y (i) is the fluorescence decay data, F D (i) is the fitting function and σ (i) 2 is the statistical uncertainty of the datum value Y (i) i.e., statistical deviation. In such photon counting experiments used in the present study the expected deviation is, [ ] 1 2 Y i σ () i = () (2.7) The error limits for the fluorescence lifetime values were found to be ±0.4 ~ ns. All the solutions prepare for lifetime measurements were deoxygenated by purging with an argon gas stream for about 30 min Picosecond single photon counting techniques: Fluorescence lifetime of the organic compounds having lifetime < 3 ns were measured by using three types of pulsed laser system (i ) Nd:YAG laser system, (ii ) Ti:sapphire laser system and (iii ) diode laser system. The details of the instruments are given below. (i) Using Nd:YAG laser system: The excitation was realized at 300 nm using a coherent synchronously pumped, cavity dumped rhodamine 6G dye laser (702-1) pumped by a coherent CW mode-locked Antares 76-S Nd:YAG laser. The fluorescence and phosphorescence were detected at magic angle (54:7 o ) polarization using Hamamatsu MCP photomultiplier tube (2809U). The resolution of the set-up is 0.02 ns. For room temperature luminescence decay measurements the solutions were deaerated by the freeze-pump-thaw technique. (ii) Using Ti:sapphire laser system: The second TCSPC set-up used was (Millenia (5W) CW 532 nm pumped Tsunami Ti:sapphire mode-locked laser with pico-option ( nm), SHG and THG as the pump source from Spectra Physics USA) with pulsed width < 2 ps with 250 nm excitation. IBH 5000U Fluorescence spectrophotometer used for picosecond excitation and detection system with MCP-PMT:R3809U ( nm), Polarizers, NIM

87 Experimental 67 timing electronics and PC-based MCA with utility software and window-based data analysis software (IBH software library). Iterative shift of the fitted function as part of χ 2 (error ± 6 ps, standard deviation ± 4 ps). N 2 /H 2 filled 100 khz gated lamp is also available for nanosecond measurement as optional. (iii) Using semiconductor (diode) laser system: The samples were excited at 405 nm using a picosecond diode laser (IBH Nanoled-07). The emission was collected at a magic angle polarization using a Hamamatsu MCP photomultiplier (2809U). The TCSPC set up consists of an Ortec 935 QUAD CFD and a Tennelec TC 863 TAC. The data is collected with a PCA3 card (Oxford) as a multichannel analyzer. The typical FWHM of the system response is about 80 ps Scanning electron microscopy (SEM) and field emission SEM (FESEM): The scanning electron microscope (SEM) is a type of electron microscope that images the sample surface by scanning it with a high-energy beam of electron in a raster scan pattern [9]. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition and other properties. In a typical SEM, an electron beam is thermoionically emitted from an electron gun fitted with a tungsten filament cathode. Tungsten is normally used in thermionic electron guns because it has the highest melting point and lowest vapour pressure of all metals, thereby allowing it to be heated for electron emission. It is cost effective also. Other types of electron emitters include lanthunan hexaboride (LaB 6 ) cathodes. The electron beam, which typically has an energy ranging from a few hundred ev to 40 kev, is focused by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. When the primary electron beam interacts with the sample, the electrons lose energy by repeated random scattering and absorption within a teardrop-shaped volume of the

88 Experimental 68 specimen known as the interaction volume, which extends from less than 100 nm to around 5 µm into the surface. The size of the interaction volume depends on the electron's landing energy, the atomic number of the specimen and the specimen's density. The types of signals produced by an SEM include secondary electrons, back scattered electrons (BSE), charecteristics X-rays, light (cathodoluminescence), specimen current and transmitted electrons. Back-scattered electrons (BSE) are beam electrons that are reflected from the sample by elastic scattering. BSE are often used in analytical SEM along with the spectra made from the characteristic x-rays. Because the intensity of the BSE signal is strongly related to the atomic number (Z) of the specimen, BSE images can provide information about the distribution of different elements in the sample. The beam current absorbed by the specimen can also be detected and used to create images of the distribution of specimen current. Electronic amplifires of various types are used amplify the signals which are displayed as variations in brightness on a cathode ray tube. The raster scanning of the CRT display is synchronised with that of the beam on the specimen in the microscope, and the resulting image is therefore a distribution map of the intensity of the signal being emitted from the scanned area of the specimen. The image may be captured by photography from a high resolution cathode ray tube, but in modern machines is digitally captured and displayed on a computer monitor and saved to a computer's hard disk. In FESEM, a field-emission cathode in the electron gun of a scannning electron microscope provides narrower probing beams at low as well as high electron energy, resulting in both improved spatial resolution and minimized sample charging and damage [10]. FESEM produces clearer, less electrostatically distorted images with spatial resolution down to 1-1/2 nm, which is 3 to 6 times better than conventional SEM. High quality, low votage images are obtained with negligible electrical charging of samples. (Accelerating

89 Experimental 69 voltage range from 0.5 to 30 kv). Figure 4 shows a schematic diagram of scanning electron microscope. Here, JEOL, JSM-6700F FESEM is used. Figure 2.5: Sketch of a SEM Circular Dichroism (CD): Circular Dichroism (CD) is observed when optically active matter absorbs left and right hand circular polarized light slightly differently. It is measured with a CD spectropolarimeter. The instrument needs to be able to measure accurately in the far UV at wavelengths down to nm. In addition, the difference in left and right handed absorbance A(l)- A(r) is very small (usually in the range of ) corresponding to an ellipticity of a few 1/100th of a degree. The CD is a function of wavelength. CD spectra for distinct types of secondary structure present in peptides, proteins and nucleic acids are different. The analysis of CD spectra can therefore yield valuable information about secondary structure of biological macromolecules [11]. Physics of CD: Linear polarized light can be viewed as a superposition of opposite circular polarized light of equal amplitude and phase. A projection of the combined amplitudes perpendicular to the propagation direction thus yields a line (figure 2.6). When this light passes through an optically active sample with a different absorbance A for the two

90 Experimental 70 components, the amplitude of the stronger absorbed component will smaller than that of the less absorbed component. The consequence is that a projection of the resulting amplitude now yields an ellipse instead of the usual line (draw on a sheet of paper and check). Note that the polarization direction has not changed. The occurrence of ellipticity is called Circular Dichroism - it is not the same as optical rotation. Rotation of the polarization plane (or the axes of the dichroic ellipse) by a small angle α occurs when the phases for the 2 circular components become different, which requires a difference in the refractive index n. This effect is called circular birefringence. It can be shown that when CD exists, optical rotation must exist as well, and they are directly related by a Kronig-Kramers transformation. The change of optical rotation with wavelength is called optical rotary dispersion, ORD [12]. CD spectra was recorded on a J-815 Jasco spectropolarimer at 25 0 C. A rectangular quartz cell of path length was used to obtain spectra with scanning speed of 50 nm/min. the optical chamber of the CD spectrometer was deoxygenated with dry nitrogen before used and kept in a nitrogen atmosphere during the experiments. Figure 2.6: (a) Linear polarized light can be viewed as a superposition of opposite circular polarized light of equal amplitude and phase. (b): different absorption of the left- and right hand polarized component leads to ellipticity (CD) and optical rotation (OR). The actual effect is minute and using actual numbers the ellipse would still resemble a line.

91 Experimental Dynamic Light Scattering: The Nano-ZS (Malvern) instrument we have used for our experiment, is equipped with a 4 MW He Ne Laser (k =632 nm). The sample is poured in a 3 ml glass cuvette (path length 1 cm) with all transparent walls. Prior to the DLS study, protein samples were passed through a 2 Am filter. The operating procedure was programmed (using the DTS software supplied with the instrument) such that there are average 20 runs, each being averaged for 10 s, and a particular Rh is computed in each case and ultimately the result is presented as the distribution of Rh. In DLS one intends to measure the three dimensional pdf (probability distribution function) for diffusion process P, a general expression is given by P(r,t/0,0) = (4πDt) -3/2 exp(-r 2 /4Dt). Since this function only depends on D, the diffusion constant of the system, this allows us to obtain the value for the Stokes radius Rh=a, if the pdf can be measured. The Stokes relation can compute the diameter of the scattering particle, D = F/6πηa The link between the pdf and the power spectrum is a consequence of the translation of the relative motion of the scattering particles in to phase differences of the scattered light. So, if I(t) is the intensity of the scattered light, then C(τ) satisfies the following relation, C(τ) = <I(t)I(t+ τ)> t exp(τ /D) where, C(τ) is the auto correlation function. It is then straightforward to measure D from the slope of the logc- τ plot, and the hydrodynamic size (dh) follows from the Stoke s relation. A particular dh is evaluated several times and the result is presented in terms of a distribution of the hydration diameter. The instrument provided the size distribution in (a) Intensity mode (b) Number mode. While the first, providing the size distribution of scattered intensity is more

92 Experimental 72 sensitive to alteration in size (intensity varying as ~r 6 ), the second mode provides size distribution of number of particles in the light path. For monitoring the population of aggregates (whose numbers are appreciably high in some cases) the multimode intensity distribution was used. 2.2 Methods of Calculations: For measuring the degree of polarization for fluorescence emission or excitation spectra and also to measure the quantum yield of fluorescence or phosphorescence emission, the adopted methods of calculation are discussed below Polarization Spectra: The polarization of the emitted luminescence radiations is characterized by a parameter known as the degree of polarization (P), defined by I // ( λ) I ( λ) P ( λ) = (2.8) I ( λ) + I ( λ) // where I // and I correspond, respectively, to the components of emission intensities parallel and perpendicular to the exciting light. The significance of P lies in the fact that it is a measure of the extent to which the emitted light has its electric vector pointing in the same direction as that of the exciting light. If the polarization (P) of the emitted radiations is plotted as a function of wavelength or frequency, the resulting curve gives the polarization of the emission spectrum. In the same way, one can obtain the polarization of the excitation spectrum of the sample. As the optical elements such as the mirrors and gratings of the instrument introduced partial polarization effects, a correction factor is introduced in Eq. 2.4 as I// ( λ) GI ( λ) P ( λ) = (2.9) I ( λ) + GI ( λ) //

93 Experimental 73 Where G is an instrumental factor (polarization characteristic of the photometric system) and is measured by using the relation i// G = (2.10) i Where i is the emission intensity with the polarizer set at 90 0 (horizontal in the lab-axis) and analyzer set at 0 0 (vertical in the lab-axis) while i // is the emission intensity with the polarizer set at 90 0 (horizontal in the lab-axis) and analyzer set at 90 0 (horizontal in the lab-axis). The polarized emission and excitation spectra, in the present research work, were measured by using UV-VIS polarizer accessories UV Linear Dichroic polarizer, wavelength range: nm, purchased from Oriel Instruments, USA, fitted to a Hitachi F-4500 fluorescence spectrophotometer equipped with a 150 W xenon lamp. While measuring the polarization spectra, equations 2.4 and 2.5 were employed. The resolution of the spectrophotometer is about 1 nm and the respond time is approximately 4 ms. Theoretically, the degree of polarization (P) can take on values from -1 to +1. However, in solutions where the molecules are randomly oriented but their reorientation are inhibited in the interval between absorption and emission, the expression for P is given by 2 3cos β 1 P = (2.11) 2 cos β + 3 where β is the angle between the directions of the absorption and emission dipole moments of the solute molecules. The maximum value of P is 0.5 for coincident absorption and emission transition moments of the molecule and minimum value is for perpendicular absorption and emission transition moments. Since the solutions having low viscosity and high concentration of the solute molecules produce depolarizing effects, solutions of high viscosity (using glycerol and ethanol mixture in the ratio 3:1) and low concentration of the solute molecules were employed for degree of polarization.

94 Experimental Quantum Yield: The luminescence quantum yield (quantum efficiency) is defined as the ratio of the rate of number of photons emitted by the luminescent molecule and the rate of absorption. As the measurements of absolute quantum yield present many difficulties [13-16], it is measured experimentally by comparing the integrated emission (fluorescence or phosphorescence) intensity (area under the corrected spectrum) and the absorbance at the wavelength of excitation of the sample to the corresponding quantities belonging to a solution of reference compound of known quantum yield under identical conditions [17-19]. The fluorescence or phosphorescence quantum yields of the samples are computed using the relation [20] ' 2 ' Af OD n E ' ϕ f = ϕ f (2.12) ' 2 A ODn' E f Where primed unprimed symbols refer to the standard and unknown molecule respectively; ϕ f s are the fluorescence or phosphorescence quantum yields; A f s are the integrated areas under the corrected emission spectra; OD s are the optical density at the same excitation wavelength; n s are the refractive indices of the respective solutions and E E ' is the spectral energy distribution of the excitation monochromator and is reduced to unity for corrected emission spectra. Equation 2.7 is strictly valid for 0.02 OD s. 2.3 Purification of Samples: The compounds derived from natural sources are not pure. Similarly the compounds prepared in the laboratory are generally mixed with the products which may also have been formed during the course of reaction. In order to study its properties, a given substance must be first of all obtained in a state of purity. Here also several processes are employed to purify the substances used for providing its characteristics in real sense.

95 Experimental Crystallization: The most general method for the purification of solid organic substances is crystallization. For that process first of all a solvent is selected for making solutions in which the solvent does not react with compound and in which the compound is more soluble at higher temperature. The solvent is slightly heated with excess of solid compound. The saturated solution thus prepared is filtered while still hot. As the filtrate cools, the pure solid crystals separated which may be removed by filtration. Then the filtration is allowed to cool undisturbed in a beaker or a dish. After some time the solid substance separates in beautiful geometrical forms called crystals Distillation: The operation of distillation is employed for the purification of the liquids from non-volatile impurities. The impure liquid is boiled in a flask and the vapors so formed is collected and condensed to give back the pure liquids in another vessel. The non-volatile impurities are left behind in the flask. The straight distillation is suitable only for liquids, which boil without decomposition. In case of liquids which decompose before their boiling point is reached, the distillation is carried under reduced pressure, in that condition the liquid boils at a lower temperature. Here all the liquids samples, solvents used are distilled under reduced pressure Vacuum sublimation: Certain compound when heated, pass directly from the solid to the vapor state without melting. The vapors when cooled give back the solid compound. It is very helpful for separating volatile from nonvolatile solid. The impure substance is heated taking in a test tube of sublimation set in reduced pressure. The vapor rising from the solid passes through another test tube and condensed to form pure compound. In all the work carried out here the

96 Experimental 76 purification of solid samples was done by vacuum sublimation and for liquid samples fractional distillation techniques was used. Also the solvents were purified and all the work was done under the nitrogen atmosphere by purging nitrogen through it. 2.4 Chemicals and solvents used throughout the work: Name of the compound Purity/Grade Source 2-(4-(dimethylamino) styryl)-1-methylpyridinium iodide (DASPMI) 99% Aldrich β-napthol Analytic grade Fluka β-cyclodextrin Analytic grade Aldrich α-cyclodextrin Analytic grade Aldrich γ-cyclodextrin Analytic grade Aldrich Sodium dodecyl sulfate (SDS) Analytic grade Aldrich Cetyltrimethyl ammonium bromide (CTAB) Analytic grade Aldrich Tween 80 (Tw-80) Analytic grade Fluka Sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT) Analytic grade Fluka Benzyln-hexadecyl dimethylammonium chloride (BHDC) Analytic grade Fluka Polyethylene-b-polyethylene glycol (PE-b-PEG) Analytic grade Aldrich Protein Analytic grade Aldrich DNA Analytic grade Aldrich Acetonitrile (ACN) Spec. grade E. Merck Ethanol (EtOH) Spec. grade E. Merck Methanol (MeOH) Spec. grade E. Merck

97 Experimental 77 Cyclohexane Spec. grade E. Merck n-heptane Spec. grade E. Merck Methyle Cyclohexen (MCH) Spec. grade E. Merck Chloroform (CHCl 3 ) Spec. grade E. Merck Urea Spec. grade Spectrochem Hydrocloric Acid (H 2 SO 4 ) Spec. grade E. Merck Acetone Spec. grade E. Merck Glycerol Spec. grade E. Merck Methyle Cyclohexen (MCH) Spec. grade E. Merck Kcl Spec. grade Spectrochem Nacl Spec. grade Spectrochem 2.5 Computational Methods: The (gas phase) ground state geometry of the molecule was fully optimized, without symmetry constraints, using the B3LYP hybrid functional [21] and the 6-31G(d) basis set [22]. Gas phase vertical excitation energies, using the optimized geometries for the molecule, were calculated using both the semi-empirical ZINDO method (using the spectroscopic parameter set) [23] and the time dependent DFT (TD-DFT) method, using the B3LYP hybrid functional and the G(d,p) basis set [24]. The effect of solvation on the computed TD-DFT vertical excitation energies was investigated using a continuum solvation model, specifically, the polarization continuum model (PCM) implemented for excited states [25]. Because we are interested in vertical excitation energies, the PCM-TD-DFT calculations were carried out using non-equilibrium salvation conditions. It is stressed that, being a continuum solvation model, specific solvent effects, such as solvent-solute H-bonding interactions, are not treated. This may be a problem in the case of ethanol solvent because of the possibility of

98 Experimental 78 the presence of solute-solvent H-bonding interaction. The frozen core approximation was used for all TD-DFT calculations; that is, MOs 15 to 270 were used in the correlation. All 46 MOs were used in the in the ZINDSO/S CI singles calculations. All calculations were carried out using the Gaussian 98 and Gaussian 03 programs [25,26] Gaussian programs: Gaussian 98 (or Gaussian 03) is a program able to perform various semi-empirical and ab initio calculations. It is useful for calculating molecular geometries and energies in different electronic states, for predicting IR, Raman, and UV spectra, and for estimating vibrational and NMR spectral patterns. The input structures and files are most conveniently manipulated from the supporting program GaussView, as well as by way of pdb files. The methods of calculations are easily defined using the GaussView sub-program, but sometimes we need to manually edit the input file. It is important to provide sufficient information in the input files, which is done via comment boxes, to clarify what the calculation is intended to do. The complete methods available in this program are given elsewhere ( To build and optimize the Cyclodextrin structure we used PM3 method. The glycoside oxygen atoms are considered to be into the XY plane, and their center is defined as the center of the coordinate system. The secondary hydroxyl groups are placed pointing toward the positive Z-axis. The longer dimension of the guest molecule is initially placed along the Z- axis. The geometry of the guest-host complex is completely optimized by PM3 method without any restriction Molecular modeling studies: The crystal structure of B-DNA used for docking was extracted from the structure having Protein Data Bank [27] identifier 1DCV. The initial geometry of o-daspmi was generated in Sybyl 6.92 (Tripos Inc.) and was minimized by the MMFF94 force field. Waters were removed from the DNA PDB file. Amber charges were added to prepare the DNA molecule

