SYNTHESIS, CHARACTERIZATION AND APPLICATIONS OF BIOLOGICAILY SIGNIFICANT

COORDINATION COMPOUNDS

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

Maittx of ^l)iIos!opl)p IN

CHEMISTRY

BY

HINA ZAFAR

DEPARTMENT OF CHEMISTRY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

2009

^

,A»^Jii*i.^^<

1 8 StP W^2

DS4007

DEDICATED TO MY

HUSBAND & SON

Dr. (Mrs.) Arunima Lai Professor & Chairman Ph: (0571) 2703515

(0571) 2700920, Ext. 3350, 3351 E-mail: arunima_49@rediffmail.com Fax : (0571) 2703515

Department of Chemistry Aligarh Muslim University Aligarh - 202002 (U.P)., INDIA

Certificate Certified that the work embodied in this dissertation entitled "Synthesis,

Characterization and Applications of Biologically Significant

Coordination Compounds." is the result of original research carried out

by Mrs. Hina Zafar under my supervision and is suitable for the

award of M. Phil degree (Chemistry) of Aligarh Muslim University,

Aligarh, India.

— P (Prof. Arunima Lai)

A cknowledsement

All praises and thanks are to Almighty "Allah " who only enabled me to complete this cherished dream.

I owe my most sincere gratitude to Professor Mohammed Shakir who gave me the opportunity to start the work under his esteemed guidance. His wide knowledge and logical way of thinking have been of great value for me and will continue to be so in future too. His extensive discussions, interesting explorations and ever ready helping attitude have been a great support in this work.

My heartfelt thanks to my supervisor Professor Arunima Lai who provided me the chance to continue my research work after Professor Mohammed Shakir went on long leave in January 2007. Her understanding, encouragement and guidance have helped me to complete my work.

Its my pleasure to thank Dr. Tahir All Khan for his moral support.

I express my sincere thanks to DR. Farha Firdaus for the continuous help and guidance. Her thoughtful advice served to give me a sense of direction and vision during my M. Phil studies.

I acknowledge the great support and facilities which Chairperson, Department of chemistry, provided during the course of my research work.

I am obliged to Miss Sultana Naseemfor her valuable suggestions,and kind support.. She was a continuous source of inspiration during my work progress.

I thank Mr. Mohd. Azam for his untiring help at every step of my dissertation work

I thank Miss Kaneez Fatimafor her persistent support and encouragement. I thank my juniors Miss Naushaba, Miss Nida, Miss Sadiqa Khanum, and Mrs. Ambreen Abbasi.

In the end I acknowledg the valuable con of my family. My parents DR. Zafar mohd khan and Mrs. Haseena Begum have made extraordinary efforts and sacrifices in nourishing my career and me as well. I thank my sister Dr. Fehmina Zafar for her possible help and support. I thank my brothers Mohd Ather khan, Mohd Bader khan and Mohd Adil khan for their emotional support to this endevour. I Thank late Mirza Azhar Beg (Father-in-Law) and late Mrs. Afsari Begum (Mother-in-Law). I thank Mirza Obaid Beg, Mirza Navaid Beg, and Mohd Saquib for their love, care and support.

Most Importantly, I cannot end without stating the name of my husband Mirza Junaid Beg for being so loving, caring, and the ultimate source of strength for me. Last but not the least my love and thanks to my son Mirza Humaid Beg for his coopration.

I thank Mr. Shadab Qamar for giving final shape to my dissertation.

HINA ZAFAR

CONTENTS

Title Page NO.

Introduction 1-14

Present work 15-16

Experimental Methods 17-37

Experimental 38-44

Results and Discussion 45-57

References 58-66

INTRODUCTION

INTRODUCTION

The long awaited renaissance in inorganic chemistry finally arrived during

1940s and since that time there has been great activity in all branches of

science, particularly in the chemistry of coordination compounds. The concept

of coordination chemistry was associated with complexation of metal cations

(Lewis acids) by a ligand (Lewis bases). Earlier workers considered and

studied the coordination compounds of only a few metals e.g., Pt, Co and Cr

and coordination numbers of four and six. Most recent research on

coordination compounds has been concerned with nearly all the metal of the

periodic table with coordination numbers from two to twelve and in different

oxidation states [1,2]. A proliferation in this area of research is not only due

to design of ligand systems with varying fiinctionalities [3], synthetic strategies

or advancement in techniques but also due to multiple applications of these

compounds in areas viz; medicinal [4], biochemical [5], bioinorganic [6],

environmental [7], industrial [8] photochemical [9], photophysical [10],

photoelectronic [11] etc. The pioneering work carried on both trace as well as

abundant elements generated coordination compounds which mimic the

biologically significant metalloenzymes and metalloproteins [4, 7], act as

models to understand bio chemical effect in vitro or in vivo [12], extending

hope and promise for treatment of various diseases [4], developing

photochemically driven molecular devices [13], fiinctional model for water

oxidation catalysis in photosystem-II [14] and in catalytic photocleavage of

water [15], design of multinuclear structures capable of directing and

modulating electron and energy transfer processes [13].

A tremendous amount of expansion and development in the area of

coordination chemistry has exposed various facets including macrocyclic

chemistry. The chemists all over the world are busy in exploring the

coordination chemistry of macrocycles porphyrins and related systems so as to

mimic them for their benefits. This has led to the development of a new field of

coordination chemistry known as macrocyclic chemistry. A macrocyclic is as

defined by lUPAC. "A cyclic macromolecule." Generally however, when

speaking of a macrocycle, coordination chemists define macrocycle as a cyclic

compound having nine or more hetero-atomic members and with three or more

ligating groups [16-17]. Macrocyclic compounds display unique and exciting

chemistry because they offer a wide variety of donor atoms, ionic charges,

coordination numbers and geometries of the resultant complexes [18]. It would

not be an exaggeration to state that macrocyclic ligands lie at the centre of life

particularly with regard to the role of such systems in understanding and

explaining the mechanism of photosynthesis [19], transport of oxygen in

mammalian and other respiratory systems and in the potency towards DNA

binders with a high potential in anti-tumor therapy [20], as sensitizer for

photodynamic therapy [21], (PDT) in cancers, as therapeutic reagents [22], as

efficient contrast agents for magnetic resonance imaging (MRI) [23]. The

macrocycycles have also been used for the treatment of AIDS [16]. A part from

the biological implications, macrocyclic chemistry plays a dynamic role in the

other fields, where novel macrocyclic ligand act as model to study magnetic

exchange phenomenon [24], in chelate therapy for the treatment of metal ion

toxication, as synthetic ionophores [25], as novel antibiotics that owe their

antibiotic action to specific metal complexation [26], stem cell mobilization

[16], to study the host-gust interactions and in catalysis [27], as metal

extractants [28] and as luminescent probes. Macrocyclic molecules can also

fiinction as receptors for substrates of widely differering physical and chemical

properties, which can be drastically altered upon complexation.

Several classes of macrocyclic ligands have been synthesized with varying

combination of aza (N), oxa (0), sulpha (S) and phospha (P) donor atoms.

Interaction between the macrocyclic ligand and the substrates can be fine-

tuned by appropriate selection of the binding site, and over all ligand topology

such as nature of donor atoms, donor set, donor array, ligand substitution and

nature of the ligand backbone. Other factors considered are as electronic

effects, structural effects, conformation, shaping groups and cavity size.

