1

INTRODUCTION

CHAPTER-I

Much attention has been devoted by bioinorganic as well as medicinal

chemists to present the relationship between the metal ions and their complexes as

antitumour [1] and antibacterial [2] agents. Several reviews showed that the

coordination chemistry of many compounds greatly influence their biological

action highlighting the catalytic function of metal in many biological processes.

Metal complexes have shown many useful evidences in medicinal aspects. This

thesis contributes a piece of work with a fact of usefulness of coordination

complexes.

1.1. Coordination complex

In chemistry, a coordination complex or metal complex, is a structure

consisting of a central atom or ion, bonded to a surrounding array of molecules or

anions (ligands, complexing agents). The atom within a ligand that is directly

bonded to the central atom or ion is called the donor atom. Polydentate ligands can

form a chelate complex. Compounds that contain a coordination complex are

called coordination compounds. The central atom or ion, together with all ligands

forms the coordination sphere. Coordination refers to the coordinate covalent

bonds between the ligands and the central atom. Originally, a complex implied a

reversible association of molecules, atoms or ions through such weak chemical

bonds. As applied to coordination chemistry, this meaning has evolved. Some

metal complexes are formed virtually irreversibly and many are bound together by

bonds that are quite strong [3].

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1.1.1. History of coordination complexes

Coordination complexes were known although not understood in any sense

since the beginning of chemistry, e.g. Prussian blue and copper vitriol. The key

breakthrough occurred when Alfred Werner proposed in 1893, that Co(III) bears

six ligands in an octahedral geometry. His theory allows one to understand the

difference between coordinated and ionic in a compound, for example chloride in

the cobalt ammine chlorides and to explain many of the previously inexplicable

isomers. In 1914, Werner resolved the first coordination complex, called hexol,

into optical isomers, overthrowing the theory that chirality was necessarily

associated with carbon compounds.

1.1.2. Applications of coordination complexes

The applications of coordination complexes are of great importance. These

complexes are widely present in the mineral, plant and animal worlds are known to

play many important functions in the area of analytical chemistry, metallurgy,

biological function system, industry and medicine. A compound containing single

or additional organic bonds, which is a connection among a couple of electrons in

which together electrons are donated by one of the atoms.

The interest of the coordination complexes of Schiff bases, stems from their

versatile catalytic reactions for organic synthesis [4] and its use in degradation of

organic substances [5] and in radiopharmaceuticals [6] and their ability to

reversibly bind oxygen [7] and photochromic properties [8], and the complexing

ability towards transition metals.

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Importance and applications of coordination compounds are of great

importance in biological system. The pigment responsible for

photosynthesis ie., chlorophyll is a coordinated compound of magnesium.

Haemoglobin, the red pigment of blood which acts as oxygen carrier is a

coordination compound of iron. Coordination compounds are used a s

catalysts for many industrial processes.

Importance and applications of coordination compounds find use in many

qualitative and quantitative chemical analyses. Many familiar color

reactions are given by metal ions with number of ligands. Similarly

purification of metal can be achieved through formation and subsequence

decomposition of their coordination compounds.

There is a growing interest in the user of chelate therapy in medicinal

chemistry. An example is the treatment of problem caused by the presence

of metal in toxic proportion in plant and animal. Some coordination

compounds of platinum effectively inhibit the growth of tumours.

1.2. Ligand

In coordination chemistry, a ligand is an ion or molecule that binds to a

central metal atom to form a coordination complex. The bonding between metal

and ligand generally involves formal donation of one or more of the ligand's

electron pairs. The nature of metal-ligand bonding can range from covalent to

ionic. Furthermore, the metal-ligand bond order can range from one to three.

Ligands are viewed as Lewis bases, although rare cases are known involving Lewis

acidic ligands. Metal and metalloids are bound to ligands virtually in all

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circumstances, although gaseous naked metal ions can be generated in high

vacuum. Ligands in a complex dictate the reactivity of the central atom, including

ligand substitution rates, the reactivity of the ligands themselves, and redox.