99 Experimental 79 for docking and Gasteiger Hückel method was applied to calculate the partial atomic charges for the ligands. Rotatable bonds in the ligands were assigned with Auto Dock Tools in AutoDock. Ligand docking was carried out with the AutoDock Lamarckian Genetic Algorithm (GA) [28,29]. Other miscellaneous parameters were assigned the default values given by the AutoDock program. The output from AutoDock was render with PyMol [30]

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102 Experimental 82 Gonzalez, M. Head-Gordon, E. S. Replogle, J. A. Pople, Gaussian 98; Gaussian Inc: Pittsbugh, PA. [26] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, Jr. J. A. Montgomery, T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian, Inc., Pittsburgh PA, [27] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res. 28 (2000) 235. [28] G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew, A. J. Olson, J. Comput. Chem. 19 (1998) [29] G. M. Morris, D. S. Goodsell, R. Huey, A. J. Olson, J. Comput.-Aided Mol. Des. 10 (1996) 293. [30] W. L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA, 2004,

103 Chapter Introduction: The environmental polarity sensitiveness of TICT state make the fluorophore very useful in probing both the bulk and the microenvironments of different media, such as micelle [1] which are known to form micro-heterogeneous compartmentalized structure in aqueous solution. In 2(4-(dimethylamino) styryl)-1-methylpyridinium iodide (DASPMI) the twisting of double bond and the twisting of dimethylamino group do not contribute to the TICT state only the rotation of remaining single bonds are important. These interesting features stimulated us to use this molecule as a reporter to probe different regions of non-ionic (TWEEN-80), anionic (sodium dodecyl sulfate) and cationic (cetyltrimethyl ammonium bromide) micelles and also to pinpoint the role of micellar environment in the formation of TICT state and the charge transfer emission. We will try to discuss in this chapter about the location of the probe molecule on the basis of ICT band intensity which is deactivated via dominant bond twisting (TICT state) Results and Discussion: Steady state emission and absorption results: The absorption spectra of DASPMI in aqueous solution with varying concentration of different surfactants (SDS, CTAB and TW-80) are shown in Figure 3.1 keeping DASPMI concentration fixed.

104 Chapter 3 84 Absorbance mm 13 mm 7 mm 4 mm 2 mm 0 mm 6 1 (a) Wavelength(nm) Absorbance mm 52 mm 26 mm 13 mm 6 mm 0 mm 6 1 (b) Wavelength(nm)

105 Chapter 3 85 Absorbance mm 1.0 mm 0.6 mm 0.2 mm 0.1 mm 0.0 mm 1 6 (c) Wavelength (nm) Figure 3.1: Absorption spectra of DASPMI in aqueous solution with varying concentration of (a) CTAB (b) SDS (c) TW-80 The absorption bands of DASPMI in aqueous solution are at 280 and 440nm and addition of CTAB causes a little enhancement in both the bands with a small red shift in 440 nm band, whereas, with addition of SDS a large red shift of 440 nm band along with enhancement could be observed. However, addition of TW-80 causes a little enhancement of the 440 nm band with no band shift and the 280 nm band gradually diminishes with an isosbestic point around 380nm. The 570 nm ICT band (λ ex =440nm)of DASPMI (Figure 3.2) gets blue-shifted to 562 nm in CTAB and SDS micelles and also the band gets enhanced in both the micelles, though in SDS the amount of enhancement is more. In TW-80 the ICT band is also blue-shifted to 561nm with an enhancement larger than ionic micelles. In water emission at 570nm may be attributed from ICT state which is involved with an additional fast nonradiative de-excitation channel via the (ICT TICT ground state) nonradiative conversion.

106 Chapter 3 86 Emission Intensity in a.u mm 13 mm 6.7 mm 3.8 mm 1.9 mm 0.9 mm 0.7 mm 0.5 mm 0.3 mm 0.1 mm 0.0 mm 11 1 (a) Wavelength in nm Emission intensity in a.u mm 52 mm 26 mm 13 mm 7.7 mm 3.7 mm 1.3 mm 0.6 mm 0 mm 9 1 (b) Wavelength (nm)

107 Chapter Emission Intensity (a.u.) mm 2.2 mm 1.6 mm 0.8 mm 0.4 mm 0.2 mm 0.1 mm 0.05 mm 0.0mm (c) Emission Wavelength (nm) Figure 3.2: Emission spectra of DASPMI (λ ex =440nm) in aqueous solution with varying concentration of (a) CTAB (b) SDS (c) TW-80 The increase in fluorescence intensity along with blue shift in all micelle compared to that of water can be attributed by the following reasons: The dipole moment of singlet excited ICT state and ground singlet state are estimated as 4D and 2D respectively, which indicates that the ICT state is much more polar than ground state. So ICT state is less stabilized than the corresponding ground state in the less polar hydrophobic micelles than in water and resulting in an increase of energy gap between ICT state and ground state with a consequent blue shift in the ICT emission. This increase of energy gap between ICT and ground state in hydrophobic interior of micelles compared to bulk water would trammel the rate of nonradiative deactivation via internal conversion in micelles (ICT ground state) and thereby causing enhancement of the intensity of emission in micelle. Moreover, due to the relaxation of the molecule in low polarity solvent the gap between ICT and TICT state may increase causing a decrement of radiationless deactivation via TICT and consequently the ICT emission increases.

108 Chapter 3 88 Another important factor in the form of hydrogen bonding with water around could increase nonradiative decay and the emission is quenched. As the probe DASPMI enters the micelle from bulk water the hydrogen bond gets stymied in less polar hydrophobic environment and thereby the radiationless transition becomes less active and enhancement of ICT emission in all micelles ensues. So the enhancement of the emission band may be attributed to two factors. One, the reduction of non-radiative path from ICT state to TICT state due to increase in the energy gap between them and the other is the reduction of hydrogen bond formation inside the micelle [2]. From figure 3.2 it may be inferred that the enhanced intensity of ICT emission with the hydrophobicity of surrounding environment may be considered as an index of hydrophobicity of the environment. From the above observation we may conclude that in all three micelles DASPMI would try to go into the core due to the hydrophobic force acting on it but the electrostatic field in the ionic micelles would restrict DASPMI to go to the core region. The formation of hydrogen bond with water molecule is still possible in the boundary region so the less enhancement and blue shift of ICT band in ionic micelle compared to non-ionic micelle would ensue In the presence of surfactant the variation of spectral bands of DASPMI is very small for low concentration of surfactant but this band gets affected remarkably after the concentration reaches a particular value, which represents the onset of micellar aggregation. At maximum concentration of the surfactant the relative intensity (I ICT/ I LE ) is very high in nonionic micelle than that in ionic micelles. The critical micellar concentration (CMC) of each micelle can be calculated from the variation of band intensity with the surfactant concentration. Table 3.1 shows the measured value of CMC of different micelle which incidentally are in good agreement with literature value and the above findings help us to use DASPMI as a fluorescent probe for micellar onset in aqueous solution.

109 N Chapter 3 89 Table 3.1: Absorption and emission (λ ex =440) data of DASPMI in different micelle at fully micellized condition Media Absorption Emission band decay times Binding Critical micellar G 0 / band maxima maxima (nm) (ns) constants concentration(m) kj mol -1 (nm) ICT LE ICT (M -1 ) Measured Literature band emission emission value value Water (67%) 1.4(28%) CTAB (55%) 7.2(44%) a SDS (70%) 10.6(28%) a TW (52%) 1.3(45%) b a Ref. (6), b Ref.(7) CH 3 N CH 3 CH I- 3 + molecular direction Figure 3.3: Approximate representation of the molecule DASPMI in aqueous solution of anionic and cationic micelles.

110 Chapter 3 90 For DASPMI the charge is transferred from the donor moiety to the acceptor moiety, making the donor group (NMe 2 group) more positive and acceptor group (NMe group) more negative. The calculated dipole moment of ICT band of DASPMI is (~4D). In the ionic micelle there are two layers - Stern layer and Gouy-Chapman layer. For cationic micelle the Stern layer contains positive charges, whereas the Gouy-Chapman layer contains negative charges. So in this case the electron acceptor part (NMe) of the guest would try to reside near the positively charged layer and the electron donor part (NMe 2 ) would expected to be located near the Gouy-Chapman layer. But in the case of anionic micelle the Stern layer contains negative ions and Gouy-Chapman layer contains positive counterions. So the electron donor group of DASPMI will try to reside near Stern layer and the acceptor group will try to orient towards the G-C layer due to the columbic interaction (Figure 3.3). In the case of TW-80 there is no electrostatic interaction so the DASPMI may go to core of the micelle. We can qualitatively estimate the binding constants (K s) between the emitting species with the micelles from the fluorescence intensity dependence data on surfactant concentration. The binding constants K s of the species to the surfactants, were measured by a method applied in cyclodextrin system [3,4]. For these measurements the micelle is taken as a host and K s are determined from the fluorescence intensities of the emitting species (F) in the absence of micelle, in the presence of given amount of micelle and when the species is completely bound to the micelle (M) and taking average aggregation number N for SDS, CTAB, TW-80 to be 60, 62, 83 respectively [5]. The typical value for binding constants of DASPMI in all the micelles is calculated and is shown in Table 3.1. From the K values, the free energy changes ( G 0 ) for the probe-micelle binding process for different micellar systems have been calculated at ambient temperature. The values are presented in Table 3.1. It is noticeable that the binding constants measured in this work are comparable with already published data [6,7] and it is very high in

111 Chapter 3 91 non-ionic micelle compared to the one for ionic micelle. The value of G 0 is maximum in nonionic micelle where the binding constant (K) seems to have a high value. In Dynamic light scattering (DLS) experiments it is observed that in ionic micelle the probe molecule resides inside as well as outside the micelle as evinced by the two peaks. For SDS the values are Ǻ and 12.9 Ǻ, whereas for CTAB these values are Ǻ and 12.9 Ǻ, which suggests that even after entering probe molecules inside the micellar environment some of them remain in the aqueous part. The 12.9 Ǻ size is possibly due to DASPMI in aqueous solution and Ǻ or Ǻ are the sizes of micelles with hydration layer around. But in non-ionic micelle only one peak at Ǻ suggests that the probe molecule resides inside the micelle fully. Form the binding energy calculation (Table 3.1) it is also clear that in nonionic micelle the probe can reside fully inside due to strong binding energy whereas for ionic micelle it may not be true completely Effect of inorganic salt: In order to estimate the possible location and orientation of DASPMI inside the micelles the emission properties of DASPMI were observed with addition of inorganic salt in fully micellized state. In the aqueous and interfacial region of the micelles quenchers are available copiously and the molecules remain outside the region of the micelle emission is affected by the molecules. The ICT emission of DASPMI in TW 80 is not affected by the addition of inorganic salt. This observation leads us to infer that in TW 80 no emitting species is available in the water-micellar interfacial region to be affected by the salt addition. But a sharp change in emission is observed in ionic micelles.

112 Chapter 3 92 Emission Intensity (a.u.) mM 1.0mM 0.75mM 0.5mM 0.25mM 0.0mM (a) Emission Wavelength (nm) 900 Emission Intensity in a.u mM 1.0mM 0.5mM 0.25mM 0.0mM (b) Emission Wavelength in nm Figure 3.4: Variation of emission of DASPMI (λ ex =440nm) in addition of varying concentration of inorganic salt (a) KCl in CTAB solution (b) NaCl in SDS solution.

113 Chapter 3 93 Figure 3.4a shows the KCl concentration dependence of emission spectra of DASPMI at fully micellized condition in CTAB. The ICT band intensity increases with the increase of KCl concentration. Also in SDS, addition of NaCl, the ICT band intensity of DASPMI increases remarkably (Figure 3.4b). It is reported in literature that the size and hydration of SDS micelle increases significantly with the salt addition [8,9]. The addition of salt causes increased micellar aggregation number and the content of mechanically trapped water in the Stern layer [10,11] along with increase in micellar hydration layer. Though there is an increase of hydration in the Stern Layer but around the ions present a kind of clustering occurs [10, 11] which give rise to microviscosity. This increase in microviscosity causes an abatement of TICT formation and a decrease in radiationless deactivation via TICT resulting in increased ICT emission. It is well known that in the ICT state the electron donor NMe 2 group will have more positive charge and the electron acceptor NMe group will have more negative charge. So it is expected that for anionic micelle the donor to be in Stern layer and the acceptor group in the Gouy-Chapman layer. The greater change in ICT emission band intensity in the anionic micelle due to the addition of inorganic salt implies that the donor group along with two free rotating single bonds of DASPMI resides inside the Stern layer. Before addition of salt electron acceptor -NMe group binds with the counterions on GC layer. After addition of salt the increment of emission intensity implied that the salt increase the microviscosity of the Stern layer which consequently decrease the formation of TICT state (nonradiative sate) via two single bond rotation and hence increase ICT emission. In cationic micelle the ICT band intensity increases gradually with the increase of the concentration of inorganic salt. We know that [12] in DASPMI two single bond rotations are important for nonradiative TICT state rather than the NMe 2 group rotation. Before addition of salt the electric field around the guest would restrict the free rotation of one or both the single bonds responsible for TICT state due to binding of counterions in the Gouy-Chapman layer.

114 Chapter 3 94 With addition of inorganic salt the microviscosity of the Stern layer increased and the rotation of single bond attached to -NMe group is restricted which consequently helps to decrease the non radiative deactivation via TICT state and increase the ICT emission. So the location of the probe molecule in the cationic micelle may accurately be described as inside G-C layer with one or both the single bonds along with NMe 2 part and only a small part is directed towards the Stern layer Time resolved emission: In aqueous solution of DASPMI the emission decay (λ ex =440, monitored at 560nm) shows three components but we neglect the component with very low amplitude (~5% or less). The life time (~0.6ns) may be due to LE emission and the ICT band also has a long lifetime (1.4ns), Table 3.1 as the long life time has been attributed as ICT emission. The fluorescence decay time for DASPMI for both LE and ICT bands in three micelles may help in understanding the difference in dynamics in three different micelles. Usually the LE band has two components; one is the faster component, the rising time (10-20 ps) of ICT state but this is too fast to be resolved at our instrument response time (100ps). Thus we could only measure the slower component of the LE decay profile (Table 3.1). Compared to water the large decay times in micelles indicate that the probe molecule resides inside the more viscous micelles. The LE emission decay time is slightly increased in micelles compared to that in water but the ICT decay time in ionic micelles increases enormously (5 to 7 times). One of the factors for enhancement of ICT emission is obviously due to the reduction in the nonradiative rates of ICT inside the micelles. Of the three micelles the LE band decay time (Table 3.1) in TW 80 increases to 1.9ns but in SDS and CTAB (Figure 3.5) the decay time is ~1ns. Comparatively large LE emission decay and corresponding lower value of ICT decay in TW 80 may point that the non-radiative transfer from LE to ICT is less due to movement of the probe molecule deep inside less polar region and consequently an upshift of

115 Chapter 3 95 ICT state. That is also reflected from the blue shift of ICT band observed in steady state emission counts Channel No Figure 3.5: Time resolved fluorescence decay curve of DASPMI in CTAB micelle (λ ex = 440 nm, monitor at 560nm) The plausible reason for larger decay time of ICT emission time in ionic micelles compared to nonionic micelle is due to counterion binding with the probe molecule originated from micelle water interface electric field. Of the two ionic micelles, large lifetime of ICT band in anionic

116 Chapter 3 96 micelle may be due to the fact that the molecule resides deep inside which was also inferred from the steady state measurements (Section 3.2.1). In water ICT decay time is ~1 ns and the value gets enhanced by 5 to 7 times in ionic micelles due to counterion binding. With salt addition the ICT decay time increases even more (~7 to 10 times) in ionic micelles, which may be attributed to increased microviscosity in micelles due to clustering (vide supra) and restriction of possible rotation of single bond and abatement of the TICT state (non-radiative) I(t) Wavelength in nm Figure 3.6: Normalized TRES of DASPMI in [SDS] = 27 mm.the times are 0.3, 2.0, 7.0, 10.0 and 15.0 ns. The spectrum moves towards lower energies with the passage of time. Decay parameters were calculated from the mono and biexponential fitting procedure. They were used together with the steady-state intensities at the corresponding wavelengths to calculate the Time resolved emission spectra (TRES). Figure 3.6 shows that for SDS micelle the emission spectra shift progressively to longer wavelengths at longer times and band shape shows no

117 Chapter 3 97 variation with time variation 0.3 ns to 15 ns. The same nature was shown for other micelles also. The solvent relaxation time is comparable to the fluorescence life time of the fluorophore, which produces the emission band shift to the lower energies at longer times. These facts are in agreement with the continuous model for a spectral relaxation, which can explain TRES that comes from a multitude of solvent fluorophore interaction [13] 3.3 Conclusion: The ICT emission of DASPMI inside micelles has been explained considering the nonradiative deactivation through ICT TICT ground state and also the hydrogen bonding channel. With salt addition increase in aggregation number, mechanically trapped water and the microviscosity contribute a lot in ICT TICT conversion and modulation of ICT emission. From the observed data it may be guessed that the donor group (NMe 2 ) of the probe is in Gouy-Chapman layer and the acceptor group resides in the Stern layer for CTAB micelle. But for SDS the donor group lies in the Stern layer and the acceptor group resides in Gouy-Chapman layer. In ionic micelles slowing down of ICT decay was attributed to mechanically trapped water and the increased microviscosity thereof. For nonionic micelle the probe enters the micellar environment fully due to strong binding force, but for ionic micelle some molecules remain outside the micellar environment.