The extensive series of macrocyclic ligands have been classified as saturated

polyazamacrocycles [29] (Figure 1), polyethermacrocycles [30] (Figure 2),

polyazathiamacrocycles [31 ] (Figure 3), polyoxathiamacrocycles [32] (Figure

4), mixed donor atom macrocycles [33] (Figure 5), polyphosphamacrocycles

[34] (Figure 6), polythia macrocycles [35] (Figure 7).

2+

O

,0

"o o

.0 .

Figure 1 Figure 2

.NH HN.

\ NH HN

Figure 3

o

\ s s

Figure 4

N

•°v_/°'

Ph / \ Ph

Mi

Ph \ / Ph

Figure 5 Figure 6

Figure 7

Macrocyclic ligands are far more stable as compared to their open chain

analogues as they have stereochemical constraints associated with their cyclic

nature, which may influence their potential for metal ion recognition [36]. The

several ligands coordinated to a metal ion are held in specific geometric

orientations and recognition of this fact led to the "coordination template

hypothesis" [37]. The enhanced stability of metal complexes of macrocyclic

ligands over other polydentate ligands is attributed to various structural effects

are, chelate effect, macrocyclic effect, cryptate effect. These effects which

have been found to give stable complexes arise from the structural factors,

viz., size, shape or geometry, connectedness or topology and rigidity of the

macrocycle. (Figure 8) displays the general observation that the affinity

between the ligands of a particular kind, e.g., amines for a given metal

increases with the increasing topological constraint [38]. The topological

constraint is in the order, simple coordination < chelation < macrocyclic effect

< cryptate effect.

Increasing Topological Constraint

Coordination Chelation Macrocyclic Effect Cryptate Effect

NH,

H,N

H,N

HjN

H

H

.NH,

N H

NH,

NH HN

NH HN

,NH,

NH " ^ N H N

NH HN HN

Figure 8. Topology and the Chelate, Macrocyclic and Cryptate Effect.

The synthesis of macrocycles is an art in itself. There are four main approaches

to prepare such macrocyclic ligand systems:

1. Conventional organic synthesis.

2. Metal ion promoted reactions, involving condensation of noncyclic

components in the presence of suitable metal ion (Template effect).

3. Modification of a compound prepared by methods a and b.

4. High dilution technique.

A variety of crown ethers and mixed oxathia crown have been prepared

mainly by the direct synthesis [32,39] (Scheme 1 and 2).

,0H

. 2 /~ \ rA , CI O CI

OH

Scheme 1

. 0

^o'

o.

"O O'

.0.

CI 0 0 0 CI HS SH

^O

0.

Scheme 2

-s^^^s-

Polyazathia macrocycles have been synthesized by reacting an appropriate

polythiane with a dibromoalkane. The reaction may be sometimes aided by

metal template [37] (Scheme 3).

^ ° /S 0

HjN SH (i)Ni

HS NH2 rt-'X'^^'"

\J^ Br

Sheme 3

Azamacrocyclic complexes have been synthesized [40], by the template

condensation reaction (Scheme 4).

4NH2—NH2H2O + 2HCH0 + MX2 + 2Br(CH2)nBr

MeOH

H

H. HN N

M

X

HN N ^ N NH

N NH

HN NH ^ HN-

/ ^ ^ ^

\ H/|\ PIN N j^ PIN-

-NH

-NH

Where M = Co(II), Ni(II), Cu(II) and Zn(II)

X = ClorN03;n = 2or3

Scheme 4

The coordination chemistry of polyaza, polythia and mixed azathia

macrocyclic ligands has attracted much attention in recent time. These

macrocycles show the remarkable ability to form stable and inert complexes

with a wide range of d- and p- block metals, in many cases forcing the metal

centere to adopt unusual coordination geometries or oxidation state [41]. The

presence of sulfur donor as a part of macrocyclic ligand has been shown to

stabilize low valent metal complexes as well as to have a marked influence on

the coordination geometry at the metal centre. Ligand incorporating sulfur

donors show a preference to bind transition metal rather than alkali and

alkaline earth ions due to the soft nature of sulfur [42].

Recently there has been quickening interest in them due in part to the

recognition of coordinated methionine sulfiir [43] in "blue" copper proteins and

in part to an enhanced awareness of applications for sulfur compounds in a

variety of technologies and medicine [44]. Crown thioethers have seen

extensive use as bioinorganic model systems, binucleating ligands, chelators

for specific metal ion, and phase transfer catalysts. For these purpose they have

several advantages. They permit control of the coordination environment

(donor atoms) and, in principle, the stereochemistry at a metal ion [45].

Macrocyclic tetrathioether complexes of copper(ll) even those with

undistorted tetragonal coordination geometry about the copper atom [35], show

the same unusual spectroscopic [46] and electrochemical properties [47] as the

blue copper proteins, such as plastocyanin [48], stellacyanin [49], and

rusticyanine [50], etc. Coordination of all four sulfur atom of a macrocyclic

tetrathioether such as 1, 4, 8 ,11-tetra thiacyclotetradecane or (|14|aneS4) by a

single metal atom requires a change in conformation of the ligand [51]. An

important aspect of the chemistry of these ligands pertains to the preference of

the free ligand for exodentate conformations in which the sulfur atom lone

pairs are directed outwards [42] (Figure 9).

Figure 9

Sulfur acts as a very good ligating atom when in the form of the sulfide ion or

as mercaptide ion, but complexes of sulfur as a thioether are much less

abundant[52], though in cyclic polyether analogs, the metal ion could be

coordinated in the expected enclosed fashion for several metal ions including

the nickel(II) complex [53] (Figure 10) or in exodentate form a found for

niobium(IV) and mercury(II) choloride [54, 55], respectively (Figure 11 and

Figure 12).

Figure 10

10

CI '/,,

CI

Nb

,01

'CI

CI

Figure 11 Figure 12

Polythia macrocycles, have been synthesized by reacting an appropriate

polythaiane with a dibromoalkane [56] (Scheme 5).

SH HS' Br Br

Scheme 5

.S S.

S S'

11

Rosen and Busch [57] have been synthesized of Nickel(II) complexes of some

tetradentate thioethers as the exclusive donors. These new materials

include the first lov^-spin nickel(II) complexes having four thioethers

as the donors, and a number of tetragonal complexes formed from one of these

low- spin complexes by coordination of various axial ligands (scheme 6).

,s s.

SH US^

NaoEtOH

^S" -S

Scheme 6

,S S,

N s^

\ /

Broer de Groot et al. [58], have been synthesized of large ring meta- and ortho-

thiacyclophanes ditopic Macrocycles Cu(I) and Ag(I) complexes, containing

six sulfur atoms (Scheme 7).

12

Scheme 7 V ^

13

In many cases thioether complexes can be obtained in a direct reaction of aqua

or halogen complexes with the thioether in aqueous or alcoholic solution by

simple addition or substitution [59].

Other crown thioether ligands containing six or more sulfur donor atoms have

the potential to coordinate two metals center [60] there by acting as ditopic

4-7

ligand and is also capable of fixing two metal ions e.g., Ni , in a square

planar coordination [61] (Figure 13).

Figure 13

The M.Phil dissertation deals with the synthesis, physico-chemical

characterization of thiaether macrocycle complexes encapsulating first row

transition metal ions. DNA binding study on [CuLC^] complexes.

