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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: [email protected] 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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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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