118 Chapter 3 98 Bibliography: [1] D. Severino, H. C. Junqueira, M. Gugliotti, D. S. Gabrielli, S. Baptista, Photochem. Photobiol. 77 (2003) 459 [2] A.C. Testa, J. Lumin 50(1991) 243. [3] A. Munoz de la Pena, T.T. Ndou, J.B. Zung, K.L. Greene, D.H. Live,I.M. Warner, J. Am. Chem. Soc.113 (1991) [4] G. C. Catena, F.V. Bright, Anal. Chem. 61 (1989) 905 [5] M. E. Haque, A. R. Das,S. P. Moulick, J. Colloid Interf. Sci. 217 (1999) 1 and references cited therein. [6] S. K. Das,S. K. Dogra, J. Colloid Interf. Sci. 205 (1998) 443 [7] A. Mohamed,A. M. Mafoodh, Colloids Surf A: Physicochemical and Engineering Aspects 87 (2006) 44. [8] Y. harbi, L. hen,m. A. Winnik, J. Am.Chem.Soc. 126( 2004) 6025 [9] B. G. Dutt, J. van Stam,F. C. De schryver,langmuir,13 (1997) [10] M. Kumbhakar, T. Goel, T. Mukherjee, H. Pal, J. Phys. Chem. B. 109 (2005) [11] J. A. Milina-Bolvar, J. Aguiar,C. C. Ruiz J. Phys. Chem. B 106(2002) 870. [12] B.Strehmel, H. Seifert,W. Rettig J. Phys. Chem. B 101(1997) 2232 [13] J. R. Lakowich, Principles of Fluorescence Spectroscopy; Plenum Press: New York, (1983)

119 Chapter Introduction: Dynamical processes in RMs often occur more slowly than in bulk water as observed previously by several time-resolved studies like solvation dynamics[1-3], internal charge transfer,[4-6] isomerization, dielectric relaxation[7,8] and transient infrared spectroscopy[9,10]. The RMs can be made by using cationic, anionic, or non-ionic surfactants. The correlation between spectral shifts and dynamics of the probe species with the surfactant charge, specially charged solute species [1,2] are well reported fact relating to the location of the probe within the RMs interior and also how it depends on the surfactant charge. Ramadass et. al. proposed excited state kinetics of DASPMI as a three-state model of LE, ICT and TICT states. Fluorescence intensity of DASPMI is a dynamic measure for the membrane potential of mitochondria [11]. In living cells, uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation such as CCCP (carbonylcyanide-mchlorophenylhydrazone). The simultaneous dependence of styryl dye photophysics on viscosity and polarity has offered several applications in polymer science and cell biology. The dependence of photophysics of these molecules on polarity and viscosity in unison opened up the possibility of different applications [12,13]. DASPMI being a popular and important staining agent for mitochondria we were intrigued in using it in biomimic membrane systems - reverse micelles before moving to real life situations. The basic intension of this chapter is to investigate thoroughly the photophysical property change in DASPMI embedded in reverse micelles containing the anionic surfactant sodium AOT (sodium 1,4-bis-2-ethylhexylsulfosuccinate) which has the ability to solubilize a large amount of water with values of W and also in cationic surfactant BHDC (Benzyl-nhexadecyl dimethyammonium chloride) [14] reverse micelles as well as the in-situ measurement of micropolarity and microviscosity inside the RMs. Position and orientation of the probe in RMs will also be investigated in this chapter.

120 Chapter Results and Discussion: Absorption and Emission of DASPMI in n-heptane/aot and benzene/bhdc reverse micelle: DASPMI in n- heptane exhibits an absorption maximum at 410 nm (Figure 4.1a). Addition of AOT to an n-heptane solution DASPMI results a decrease in absorbance and hypsochromic shift of absorbance peak. For BHDC cationic reverse micelle the absorption maximum of DASPMI is also blue-shifted with the increase of reverse micelle concentration (Figure 4.2a). The relative contribution of resonance hybrids of the probe, i.e., benzenoid and the quinoid forms is responsible for solvent induced polarity-dependent shift. Because of being more localized π- electrons the benzenoid form becomes more polar and makes it more favorable in polar solvents and a consequent blue shift is observed in the absorption spectrum in both the reverse micelles. Moreover the blue shift in absorption spectra along with the decrease in quantum yield in reverse micelles may be interpreted as the dissipation of energy by the formation of charge transfer state. 0.6 Absorbance [AOT]/M 0 M 0.06 M 0.1 M 0.2 M 0.3 M (a) Wavelength in nm

121 Chapter Emission intensity (a.u) [AOT] 0 M 0.06 M 0.1 M 0.2 M 0.3 M (b) Wavelength in nm Figure 4.1: (a) Absorption spectra of DASPMI at different concentrations of AOT in n- heptane/aot solution at W=0. (b) Fluorescence spectra of DASPMI at different concentrations of AOT in n-heptane/aot solutions at W=0 DASPMI in n-heptane shows an emission peak at 536 nm (Figure 4.1b). With increase in concentration of AOT from 0 to 0.2M, a gradual blue shift in the fluorescence spectrum is observed. Changing excitation wavelength does not affect the fluorescence spectrum of DASPMI in binary mixture. The above observation of blue shift indicates that the benzenoid form of the probe molecule is sensing an increasingly polar microenvironment in both ground and excited states. In benzene DASPMI shows an emission peak at 558 nm and with increasing the concentration of BHDC a spectral increment with blue shift is observed and finally the peak is observed at 554 nm with BHDC ~ 0.2 M (Figure 4.2b).

122 Chapter Absorbance [BHDC] 0 M 0.05 M 0.1 M 0.2 M 0.3 M (a) Wavelangth in nm Emission intensity (a.u) [BHDC]/M 0 M 0.05 M 0.1 M 0.2 M 0.3 M (b) Wavelength in nm Figure 4.2: (a) Absorption spectra of DASPMI at different concentrations of BHDC in benzene/bhdc solution at W=0. [DASPMI]= M (b) Fluorescence spectra of DASPMI at different concentrations of BHDC in benzene/bhdc solutions at W=0. [DASPMI]= M

123 Chapter The hypsochromatic shift of emission maximum observed by increasing the surfactant concentration is due to the higher polarity sensed by DASPMI when aggregates are forming. This apparent dependence on the solubility behavior of DASPMI with the surfactant concentration is the characteristic feature of the system. For both in AOT and BHDC RMs the emission peak is blue shifted which most probably indicate that emission is only from LE (locally excited state) state. In the binary mixture DASPMI emits from locally excited state (LE) only Time resolved emission in AOT/n-heptane and BHDC/benzene: The fluorescence lifetime of DASPMI in the range of 0.03M and 0.1M AOT concentration monitored at 560nm is shown in Table 4.1. The decay of DASPMI (excitation at 440 nm) in AOT/n-heptane binary solution can be fitted as bi-exponential functions with time constants 970 ps and 1750 ps. The contribution of shorter lifetime (τ 1 ) is due to the fluorescence life time of DASPMI in n-heptane and longer component (τ 2 ) corresponds to the probe in the AOT reverse micelle (RMs) interface. Also for cationic micelle (BHDC) the emission is fitted to double exponential decay and with increase of micellar concentration the contribution for reverse micelle is increased. TABLE 4.1: Fluorescence lifetime (τ) of DASPMI in AOT/n-heptane and BHDC/benzene a. Conc. In AOT In BHDC in M a 1 τ 1 (ps) a 2 τ 2 (ps) a 1 τ 1 (ps) a 2 τ 2 (ps) a [DASPMI]= The above observation suggests that at higher AOT concentrations both the nonpolar solvent phase and the micellar (RMs) phase are being sensed by the dye. In the absence of ionic

124 Chapter interaction, the driving force in the form of hydrophobic interaction between the dye and hydrophobic tail of AOT possibly helps to associate the dye in the micellar phase. Apart from hydrophobic force, the dipole dipole interaction [15,16] also comes into play as the electron donor (dimethylamino) group of DASPMI may expected to be aligned close to polar head group of the surfactant. Consequently this can lead to the presence of dye molecules in the interface of the reverse micelles. For BHDC the hydrophobic interaction is same as that of AOT, but the electron donor (dimethylamino) group of DASPMI may be expected to be aligned far from the positive polar head of the surfactant. The pseudophase model [17] which considers only changing W value in reverse micelles and independent of AOT concentration was invoked while measuring the partition function (K p ) of DASPMI between the AOT or BHDC RMs and the external solvent. Considering two solubilization sites, one the external solvent pseudophase and other the RMs interface i.e., the distribution of DASPMI between the micelles and external solvent the partition function (K p ) of DASPMI can be determined from the following relation K p = [DASPMI] b /[DASPMI] f [Surf] (4.1) where [DASPMI] b is the analytical concentration of the substrate incorporated in the RMs, [DASPMI] f is the concentration of substrate in the organic solvent, and [Surf] is the micellized surfactant (total AOT concentration minus the operational CMC 10-4 M obtained using the absorption or emission bands shift with the AOT concentration of different molecular probes at different water content) [18,19]. This relation is applicable at a fixed value of W and at probe analytical concentration [DASPMI] T <<[Surf]. From the fluorescence intensity change of the probe with the surfactant concentration at a given wavelength we can measure the value of K p. If the analytical concentration of the probe is kept constant and the absorbance of the sample at the working excitation wavelength is low we arrive at the following equation [20]

125 Chapter I = I ( ϕ + ϕ K 0 f (1 + K P b P [ Surf ] [ Surf ]) (4.2) where I 0 is the incident light, I is the fluorescence intensity measured at the surfactant concentration considered (0.3 M), and φ f and φ b are the fluorescence quantum yields of DASPMI in the organic solvent and bound to the RMs interface, respectively. As the concentration of RMs is increased the steady state fluorescence anisotropy increases which indicates that the probe senses a higher microviscosity inside the RMs pseudophase than that in the bulk solvent. The relationship between steady state anisotropy <r> and the surfactant concentration as obtained from the additive law of anisotropy [21] gives us the value of K p < rf > + < rb > K P[ Surf ] < r >= (4.3) (1 + K [ Surf ]) P where <r> is the anisotropy of the mixture (steady state), <r f > and <r b > are the anisotropies of the free and bound DASPMI species, respectively, [DASPMI ] T is the analytical probe concentration. The values of K p in both the RMs (Table 4.2) are in good agreement between the values obtained from the emission and anisotropy data. It is interesting to observe that the value of Kp in BHDC/benzene is slightly higher than that in AOT/n-heptane RMs which may be attributed to greater specific interaction between the cationic polar head of the surfactant and the probe through aromatic π electron cloud. This specific interaction is an effective driving force for the molecular probe to reach the cationic RMs interface.

126 Chapter TABLE 4.2: Equilibrium rate constants (K P ) for partition function of DASPMI in AOT and BHDC reverse micelles as a function of W In AOT In BHDC W K Em P /M -1a K An P/M -1b K Em P /M -1a K An P/M -1b ± ± ±0.2 13± ±0.2 14± ±0.1 16±0.1 a Equation (4.2). b Equation (4.3) Emission of DASPMI in n-heptane/aot/water and benzene/bhdc/water: Upon increasing the water content the pool size swells and the polarity of the microenvironments increases [22,23] resulting in red-shifted emission maximum (Figure 4.3a) Emission intensity (a.u) W (a) Wavelength in nm

127 Chapter Emission intensity (a.u) W (b) Wavelength in nm Figure 4.3: (a) Fluorescence emission spectra of DASPMI at different values of W in AOT reverse micelles concentration of [AOT] =0.1M. (b) Fluorescence emission spectra of DASPMI at different values of W in BHDC reverse micelles. [BHDC]=0.1M. [DASPMI]= M In the present case for anionic RMs (0.1 M AOT), as we go from W=0 to higher W (W=[H 2 O]/[AOT]) values, the emission maximum goes towards red as compared to that in n- heptane. The observation reflects that the microenvironment around the probe gets modified as we move from lower W to higher W. In such a microemulsion of W= 25, DASPMI exhibits redshifted emission maximum at 562 nm compared to that in n-heptane but this value of emission maximum seems to be blue-shifted by about 13 nm from that of DASPMI in water (575 nm). The observation of blue-shifted emission maximum in the reverse micellar environments compared to that of bulk water is quite indicative of significantly different environment around DASPMI compared to bulk water. The shift in emission maximum due to the change in polarity experienced in anisotropic assemblies depends on the position (location) of the probe molecule. Kelker et al [24] explained this issue using anthroloxy probes of different chain length and showed that the shift in the emission maximum decreases as the distance of the fluorophore

128 Chapter increases from the water pool. Using 7-nitrobenz-2oxa-1,3 diazol-4-yl(nbd)-cholesterol they found that the emission maxima were invariant with increased W number, indicating that the probe molecule is located in a region far from the water pool of the RMs. In our present work the emission maximum is sensitive to W up to a certain level, so following Kelker et al [24] we can easily rule out the possibility of the location of the probe molecule to be in a region far from the reverse micellar core. The noticeable difference in the emission maximum in RMs and bulk water indicates that the probe molecule does not reside (or only a part of it resides) in the core region. The spectral changes could be due to the formation of hydrogen bond between the acceptor N-CH 3 and the water molecule in reverse micelles. The fluorescence quenching of many molecules having ICT state [25] occurs due to deactivation via the internal conversion (IC) resulting from hydrogen bonding [26]. Very recently Kwok et al [27] showed that IC deexcitation rate of HICT (hydrogen bonded ICT) state is much larger than that of ICT state and it may be mainly responsible for the reduced quantum yield of DASPMI in water (Table 4.3). Thus the deactivation channel (via IC) from the hydrogen bonded ICT (HICT) of DASPMI may be responsible for the observed reduced emission in water reverse micelles. TABLE 4.3: Radiative and Nonradiative rate constants of DASPMI in AOT reverse micelles as a function of W W φ f k r 10-8 (s -1 ) k nr 10-8 (s -1 ) water

129 Chapter Emission Intensity (a.u) % of water Wavelength in nm Figure 4.4: Variation of emission spectra of DASPMI in acetonitrile with increase in water concentration. [DASPMI]= M In order to verify the hydrogen bonding effect we studied the effect of adding water in the emission spectra of DASPMI in acetonitrile solvent. As acetonitrile is a polar aprotic solvent so hydrogen bond formation between the solute and solvent is not possible. Now with addition of water in the acetonitrile solution the hydrogen bond formation between solute and solvent is possible. A sharp decrease (Figure 4.4) in ICT emission spectra of DASPMI with the increase of water concentration is observed. So this observation confirms the involvement of hydrogen bonded ICT state of DASPMI in the case of water reverse micelles. It has been already predicted (vide supra) that the probe molecules are located at the interface of the reverse micelles. On the basis of the hydrogen bonding between the acceptor group of the probe molecule and co-solvents for observed ICT behavior in water reverse micelles we can very well predict that the probe molecules are oriented at the interface of reverse micelles with acceptor group towards the water pool while NMe 2 group remains buried at the interface ( Scheme 4.1).

130 Chapter CH 3 CH 3 O O CH CH 3 3 N O CH 3 + S - O Na + OH 2 O - O O Na + O S O O O N O O S O O -O Na + OH2 CH 3 Pool N CH 3 CH 3 + N N O O O N + CH 3 S O OH Na + 2 O- CH 3 Na + OH O - 2 OH O 2 O S O O O N + O O CH 3 N CH 3 Scheme 4.1: Approximate presentation of orientation of DASPMI within AOT-n-heptane reverse micelle In the case of cationic micelle (BHDC), having micellar concentration 0.1 M the emission peak is observed at 552 nm (Figure 4.3b). As we go from W=0 to higher W values the emission maximum decreases and moves slightly towards blue. Normally TICT emission is very much dependent on polarity of the medium and the TICT emission moves towards blue as the polarity decreases due to upward movement of TICT state. This will consequently decrease the population in the TICT state and thereby a decrease in observed emission intensity with increase in W. This strongly suggests that the probe goes far from the core (polar) region as the hydration increases Steady state fluorescence anisotropy: The fluorescence anisotropy indicates the extent of restriction imposed by the microenvironment on the dynamical properties of the probe. As any factor that affects size, shape, or segmental flexibility of molecule will be reflected in the observed anisotropy [24] so the measurement of fluorescence anisotropy has an important role for its tremendous potential in biochemical

131 Chapter research. The steady state fluorescence anisotropy (r) was calculated using the relation given below r = (I VV -GI VH )/(I VV +2GI VH ) (4.4) where G is the correction factor for detector sensitivity to the polarizer direction of emission and I VV and I VH represents the vertically and horizontally polarized emission intensity obtained on excitation with vertically polarized light. The variation of fluorescence anisotropy of DASPMI in AOT reverse micelle as a function of water pool size W is shown in Figure 4.5a suggests that the probe molecule experiences freedom in motion with bigger pool size. We also observe here that the value of r decreases rapidly until W=16 and after that it levels off gradually. It is a known fact [28] the water relaxation rate in reverse micelles is faster with the increase of W. The reduced anisotropy for the probe in the reverse micellar interface and water pool suggests the increased reorientation rate. At highest W (W=25) the value of anisotropy (r=0.1) is still higher than the anisotropy in pure water(r=0.04, nearly isotropic), which indicates that the probe molecule in RMs still experiences a reasonable restriction compared to the situation in the bulk water. Fluorescemce anisotropy (r) Rotational corelation time (ps) 800 (a) W W

132 Chapter (b) Fluorescence anisotropy (r) W Figure 4.5: (a) Steady-state fluorescence anisotropy of DASPMI plotted against W in AOT reverse micelles at room temperature. Inset shows the variation of rotational correlation time as a function of W. (b) Steady-state fluorescence anisotropy of DASPMI plotted against W in BHDC reverse micelles at room temperature. [DASPMI]= M The above observations point that the probe molecule may be situated at the water/surfactant interface of the AOT reverse micelles. For cationic reverse micelles (BHDC) the anisotropy increases slightly as we increase the value of W (Figure 4.5b), which possibly indicates that the probe molecule moves far away from the water core as the pool size increases Micropolarity around the Fluorophore: Due to the great importance of the in-situ determination of microscopic polarity of biological system using fluorescence probes attention has been drawn for the past few decades in this direction. The micropolarity of a biological system like protein, membrane and reverse micelles can be estimated by comparison of spectral properties of a fluorophore in that environment with those of the probe in solvents of known polarity [29-31]. The empirical solvent polarity