14

PRESENT WORK

The design and study of well-arranged metal containing macrocycles have

gained an accelerated research interest in resent years and has been a subject of

numerous reports [62] because of their unique coordination chemistry [63, 64]

and their relevance to the models for charge transfer or electron transport and

allosteric behavior in bioinorganic systems as well as their potential

applications in catalysis [65]. Such compounds usually behave as model

ligands for metal enzymes, as metal ion selective ligands and as metal chelating

agents for medical purposes and are also significant for the development of

new methodologies in separation science [66]. This may represent very

promising new synthetic approaches to design the macrocyclic ligands whose

metal binding sites are extremely effective in terms of cavity size, geometrical

requirements, coordination number and nature of the donor atoms to form

complexes selectively [67]. A rapidly emerging area of chemical interest in

recent years is the synthesis of polyaza, polythia and mixed azathia

macrocyclic ligands and their metal complexes because of presence of several

potential donor atoms, their flexibility and ability to coordinate with several

metal ions [68, 69]. These macrocycles show the remarkable ability to form

stable and inert complexes with a wide range of d- and p- block metals, in

many cases forcing the metal centre to adopt unusual coordination geometries

or oxidation states [70-71]. The presence of sulfur as a donor atom for

transition metals is a well known phenomena [59]. Although sulfides are

generally regarded as very poor ligand to transition metal centers, recent

studies have shown that macrocyclic sulfides readily bind to certain metal ions

15

to form highly stable complexes [72-74] and have received great attention of

their biological activities including antitumar, antibacterial, antiviral,

antifungal and anticarcinogenic properties [75-79]. Macrocyclic complexes of

Cu(II) have been reported to interact with DNA by different binding modes and

exhibited efficient nuclease activity [80, 81] but unfortunately have received

little attention. However, the exact mode and extent of binding and cleavage

mechanism still remain unknown leaving behind a scope for an extensive

studies involving macrocycles with different structures to evaluate and

understand the feature that determine the mode of the binding interaction with

DNA and mechanism. All the above facts inspired to initiate the work on

macrocyclic complexes and the present work deals with the synthesis and

characterization of tetrathia macrocyclic complexes by template condensation

reaction of a,a'-dibromo-o-xylene and 1,3-propanedithiol in 1:2:2 molar ratio

and DNA binding studies on [CuLCb] complex with Calf thymus DNA by

fluorescence measurement and absorption spectroscopy.

16

EXPERIMENTAL METHODS

EXPERIMENTAL METHODS

There are several physico-chemical methods available for the study of

coordination compounds and a brief discussion of the techniques used

in the investigation of the newly synthesized Schiff base complexes

described in the present work are given below:

1- Infrared Spectroscopy

2- Nuclear Magnetic Resonance Spectroscopy

3- Electron Paramagnetic Resonance Spectroscopy

4- Ultraviolet and Visible (Ligand Field) Spectroscopy

5- Magnetic Susceptibility Measurements

6- Mass Spectrometry

7- Molar Conductance Measurements

8- Elemental Analysis

9- Fluorescence study

1. INFRARED SPECTROSCOPY

When Infrared light is passed through a sample, some of the

frequencies are absorbed while other frequencies are transmitted through

the sample without being absorbed. The plot of the percent absorbance or

percent transmittance against frequency, the result is an infrared spectrum.

The term "infrared" covers the range of the electromagnetic spectrum between

0.78 and lOOO^m. In the infrared spectroscopy, wavelength is measured in

"wavenumbers", Which has unit as cm''.

Wave number = 1/ wavelength in centimeters

17

It is useful to divide the infrared region in to three regions; near, mid and far

infrared.

Region Wave Length Range (^m) Wave Number Range (cm')

Near 0.78-2.5 12800-4000

Middle 2.5-50 4000-200

Far 50-1000 200-10

Theory of Infrared Absorption

The IR radiation dose not have enough energy to induce electronic

transitions but has enough energy to induce vibrational and rotational

transitions having small energy difference between vibrational and

rotational states in the molecule.

For a molecule to absorbed IR radiations, the vibrations or rotations

within a molecule must cause a net change in the dipole moment of

the molecule. The alternating electrical field of the radiation

(electromagnetic radiation consists of an oscillating electrical field and

an oscillating magnetic field, perpendicular to each other) interacts with

fluctuation in the dipole moment of the molecule. If the frequency of the

radiation matches the vibrational frequency of the molecule then radiation

will be absorbed, causing the change in the amplitude of molecular

vibration. In the absorption of the radiation, only transition for which

change in the vibrational energy level is AV = 1 can occur, since most

of the transition will occur from stable VQ to Vi the frequency

corresponding to its energy is called the fiindamental frequency.

18

The group frequencies of certain groups characterize the group

irrespective of the molecule in which these groups are attached. The

absence of any band in the predicted region for the group indicates the

absence of that particular group in the molecule.

Molecular rotation

Rotational levels are quantized, and absorption of IR by gases yields line

spectra. However, in solids, these lines broaden in to a continuum due

to molecular collisions and other interactions.

Molecular vibrations

The position of atoms in a molecule is not fixed; they are subject to a

number of different vibrations. Vibrations fall in to the two main categories

of stretching and bending.

Stretching:- Change in inter-atomic distance along bond axis

1. Symmetric

2. Asymmetric

Bending: - Change in angle between two bands. There are four types of bend.

1. Rocking

2. Scissoring

3. Wagging

4. Twisting

Vibrational coupling

In addition to the vibrations mentioned above, interaction between

vibrations can occure (coupling) if the vibrating bonds are joined to a

19

single, central atom. Vibrational coupling is influenced by a number of

factors viz., strong coupling of stretching vibrations occurs when there is a

common atom between the two vibrating bonds, coupling of bending

vibrations occurs when there is a common bond between vibrating groups,

coupling between a stretching vibration and a bending vibration occurs if

the stretching bond is one side of an angle varied by bending vibration,

coupling is greatest when the coupled groups have approximately equal

energies, no coupling is seen between groups separated by two or more

bonds.

Important Group Frequencies in the IR Spectra Pertinent to the

Discussion of the Newly Synthesized Compounds.

1) M-X Stretching Frequency

Metal-halogen stretching bands appear [82] in the region of 500-750

cm"' for M-F, 200-400 cm''for M-Cl, 200-300 cm'' for M-Br and 100-

200 cm"' for M-I.

2) M-O Stretching Frequency

Metal-oxygen stretching frequency has been reported [83] to appear

in different region for different metal complexes. The M-0 stretching

frequency has been reported to appear in different region for

different metal complexes. The M-0 stretching frequency of nitrato

complexes lie in the range 250-350 cm"'.

20

3) S-H Stretching Frequency

The S-H stretching vibration in mercaptans are usually observed in the

range 2500-2600 cm''. The S-H absorption is not inherently strong and is

often difficult to detect in dilution solution or in samples examined in very

thin cells.

4) C-S Stretching Frequency

The C-S stretching frequency generally appear [84] as a band of weak or

moderate intensity in the range 570-750 cm"'. There appears to be a

progressive decrease in the frequency in the order, primary, secondary

and tertiary C-S. In aromatic derivaties the C-S frequency is higher due

to the presence of the intense C-H out of plane defor mation band in this

region. In phenyl, sulphonyl, halide the C-S vibration occurs between

706-715 cm"'.

5) M - S Stretching Frequency

The metal sulfure stretching frequentcy is interesting , as it gives a direct

evidence for coordination through the sulfur atom in metal mercapto

complexes. It has been reported [82] that M-S appear in the region 325 -

390 cm-'.

2) NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

Nuclear magnetic resonance (or NMR) is concerned with the magnetic

properties of certain nuclei e.g. 'H , '^C etc. The nuclei for which spin

quantum number, I > 0 exhibits the NMR phenomenon where I is

21

associated with the mass number and atomic number of the nuclei as

shown below:

MASS NUMBER ATOMIC NUMBER SPIN QUANTUM NUMBER

Odd Odd or Even 1/2, 3/2, 5/2

Even Even 0

Even Odd 1, 2, 3

Since atomic nuclei are associated with charge, a spinning nucleus

generates a small electric current and has a finite magnetic field

associated with it. When a spinning nucleus is placed in a magnetic

field, the nuclear magnet experiences a torque which tend to align it with

the external field. The number of possible orientafions for a magnefic

nucleus under the influence of an external magnetic field as given by

(21 + 1) so that for nuclei with spin 'h ( 'H, '^C, '^F etc.) only two

orientations are allowed, parallel to the field (low energy) and against the

field (high energy). If the precessing nuclei are irradiated with a beam of

radiofrequency energy of the correct frequency , the low energy nuclei

may absorb this energy and move to a higher energy state. The

precessing nuclei will only absorb energy from the radiofrequency

source if the precessing frequency is the same as the frequency of the

radiofrequency beam, the nucleus and the radiofrequency beam are said to

be in resonance, hence the term nuclear magnefic resonance .

The Precessional frequency, D is directly proportional to the strength

of the external field, BQ, i.e.

22

1) a Bo

This is the most relationships in NMR spectroscopy.

A proton exposed to an external magnetic force 1.4 1(14,000 guass)

will precess = 60 million times per second, so that v = 60MHz. The

precessional frequency of all protons in same external applied field is not,

however, the same, and the precise value for any proton depends on a

number of factors such as electronegativity of the attached groups, van der

waals desheilding and anisotropic effects explained by Packard in 1951,

who detected three different values for the precessional frequencies of the

protons in ethanol (CH3, CH2, OH) since then NMR has become an important

tool for the chemist. As the shift in frequency depends on chemical

environment, the terms chemical shift was coined . Chemical shift positions

are normally expressed in 8 (delta units) which are defined as proportional

differences in parts per million (ppm) from an appropriate refemce standard

(TMS in the case of proton and carbon -13 NMR). Siince the 5 unit is

proportionality, it is a dimensionless number it is independent of field strength.

There are two types of NMR spectrometers :-

1) Continuous wave (CW)

2) Pulsed or Fourier transform (FT-NMR)

These instrument use very powerful magnets and irradiate the sample with

electromagnetic radiation in the radio frequency region .

23

NMR spectra can be recorded by either holding the magnetic field

constant or by keeping the radiofrequency constant and varying the

magnetic field .

3) ELECTRON SPIN RESONANCE SPECTROSCOPY

Electron spin resonance is a branch of absorption spectroscopy in which

radiation of microwave frequency is absorbed by molecules possessing

electrons with unpaired spins.

Gorter demonstrated [85] that a paramagnetic salt when placed in a high

frequency alternating magnetic field absorbs energy, which is influenced by the

application of a static magnetic field either parallel or perpendicular to the

alternating magnefic field. The degeneracy of a paramagnetic ion is lifted in a

strong stafic magnetic field and the energy levels undergo Zeeman splitting.

Applicaction of an oscillating magnetic field of appropriate frequency will

induce transitions between the Zeemen levels and the energy is absorbed from

the electromagnefic field. If the static magnetic field is slowly varied, the

absorption shows a series of maxima. The plot between the absorbed energy

and the magnetic field is called the electron paramagnetic resonance spectrum.

A system exhibit paramagnetism wherever it has a resuhant angular momentum

Such paramagnetic system includes, elements containing 3d, 4d, 4f, 5d, 5f, 6d

etc. electrons, atom having an odd number of electrons like hydrogen,

molecules containing odd number of electrons such as NO2, NO etc. and free

radicals which possess an unpaired electron like methyl free radical

diphenylpicryl hydrazyl free radical etc. are among the suitable reagents for

24

EPR investigation. Spilitting of energy levels in EPR occurs under the

effect of two type of fields, namely the internal crystalline field and

applied magnetic field. While studying a paramagnetic ion in a diamagnetic

crystal lattice, two type of interactions are observed, i.e., interactions between

the paramagnetic ion called dipolar interaction and the interactions between

paramagnetic ion and the diamagnetic neighbour called crystal field interaction.

For small doping amount of paramagnetic ion in the diamagnetic host, the

dipolar interaction will be negligibly small. The later interaction of

paramagnetic ions with diamagnetic ligands modifies the magnetic properties

the paramagnefic ion. According to crystal field theory, the ligand influences

the magnetic ion through the electric field, which they produce at its site and

their orbital mofion gets modified. The crystal field interaction is affected by

the outer electronic shells.

The dipole-diapole interaction arises from the infiuence of magnetic field of

one paramagnetic ion on the dipole moments of the similar neighboring ions.

The local field at any given site will depend on the arrangements of the

neighbors and the direction of their dipole moments. Thus resultant field varies

from site to site giving a random displacement of the resonance frequency of

each ions and thus broadening the line widths.

Hyperfine interactions are mainly magnefic dipole interactions between the

electronic magnetic moment and the nuclear magnetic moment of the

paramagnetic ion. The quartet structure in the EPR of vanadyl ion is the results

of hyperfine interactions. The origin of this can be understood simply by

25

assuming that the nuclear moment produces a magnetic field, BN at the

magnetic electrons and the modified resonance condition will be E = hv = gP

|B + BNI where BN takes up 21+1, where I is the nuclear spin. There may be an

additional hyperfine structure also due to interaction between magnectic

electrons and the surrounding nuclei called superhyperfine structure. The effect

was first observed by Owens and Stevens in ammonium hexachloroiridate

[86] and subsequently for a number of transition metal ion in various hosts

[87].

4) ULTRAVIOLET AND VISIBLE SPECTROSCOPY

When a molecule absorbs ultraviolet / visible light of a particular energy ,

we assume as a first approximafion that only one electron is promoted to

a higher energy level and that all other electrons are unaffected. The

excited state thus produced is formed in a very short time (of the order

of 10"' s) and a consequence is that during electronic excitation the

atoms of the molecule do not move( the Franck Condon Principle).

The most probable AE transition would appear to involve the promotion of

one electron from the highest occupied molecular orbital to the lowest

available unfilled orbital, but in many cases several transitions can be

obererved, giving several absorpfion bands in sepectrum. Not all transifions

from filled to unfilled orbitals are allowed, the symmetry relationship between

the two orbitals being important.

Where a transition is 'forbidden', the probability of that transidon occurring is

low, and correspondingly the intensity of the associated absorpfion band is also

26

low. These two early empirical laws govern the absorption of light by

molecules :

Beer's Law relates the absorption to the concentration of absorbing solute,and

Lambert's Law relates the total absorption to the optical path length.

They are most conveniently used as the combined Beer-Lambert Law :

Log (Wl) = e c l o r e = A / c l

Where, lo is the intensity of the incident light (or the intensity passing through

a reference cell), I is the light transmitted through the sample solution, Log

(lo/I) is the absorbance (A) of the solution (formerly called the optical density,

1 T

OD), c is the concentration of solute (in mol 1", mol dm"), 1 is the path length

of the sample (in cm, m xlO") and G is the molar absorptivity (formerly called

the molecular extinction coefficient, in 1 mol" cm", m mol" xlO".