133 Chapter parameter, E T (30) based on transition energy for the solvatochromatic intramolecular charge transfer absorption of the betaine dye 2,6-diphenyl-4-(2,4,6 triphenyl-1-pyridono) phenolate as developed by Reichardt, was used [32] to get a quantitative measure of the polarity of the local environment of DASPMI in reverse micelles taking advantage of polarity-sensitive fluorescence property of DASPMI. We have determined the micropolarity around the probe in the reverse micelles with various W values in terms of fluorescence maximum of DASPMI from the calibration plot of emission maxima of DASPMI versus known values of E T (30) (Figure 4.6). Though only 78% of the probe is incorporated inside the micelle with the AOT concentration used (0.3 M) we thought of getting an in-situ estimate of the micro-properties here in the line of method described [33]. Figure 4.7 shows the variation of micropolarity of DASPMI in reverse micellar environments as a function of W. The Figure 4.7 clearly shows that with increasing W, E T (30) increases rapidly until W=16 and levels off beyond that water (nm) 560 MeOH max λ em 550 DMF DMSO 540 CHCl n-hexane E T (30) kcal mol -1 Figure 4.6: Variation of emission maximum of DASPMI in different solvents as a function of E T (30)

134 Chapter E T (30)kcal mol E T (30) kcal mol W W Figure 4.7: Plot of E T (30) against W of DASPMI in AOT/n-heptane reverse micelles. Inset plot of E T (30) against W of DASPMI in BHDC/benzene reverse micelles. At the highest W (W=25), the E T (30) value is 53, indicating that the average environment of the probe molecule is still less polar than that of bulk water which vindicates our earlier inference from steady state fluorescence anisotropy study that the probe resides at the water-surfactant interface. On the other hand for BHDC reverse micelles E T (30) varies very little and its overall values decrease as W increases gradually ( inset Figure 4.7). So the decrease in polarity indicates that the probe molecule goes far from the micellar core as the pool size increases and after a limit it eventually remains the same, which indicates that further translation does not occur even if we increase the pool size Microviscosity around the Fluorophore: The viscosity around the fluorophore influences the fluorescence anisotropy. So the in-situ microviscosity at a fixed temperature is often estimated by comparing the fluorescence anisotropy of a fluorophore in an environment with those of the probe in the solvents of known

135 Chapter viscosity [34,35]. The variation of viscosity as a function of W in the reverse micellar environments has been measured in the usual way [35]. Glycerol and water have nearly the same polarity so the mixture of them is taken for changing viscosity of the medium without changing the polarity. Fluorescence anisotropy (r) Viscosity (cp) W Weight percentage of glycerol Figure 4.8: Variation of fluorescence anisotropy as a function of composition of glycerol-water mixture. Inset shows the variation of microviscosity of DASPMI in AOT reverse micelles plotted against W Figure 4.8 shows the calibration curve monitoring the fluorescence anisotropy of DASPMI in glycerol-water mixtures against the weight percentage composition of glycerol. The microviscosity (error ~10%) is determined in the reverse micelles of different W and the variation is presented in the inset of Figure 4.8. It is observed from the Figure 4.8 that the microviscosity decreases rapidly upto W=16 and thereafter it gradually decreases until the solution becomes turbid above W=25, because of the limit of water solubilization. The expected

136 Chapter high viscosity at low W confirms that the water molecules are tightly bound to sulfonate head group of AOT. Table 4.4 also indicates that the microviscosity in the vicinity of water/surfactant interface is very much higher than that of in bulk water even in fully expanded state (microemulsion). Here we find that the properties of water in the reverse micelles of AOT are quite different from that of bulk water [36]. It was suggested earlier that even at high water content the apparent viscosity is 6-8 times greater than that of free water molecules [37]. In our case at the highest W the estimated value of microviscosity is very much close to the value reported earlier [37]. So above result confirms that we can measure the in-situ microviscosity of AOT reverse micelles, particularly in the interfacial region, of different dimension with the help of our probe DASPMI. TABLE 4.4: Values of Micropolarity, Anisotropy and Microviscosity of DASPMI as a function of W W emission max (nm) E T (30)(kcal mol -1 ) r η(cp) water So the most plausible mechanism in the DASPMI excited state in the reverse micellar medium is the solvent relaxation where the initially excited state relaxes to a solvent relaxed state. In general medium polarity is more effective than viscosity of the medium in TICT process. So we

137 Chapter can say that high polarity medium is the favorable for TICT process and high viscosity medium is favorable for solvent relaxation process. The micropolarity increases with the water addition up to a W value around 16 and then remains constant Metal-Induced Fluorescence Quenching: To get the probable location of the probe we have studied the quenching effect of DASPMI with the ionic quencher Cu 2+ (CuCl 2 ) as a function of W and also we wanted to see how the accessibility of the probe molecule to the quencher depends on W. As we observed a drastic major change in position and intensity of emission of DASPMI with the gradual formation of water pool inside the AOT we expected all or nearly all probe molecules were within the pool and no probe molecules were present in n-heptane. With this understanding we invoked the Stern-Volmer plot of experimental results for quenching of DASPMI by Cu 2+ in Figure 4.9. The slope (K SV ) in the Stern-Volmer plot defines the degree of accessibility of the probe to the aqueous phase; generally higher the slope greater is the degree of accessibility [34,38]. Figure 4.9 shows that the accessibility of DASPMI molecule to Cu 2+ increases as the value of W increases and so is the increased quenching by Cu 2+. Any decrement of quenching process indicates the decrement of fluidity of the medium, which also slows the rate of diffusion of the interacting species. The quenching of fluorescence of different fluorophores by Cu 2+ ion has been studied by Panda et. al.[39] in aqueous and oil/water microemulsion. They showed that the relatively lower value of K SV in microemulsion than water is due to interfacial barrier (by the way of solvation and separation) for the interaction of fluorophore and quencher which are populated in the oil and water, respectively. So in the light of above discussion it may be concluded that the interfacial barrier and regional fluidity influence the photophysical process involving the fluorophore and the quencher. In our present case the probe is most likely to be at the interfacial region (vide supra) and the quencher Cu 2+ (formed from the copper chloride salt inside the water pool after it overcomes the interfacial barrier) resides in the interior of water

138 Chapter pool of the reverse micelles. So the interaction propensity is guided by both the interfacial barrier and fluidity of water inside the reverse micelles. The solvation of interfacially absorbed AOT molecules in the water pool (to the extent of 1:6 as AOT:water) [38] at low W makes the pool fluidity lower and hence reduces K SV. So our results reflect that the polarity of microenvironment increases with increasing W and that also induces increased hydration. The steady state anisotropy experiment suggested that as the core size increases the probe molecule is relatively free in motion. The binding of Cu 2+ ions with the AOT reverse micelles by displacing Na + (counterion exchange) at the micellar interface [40,41] is expected to increase the local concentration of the quencher around the fluorophore bound to the interfacial region within the reverse micelle which leads to increase the value of K SV W=16 F 0 /F 6 4 W=10 W=6 2 W= [Cu 2+ ] 10 3 M Figure 4.9: Variation of relative fluorescence intensity (F 0 /F) of DASPMI in AOT reverse micelles as a function of [Cu 2+ ]. [DASPMI]= M For cationic reverse micelle (BHDC) the quenching effect is different from AOT reverse micelles. In this case upto W=2 the quenching effect is observed a little but as we increase the

139 Chapter value of W there is practically no quenching at all (not shown). This result suggests that as we increase the value of W the probe molecule is moved from the core region gradually, which was predicted from other experiments (vide supra) Time resolved studies of DASPMI in n-heptane/aot/water and benzene/bhdc/water: Fluorescence life time acts as a sensitive parameter for exploring the local environment around a fluorophore [42,43] and it is sensitive to excited state interactions. Different extents of solvent relaxations around a fluorophore could also be expected to give rise to differences in its lifetime. In the case of DASPMI in RMs the decay becomes multi-exponential and the lifetime values are observed to be longer than in pure aqueous solution. Certainly it is difficult to extract a meaningful rate constant in such a heterogeneous system. Robinson et al [35] pointed out that if the diffusions coefficient (D) of the probe in the water pool is the same as that of an aromatic molecule in ordinary water (0.05 Å 2 ps -1 ) the probe molecule moves about 10 Å/ns in a direction normal to the surface (Z 2 = 2D<t>). Thus the DASPMI molecule passes through several water layers within the observed lifetime of ~ 1 ns. The multi-exponential decay originates from the different polarity regions experienced by the molecule within its life time. If the diffusion coefficient inside the water pool is smaller by one order of magnitude compared to that of pure aqueous medium every DASPMI molecule would be more or less confined to one layer of water molecules. Even then there is a possibility of different DASPMI molecules in different polarity regions being excited simultaneously. That superposition of many decays of slightly different lifetimes looks like bi-exponential decays, as discussed by many workers [44,45]. In Figure 4.10 the bi-exponential decay profiles of DASPMI in AOT reverse micelles are shown. In Table 4.5 the fluorescence lifetimes of DASPMI in AOT reverse micelles obtained as a function of W are shown. A negative pre-exponential factor which is a characteristic of an excited state process should be observed in lifetime values but we could not observe the rise time

140 Chapter of the fluorescing state because of our instrumental limitations. We have chosen the mean fluorescence lifetime defined by equation below as an important parameter for exploiting the behavior of DASPMI molecule bound to AOT reverse micelles instead of placing too much emphasis on the magnitude of individual decay constants in such bi-exponential decays. <τ f >= a 1 τ 1 + a 2 τ 2 (4.5) The average lifetime values are given in Table 4.5. The most interesting feature of our investigation with a probe having ICT state is the increase in the mean fluorescence life time with increase water content in the micelle W=2 W=8 W= Counts Time (ns) Figure 4.10: Time resolved fluorescence intensity decay of DASPMI in AOT reverse micelles in different W(λ exc =440). [DASPMI]= M

141 Chapter TABLE 4.5: Life time data of DASPMI in AOT reverse micelle as a function of W W a 1 τ 1 (ps) a 2 τ 2 (ps) <τ f >(ps) τ c (ps) water In very low polarity solvent like heptane DASPMI shows very small ICT emission, because of such low polarity the barrier of formation of ICT from the LE state is large enough. In going form heptane to reverse micelles the polarity increases which favors the enhanced rate of the conversion from LE to ICT state so in RMs with increasing W value the ICT state is the only emitting state. The lifetime of ICT state also increases from low W value to high W value in reverse micelle. To explain this it is considered that there are two competing processes: formation of the ICT state and subsequent nonradiative decay of the ICT state. As the solvent polarity increases the rate of formation of ICT state increases. Again, with increasing solvation of the ICT state the energy gap between the ICT state and low-laying dark triplet and/or ground state decreases. According to the energy gap law of nonradiative transitions a decrease in energy gap results in an increase in the nonradiative rates, hence a decrease in the yield of the ICT state would ensue. In pure water DASPMI lifetime reduces to ~840 ps which has been attributed to the stabilized CT state accompanied by an increase in the non radiative decay. We can calculate the radiative and non-radiative rate constants for the ICT process, from the observed φ f and τ f, using the following relations (vi) and (vii) which is more supporting and explaining the above observation. K r = φ f / τ f (4.6)

142 Chapter / τ f = K r + K nr (4.7) where φ f, τ f, K r and K nr are the fluorescence quantum yield, mean fluorescence life time, radiative rate constant, and non-radiative rate constants respectively. It may be observed from the Table 4.3 that with the increase of W the non-radiative rate constant K nr increases. We compared the non-radiative rate constant of ICT process in reverse micelles with that in pure water. The K nr in water is remarkably higher than that in reverse micelles. In different RMs the variation of non-radiative rate constants for ICT process is dependant on the location of the probe [46]. TABLE 4.6: Life time data and anisotropy data of DASPMI in BHDC reverse micelle as a function of W W emission max (nm) a 1 τ 1 (ps) a 2 τ 2 (ps) <τ f >(ps) r The observed spectral variation of emission of DASPMI with increased hydration negates the possibility of finding the probe near n-heptane region. From the difference of the parameters (steady state fluorescence, anisotropy, fluorescence lifetime, and non-radiative rate constants) of the probe in the reverse micelles and the bulk water we can rule out the possibility of the probe staying in the core region of the micelle. So it is very logical to say that the probe molecule resides at the AOT/water interface (Scheme 4.1). The spectral variation of emission spectra (Table 4.6) in BHDC with increased hydration layer evinces that the possibility of finding the probe near core region is very thin. The fluorescence lifetime and the steady state anisotropy value (Table 4.6) confirm that the probe is far from the core region in the reverse micelle but it is not that far to go in n-heptane region.

143 Chapter Pico-second Anisotropy Decay: Fluorescence anisotropy is a property which is dependent upon rotational diffusion of the fluorophore and the fluorescence lifetime. The steady state anisotropy change of DASPMI as a function of W is not due to any change in lifetime, the rotational correlation times for DASPMI in AOT reverse micelles with increasing hydration were calculated using Perrin s equation [34] τ c = (<τ f >.r)/(r 0 -r) (4.8) where <τ f >, r, r 0 are mean fluorescence life time, steady state anisotropy, limiting anisotropy of DASPMI, respectively. Here as Perrin s equation is not strictly valid for heterogeneous medium we use mean fluorescence lifetime to get the approximate result. The rotational correlation time is determined from the above equation taking r 0 =0.38 (from time resolved anisotropy study) with varying W. Figure 4.5a shows significant decrease in rotational correlation time with W which confirms that with increasing hydration there is a decrease in rotational restriction experienced by the probe molecules. 4.3 Conclusion: The present work evinces that the photophysical properties of DASPMI are quite different in biomimetic reverse micelle than those in bulk water. This polarity sensitive probe was used to determine the in-situ micropolarity as well as microviscosity at the probe binding site i.e. micellar interface as a function of micellar core size (W). This is a veritable study in locating the probe along with the orientation in cationic and anionic reverse micelles. The values of partition constants in RMs correlate well with the position of emitting probe and the affinity for the probe to enter BHDC has been explained. The knowledge of DASPMI photophysics in microenvironment gives a significant knowledge from the biological point of view to understand microheterogeneous environments like protein and allied enzymes.

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147 Chapter Introduction: The rapid development of supramolecular chemistry over the past decade has resulted in some understanding in host-guest interactions [1]. The nature and structure of host-guest complexation is of fundamental interest in the field of molecular recognition and is of increasing importance to the design of host-guest systems for different applications. Understanding the dynamic aspects of caged drugs into nanocavities can be used to gain information for new formulations and better bioavailability of the drug. Through the study of fast dynamic aspects of different media, including CD cavities[2] can be explored by the molecular mechanism of the drug phototoxicity. CD cavity has the ability to influence the fate of the reaction intermediates and the deactivation pathways of the excited caged drugs [2, 3]. It is reported that all the three isomers of (4-(dimethylamino) styryl)-1-methylpyridinium iodide (DASPMI) possess interesting excited state photophysical properties [4,5]. The drug delivery by complexation with CD is yet another possible potential application. These molecules interestingly have the possibility of multiple single-bonds and a double-bond twisting involvement towards the TICT emission and in ortho-isomer (DASPMI) the twisting of double bond and the twisting of dimethylamino group do not contribute to the TICT emission only the twists of remaining single bonds are important. With all these things in mind in this chapter we will discuss the modulation of photophysics of DASPMI inside the cyclodextrin cavities (α, β and γ) and also to find possible orientation of guest inside the CD cavity both from theoretical and steady state and time-resolved emission spectroscopy in view of its potential application in drug delivery. 127

148 Chapter Results and Discussion: Spectroscopic and photophysical properties of complexes: The absorption and fluorescence intensity changes upon complexation with α, β and γ- CD s with DASPMI. Figure 5.1 shows UV-visible absorption spectra of DASPMI in neat water and in the presence of β-cd and the binding efficiency of ground state molecule to CD s have been determined from the gradual change in intensity upon complexation with CD s Absorbance Wavelength/nm Figure 5.1: β-cd concentration dependent absorption spectra of M DASPMI in aqueous solution: (1) 0mM, (2) 2mM, (3) 4mM, (4) 6mM, (5) 8mM, (6) 12mM, (7) 15mM of β-cd concentration. The change in spectra upon addition of β-cd, and the existence of an isosbestic point at 490 nm is indicative of formation well-defined 1:1 complex between β-cd and DASPMI. in the ground state. Addition of α-cd however, does not cause any significant change in absorption spectra. 128

149 Chapter Figure 5.2 shows the steady state emission spectra (λ excitation ~440 nm) of neat aqueous solution of DASPMI and also in the presence of different β-cd concentration. The emission peak of DASPMI in pure water is at 570nm. The spectrum gradually exhibits hypsochromatic shift on successive addition of β-cd and finally at 15mM of β-cd the peak maximum is at 550nm. Emission intensity in a.u I/(I-I 0 ) /[β CD]Μ Wavelength/nm Figure 5.2: β-cd concentration dependent spectra emission spectra of M DASPMI in aqueous solution (excitation wavelength 440nm); (1) 0mM, (2) 1mM, (3) 2mM, (4) 4mM, (5) 6mM, (6) 8mM, (7) 10mM, (8) 12mM, (9) 15mM of β-cd concentration. Inset is the double reciprocal plot of I-I 0 against concentration of β-cd The blue shift in the emission spectra (Figure 5.2) and increase in fluorescence quantum yield indicates the formation of inclusion complex between DASPMI and β-cd (Scheme 5.1). DASPMI is very weakly fluorescent in water but on addition of β-cd the fluorescence quantum yield increases significantly (φ f ~ 0.08). TICT emission being polarity sensitive, the blue shift of 129

150 Chapter the TICT emission with increase of β-cd concentration indicates that DASPMI experiences less polar environment inside the cavity compared to the aqueous bulk medium. CH 3 CH 3 N + N CH3 CD o-daspmi N + CH 3 CH 3 N CH 3 θ o-daspmi:cd Scheme 5.1: Schematic representation of the 1:1 inclusion complex between DASPMI and β- CD cavity in water. The enormously large (~4 times) value of quantum yield of DASPMI:β-CD complex compared to that in neat aqueous solution (Table1) could well be explained as the decrease in non-radiative process due to encapsulation of DASPMI by β-cd. A reported value of ~7 times [6] increase in quantum yield in β-cd from that in water also indicates the decrease in non-radiative process on encapsulation. In water H-bonds and the twisting motion of two single C-C bond connecting two aromatic moieties are the two important components to affect the photophysics of DASPMI. It is known that the hydrogen bond formation precedes the TICT formation [7] and acts as nonradiative channel to trammel [8] TICT formation. On inclusion of DASPMI by β-cd reduces the efficiency of the above mentioned non-radiative channel due to hydrogen bond and an increase in fluorescence quantum yield ensues. 130

151 Chapter Analysis of the emission data (Figure 5.2) assuming a 1:1 stoichiometry of the complex was performed using the following relation [9] I =(1+KG(Φc /Φ 0 )[CD])/(1+K[CD]) (5.1) I 0 where I and I 0 are the emission intensities with and without CD, respectively. G is the ratio of molar absorption coefficients (Є complex / Є free ) of the molecule at the excitation wavelength 440nm. Φ 0 and Φc are the emission quantum yield of the free and complexed molecule, respectively. Here in our study we used [β-cd] >> [DASPMI]. The value of K comes out to be 99±5M -1 from the relation (1) using the value of G as 1.1, computed from the absorption spectra. The above equation is based on assumption that stoichiometry of the inclusion complex is 1:1. The linear double reciprocal (inset figure 5.2) Benesi-Hildebrandt of 1/(I-I 0 ) versus 1/[CD] confirms that the formation of 1:1 inclusion complex in the excited state Emission intensity in a.u e I/(I-I 0 ) /[CD]M -1 d 0 a Wavelength/nm Figure 5.3: Steady state emission spectra of M DASPMI in water (a) and in presence of γ-cd (b-d) containing 5mM (b), 9mM (c), 13mM (d) γ-cd. (e) Represents emission spectrum of DASPMI in presence of 13mM γ-cd and 7M urea. Inset is the double reciprocal plot of I-I 0 against concentration of γ-cd. 131

152 Chapter On addition of γ-cd in the aqueous solution of DASPMI a little change in the intensity and quantum yield (Table 5.1) could be observed. A representative fluorescence spectrum of DASPMI in aqueous solution on addition of γ-cd is shown in Figure 3. But the plot of 1/(I-I 0 ) against the concentration of γ-cd (inset figure 5.3) could not be fitted as a straight line and the plot exhibits an upward concave curvature. This suggests that 1:1 host: guest complex formation is not possible rather a higher order complex may form. There is no significant change in emission of DASPMI with gradual addition of α-cd, which indicates that a possible inclusion complex with α-cd is hindered due to size constraint of the α-cd compared to the molecule. TABLE 5.1 Values of the emission Lifetimes (τ i ) and Normalized Pre-exponential Factors (a i ) from Multi-exponential Fit to the Fluorescence Decays of M DASPMI in Different Media and also the Values of Wavelengths corresponding to the Maximum of the UV-visible absorption (λ A ) and Emission (λ F ) Bands Solvent [CD]/mM φ f τ 1 /ns a 1 τ 2 /ns a 2 τ 3 /ns a 3 λ A /nm λ F /nm β-cd , β-cd , γ-cd , water , The cavity diameter of γ-cd is much larger (~9.5 A 0 ) compared to β-cd (~8 A 0 ). Normally one would expect the possibility of inclusion of o-daspmi in the γ-cd to be much higher compared to that by β-cd. But surprisingly the increase in fluorescence quantum yield of DASPMI is much less than the addition of γ-cd in comparison to β-cd. This is contrary to the above 132

153 Chapter emission of DASPMI:β-CD result. Recently Ramadass et al [5] showed that viscosity barely influences the fluorescence behavior of DASPMI. It was shown that with increasing hydrogen bond donating capacity of the medium, the fluorescence quantum yield and lifetime were found to be diminishing significantly. So in presence of γ-cd the hydrogen bonding between the molecule and γ-cd may play a role to control the TICT process. Further, in β-cd the intramolecular hydrogen bonding is very strong between the adjacent OH groups. So β-cd is more rigid structure and hydrogen bond between the adjacent OH groups make a complete secondary belt. But in the case of γ-cd this type of hydrogen belt is not possible as it is a noncoplanar molecule. So in presence of hydrogen bonding agent available at the rim of γ-cd the intermolecular hydrogen bonding with external guest molecule is more favorable than β-cd. Emission intensity in a.u ph= 6.5, 5.5, 4.5, 3, 2.5, 2, Wavelength/nm Figure 5.4: Variation of emission spectra of M DASPMI in aqueous β-cd solution with varying ph with the addition of (H 2 SO 4 ); (1-7) ph= 6.5, 5.5, 4.5, 3, 2.5, 2, 1.5 To verify the effect of hydrogen bond of DASPMI: β-cd and DASPMI: γ-cd complexes, urea, a strong hydrogen bond breaker [10,11] was added (~7M) to the solution and there was a sharp 133

154 Chapter increase in fluorescence quantum yield in γ-cd solution containing DASPMI (Figure 5.3). This increment on quantum yield ( φ f =0.05) upon addition of urea suggests that urea breaks the hydrogen bond between DASPMI and OH group of γ-cd at the rim. Consequently, the guest molecules have easier access to the less polar cavity of the γ-cd and an increase in quantum yield ensues. But there is little change in quantum yield ( φ f =0.004) for DASPMI: β-cd complex with addition of urea which indicates that the most of the DASPMI molecules were already included in the β-cd cavity before urea addition. We have already noted that the linear fit in the plot of 1/(I-I 0 ) against (1/[β-CD]) (Figure 5.4) confirms the formation of 1:1 inclusion complex formation between DASPMI and β-cd in the excited state (Scheme 5.1) but the possibility of formation of 2:1 (host:guest) complex may be ruled out as 1/(I-I 0 ) vs 1/[β-CD] 2 plot gives a nonlinear fit. In the absorption spectra of DASPMI with addition of γ-cd the intensity increases much in comparison to the addition of β-cd, which appears that the molecule feels less polar environment in presence of γ-cd. The nonlinear fit in figure of 1/(I-I 0 ) against (1/[ γ-cd]) removes the possibility of 1:1 inclusion complex but it may hint at higher order complex formation. But small enhancement in fluorescence quantum yield with excessive addition of γ-cd rules out the possibility of 2:1 (host: guest) complex formation. The formation of 1:2 (host: guest) inclusion complex should be reflected by the decrease in fluorescence quantum yield (φ f ) compared to DASPMI in aqueous solution due to self quenching inside the γ-cd cavity. But we observed a slight increase in fluorescence quantum yield (φ f ) compared to DASPMI in aqueous solution, which rules out the possibility of 1:2 formation. As there is a small enhancement of emission intensity of DASPMI in presence of γ-cd, there is a possibility of association due to hydrogen bonding at one (1:1) or both (1:2), capping complex at the rims rather than one inclusion complex. With urea addition in DASPMI and γ-cd aqueous solution the emission intensity increases significantly (to the level of DASPMI:β-CD complex emission) indicating the formation of inclusion complex, Scheme 5.2. Urea possibly breaks the 134

155 Chapter hydrogen bonding formed between the guest and the rim of the γ-cd, which helps the guest to go inside the CD cavity. The effect of ph change due to urea addition (ph~8.5) may be ruled out for the cause of inclusion of DASPMI as this amount of ph change rather decreases the intensity. This again confirms the formation of strong hydrogen bonds between DASPMI and OH group of γ-cd before adding urea. CH 3 N CH 3 CH 3 N CH 3 OH N CH 3 OH OH OH OH N OH HO CH 3 OH HO OH OH H 3 C N OH CH 3 N CH 3 OH OH OH OH 1:2 1:1 UREA H 3 C N CH 3 CH 3 N + Scheme 5.2: The pattern of capped complexes between DASPMI and γ-cd and also inclusion in presence of urea. To determine the orientation of the inclusion complex the incremental addition of mild acid (H 2 SO 4 ) was made to the aqueous solution of DASPMI and β-cd and a gradual quenching of 135

156 Chapter TICT emission with lowering of ph (Figure 5.4) could be observed. This experiment was repeated with acetic acid but the result was the same. This observation is only possible if the orientation of DASPMI: β-cd complex is such that the donor part N atom sticks outside and gets protonated in mild acid [12, 13]. The charge transfer is not possible from protonated donor and quenching of TICT emission ensues. The possible orientation of DASPMI has been shown in Scheme Emission Intensity in a.u c b a Wavelength/ nm Figure 5.5: Excitation spectra of M DASPMI monitored 560nm at in water (a) and in presence of 12mM γ-cd (b) and 15mM of β-cd (c). The fluorescence excitation spectrum of 560 nm emission band of molecule in water and different CD s are given in Figure 5.5. The shape of spectrum does not depend on the emission wavelength. The excitation spectrum for neat water (445nm) matched well with absorption 136

157 Chapter spectrum. However, in case of β-cd the maximum is shifted by 11 nm to 456nm and for γ-cd it is shifted to 450 nm. This change indicates that there is a change in electronic structure due to the more stabilized structure upon encapsulation of the caged molecule. Another possible explanation of this bathochromic shift upon encapsulation may be due to the formation of the planar conformation in which a larger electronic delocalization makes this state lower in energy Pico-second time resolved emission and anisotropy decay: The decay of DASPMI (excitation at 440 nm) in absence of CD may be fitted as bi-exponential functions with time constants 0.6 ns and 1.4 ns. The contribution of longer life time is due to emission of TICT state of the molecule. On addition of β-cd, the molecular decay is triple exponential with time constants 0.7 ns, 1.3 ns and 2.2 ns (Figure 5.6). On comparing with lifetime in neat water the extra component 2.2 ns may be attributed to the lifetime of the caged molecule. As expected, the values of pre-exponential factors (a 3 ) of the inclusion complex emission component increase with gradual addition of β-cd, while that of free molecule (a 1 ) decreases. With γ-cd, the decays of the molecule are the three components 0.5 ns, 1.3 ns and 1.9 ns (Table 5.1). In the case of DASPMI: β-cd inclusion complex, the rotational freedom of at least one of the single bond is hindered and DASPMI molecule experiences a less polar environment inside the cavity of β-cd. Hence the formation of TICT state of DASPMI is to some extent prevented in presence of β-cd. Consequently the non-radiative rate of DASPMI is retarded in presence of β-cd. Within β-cd, the molecule is not fully encapsulated into the nanocavity and some part of the guest is exposed to the water molecules. The guest may be subjected to twisting motion of the single bond connected to the donor part and therefore short emission life time is expected. The larger component of decay (2.2 ns) suggests that the corresponding ICT state is sensitive to a further twisting motion of the donor group. 137

158 Chapter Counts 1000 IRF Channel No 2 1 Residues Figure 5.6: Tri-exponential fit of DASPMI upon excitation of 440nM in 15mM solution of β- CD with corresponding residuals. To get the information on rotational times (φ) of the cyclodextrin inclusion complex, the time resolved anisotropy; (r(t)) measurements were done by exciting at 440 nm. In pure water the decay time fitted to a single exponential function give a rotational time τ as 120±10 ps. In presence of β-cd the rotational relaxation of DASPMI is biexponential, having time constants 100±10 (ps) and 1500±20 (ps) (Figure 5.7). The presence of long relaxation time (1500±20 ps) in β-cd suggests that most of the molecules are caged in the cavity and the short component arises 138

159 Chapter due to a significant contribution from the rotational time in pure water (120±10 ps). In the case of DASPMI in γ-cd, the anisotropy decay is fitted by single exponential function with a rotational relaxation time constant (147±10 ps) (Table 5.2) Anisotropy Channel No Figure 5.7: Anisotropy (r(t)) decay of DASPMI in an aqueous solution of 15mM of β-cd upon excitation at 440nm and monitored at 560nm. TABLE 5.2 Values of Rotational Time (τ rot ) and Normalized Pre-exponential Factors (a i ) of Fluorescence Anisotropy Decay Fitting of DASPMI in Water and CDs a Solvent τ rot1 /ps a 1 τ rot2 /ps a 2 β-cd 100± ± γ-cd 147±10 1 water 120 ±10 a The excitation wavelength is at 440nm and observation wavelength is at 560nm The slight increase in relaxation time may be due to the possibility of hydrogen bond of DASPMI molecule with the OH group of the rim of γ-cd. The initial value of r(t) for the 139

160 Chapter complexes r(0)~0.39, very close to the ideal one 0.4. Considering DASPMI and the complex as a prolate ellipsoid and nonhydrated rotator, the rotational relaxation of the overall host-guest complex can be described by the hydrodynamic model. The Debye-Stokes-Einstein (DSE) [14] model expresses the rotational relaxation time as τ rot =ηvfc/k B T (5.2) where v is the hydrodynamic volume of the solute, η is the viscosity of the solution, c is a parameter that describes the shape of the solute and the boundary condition (slip or stick). The parameter f is a factor to account for the shape of the solute [15]. For non-spherical molecule, f>1 and the deviation in magnitude of f from unity describes the degree of the non-spherical nature of the solute. The value of f under stick limit conditions is determined from the molecular dimensions using the following formula.[16,17] f = β 2 4 (2β β ) ln[ 1 (1 + 2 (1 β β β )] β where β=b/a and a, b are the semi-major and semi-minor axis respectively. The volume of a single molecule of β-cd is estimated from its dimensions to be 4147 Å 3 and the value of f for β-cd complex is computed as 2.1. We may take the value of c=1 as the volume of DASPMI:CD complex is much larger than that of water molecules and η (293K) ~1.1 cp [18] to find the value of τ rot from equation 2. For β-cd complex we obtain theoretically computed value τ rot =1.2 ns which is very close to the experimental value (τ rot =1.5 ns) measured from observed spectra, emission lifetime and rotational time constant. Host-guest orientational dynamics: Since guest is included relatively deeply inside β-cd, its internal motion is restricted by the cavity of CD. To describe the motion of the guest inside the cavity diffusion in a cone model is used [19]. In the diffusion in a cone model the unit vector µ with orientation Ω= (θ,φ) diffuses freely in the angular region 0 0 θ θ max and 0 0 φ 2π with a 140

161 Chapter diffusion coefficient D w. In this case the amplitude of the short component which characterizes the amount of anisotropy loss from the internal motion can be used to estimate the angle of the cone. Considering the transition dipole of the chromophore to be directed along the long symmetry axis of the guest and also taking guest s long axis to be perpendicular to the β-cd s open end (scheme 5.3) and along the axis of CD, it can be written as r( ) = r(0) A short A long + A long = 1 cos 4 2 θ (1 + cosθ max max ) 2 From rotational relaxation data A long / (A long +A short ) =0.94 for β-cd. Using the above equation we find θmax=12 for β-cd. Using γ-cd solution the same equation gives θ max is either 0 or 180. It again supports the formation of capped complexes for DASPMI in presence of γ-cd. Theoretical modeling: We performed PM3 calculations to get information on the conformation of complexes. There are two possibilities for the inclusion of the guest molecule into the host. One is donor dimethylamino group inside the cavity and the other is opposite, the donor group is outside the cavity. But the first conformer of dimethylamino group being inside the CD is not stable and cannot be optimized. The stable structure is shown in the Scheme 5.3. Experiments also corroborate the formation of stable inclusion complex as one of the single bonds which is responsible for TICT emission is sticking outside the host. The maximum rotation angle (θ) between the plane of the molecule and perpendicular to the gate of CD to be ~10 (the experimentally observed value is ~12, vide supra). Furthermore for β-cd inclusion complex a length of ~5Å of the dimethyl part is exposed to the water. Figure 5.8 shows the dependence of the energy of the formation of the host-guest complex on the distance between the centers of mass of the mass of two molecules. The minimum energy structure of the complex is found at a distance of ~ 2 Å. The orientation of 141

162 Chapter inclusion complex thus computed corresponds nicely to the partial inclusion of the DASPMI into the β-cd found experimentally Scheme 5.3: Structure of the DASPMI-β-CD complex with minimum energy obtained by PM3 calculation C H 3 N C H Energy in a.u N + C H Distance/A 0 Figure 5.8: Dependence of the energy of the complex on the distance between the centers mass of the host and guest for DASPMI-β-CD complex 142

163 Chapter Conclusion: The exploration of structure and dynamics of α, β and γ-cd cavity inclusion complex of an excited state charge transfer compound (DASPMI) have been made with steady state and timeresolved emission supported by quantum mechanical calculation. In α-cd formation of inclusion complex could not be possible possibly due to size restrictions. In β-cd nice 1:1 inclusion complex in the excited state could be found with the dimethylamino group of the molecule sticking out. The caged molecule seems to have a longer decay time compared to the molecule in water. Anisotropy decay shows that the molecule enters at an angle of 12 which is corroborated by semiemperical PM3 calculations (~10 ). The minimum energy of the inclusion complex shows that nearly 5 Å length of the molecule with dimethylamino part sticks out in the bulk water. The rotational dynamics of the guest inside β-cd has been demonstrated. In γ-cd the DASPMI molecules remain hydrogen bonded with the rim as 1:1 or 1:2 association and enter the γ-cd cavity after urea breaks the hydrogen bonds. In γ-cd the orientation of DASPMI is along the axis of the cavity and the long axis of the molecule is perpendicular to the opening of the cavity. 143