5) MAGNETIC SUSCEPTIBILITY MEASUREMENTS

The determination of magnetic moments of transition metal complexes have

been found to provide ample information in assigning their structure. The main

contribution to bulk magnetic properties arises from magnetic moment

resulting from the motion of electrons. It is possible to calculate the

magnetic moments of known compounds from the measured values of

magnetic susceptibility.

There are several kinds of magnetism in substances viz., diamagnetism,

paramagnetism and ferromagnetism or antiferromagnetism. Mostly compounds

of the transition elements are paramagnetic. Daimagnetism is attributable

to the closed shell electrons with an applied magnetic field. In the closed

27

shell the electron spin moment and orbital moment of individual electrons

balance one another so that there is no magnetic moment. Ferromagnetism

and antiferromagnetism arise as a result of interaction between dipoles of

neighboring atoms.

If a substance is placed in a magnetic field H, the magnetic indection B

with the substance is given by

B = H +47il

Where I is the intensity of magnetization. The ratio B/ H is called

magnetic permeability of the material and is given by

B/H I + 4 71 (I/H ) = I + 4 7t K

Where K is called the magnetic susceptibility per unit volume or volume

susceptibility. B/H is the ratio of the density of lines of force within the

substance to the density of such line in the same region in the absence

of sample. Thus the volume susceptibility of a vacuum is by definition

zero since in vacuum B/H = 1.

When a magnetic susceptibility is considered on the weight basis , the

gram susceptibility (Xg) is used instead of volume susceptibility. The jiefr

value can then be calculated from susceptibility multiplied by the

molecular weight and corrected for diamagnetic value as

28

Where, T is the absolute temperature at which the experiment is

performed . The magnetic properties of any individual atom or ion will

result from some combination of these two properties that is the inherent

spin moment of the electron and the orbital moment resulting from the

motion of the electron around the nucleus. The magnetic moments are

usually expressed in Bohr Magnetons (BM). The magnetic moment of a

single electron is given by

^s= gVS(S + l) BM

Where S is the spin quantum number and g is the gyromagnetic ratio. For

Mn , Fe ^ and other ions whose ground states are S states there is no

orbital angular momentum. In general, the transition metal ions in their

ground state D or F being most common do possess orbital angular

momentum. For ions such as Co ^ and Ni , the magnetic moment is

given by

[i(s+ L) = V4S(S + 1) + L(L + 1)

In which L represents the orbital angular momentum quantum number for

the ion.

The spin magnetic moment is insensitive to the environment of the metal

ion, the orbital magnetic moment is not. In order for an electron to have

an orbital angular momentum and thereby an orbital magnetic moment

with reference to a given axis, it must be possible to transform the

orbital into a fiilly equivalent orbital by rotation about that axis.

29

For octahedral complexes the orbital angular momentum is absent for

Aig, A2g and Eg terms, but can be present for Tig and Tig terms. The

magnetic moments of the complexes possessing T ground terms usually

differ from the high spin value and vary with temperature. The magnetic

moments of the complexes having a ^Aig ground term are very close to

the spin-only value and are independent of the temperature.

For octahedral and tetrahedral complexes in which spin-orbit coupling

causes a spilit in the ground state an orbital moment contribution is

expected. Even no spiliting in the ground state appears in cases having no

orbital moment contribution, an interaction with higher states can appear

due to spin-orbit coupling giving an orbital moment contribution.

Practically the magnetic moment value of an unknown complex is

obtained on Gouy Magnetic Balance. Faraday method can also be applied for

the magnetic susceptibility measurements of small quantity of solid

samples. The gram susceptibility is measured by the following formula

AW W',, ' W AW,"'

std

Where Xg ^ Gram Susceptibility

AW = Change in weight of unknown sample with magnet on and off.

W = Weight of the known sample

A Wstd = Change in weight of standard sample with magnet on and off.

Wstd = Weight of standard sample.

Xstd = Gram susceptibility of the standard sample.

30

6) MASS SPECTROMETRY

In mass spectrometry, a substance is bombarded with an electron beam

having sufficient energy to fragment the molecule. The positive fragments

which are produce (cations and radical cations) are accelerated in a vacuum

through a magnetic field and are sorted based on the mass-to-charge ratio.

Since the bulk of the ions produced in the mass spectrometer carry a unit

possitive charge, the value m/e is equivalent to the molecular weight of the

fragment. The analysis of mass spectroscopy information involves the re­

assembling of fragments, working backwards to generate the original

molecule. A very low concentration of sample molecules is allowed to leak

in to the ionization chamber (which is under a very high vacuum) where

they are bombarded by a high-energy electron beam. The molecules

fragment and the positive ions produced are accelerated through a charged

array into an analyzing tube. The path of the charged molecules is bent by

an applied magnetic field. Ions having low mass (low momentum) will be

deflected most by this field and will collide with the walls of analyzer.

Likewise, high momentum ions will not be deflected enough and will also

collide with the analyzer wall. Ions having the proper mass-to-charge ratio,

however, will follow the path of analyzer, exit through the slit and collide

with the collector. This generates an electric current, which is then amplified

and detected. By varying the strength of magnetic field, the mass-to-charge

ratio which is analyzed can be continuously varied.

31

The output of the mass spectrometer shows a plot of relative intensity Vs

the mass -to-charge ratio (m/e). The most intense peak in the spectrum is

termed the base peak and all others are reported relative to it's intensity.

The peaks themselves are typically very sharp, and are often simply

represented as vertical lines.

The process of fragmentation follows simple and predictable chemical

pathways and the ions, which are formed, will reflect the most stable

cations and radicals cations, which that molecule can form. The highest

molecular weight peak observed in a spectrum will typically represent the

parent molecule, minus an electron, and is termed the molecular ion (M" ).

Generally, small peaks are also observed above the calculated molecular

weight due to the natural isotopic abundance of C, H, etc. Many

molecules with especially labile protons do not display molecular ions;

an example of this is alcohol, where the highest molecular weight peak

occurs at m/e one less than the molecular ion (m-1). Fragments can be

identified by their mass-to-charge ratio, but it is often more informative to

identify them by the mass which has been loss. That is, loss of a methyl

group will generate a peak at m-15; loss of an ethyle, m-29, etc.

7) CONDUCTIVITY

The resistance of a sample of an electrolytic solution is defined by

R = p [1/A]

where 1 is the length of a sample of electrolyte and A is the cross sectional

area. The symbol p is the proportionality constant and is a property of a

32

solution. This property is called resistivity or specific resistance. The reciprocal

of resistivity is called conductivity, K

K = 1/p 1/RA

Since 1 is in cm, A is in cm^ and R in ohms (Q), the units of K are Q"'cm"' or S

cm"' (Siemens per cm).

MOLAR CONDUCTIVITY

If the conductivity K is in Q"' cm"' and the concentration C is in mol cm", then

the molar conductivity A is in Q"' cm^ mol"' and is define by

A = K / C

Where C is the concentration of solute in mol cm"

Conventionally solutions of 10' M concentration are used for the conductance

measurements. Molar conductance values of different types of electrolytes in

a few solvents given below :

A 1: 1 electrolyte may have a value of 70-95 Q' cm mol" in nitromethane,

50-75 Q"' cm^ mol"' in dimethyl formamide and 100-160 Q"' cm^ mol"' in

methyl cyanide. Similarly a solution of 2:1 electrolyte may have a value of

150-180 Q"' cm^ mof' in nitromethane, 130-170 Q"' cm^ mol"' in dimethyl

formamide and 140-220 Q"' cm^ mol"' in methyl cyanide [88].