164 Chapter Bibliography: [1] (a) H. J. Schneider, H. Du rr, Eds. Frontiers in Supramolecular Chemistry and Photochemistry; VCH: Weinheim, (b)v. Balzani, L. DeCola, Supramolecular Chemistry; Kluwer Academic Publishers:Dordrecht, The Netherlands, (c) M. Komiyama, H. Shigekawa, In Comprehensive Supramolecular Chemistry, J. L. Atwood, J. E. D. Davies, D. D. MacNicol, Vögtle, Eds.; Pergamon: New York, 1996; Vol. 3. [2] M. El-Kemary, A. Douhal, Photochemistry and photophysics of cyclodextrin caged drugs. In Cyclodextrin materials photochemistry, photophysics and photobiology; A.Douhal,, Ed.; Elsevier: 2006; Chapter 4. [3] (a) P. Bortolus, S. Monti, Adv. Photochem. 21 (1996) 1 (b) K. Hamasaki, H. Ikeda, A. Nakamura, A. Uens, F. Toda, I. Suzuki, T. Osa, J. Am. Chem. Soc. 115 (1993) 5786 [4] B. Strehmel, W. Rettig, J. Biomed. Optics. 1 (1996) 98 [5] R. Ramadass, J. B-Hahn J. Phys. Chem. B. 111 (2007) 7681 [6] J. W. Park and K. H. Park Journal of Inclusion Phenomena and molecular Recognition in Chemistry. 17 (1994) 277 [7] (a) W. M. Kwok, M. W. George, D. C. Grills, C. Ma, P. Matousek, A. W. Parker, D. Phillips, W. T. Toner, M. Towrie, Angew. Chem. Int. Ed., 42 (2003) (b) Y. H. Kim, D. W. Cho, M. Yoon, D. Kim, J. Phys. Chem., 100 (1996) [8] P. R. Bangal, S. Panja, S. Chakravorti, J. Photochem.Photobiol. A. 13 (2001) 5 and references cited therein. [9] K.A. Comors, Binding constants: The measurement of molecular stability : John Wiley and Sons : New York, 1987;chepter 12 [10] R. Breslow, Acc.Chem.Res. 28 (1995) 146 [11] R. A. Kuharski, P. J. Rossky, J. Am. Chem. Soc. 106 (1984) 5786 [12] S. Panja, P. R. Bangal, S. Chakravorti, Chem. Phys. Lett. 329 (2000)

165 Chapter [13] S. Panja, P. Chowdhury, S. Chakravorti, Chem. Phys9. Lett. 368 (2003) 27 [14] G. R. Fleming, Chemical applications of Ultrafast Spectroscopy; Oxford: New York, 1986 [15] B. Kalman, N. Clark, L. B. A. Johanson, J. Phys. Chem. 93 (1989) [16] J. S. Baskin, A. H. Zewail, J. Phys. Chem. A. 105 (2001) 3680 [17] F. Perrin, J. Phys. Radium. 5 (1934) 497 [18] P. Sen, D. Roy, S. Kumar, M. K. Sahu, S. Ghosh, K. Bhattacharyya, J. Phys. Chem. A. 109 (2005) [19] (a) G. Lipari, A. Szabo, J. Chem. Phys.75 (1981) 2971 ; J. M. Schurr, Chem. Phys., 65 (1982) 417. (b) J. Fisz, J. Chem. Phys. 181 (1994)

166 Chapter Introduction: The Interaction of polymer with surfactants in aqueous solution is an interesting and engaging subject as many formulations and industrial process, like formulations in skin care and cosmetic product and pharmaceutical compounds [1-5] make simultaneous use of polymers and surfactants for their complementary or even synergistic roles [6]. The detailed investigation about this interaction using fluorescence correlation spectroscopy [7] conductivity [8] fluorescence light scattering [8-11] NMR [12] and neutron scattering [13] has been done by several groups. The first quantitative model of polymer surfactant interaction [14, 15] was based on two independent co operative equilibrium of the surfactant, viz. surfactant aggregate formation on the polymer chain and free micelle formation. It is well established [16] that if ionic surfactant is added to the aqueous micellar solutions of amphiphilic diblock co-polymers (PE-b- PEG) the surfactants will penetrate into the hydrophobic core of the block co-polymers (Scheme 6.1). Scheme 6.1 Schematic image of the PE-b-PEG and SDS micellar aggregates

167 Chapter The interaction between hydrophilic homopolymer and SDS can be strengthened by hydrophobic modification [17]. Because of hydrophobic effect [18] the polymer can readily form aggregates with surfactant in aqueous solution above a particular surfactant concentration, known as critical aggregation concentration (CAC). The value of CAC is much smaller than CMC (critical micellar concentration) of surfactant [8, 9, 12, 19-22]. Due to this interaction the environments around the micelles get modified by the co-polymer in the shape of a bead [23], as shown in Scheme 6.1. The mixed micellar structure thus formed by polymer and SDS micelle creates a hydrophobic region than that by SDS alone and the probe in this region can experience more restricted regime. This type of formation of aggregation co-polymer is more advantageous than polymer because surfactant-copolymer mixtures may associate into different nano structures, which can be designed by simply changing the composition of the species of the system or the medium condition. The fluorescence anisotropy decay of the dye molecule in a micelle can be described by wobbling-in-cone model according to which there are three independent motions: (i) translational motion of dye on the surface (ii) wobbling of the probe in a cone of angle θ (iii) overall rotation of the whole micelle. For polymer-surfactant medium the values of these motions could be varied. The microscopic friction in many organized assemblies such as DNA and protein [24-26] micelles [27-29], reverse micelle [30] and cyclodextrins [31,32] have been studied using rotational relaxation of suitable probe. Despite some work in this emerging area of surfactant- copolymer interaction no work regarding the molecular behavior inside the core area of surfactant-copolymer aggregate wrapped by block copolymer could be observed. We were really intrigued to explore in this chapter the behavior of 2-(4-(dimethylamino) styryl)-1- methylpyridinium iodide (DASPMI) in the new supramolecular assembly of anionic micelle (SDS) in presence of amphiphilic block copolymers polyethylene-b-polyethylene glycol (PE-b-

168 Chapter PEG) and also the nature of the assembly through the spectra and dynamics of the probe, as revealed from steady state and time-resolved emission studies. 6.2 Results and Discussion: Steady state absorption and emission spectra: The absorption spectra of DASPMI in water, SDS micelles and in polymer-surfactant aggregates are shown in figure 6.1. In aqueous solution DASPMI exhibits an absorption band at 435 nm. On SDS addition we observe a red shift (~462nm) in the absorption spectra possibly due to excess excited state stabilization compared to ground state in SDS. The red shift may be due to hydrogen bonding between the surfactant head group and NH 2 group. In the absence of SDS the solution containing 1mg/ml polymer shows a peak which was very similar with water solution (at ~435 nm), possibly here the dye does not go into the copolymer. But the solution containing SDS and 1mg/ml polymer absorption spectrum shows an increased peak at ~462nm. 1.0 (3) Absorbance(a.u) (1) (2) Wavelength(nm) Figure 6.1: Absorption spectra of DASPMI ( M) (1) in aqueous solution (2) in SDS (15 mm) (3) in diblock co-polymer (1 mg/ml) and SDS (15 mm)

169 Chapter Emission Intensity(a.u.) (4) (3) (2) (1) Wavelength(nm) Figure 6.2: Emission spectra of DASPMI ( M) (1) in aqueous solution (2) in diblock copolymer (1 mg/ml) only (3) in SDS micelle (15 mm) only (4) in diblock co-polymer (1 mg/ml) and SDS (15 mm) Like absorption spectra, when the copolymer is added in water solution of DASPMI the intensity of the charge transfer emission band at 570nm remains unchanged, although the diblock copolymer form micelle keeping hydrophobic part (poly ethylene) in the core region and hydrophilic part (poly-ethylene glycol) in water environments. But surprisingly the intensity of the 570 nm emission band gradually increases with SDS concentration in presence of copolymer (1mg/ml). This enhancement of intensity maximum is enormous at 15 mm of SDS (Figure 6.2). The magnitude of emission enhancement caused by 15 mm of SDS alone is much less than that in the presence of both polymer and SDS. It is to be mentioned here that in absence of copolymer, the emission intensity of DASPMI shows a jump at 8mM of SDS [33] indicating CMC (critical micellar concentration) of SDS. In polymer solution of DASPMI with addition of SDS, the emission intensity starts to increase (critical aggregation concentration,

170 Chapter CAC, for the polymer-surfactant system) at a concentration at least 10 times smaller than the CMC of the surfactant SDS. In present system the value of CAC is 0.7 mm. However, for high SDS concentration in polymeric micellar solution the rate of increment of fluorescence intensity of DASPMI gradually decreases compared to the same concentration of SDS solution alone. This result indicates that in the presence of copolymer, DASPMI experiences a microenvironment which is very different from that in neat SDS micellar aggregates. Dramatic increase in fluorescence intensity in the complex micelle-copolymer system than that in micellar environment alone may be attributed to the drastic cut in nonradiative channel in absence of water [34]. So this composite system (supramolecular assembly) creates a water tight environment in which the probe dye shows a completely different emission characteristics than that only in neat water, micelle and in PE-b-PEG polymer alone. When SDS concentration increases beyond CAC value it may be surmised that a new type of aggregate (supramolecular assembly) due to the interaction of the PEG chains in corona region and SDS micelles is formed. As SDS is added gradually in the mixed polymer-surfactant micelles, the disruption of the mixed polymer-surfactant micelles would arise and this disruption would lead to a decrease of the binding rate due to electrostatic repulsions of SDS head groups resulting in the change in emission intensity. The formation of supramolecular assembly is confirmed by the SEM picture (Figure 6.3), like the simulated picture of diblock copolymer [35]. In Figure 6.3b it is shown that the diameter of polymer-surfactant aggregates is 15nm which is also corroborated by the DLS measurement (14.2nm).The very small blue shift in polymer-surfactant environment than that in micelles alone indicates that an environment in polymer-surfactant aggregates is slightly less polar compared to that of micelles. This extra blue shift (~3 nm) in presence of polymer may be due to a new supramolecular formation around the Stern layer, which induces an additional nonpolarity around the micellar surface. After certain limit beyond CAC the excessive amount of SDS molecules can not interact with PE-b-PEG which may be inferred from the gradual decrease

171 Chapter in the rate of increment of emission intensity in high concentration of SDS. This inclusion of surfactant by PE-b-PEG micelles can possibly influence the molecular motion of SDS alkyl chains, which may be understood from the following results. (a) (b) Figure 6.3: SEM picture of SDS and PE-b-PEG di-block copolymer aggregates (diameter ~ 15 nm) (a) in µm scale (b) in nm scale Time resolved emission studies: Fluorescence life time acts as a sensitive parameter for exploring the local environment around a fluorophore [36] and it is sensitive to excited state interactions. Different extent of solvent relaxations around a fluorophore could also be expected to give rise to differences in its lifetime. The increase in average lifetime values of multi-exponential decay of DASPMI in the composite medium compared to SDS is ~2 times (Table 6.1). The multi-exponential decay arises due to the superposition of many decays of slightly different lifetimes, as discussed by many workers [37,38]. To get an average picture we fitted the fluorescence decays to a bi-exponential, e.g. t t a 1 exp( ) + a2 exp( ) and the averaged lifetime comes as, < τ f a1τ 1 + a2τ 2. τ τ 1 2 The fluorescence life times, amplitudes, of the probe in different media are shown in Table 6.1(Figure 6.4).

172 Chapter Table 6.1 Fluorescence lifetime decay parameters of Dye in micelle and in polymer-surfactant aggregates. System φ f τ 1 (ns) a 1 τ 2 (ns) a 2 τ r (ns) χ 2 Dye+ water Dye+ 20mM SDS Dye+ 20mM SDS poly(1mg/ml) IRF In micelle In polymer + micelle Counts Time in ns Figure 6.4: Fluorescence decays of M aqueous DASPMI solution containing SDS (15 mm) and diblock co-polymer (1 mg/ml) and SDS (15 mm) The average fluorescence lifetime of DASPMI in polymer-surfactant aggregate is nearly double of that in SDS alone, possibly due to the supramolecular assembly between polymer and micelle and complete isolation of the supramolecular assembly from water. The dramatic increase in emission intensity, as revealed from the steady state experiments due to diminished nonradiative

173 Chapter hydrogen bonding channel caused by estrangement of DASPMI from water also corroborates the increase in lifetime. The location of the probe molecules can be measured by the time resolved anisotropy measurement in water molecules and in polymer-surfactant aggregates. Decay parameters were calculated from the mono and biexponential fitting procedure. They were used together with the steady-state intensities at the corresponding wavelengths to calculate the TRES. Figure 6.5 shows that the emission spectra shift progressively to longer wavelengths at longer times and band shape shows no variation with time variation 0.3 ns to 15 ns. The solvent relaxation time is comparable to the fluorescence life time of the fluorophore, which produces the emission band shift to the lower energies at longer times. These facts are in agreement with the continuous model for a spectral relaxation, which can explain TRES that comes from a multitude of solvent fluorophore interaction [39] I(t) Wavelength in nm Figure 6.5: The peak normalized components of time-resolved emission of DASPMI in diblock co-polymer (1 mg/ml) and SDS (15 mm). The times are 0.3, 1.3, 4.0, 7.0, 10.0 and 15.0 ns. The spectrum moves towards lower energies with the passage of time.

174 Chapter Time resolved fluorescence anisotropy measurement: The fluorescence anisotropy decay r(t) is expressed as t τ t 1r τ 2r r( t) = a1 r e + a2r e = r0 [ β exp( τ t slow t ) + (1 β ) exp( τ fast )] (6.1) where a 1 r and a 2 r = β are the fast and the slow components with time constant τ 1r = τ fast and τ 2r = τ slow respectively. Figure 6.6 shows the fluorescence anisotropy decay of DASPMI in micelle and polymer-surfactant aggregates. In Table 6.2 the fitted results of fluorescence anisotropy decays of DASPMI in pure water, micelles and polymer-surfactant aggregates are shown (2) r(t) 0.2 (1) Channel No Figure 6.6: Fluorescence anisotropy decay of DASPMI ( M) in (1) SDS (15 mm) ; value of χ 2 =1.01 and (2) diblock co-polymer (1 mg/ml) and SDS (15 mm) ;value of χ 2 =1.06

175 Chapter The Table 6.2 shows that the molecules have higher rotational relaxation time in micelle and polymer-surfactant aggregates compared to that of water. The large value in rotational relaxation time in micelle and polymer-surfactant aggregates indicates that the molecule bound to the Stern layer experiences a restricted environment. It is interesting that the rotational relaxation time in polymer-surfactant aggregates is larger than in micelle which indicates a more restricted environment due to presence of the excess polymer surrounding the micelle and thus shielded from outside water Table 6.2 Initial anisotropy (r 0 ) and rotational relaxation time of Dye in micelle and in polymersurfactant aggregates. System r 0 a 1r τ 1r (ns) a 2r τ 2r (ns) τ r (ns) Dye+ water Dye+ 20mM SDS Dye+ 20mM SDS poly(1mg/ml) It has been well established fact from numerous studies available in the literature [27,30,40] that the bi-exponential anisotropy decay of the probe observed in micellar system is neither due to the fact that the probe is solubilized in two distinct regions of the micelle nor it is due to anisotropic rotations of the probe. On the other hand in micelles and micelle-polymer aggregates the bi-exponential rotational relaxation time is a consequence of three independent depolarizing motions [27,28,40] viz, (1) the translational motion r t (t) of the dye; (2) wobbling dynamics (t) of the dye about the local symmetry axis in the micelle [41,42]; (3) the rotational dynamics r m (t) of the spherical micelle as a whole. r w

176 Chapter The decay time constant associated with the three motions are τ tr (translational diffusion), τ w (wobbling dynamics) and τ m (rotation of the micelle) In the present case the rotational motion of SDS micelle is hindered by polymeric micelle and apart from the above three motion there is an additional motion r A (t) due to the overall rotation of the polymer-surfactant aggregates (Scheme 6.2) θ Scheme 6.2: Model of the rotational dynamics in di-block copolymer and surfactant aggregates So we can write r (t) = r w (t) r t (t) r A (t) (6.2) For the description of the rotational motion in polymer-surfactant aggregates we use the following equation from the bi-exponential anisotropy decay (Figure 6.5) 2 2 t 1 1 r( t) = r0 [ S + (1 + S )exp( )]exp{ t( + )} (6.3) τ ω τ τ tr A where r 0 denotes initial anisotropy and τ A is the time constant of the overall rotation of the aggregates. Comparing this equation with equation (6.1) we get

177 Chapter τ slow = = + (6.4) τ τ τ 2r tr A 1 τ fast = = + + (6.5) τ τ τ τ 1r tr ω A So the wobbling time of the dye may be obtained from the following equation = (6.6) τ ω τ fast τ slow Here we assumed that the probe molecules are attached to the surface of the micelles. The micellar rotation for SDS micelle alone ( τ m ) can be obtained from the Stokes-Einstein-Debye relation [43] with stick boundary condition 3 4πηr τ h m = (6.7) 3KT where r h is the hydrodynamic radius of the micelles, η is the viscosity of the water, and K and T are the Boltzmann constant and absolute temperature, respectively. The hydrodynamic radius of SDS micelles is found to be 21 Ǻ [44]. Using this value the SDS micellar rotational time τ m is 8.09 ns. The order parameter S is a measure of the equilibrium orientational distribution of the probe and satisfies the inequalities 0 S 2 1. If the fast motion is isotropic [27] S=0 and if it is completely restricted S =1. S also can be defined as, S 2 = a 2 = β (magnitude of slow component) (6.8) r In the present case the value of S 0.87 for polymer-surfactant aggregates and S 0.8 for only micellar medium. This indicates that the probe molecules face more restricted environment

178 Chapter inside the polymer-surfactant aggregates than micellar media. The order parameter is related to the cone semi-angle θ 0 = cos [ {(1 + 8 S ) 1}] (6.9) 2 The dynamic light scattering experiment reveals that the hydrodynamic radius of polymer is approximately 71A 0. For the rotation of polymer-surfactant aggregates as a whole ( r A ) may be written as poly SDS m τ / τ SDS m (71/21) ns 312 ns. So now we can get that the values of τ A, τ tr and τ w for polymer-surfactant aggregates (Table 6.3) Table 6.3 Analytical rotational parameters for Dye in micelle and in polymer-surfactant aggregates System τ m (ns) τ A (ns) τ W (ps) τ tr (ns) D W 10-8 (S -1 ) D t (m 2 S -1 ) θ Dye+ 20mM SDS O Dye+ 20mM SDS O +poly(1mg/ml) The translational diffusion co-efficient ( D t ) [28] is related to τ tr by the following equation D t 2 rh = (6.10) 6τ tr Now wobbling diffusion co-efficient [45] is obtained from the following relations:

179 Chapter Dωτ ω (1 S 2 ) = x 2 0 (1 + x 0 2 ) [log{(1 + x 0 ) / 2} + (1 x 0 ) / 2]/{2(1 x 0 )} + (1 x 0 )(6 + 8x 0 x x0 7x (6.11) 4 0 ) / 24 where x 0 = cosθ0 The values of D t, Dw and 0 θ that were calculated using the above equations is given in Table 6.3. From Table 6.3 it is evident that because of the interaction with the polymer chains the wobbling motion of the dye molecule at the surface of the micelle is retarded by a factor of about 1.5, as indicated by the increase in τ tr and decrease in D w. It is evident that in the polymer-surfactant aggregates the presence of the polymer chains around the spherical SDS micelles hinder both the wobbling and translational motion of the probe. It should be mentioned here that we have assumed that the orientation of the transition dipole of the dye is normal to the micelle surface. 6.3 Conclusion: So in conclusion, we have created a completely new water tight compartment of micelle-polymer supramolecular assembly where a different photophysics of dye due to significant decrease in nonradiative decay through hydrogen bonding could be seen. The aggregation of polymer and micelle creates a water tight environment in the core of the micelle. The SEM picture also confirmed the formation of assembly between the polymer and the micelle. In polymersurfactant aggregates the presence of the polymer chains around the spherical SDS micelles hinder both the wobbling and translational motion of the probe. The composite polymer micelle system has got a solvent reorientation time.

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183 Chapter Introduction: Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. Another important thing is albumins, the most abundant proteins in plasma, have the ability to carry drugs as well as endogenous and exogenous substances. Bovine Serum albumin (BSA) has three major domains, each with two subdomains. Major binding sites, namely site I and site II are located at subdomain IIA and IIIA.[1] BSA has three intrinsic fluorophores: tryptophan (Trp), tyrosine (Tyr), and phenylalanine of which Trp 214 is located in site I (subdomain IIA) and Tyr 411 is in site II (subdomain IIIA). Of the three chromophores of BSA, Trp is the key player, phenylalanine has feeble fluorescence and the fluorescence of Tyr is almost totally quenched if it is ionized or near an amino group, a carboxyl group or a tryptophan residue [1] The surfactant based nanoparticles have been employed to investigate fundamental physical properties of biomolecules [2]. The encapsulation of molecules and particles by reverse micelles can provide separation media and study of these systems are useful for information in drug designing and delivery [2]. For example: biphasic extraction of amino acids between bulk water solutions and reverse microemulsions, as well as their solubilization in the reverse micellar phase; reverse micelles of AOT (bis(2-ethylhexyl) sodium succinate) for the selective transport of tryptophan and p-iodophenylalanine; liquid-liquid extraction of proteins using reverse micelles; hydrophilic bulky proteins with molecular weights larger than 60 kda, such as catalase, beta.-galactoside, bovine serum albumin, and hemoglobin, can be solubilized into a micro-water

184 Chapter pool of AOT reverse micelles by an injection method, because proteins and enzymes solubilized into reverse micelles maintain their activities and native structures, and can be back-extracted. Ionic styryl dyes are known to respond to changes in transmembrane potential by a fast electrostatic mechanism [3]. The shift of charge of the ionic dyes from ground state to excited state coupled with electric field within a cell membrane results in electrochromism. Among these dyes the fluorescence intensity of 2-(4-(dimethylamino) styryl)-1- methylpyridinium iodide (DASPMI) is a dynamic measure for the membrane potential of mitochondria [4,5] in living cell. This dye also used in polymer science and in cell biology [6] because of the strong dependence of its photophysics on viscosity and polarity. The qualitative and quantitative detection of binding site may help in designing efficient drug sensitizers for photodynamic therapy (PDT) [7]. The extended photodynamic action depends on the biodistribution of the probe molecule in the cytoplasmic and mitochondrial membranes, the retention and the nature of the binding inside the cell. In this article we intend to investigate the binding mechanism and efficiency of attachment of the dye with different sites BSA in buffer solution and compare those information with that inside RM, the most practicable biomimetic model. 7.2 Results and Discussions: The effect of BSA on the fluorescence of DASPMI in 0.01M phosphate buffer at 26 0 C is shown in figure 7.1. The increase in DASPMI fluorescence as a function of protein concentration reaches a plateau, which indicates the increment of rigidity of the surrounding microenvironments.

185 Chapter Emission Intensity (F α -F 0 )/(F x -F 0 ) [L] -1 x Wavelength in nm Figure 7.1: Emission spectra of DASPMI as a function of BSA concentration (1) 0 µm (2) 2 µm (3) 7 µm (4) 10 µm (5) 14 µm (6) 15 µm (7) 17 µm. Inset shows the plot of (Fα-F 0 )/(F x -F 0 ) against [L] -1 for BSA The BSA binding sites are very effective in preventing free rotator motions in DASPMI moiety. This decreases the efficiency of radiationless deactivation of DASPMI, a process that involves a low-lying twisted intramolecular charge transfer state (TICT) [8] and leads to a remarkable enhancements in its fluorescence quantum yield. Pico-second time resolved fluorescence analysis indicates that in buffer, at ph 7, DASPMI exhibits a bi-exponential decay with a life time 1.4 ns (20%) and 0.06 ns (80%), but addition of BSA causes a change in decay time as 4.8ns (30%) and 2.4ns (70%)( Figure 7.2).

186 Chapter Counts Time in ns Figure 7.2: Time-resolved fluorescence decays of DASPMI (1) in aqueous buffer (2) with BSA in AOT solution (3) in BSA of buffer solution. [BSA] =15µM Calculation of radiative and non-radiative rate constant: In order to calculate the radiative decay rate constant (K r ), the equation 7.1 was used [9] K r = (7.1) where k r is radiative decay rate constant, = fluorescence quantum yield of DASPMI and τ f is lifetime of DASPMI. Non-radiative decay rate constant k nr can be determined using the equation 7.2 k nr = k r [ - 1] (7.2) The computed non-radiative and radiative decay constant of DASPMI from the quantum yield and life times are found to be and in absence of BSA and in presence of BSA they are and respectively (Table 7.1).

187 Chapter Table 7.1 Photophysical properties and binding constant of DASPMI in buffer and AOT solution in absence and presence of BSA( [BSA]= 15µM) Φ f τ 1 τ 2 K r 10-9 K nr 10-9 K b (ns) (ns) (S -1 ) (S -1 ) (M -1 ) DASPMI (20%) 0.06(80%) DASPMI+BSA (30%) 2.4(70%) In buffer DASPMI+BSA (60%) 2.2(40%) In AOT Calculation of binding constant: The binding constant values have been determined from the fluorescence intensity data considering the following rearranged equation of the original one developed by Benesi and Hildebrand based on 1:1 probe protein complexation [10] [(F - F 0 )/( F x -F 0 )] 1 = (K b [L]) -1 (7.3) Where F 0, F x, F are the fluorescence intensities of DASPMI in absence of protein, at an intermediate protein concentration and an protein concentration when the interaction is complete respectively. K b is the binding constant and [L] is the free concentration of protein. Plot of [(F - F 0 )/( F x -F 0 )] against [L] -1 for BSA shows linear variations (Inset Figure 7.1), justifying the validity of above equation and hence confirming one-to-one interaction between the probe and proteins. The binding constant is determined from the slope of the plot. To get the free protein concentration from total protein concentration we adopted selfconsistent approach similar to the one used during the orbital calculations.

188 Chapter Calculation of thermodynamic parameters: Thermodynamic parameter associated with the complexation between SQ and BSA, was determined using the following equations [11] lnk = - H/(RT) + S/R (7.4) G = H - T S = -RTlnK (7.5) Where H is enthalpy change, S is entropy change, G is change in free energy and K is the binding constant. Considering 1:1 stoichiometry for the complex formation the binding constant between DASPMI and protein (BSA), is calculated to be M -1 and corresponding free energy change is KJM -1. The H 0 and S 0 values for the binding reaction between DASPMI and BSA were found to be KJM -1 and JM -1 K -1. The negative G means that the binding process was spontaneous and formation of DASPMI-BSA coordination compound was an exothermic reaction accompanied by positive S 0 value. However H 0 might play a role in electrostatic reaction. So binding process of DASPMI to BSA involve hydrophobic interaction strongly as evinced by positive values of S 0 but electrostatic interaction could also not be excluded. (a) (b) Figure 7.3: SEM images of BSA (15µM) alone (a) and in presence of DASPMI (b)

189 Chapter Current in µa Voltage in mv Figure 7.4: Cyclic voltammograme (CV) of (1) DASPMI (0.05 mm) alone (2) DASPMI in presence of BSA (15µM) The binding of DASPMI with BSA is further confirmed by Field Emission Scanning Microscope (FESEM), Cyclic Voltametry (CV), Circular Dichroic Spectra (CD) and 1 H NMR techniques. The FESEM images show that BSA alone has a regular structure and this drastically changes upon addition of DASPMI, which is reflected in drastic increase of width of FESEM image of BSA alone of 2 ± 0.1nm to a mean width of 70±10 nm upon complexation of BSA with DASPMI (Figure 7.3). In the present case, the repulsion between DASPMI molecules is less as the cationic dye DASPMI loses its positive charges after interaction with BSA and consequently the aggregations formed (spots shown in FESEM) of protein-dye binding site by DASPMI. In CV there is a noticeable decrease in current intensity (314µA) upon addition of BSA with DASPMI compared to that of DASPMI alone (Figure 7.4). In CD spectra the significant decrease of negative elliptisity after addition of DASPMI into BSA indicates that the binding of DASPMI induces the α-helical structure of protein. The decrement of α-helics by 20% from free BSA in

190 Chapter buffer to bound BSA with DASPMI suggests that binding of DASPMI to BSA brings forth an alteration of the secondary structure of the protein significantly ( Figure 7.5). An upfield shift of about δ 0.03 ppm in 1 H NMR spectrum was observed in presence of BSA. (Figure 7.6). All these above mentioned phenomena confirm the formation of a stable and noncovalent complex between DASPMI and BSA. 5 0 d θ in mdeg c a Wavelength in nm Figure 7.5: CD spectra of BSA in buffer solution as a function of DASPMI concentration (a) 0 mm (b) 0.02 mm (c) 0.07 mm (d) DASPMI alone The anisotropy measurement of DASPMI in varying concentration of BSA also gives an indication of binding with BSA. An increment in anisotropy value of the emission of DASPMI with BSA concentration implies an imposed motional restriction on the fluorophore in the protenious environments, leading to the reduction of tumbling motion and greater binding interaction between the probe and BSA (Table 7.2).

191 Chapter Table 7.2 : Fluorescence anisotropy (r) of DASPMI in different concentration of BSA in buffer Con. of BSA (µm) Anisotropy value (r) Exploring the binding site of any biologically active probe in proteins is the crucial factor for understanding the efficiency of the probe as therapeutic agent. To know the sites in which the dye gets attached we determined the micropolarity around the probe in different states of the proteins, that is, at native (N), intermediate (Int) and unfolded (U) states. Fluerescence Intensity (a.u.) max (nm) em λ Wavelength in nm water 3 2 MeOH 1 DMF DMSO CHCl 3 n-hexane E T (30) kcal mol -1 Figure 7.7: Fluorescence spectra of BSA-bound DASPMI as a function of added urea. Curves 1 6 correspond to 0.0, 3.0, 5.0, 7.0, 8.0, 9.0 M urea. Inset shows the variation of emission maximum (λ max em ) of DASPMI in different solvent against E T (30). (1), (2), (3) give the interpolated λ max em values of native (N), intermediate (int), unfolded(u) states of BSA.

192 Chapter Table 7.3: Micropolarity values in terms of E T (30) at different states Different states of protein BSA Native (N) 51.8 Intermediate (int) 54.8 Unfolded (U) 59.1 Figure 7.7 and Table 7.3 shows that the difference in micropolarity values for the N-Int transition (involving domains I and II) is nearly same as for Int-U transition (involving domain II). The micropolarity of domain II and III is near about 51.7 in terms of E T (30) [12], which is very much close to our measured values. So it may be inferred that the probe is located in domain II and III, as they are more hydrophobic than domain I [13]. As Trp is most active moiety in BSA the Förster resonance energy transfer (FRET) from the Tryptophan (Trp) moiety in BSA to DASPMI has been computed as 27%, which suggests that probe is located near the Trp moiety of domain II. Emission Intensity in nm [BSA] in µm [DP] in mm Wavelength in nm (a)

193 Chapter Attachment of DASPMI(%) with BSA site I site II Concentration of BSA in µm (b) Figure 7.8: (a) Fluorescence spectra of DASPMI in buffer solution with the addition of BSA (1) 0µM (2) 2 µm (3) 7 µm (4) 10 µm (5) 15µM followed by the addition of DP (6) 0.01mM (7) 0.03mM (8) 0.05mM (9) 0.07mM (10) 0.08mM (11)0.09mM (b) Percentage of DASPMI attached in site I and site II of BSA in buffer solution To understand and confirm the site selective binding of DASPMI with BSA, known site selective binding ligands, dansylamide (DNSA) for site I (subdomain IIA) and dansylproline (DP) for site II( subdomain IIIA) [14] were used. Initial addition of DNSA in BSA-DASPMI complex showed a gradual decrease in fluorescence intensity and reached saturation at 0.7 mm. In this process DNSA effectively displaced 30% of DASPMI from BSA- DASPMI complex. Similar experiment was done in BSA-DASPMI complex with DP which showed a displacement of 70% of DASPMI from BSA-DASPMI complex by DP (Figure 7.8). No noticeable change in intensity due to the titration of DNSA and DP with DASPMI(Figure 7.9) indicates all the changes that were observed earlier were due to displacement of DASPMI from BSA-DASPMI complex by binding ligands. The above observation unambiguously helps us to conclude that the dye can bind with site I as well as site II of BSA. This is further

194 Chapter corroborated by the observation of bi-exponential lifetime having values 4.8 ns (30%) and 2.4 ns (70%) Fluorescence Intensity(a.u.) Wavelength in nm Fluorescence Intensity(a.u.) Wavelength in nm (a) (b) Figure 7.9: (a) Emission spectra of DASPMI as a function of dansylamide (DNSA) concentration (1) 0mM (2) 0.4 mm (3) 0.7 mm (b) Emission spectra of DASPMI as a function of dansylproline (DP) concentration (1) 0 mm (2) 0.4 mm (3) 0.7 mm The increase in fluorescence quantum yield along with a hypsochromic shift of emission spectra with the addition of BSA reflects that the microenvironments around the fluorophore in the protein solutions are quite different from the in pure aqueous solution. Possibly the small cavity size (2.53Ǻ) of the site (I) causes the formation of tight complex with DASPMI having π stacking and hydrophobic interactions. Also from life time data the 30% longer life time (4.8 ns) indicates that DASPMI binds with BSA in site I by 30%. This is further confirmed by binding of the ligand DNSA by displacing 30% of DASPMI from BSA-DASPMI complex. The relatively larger cavity size of 2.6 Ǻ of site II involve binding with DASPMI by hydrogen bonding, hydrophobic and electrostatic interaction. As there is no tryptophan residue in the site II, a relatively loose complex is formed with DASPMI at this site. This loose binding led to observe

195 Chapter the major component of 70% with a shorter life time of 2.4 ns, which is also further corroborated by the 70% displacement of DASPMI from BSA-DASPMI complex by DP. Now we want to see all the binding characteristics of DASPMI with BSA within a model biological system, AOT reverse micelle. The suitable value of water pool (W 0 ) of microemulsion for BSA s stability was found to be by trial method better for W 0 =25. Within the water pool the quenching rate of BSA with addition of DASPMI is higher than that in buffer solution (outside the RM). Interestingly a large change in spectroscopic parameters like quantum yield, lifetimes, radiative and nonradiative decay constant could be observed inside the reverse micelle compared to that in buffer solution (Table 7.1). The binding constant ( ) of BSA- DASPMI complex is higher in microemulsion than that in buffer solution, and the change in free energy is KJM -1 in microemulsion environment. The H 0 and S 0 values for the binding reaction between DASPMI and BSA in microemulsion environment were found to be KJM -1 and 58 JM -1 K -1 Fluorescence Intensity (a.u.) [BSA] in µm [DP] in mm Wavelength in nm (a)

196 Chapter Attatchment of DASPMI(%) with BSA site I site II Concentration of BSA (b) Figure 7.10: (a) Fluorescence spectra of DASPMI in AOT solution with the addition of BSA (1) 0µM (2) 2 µm (3) 7 µm (4) 10 µm (5) 15µM followed by the addition of DP (6) 0.01mM (7) 0.03mM (8) 0.05mM (9) 0.07mM (10) 0.08mM (11)0.09mM (b) Percentage of DASPMI attached in site I and site II of BSA in AOT solution To get an idea about the site selective binding of DASPMI with BSA in this environment (RM) site selective binding ligand DNSA was added to BSA-DASPMI complex in microemulsion we found an effective 60% displacement of DASPMI from BSA-DASPMI complex. Similar experiment done with dansylproline (DP) showed that about 40% of DASPMI is titrated by DP (Figure 7.10). The greater binding constant in microemulsion than that in water indicates greater accessibility of DASPMI towards BSA when BSA is encapsulated in microemulsion, which possibly is due to the different nature of water in microemulsion. In AOT microemulsion in presence of BSA, DASPMI emission shows a bi-exponential decay with life time values 4.2ns (60%) and 2.2 ns (40%) compared to the values 4.8 ns (30%) and 2.4 ns (70%) in buffer. The different attachment efficiency in two sites as the protein and dye are placed inside RM compared to that in buffer may be due to one or the combination of following reasons: (i) the

197 Chapter water activity may change due to the specific RM milieu, which may have a profound effect on different sites of protein as water is considered as an integral part of proteins, (ii) the dielectric constant of water in RM is different than that of bulk water (for W 0 =25, dielectric constant is ~23) (iii) the small scale of inner volume of reverse micelle provides a confined space effect on the cavity of different sites of protein, (iv) the concentration of ions inside the water core is higher than that of bulk water which differs the π stacking, hydrophobic interaction, hydrogen bonding and electrostatic interaction of protein. The fluorescence resonance energy transfer (FRET) from BSA to DASPMI in buffer solution is measured to be 27% whereas, for the value of FRET in microemulsion is found to be increased to 36%. The greater FRET efficiency also indicates that probe is located much more nearer to Trp of BSA and stronger binding between BSA-DASPMI complex in RM than in buffer. 7.3 Conclusion: The most important upshot of the investigation is that we have demonstrated the site selective binding of probe in both water and microemulsion and reversal of efficiency of interaction of BSA with DASPMI as BSA moves inside microemulsion, a realistic biological environment, i.e., subdomain IIA is doubly active inside RM compared to that in buffer solution and the activity of subdomain IIIA in RM is halved compared to that in buffer solution. This in situ knowledge is very important for drug designing and its delivery.