8) ELEMENTAL ANALYSIS

The chemical analysis is quite helpful in fixing the stoichiometric composition

of the ligand as well as its metal complexes. Carbon, Hydrogen, Nitrogen and

Sulfur analysis were carried out on a perkin Elmer-2400 analyzer from Central

Drug Institute, Lucknow, India. Sulfur and cholerine were analyzed by

33

conventional! method [89] where a known amount of the sample was

decomposed in a platinum crucible and dissolved in water with a little

concentrated nitric acid. The solution was then treated with silver nitrate

solution. The precipitate was then dried and weighed. For the metal estimation

[90], a known amount of complex was decomposed with a mixture of nitric,

percholric and sulphuric acids in a beaker. It was then dissolved in water and

made up to known volume so as to titrate it with standard EDTA.

9) FLUORESCENCE STUDY

With some molecules, the absorption of a photon is followed by the emission

of light of a longer wavelength (i.e. lower energy). The emission is called

fluorescence (or phosphorescence, if the emission is long lived). There are

many environmental factors that effect the fluorescence spectrum; furthermore,

fluorescence efficiency is also environmentally dependent. Because these

parameters of fluorescence are more sensitive to the environment than are those

or absorbance and because smaller amounts of material are required,

fluorescence spectroscopy is frequently of greater value than absorbance

measurement. With macromolecules, fluorescence measurements can give

information about conformation, binding sites, solvent interactions, degree of

flexibility, intramolecular distances and rotational diffusion coefficient of

macromolecules. Furthermore, with living cells, fluorescence can be used to

localize otherwise undetectable substances.

As with other physical methods, the theory of fluorescence is not yet adequate

to permit a positive correlation between fluorescent spectrum and the properties

34

of the immediate environment of the emitter; hence the utility of the procedure

is based on establishing empirical principles from studies with model

compounds.

The excited molecules do not always fluoresce. The probability of fluorescence

is described by the quantum yield, Q that is the ratio of the number of emitted

to absorbed photons. Several factors determine Q, same of these are properties

of the molecules itself (internal factors) and some are environmental.

The internal factors are not generally of interest to biochemist concerned with

the properties of macromolecules, environmental factors are more important.

The effect of the environment is primarily to provide radiation less processes

that compete with fluorescence and thereby reduce Q, this reduction in Q is

called quenching. In biological systems, quenching is usually a result of either

collisional processes (either a chemical reaction or simply collision with

exchange of energy) or a long range radiative process called resonance energy

transfer. These three factors are usually expressed in an experimental situation

involving solutions as an effect of the solvent or dissolved compounds (called

quenchers), temperature, pH, neighboring chemical groups, or the

concentration of the flour.

It is important to know that distinction between a corrected spectrum and an

uncorrected one is not often in the presentation of fluorescence spectra in

journal articles. It is common to plot a spectrum as the photomultiplier output

versus wavelength. This is an uncorrected spectrum. Plotting fluorescence

intensity or quantum yield produces a corrected spectrum. Invariably, when

35

photomultiplier output is plotted, it is incorrectly called fluorescence or

fluorescence intensity.

To measure Q requires the counting of photons because

Q = photons emitted / photons absorbed

Q is a dimensionless quantity Because the energy, E, of one photon is related to

the frequency, u of the light by the relation E = hu, a measurement of the

number of photons requires measuring the energy of the radiation and

correcting for frequency. This usual method for determining Q requires a

comparison with a flour of known Q, two solutions are prepared -one of the

sample and one of the standard fluor - and, with the same exciting source, the

integrated fluorescence (i.e. the area of the spectrum) of each is measured.

The quantum yield, Qx, of a sample X is

Vx ~ IxVsAs/lsAx

Where Qs is the quantum yield of the standard, Ix and Ig are the integrated

fluorescence intensities of the sample and the standard, respectively and Ax and

As and the percentage of absorption of each solution at the exciting

wavelength. Usually the solutions are adjusted so that Ax = Aj.

Two types of fluors are used in fluorescence analysis of macromolecules

intrinsic fluors (contained in the macromolecules themselves) and extrinsic

fluors (added to the system, usually binding to one of the components).

For proteins, there are only three intrinsic fluors - tryptophan , tyrosine and

phenylalanine. The fluorescence of each of them can be distinguished by

exciting with and observing at the appropriate wavelength. In practice,

36

tryptophan fluorescence is most commonly studied, because phenylalanine has

a very low Q and tyrocine fluorescence is frequently very weak due to

quenching. The fluorescence of tyrocine is almost totally quenched if it is

ionized, or near an amino group, a carboxyl group, or a trytophan. In special

situations, however, it can be detected by excitation at 280 nm. The principle

reason for studying the intrinsic fluorescence of protein is to obtain information

about conformation. This is possible because the fluorescence of both

tryptophan and tyrosine depends significantly on their environment (i.e.

solvent, pH and presence of a quencher, a small molecule, or a neighboring

group in the protein).

Fluorescence measurements were made on a Shimadzu RF-5301PC

spectrofluorometer, equipped with microcomputer.

37

EXPERIMENTAL

Material and Methods

Metal salts (all Merck) were commercially available pure samples. The

chemicals a,a'-dibromo-o-xylene (Aldrich) and 1,3-propanedithiol (Acros)

were used as received. Methanol was dried before use [82]. Highly

polymerized Calf-thymus DNA sodium salt (7% Na content) was purchased

from Sigma Chemical Co. Other chemicals were of reagent grade and used

without further purification.

Preparation of stock solutions

Calf thymus DNA was dissolved to 0.5% w/w, (12.5 mM DNA/phosphate) in

0.1 M sodium phosphate buffer (pH 7.40) at 310 K for 24 h with occasional

stirring to ensure formation of homogeneous solution. The purity of the DNA

solution was checked from the absorbance ratio v426o/ 28o- Since the absorption

ratio lies in the range 1.8< v426o/ 28o < 1-9, therefore no further deproteinization

of DNA was needed. The stock solution of [CULCI2] complex with 5 mg/ml

concentration was also prepared.

Etbr labeling of the DNA

External flour ethidium bromide (Etbr) was used for labeling the DNA. An

equimolar concentration of DNA was incubated with Etbr for 30 min. The

binary solution (DNA-Etbr) was centrifiiged at SOOOg and washed twice with

phosphate buffer to remove the unbound probe. The recovered pellet was

resuspended in buffer to obtain the desired concentration of DNA.

38

Synthesis of Complexes

Dichloro/dinitrato [7,8:16,17-dibenzo-l,5,10,14-tetrathiacyclooctadecane]

metal (II), [MLX2]; [M = Co(II), Ni(II), Cu(II) and Zn(II); X = CI or NO3]. To

a methanolic solution (~25cm ) of the metal salt (0.001 mol) placed in a three

necked round bottom flask was slowly added a methanolic solution (~25cm )

of a,a'-dibromo-o-xy]ene (0.002 mol, 0.527g) and 1,3-propanedithiol (0.002

mol, 0.20) simultaneously. The reaction mixture was stirred for 12h leading to

the isolation of a solid product. The solid product thus formed was filtered,

washed with methanol, and dried in vaccum.

Quenching analysis

To further elaborate the fluorescence quenching mechanism the Stem-Volmer

equation was used for data analysis [91]:

Fo/F=HK,y[Q] (1)

where Fo and F are the steady-state fluorescence intensities in the absence and

presence of quencher, respectively, K^^y the Stem-Volmer quenching constant

and [Q] is the concentration of quencher (DNA). The Ksv for the [CULCI2]

complex was found to be of the order of 10'* (9.05 x lO"* Lmol"'). The linearity

of the FQ IF versus [Q] plots for DNA-[CuLCl2] complex binding (Figure 14)

depicts that the quenching may be static or dynamic, since the characteristic

Stem-Volmer plot of combined quenching (both static and dynamic) is an

upward curvature. The procedure of quenching was further confirmed from the

values of bimolecular quenching rate constants, Kq, which are evaluated using

the equation

39

^q = K^v IT, (2)

where TQ is the lifetime of polymer without the quencher. The fluorescence

lifetime used was about 10 " [92]. The bimolecular quenching rate constant was

calculated to be 9.05 x 10 L mol" s' which is largely greater than the

maximum limiting diffusion constant Kdu of the biomolecule {K^

=2.0^10'Ymor' s'') [93]. Which suggested the high affinity and specificity of

this interaction and that the quenching was mainly arisen by the formation of

non-fluorescent complex i.e. static quenching.

Binding equilibrium

When ligand molecules bind independently to a set of equivalent sites on a

macromolecule, the equilibrium between free and bound molecules is given by

the equation [94]:

log [{F, -F)IF\ = \ogK + n log[Q] (3)

where K and n are the binding constant and the number of binding sites,

respectively. Thus, a plot of log (FQ -F)/F versus log [Q] can be used to

determine K as well as n (Figure 15). The values ofK and n were found to be

(1.28 ± 1.21) xio^ M-' and 1.04, respectively.

40

[QJ^iM

Fig .14 Stern-Volmer plot for the binding of [CuLC^] complex with DNA at 298K.

41

u.

1

o

0.4 -

0.2 -

0.0 -

-0.2 -

-0.4 -

-0.6 -

-0.8 - 1

y*

1 1

y^

.... . .

•

- r' —

-5.6 -5.4 -5.2 -5.0

log [Q]

-4.8 •4.6 -4.4

Fig. 15 Plot of log (FQ -F)IF versus log [Q], for determining the binding

constant and number of binding sites of [CULCI2] complex on DNA.

42

Physical Measurements

The elemental analyses were obtained from the Micro-analytical laboratory of

the Central Drug Research Institute (CDRI) Lucknow (India). The IR spectra

(4000-200cm"') were recorded as KBr / Csl discs on the Perkin Elmer - 2400

spectrometer. Metals and chloride were determined volumetrically [95] and

gravimetrically [82], respectively. 'H and ' C NMR spectra were recorded in

DMS0-d6 using a Bruker Avance II 400 NMR spectrometer with Me4Si as an

internal standard from SAIF, Punjab University, Chandigarh (India).

Electrospray mass spectra of the complexes were recorded on a micromass

quattro II triple quadruple mass spectrometer. Magnetic susceptibility

measurements were carried out using a Faraday balance at 25 C. The UV-

visible spectrophotometric studies of the freshly prepared 10" M DMSO

solutions of the complexes in the range 200-1100 nm were conducted using a

Cintra 5 GBC Scientific Spectrophotometer at room temperature. EPR spectra

were recorded at room temperature on a varian E-4 X-band spectrometer using

TCNE as the g-marker. The electrical conductivities (10" M solution in DMSO)

were obtained on a Systronic type 302 conductivity bridge thermostated at

o

25.00 ± 0.05 C. Fluorescence measurements were performed on a

spectrofluorimeter Model RF-5301PC (Shimadzu, Japan) equipped with a

150W Xenon lamp and a slit width of 5 nm. A 1.00 cm quartz cell was used for

measurements. For the determination of binding parameters, 33 iiM of the

labeled DNA solution was taken in a quartz cell and increasing amounts of

complex was titrated. Fluorescence spectra were recorded at temperatures 298

43

K in the range of 540-690 nm (Xex was 590 nm). The UV measurements of calf

thymus DNA were recorded on a Shimadzu double beam spectrophotometer

model-UVnOO using a cuvette of 1 cm path length. Absorbance value of DNA

in the absence and presence of complex were made in the range of 220-300

nm. DNA concentration was fixed at 33 ^M, while the complex concentration

was varied from 3 \iM to 24 |j,M.

44

RESULTS AND DISCUSSION

A series of tetrathia macrocyclic complexes of the type [MLX2]; [M = Co(II),

Ni(TT), Cu(II) and Zn(II); X = CI or NO3] have been prepared by the metal

template condensation reaction between a,a'-dibromo-o-xylene and 1,3-

propanedithiol in a 1:2:2 molar ratio in methanol (Scheme 8). The complexes

were stable in the atmosphere. The level of the purity of the complexes was

checked by T.L.C. on silica gel-coated plates. All attempts failed to obtain a

single crystal suitable for X-ray crystallography.

The formation of ligand framework and the complexes was deduced on the

basis of results of elemental analyses, molecular ion peak in ESI- mass spectra

(Table 1), Characteristic bands in the FT-TR (Table 2) and resonance signals in

1 1 "

the H and C NMR spectra. The overall geometry of the complexes was

inferred from the observed values of magnetic moments and the position of

bands in the EPR and electronic spectra (Table 3). The molar conductance

measurements of all the complexes in DMSO suggest [96] their

non-electrolytic nature.

45

MX.

HS SH

MeOH

Schemes. Formation and Suggested structure of tetrathiamacrocyclic complexes.

M = Co(II), Ni(II), Cu(II) and Zn(II); X = CI or NO3

46

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Infrared spectra

Preliminary identification of the synthesized tetrathia macrocyclic complexes

has been obtained from IR spectra (Table 2). The absence of bands

characteristics of thiol group and the appearance of the u(C-S) bands in 720-

750 cm"' region strongly suggests [97] that the macrocyclic framework has

been formed. This is further supported by the appearance of a new band in the

region 350-390 cm'' assignable [98] to u (M-S) vibration. Bands appearing in

the 1445-1485, 1070-1100 and 760-790 cm"' regions were assigned to aromatic

ring vibrations. The other absorption peaks corresponding to D ( C - H ) and

5(C-fT) appear at their proper position. The coordination of nitrato and chloro

groups has been ascertained by appearance of bands in the 230-240cm"' and

270-300cm"' regions, which may reasonably be assigned [99] to u(M-O) of

the O-NO2 group and D(M-C1) in [ML(N03)2] and [MLCI2] complexes,

respectively. The spectra of the [ML(N03)2] complexes show additional bands

in the 1230-1260, 1040-1080 and 870-890cm"' regions, consistent with

monodentate coordination of the nitrato group [100].

'H and 'C NMR spectra

The integral intensities of each signal in the ' H - N M R spectra of tetrathia

macrocyclic complexes were formed to agree with the number of different

types of protons present. The 'H NMR spectra of both Zn(II) complexes

recorded in DMS0-d6 show a sharp singlet at 4.72 and 4.79 ppm region for

[ZnL(N03)2] and [ZnLCb] respectively, which can be assigned [101] to (Ar-

CH2-S, 8H) protons. A triplet observed in the region 2.82-2.85 ppm for

48

[ZnL(N03)2] and [ZnLCla] corresponds [102] to the (S-CHj, 8H). Another

multiplet in the region 1.72-1.76 ppm may reasonably be assigned [102] to the

middle methylene protons (-SCH2CH2CH2S-) of the 1,3-propanedithiol. A

multiplet observed in the range 7.13-7.34 (Ar-H, 8H) may reasonably be

assigned to aromatic protons. However, the absence of thiol protons again

confirm the proposed macrocyclic framework.