198 Chapter Bibliography: [1] (a) X. M. He, D. C. Carter, Nature 358 (1992) 209. (b) M. Dockal, D. C. Carter, F. Ruker, J. Biol. Chem. 274 (1999) (c) A. Sulkowska, J. Mol. Struct., 614, (2002) 227. [2] (a) M. Maestro, J. Mol. Liq. 42 (1989) 71 (b) S. Prichanont, D. J. Leak, D.C. Stuckey, Colloid Surf. A Physicochem.Eng. Asp. 166 (2000) 177. (c) M. Varshney, T. Khanna, M. Changez, Colloid Surf. B Biointerfaces., 13 (1999) 1 [3] (a) L. M. Loew, G. W. Bonneville, J. Surow, Biochemistry 17 (1978) (b) L. M. Loew, S. Scully, L. Simpson, A. S. Waggoner, Nature 281 (1979) 497. (c) L. M. Loew, L. L. Simpson, Biophys. J. 34 (1981) 353. [4] (a) J. Bereiter-Hahn, Biochim. Biophys. Acta, 423 (1976) 1. (b) J. Bereiter-Hahn, K. H. Seipel, M. Vo th, J. S. Ploem, Cell Biochem. Funct., 1 (1983) 147. [5] (a) B. Strehmel, H. Seifert, W. Rettig, J. Phys. Chem. B 101 (1997) (b) B. Strehmel, W. Rettig, J. Biomed. Opt. 1, (1996) 98. [6] (a) S. P. Spooner, D. G. Whitten, In Photochemistry in Organized &Constrained Media; Ramamurthy, V., Ed.; Wiley-VCH: Weinheim,Germany, 1991; pp (b) A. Ulmann, An Introduction to Ultrathin Organic Films: FromLangmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA,1991; Chapters 3 and 5. (c) H. Ephardt, P. Fromherz, J. Phys. Chem., 95 (1991) [7] (a) K. Szacilowski, W. Macyk, A. Drzewiecka-Matuszek, M. Brindell, G. Stochel, Chem. Rev., 105 (2005) (b) R. Bonnett, Chemical Aspects of Photodynamic Therapy; Gordon and Breach Science Publishers: The Netherlands, (c) B. Henderson, T. Dougherty, Eds. Photodynamic Therapy: Basic Principles and clinical applications; Marcle Dekker Inc.: New York, 1992 [8] R. Ramadass, J. B-Hahn, J. Phys. Chem. B. 111 (2007) 7681

199 Chapter [9] P. V. Kamat, S. Das, K. G. Thomas, M. V. George, J. Phys. Chem. 96 (1992) 195 [10] M. L. Benesi, J. H. Hildebrand, J. Am. Chem. Soc. 71 (1949) [11] J. Tian, J. Liu, W. He, Z. Hu, X. Yao, X. Chen, Biomacromolecules 5 (2004) 1956 [12] A. Mallick, B. Haldar, N. Chattopadhya, J. Phys. Chem. B. 109 (2005) [13] T. Peters, Serum albumin. Advances in protein Chemistry; Academic Press: New York 1985; Vol. 37, pp [14] R. K. Pandey, S. Constantine, T. Tsuchida, G. Zheng, C. J. Medforth, M. Aoudia, A.N. Kozyrev, M. A. J. Rodgers, H. Kato, K. M. Smith, T. J. Dougherty, J. Med.Chem. 40 (1997) 2770.

200 Chapter Introduction: High sensitiveness of the fluorescence spectroscopy[1,2] makes fluorescence probes valuable tools in chemistry, materials science, biology and medicine to look into the properties of environment they are associated with. Upon association with bio-macromolecules, such as DNA or proteins the emission intensity of the probe increases, and they are useful markers in genomics and proteomics, as the binding with the host molecule enhances fluorescence emission ( light-up probes )[3]. Various structural and electronic factors [4] control the binding affinities and sequence specificity of small molecules towards the bio-macromolecules. For the development of the effective therapeutic agents in controlling gene expression [2] the interaction of the small molecules with DNA provides valuable information. The interactions between small molecules and DNA studies help us to design the new and effective drugs against the several diseases [2]. Thus the quest for new efficient probes for the fluorometric detection of DNA is on and there is an increasing interest in studying the interactions of drug and dye molecules with the various biological targets [5]. Calf thymus DNA (ct-dna) is a polymer and its DNA backbone contains alternating sugar phosphate sequence. Small molecules can bind with DNA by three dominant modes: (1) Intercalative binding, where the molecules intercalates itself within the nucleic acid base pairs [6,7] (2) Electrostatic binding between the negatively charged DNA phosphate backbone and cationic or positive end of the molecules. (3) Groove binding involving hydrogen bonding or Vander Waals interaction with the nucleic acid bases in the deep major groove or the shallow minor groove of the DNA helix. The groove binding involves docking the thin ribbon-like molecules in the DNA minor groove, in close proximity to the sugar phosphate. Considering the enormous importance of dye-dna

201 Chapter interaction in designing new drugs we intend to discuss the nature of binding of 2-(4- (dimethylamino) styryl)-1-methylpyridinium iodide (DASPMI) with ct-dna with time-resolved and steady state emission and absorption spectroscopy. Since cationic nature of dye might help in the binding due to electrostatic attraction with ct-dna. Apart from throwing light on the nature of binding efforts will be there to know about the way the dye docks into DNA from theoretical modeling. 8.2 Results and Discussion: Absorption Study: DASPMI in aqueous solution shows broad unstructured lower-energy absorption bands with maximum around 440 nm. Addition of ct-dna to the solution the absorbance decreases appreciably with a red shift. The absorption maximum was shifted from 440nm for free dye to 447 nm for the bound dye in presence of ct-dna (Figure 8.1). In general large bathochromic shift confirms the intercalation of dye into base stack of DNA, and the small shift as in this case could be due to the groove binding, rather than the intercalation of the dye. Here DNA induced spectral changes may be explained in terms of change in local polarity around the dye which in turn affects the stabilization of different energy levels. The polarity of the environment around the dye decreases with addition of DNA and the energy gap between highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the dye decreases which is reflected as bathochromism (Scheme 8.1). The absence of isosbestic point in the absorption spectra is indicative of more than one type of binding with dye-dna complex.

202 Chapter Absorbance in a.u Wavelength in nm Figure 8.1: Absorption spectra of DASPMI in presence of different ct-dna concentrations. Curves corresponds to 0, 2, 3, 8, 12, 17, 27, 37, 47, 60, 72, 80, 90 µm of DNA. LUMO Red Shift HOMO Bound DASPMI Free DASPMI Scheme 8.1: Energy Level Diagram for the Absorption of DASPMI upon Binding with ct-dna Steady State Fluorescence Spectra: The fluorescence spectrum of DASPMI in aqueous solution shows broad unstructured band around 570 nm. Addition of DNA to the solution leads to the enhancement of fluorescence intensity along with a slight red shift (5nm) (Figure 8.2). The observed increment points to binding interaction between the probe and DNA. Binding of the dye with the DNA helix hinders

203 Chapter the rotations around various bonds and thereby decreases the possible non-radiative process through TICT state [8]. This in turn increases the fluorescence intensity and quantum yield. Fluorescence Intensity (a.u) Log[(F 0 -F)/F] Log[DNA] Wavelength in nm Figure 8.2: Emission spectra of DASPMI in presence of different concentration of ct-dna. Curves corresponds to 0, 2, 3, 8, 12, 17, 27, 37, 47, 60, 72, 80, 90 µm of DNA Inset shows the linear plot of log F/F vs DNA The observed fluorescence intensities were quantified by plotting F/F 0 as a function of DNA concentration, where F 0 and F are the fluorescence intensity without and with DNA respectively. The plot indicates that fluorescence yields are highly sensitive to DNA concentration (Figure 8.3). The linear plot indicates that the DASPMI fluorescence may be used as a sensitive method to determine the concentration of DNA nucleotides by fluorimetric methods using visible light sources for the excitation of fluorescence.

204 Chapter F/F µμ [DNA]/ Figure 8.3: Stern-Volmer plot for the observed fluorescence enhancement on addition of ct- DNA to DASPMI Fluorescence Quenching Study: To obtain insight of the mode of binding of the dyes with DNA, the fluorescence quenching in the ct-dna environment was studied using potassium iodide (KI) as a quencher and the correlation between the degree of accessibility of each the molecule to the quencher and its steric bulk was examined [9]. It is well known [10-12] that intercalation of small molecules into the DNA double strands protects the entrapped molecules from an ionic quencher. On the contrary, in electrostatic binding and groove binding the probe molecules are exposed to the approach of the quencher to it [11] in aqueous phase and are likely to have no differential effect on the dye in terms of its steric bulk. Even when groove bound, the negatively charged phosphate groups are expected to repel the anionic quenchers from the helix surface. In aqueous solution iodide anions quench the fluorescence of DASPMI very efficiently indicating a possible groove binding. For groove binding, the effective quenching efficiency of the quencher towards the fluorophore in ct- DNA environment is likely to be the same like in bulk water. On the other hand the intercalative

205 Chapter binding of a fluorophore should lead to the reduction in the extent of fluorescence quenching in ct-dna environment in comparison to bulk water [13]. Fluorescence quenching of the dye by KI was studied following the Stern-Volmer equation. F/F 0 = 1+K SV [Q] (8.1) where F 0 and F are the fluorescence intensities in the absence and presence of the quencher [Q] and K SV is the Stern Volmer quenching constant. The observed quenching constants (K SV ) were 8.43 M -1 and M -1 in buffer solution and in DNA environment respectively (Figure 8.4). The quenching of the dye fluorescence is in fact enhanced by a factor of more than 3 when DASPMI is bound to the helix. This enhancement of quenching may be explained on the basis of ionic strength alone. If the probe is bound in the groove, then the increase in the ionic strength caused by the addition of the quencher is expected to release the dye from the helix [14]. The fluorescence yield of the free dye is much lower than that of bound dye, which promptly indicates the contribution of ionic strength in the enhanced No DNA (KI) CTDNA (KI) CTDNA (NaCl) (F 0 /F) [Q]/M Figure 8.4: Quenching of DASPMI fluorescence by KI and NaCl in presence of ct-dna (90µM) and by KI in the absence of ct-dna

206 Chapter Effect of Ionic Strength: As the probe carries the positive charge and DNA has a negative phosphate back bone, the effect of ionic strength on dye-dna binding has been examined studying the binding in presence of strong electrolyte NaCl to verify if there is significant electrostatic interaction between dyes and DNA [15,10]. Increased ionic strength screens the phosphate-phosphate repulsion prompting the helix to shrink due to a decrease in the unwinding tendency caused by electrostatic repulsion between the negatively charged phosphate groups [16]. Hence the bonding of positively charged fluorophore DASPMI and DNA gets weakened by the enhancement of ionic strength of the solvent. The free probe does not show any significant change in fluorescence spectra with addition of NaCl in buffer solution, whereas in presence of ct-dna there is a sharp quenching in addition of NaCl. This result indicates that quenching in presence of DNA was due to the release of the probe from DNA grooves into bulk aqueous solution in which it can undergo rapid nonradiative deactivation. In this situation iodide can efficiently quench (K SV =26.16 M -1 ) than NaCl (K SV =16.18) from free dye (K SV =8.43 M -1 ), Figure 8.4. So above observation confirmed the strong dependence of ionic strength controlling the binding of DASPMI with DNA by electrostatic interaction, which are consistent with groove binding rather than intercalation into the helix Equilibrium binding Titration: To determine the binding constant (K) and binding stoichiometry (n) for the complex formation of DASPMI with ct-dna help of fluorescence titration data have been taken. Figure 8.2 shows the fluorescence spectrum of DASPMI in presence of different concentration of ct-dna. Using this change we can estimate the binding constant (K) and n for the binding of DASPMI to ct- DNA from the following equation [17,18]

207 Chapter log [(F 0 -F)/F]=log K + n log[dna] (8.2) where F 0 and F are the fluorescence intensities of the fluorophore in the absence and present of different concentration of ct-dna, respectively. In the case of enhancement of emission intensity, F 0 <F, and the above equation becomes log[(f-f 0 )/F]= logk + nlog[dna]log( F/F) = logk + nlog[dna] (8.3) The inset of Figure 8.2 shows a linear plot of log( F/F) vs log[dna], and the values of K and n are found to be and 0.952, respectively. The value of K indicates the weak binding of the probe with ct-dna, i.e., groove binding CD Studies: The geometry of complex formation between ct-dna and DASPMI is further investigated by means of circular dichroism techniques. In CD spectra no noticeable induced CD is observed on the complex formation indicating that DASPMI bound to ct-dna does not posses a chiral center and is optically inactive (Figure 8.5). So intercalation of DASPMI may be ruled out in the binding with ct-dna. There is a slight decrease in the positive DNA dichroic signal (Figure 8.5), which is likely to be due to a transition from the extended nucleic acid double helix to the more compact form known as the ψ structure [19]. The equilibrium constant of complex formation may be estimated from the change in CD response at the fixed wavelength using Benesi- Hildebrand equation [20] 1/ A=1/[(ε b - ε f )L T ] + 1/[(ε b - ε f )L T K a ] 1/M (8.4) where ε is the extinction coefficient; subscript b, f, T denote bound free and total complex and L is the concentration of DASPMI. M is the concentration of ct-dna and A is the change of CD

208 Chapter response at a particular wavelength. The value of the association constant (K a ) for the complex formation calculated from the slope of the double reciprocal plot (inset figure 8.5) using the above equation was found to be , and the value of K a is very much comparable to the binding constant calculated from the UV-vis study. Ellipticity/mdeg /(θ 0 θ) /[DASPMI] 10-4 M Wavelength in nm Figure 8.5: The circular dichroism spectrum of ct-dna adding 2, 10, 30, 90µM DASPMI. Inset figure shows a linear plot for (1/[θ 0 -θ]) vs 1/[DASPMI] Steady State Anisotropy: Steady state fluorescence anisotropy gives significant information about the nature and physical characteristic of biological probes. In the anisotropy value many things, like change in size, shape and segmental flexibility of the molecule are reflected [9]. So the anisotropy monitoring helps us in finding the probable location of a probe in microheterogeneous environment like micelles, reverse micelles, proteins, DNA [21-23]. There is a sharp increase in fluorescence anisotropy with the increase in the concentration of ct-dna, which suggests that the fluorophores are trapped in a motionally restricted region within ct-dna compared to that in

209 Chapter buffer water. Figure 8.6 also shows that the anisotropy is level off at 0.2, which indicates that irrespective of the extent of groove binding the overall tumbling motion of dye-dna complex is also responsible for the fluorescence anisotropy. Anisotropy(r) [DNA]/ µμ Figure 8.6: Variation of fluorescence anisotropy as a function of increasing concentration of ct- DNA for DASPMI. [DASPMI]= Fluorescence Lifetime: The decay of DASPMI in buffer solution may be fitted as bi-exponential functions with time constants 0.6 ns and 1.4 ns. Table 8.1: Fitted Fluorescence Decay of DASPMI in Various Environments Systems τ 1 (ns) a 1 τ 2 (ns) a 2 χ 2 Water (ph=7.0) ct-dna

210 Chapter With addition of ct-dna, the fluorescence decay shows bi-exponential behavior (Table 8.1), but the values of decay constants and relative amplitude 0.65 ns(0.13) and 2.7 ns(0.87) change drastically (Figure 8.7). So after addition of ct-dna the life time of the dye increases due to the binding with DNA IRF DASPMI + DNA ( 90µM) DASPMI Counts Time in ns 4 2 Residue T i m e i n n s Figure 8.7: Time resolved fluorescence decay of DASPMI without and with DNA. ([DNA]=90µM)

211 Chapter Molecular modeling of DASPMI-DNA interaction: Molecular docking studies provide some insight into the interactions between the macromolecule and ligand, which can corroborate the experimental results. Though the crystal structure of the complex can provide details of the interactions, general observations may be obtained docking studies. The docked conformation (Figure 8.8) shows that the DASPMI molecule exists in crescent shape which is complementary to the natural curvature of the minor of B-DNA [24,25]. The radius of curvature of DASPMI was calculated to be Ǻ, which is very similar to the values of known minor groove binding agents [26]. Linear length of minor groove formed by 3 base pairs is ~11.5 Ǻ and the length of DASPMI molecule is 14Ǻ. Minor groove binders generally have aromatic rings connected by single bonds that allow for torsional rotation in order to fit into the narrower helical curvature of the groove comprising the A-T region with displacement of water molecules. The extensive van der Waals contacts between the docked DASPMI and the floor and walls of the minor groove point out the significant solubilization of the complex through this interaction. Figure 8.8 : Docked pose of DASPMI bound to minor grove of ct-dna