I "i

The C NMR spectra of the Zn(TI) complexes recorded in DMSO-de at room

temperature exhibits the sharp resonance signals at 33.99, and 34.14 ppm for

(S-CH2-Ar) and 32.63 and 32.54 ppm for (S-CH2). While the resonance signal

for middle methylene carbon atom (C-CH2-C) of the 1,3-propanedithiol moiety

at 28.97 and 28.17 ppm for [ZnL(N03)2] and [ZnLCl2] complexes, respectively

[101,102]. The chemical shifts of aromatic carbons appear at 136.62, 135.78,

135.24 and 136.70, 135.82, 135.18 ppm for [ZnL(N03)2] and [ZnLClj]

respectively, corresponding to the proposed macrocyclic skeleton.

EPR spectra

The EPR spectra of the powdered solid copper(II) macrocyclic complexes were

recorded on X-band at frequency 9.1GHz under the magnetic field strength

3100G scan rate 1000, recorded at room temperature. The g|| and gi values

were computed from the spectrum using TCNE free radical as 'g' marker. The

study of the spectra gives g|| values in the range 2.0889-2.1362, gi values in

the range 2.0478-2.0682 (Table 3). The observed g|| values for the complexes

are less than 2.3 in agreement with the covalent character of the metal ligand

band. The ratio g|| > gi > 2.0023 calculated for Cu(II) complexes, suggesting

49

that the unpaired electron is localized in dx -dy orbital of the Cu(II)ion and

the spectral figure are characteristic of axial symmetry and tetragonally

elongated geometry.

G = (g||-2) / (gi-2), which measure the exchange interaction between the

metal centers in a solid complex has been calculated. The observed G value of

< 4 suggest considerable exchange interaction in the solid complexes as

reported by Hathaway and Billing [103].

Electronic spectra and magnetic moments

The observed d-d transitions in the UV-visible spectra of all the macrocyclic

complexes and magnetic moment data (Table 3) are indicative of an octahedral

geometry around the metal ions.

The electronic spectra of cobalt(TI) complexes [CoL(N03)2] and [CoLCli]

showed three bands in the 9,185-9,070, 16,850-17,150 and 21,200-21,400 cm''

regions assignable to the ' TigCF) -> '*T2g(F), *Tig(F) -^ ''AigCF) and ' Tig(F) -^

'*Tig(P) transition, respectively corresponding to the octahedral geometry

around the cobalt(II)ion [104, 105]. The observed magnetic moment values

4.59 and 4.62 B.M. correspond to high spin state and further support the

octahedral geometry around Co(II)ion.

The proposed octahedral geometry around the Ni(II)ion in [NiL(N03)2] and

[NiLCl2] has been inferred from the obtained bands in the 10,030-10,150,

15,650-16,150 and 24,360-24,580 cm'' regions attributed to %g(F) -^ ^T2g(F),

A2g(F) -> Tig(F) and A2g(F) -» T|g(P) transitions, respectively, similar to

the reported earlier [106]. The observed magnetic moments of 2.92 and 2.95

50

B.M. further corroborate an octahedral environment around the Ni(II)ion in

high spin configuration [107].

The absorption spectra of the six coordinate Cu(II) complexes show three spin

allowed transitions in the 11,425-11,535, 18,750-18,688 and 27,355-27,430

cm'' range corresponding to the transitions ^Big-^ Aig ^Big -^ '^Bjg and

Big^ Eg, respectively [108]. Therefore, it may be concluded that the

complexes possess tetragonally distorted octahedral geometry. It has been

further confirmed by the magnetic moment values 1.82 and 1.97 B.M. for

[CuL(N03)2] and [CuLCIz] respectively.

51

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< N _ _ — ' ( N — — — ^ 0 0 — <N

LIT < H H < H H H H H H H < m [ if < ffl w" ^ J ^ - T ^ ^ -5- ^ CO ro CO CO m rA ->rsr!T^rV^rtr<Ni

T t T ^ X X J ^ T J^J^T r T T T T T T ,<—V r " ^ ^ " " ^ r ^ ? ^ r r ^ ' • ' " ^ ^ " " ^ " ^ " ^ ' ' " ^ '—*^ >"••"^ 00 CiO tJO 0 0 00 00

, ^ o o o o o o o o o o o i n o < / ^ i n o o o X J ' ^ o r - - > n O f n i n ' v o > ^ > n o o c N > A ) < r ) m o o f n ° ^ o o c N i o ^ ^ " ^ O v o m ^ ^ ^ > r ) T f t ^ m i n > O T f

^ o - ^ c ^ s t - ^ — i O m - ^ O v o ^ — ^ o o c ~ ~ ' - ^ o o t - -a^:^

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Fluorescence measurements

Binding property of the [CuLCh] complex with DNA

The fluorescence spectroscopy provides insight of the changes taken place in

the microenvironment of DNA-[CuLCl2] complex. The interaction of [CULCI2]

complex with calf thymus DNA was studied by monitoring the changes in the

fluorescence of DNA-Etbr system at varying concentration of the studied

compound (Figure 16), shows the representative fluorescence emission spectra

of the synthesized compound upon excitation at 280nm. The addition of

compound caused a gradual decrease in the fluorescence emission intensity of

the DNA-Etbr system with a conspicuous change in the emission spectra. It can

be seen that a higher excess of the compound led to more effective quenching

of the fluorophore molecule fluorescence. The quenching of the fluorescence

clearly indicated that the binding of the DNA to [CULCI2] complex has

changed the microenvironment of fluorophore residue. The reduction in the

DNA-Etbr fluorescence upon interaction with synthesized molecule could be

due to masking or burial of DNA bound fluorophore upon interaction with

[CuLCli] complex. The quenching of the Etbr fluorescence by the [CULCI2]

complex also suggests the same binding sites of the two and illustrates the

intercalative binding mode of the compound.

Absorption spectroscopy

UV-vis absorption studies were performed to further ascertain the DNA-

[CULCI2] complex interaction. The UV absorbance showed an increase with the

increase in drug concentration (Figure 17). Since [CULCI2] complex does not

54

show any peak in this region (Figure 17, curve x), hence the rise in the DNA

absorbance is indicative of the complex formation between DNA and [CuLCli]

complex molecules. [CULCI2] complex at 260 nm exhibited hyperchromism of

32% at 1:1 molar ratio. The K—K* orbital of the bound ligand can couple with

the 71 orbital of the base pairs, due to the decrease ro—TT* transition energy,

which results in bathochromic shift [109]. The slight shift (~2nm) in the spectra

also suggests complexation of synthesized molecule with DNA. The above

changes are indicative of the conformational alteration of DNA on SJl binding.

/ ^ • . ^ ^ ^ ^

55

200.000

100.000

S9S.0 Wavelengtii (nm)

eSQ.O

Fig. 16. Fluorescence emission spectra of DNA-Etbr complex in the

absence and presence of increasing amount of [CuLCla] complex from

(a) to (j); pH= 7.4; T= 298K.

56

o c CD

o (/) n <

240 250 260 270 280

Wavelength [nm]

290 300

Fig. 17. Absorbance spectra of DNA in the absence and presence of

different concentration of [CULCI2]. DNA concentration was 33

\iM (a). [CULCI2] complex concentration for DNA-compound

system was at 6 ^M (b), 12 \LM (C) 24 (d) and 48)j,M (e) and x

represents the [CuLCli] complex alone.

57

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