Ligand selection is a critical consideration in many practical areas, including

bioinorganic and medicinal chemistry, homogeneous catalysis and environmental

chemistry.

Ligands are classified in many ways: their charge, size (bulk), the identity

of the coordinating atom and the number of electrons donated to the metal

(denticity or hapticity). For example, a diagrammatic representation of cobalt

complex is shown below.

Figure.1.1. Cobalt complex [HCo(CO)4] with five ligands

1.2.1. Chelation

Chelation is the binding or complexation of a bi- or multidentate ligand.

These ligands, which are often organic compounds called chelants, chelators,

chelating agents or sequestering agents. Chelating agents form multiple bonds with

a single metal ion. The ligand forms a chelate complex with the substrate. The term

is reserved for complexes in which the metal ion is bound to two or more atoms of

the chelant.

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1.3. Schiff bases

Coordination compounds containing polydentate ligands are called chelates

and their formation is termed chelation. Chelates are particularly stable and very

useful. Schiff base ligand is also called as chelate type ligand. Schiff base

(azomethine), named after Hugo Schiff, is a functional group that contains a

carbon nitrogen double bond with the nitrogen atom connected to an aryl group or

an alkyl group but not hydrogen.

Schiff bases are of the general formula R1R2C=N-R3, where R3 is an aryl

or alkyl group that makes the Schiff base a stable imine. Schiff bases can be

synthesized from an aromatic amine and a carbonyl compound by nucleophilic

addition followed by a dehydration reaction to generate an imine.

1.3.1. Applications of Schiff bases

Schiff bases have a wide variety of applications in many fields, e.g.,

biological, inorganic and analytical chemistry [9]. Application of many new

analytical devices requires the presence of organic reagents as essential compounds

of the measuring system. They are used, e.g., in optical and electrochemical

sensors, as well as in various chromatographic methods, to enable detection of

enhance selectivity and sensitivity [10-12]. Among the organic reagents actually

used, Schiff bases possess excellent characteristics, structural similarities with

natural biological substances, relatively simple preparation procedures and the

synthetic flexibility that enables design of suitable structural properties [13-18].

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A large number of Schiff bases and their complexes have been investigated

for their interesting and important properties, such as their ability to reversibly bind

oxygen, catalytic activity in the hydrogenation of olefins, photochromic properties

and complexing ability towards some toxic metals. Furthermore, complexes of

Schiff bases showed promising biological activity and biological modeling

applications. The Schiff base ligands with sulphur and nitrogen donor atoms in

their structures act as good chelating agents for the transition metal ions.

Coordination of such compounds with metal ions, such as copper, nickel,

manganese and vanadium, often enhances their activities. There is a continuing

interest in metal complexes of Schiff bases. Because of the presence of both hard

nitrogen or oxygen and soft sulphur donor atoms in the backbones of these ligands,

they are readily coordinate with a wide range of transition metal ions yielding

stable and intensely coloured metal complexes. Some of which have been shown to

exhibit interesting physical and chemical properties and potentially useful

biological activities.

In recent years there has been considerable attention focused on the

chemistry of metal complexes of Schiff bases containing nitrogen and oxygen

[19, 20]. Schiff base ligands and their metal complexes have been found to have a

variety of applications in many fields including biology [21], materials synthesis,

photochemistry, magnetism [22], medical imaging [23], and industrial use as

catalysts [24] etc.,

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1.4. Transition metal complexes

Transition metals have an esteemed place in medicinal chemistry. This

property of transition metals resulted in the foundation of coordination complexes.

Metal complex or coordination compound is a structure consisting of a central

metal atom, bonded to a surrounding array of molecules or anions. Sophus

Jorgensen in Denmark synthesized metal conjugates for the first time in the mid

1870‟s. In 1893 the major breakthrough in this field occurred when Alfred Werner

investigated a series of compounds, which contained cobalt, chlorine and

ammonia. He was awarded the Noble Prize in 1913 for his work.

Transition metal complexes having unique electronic and spectroscopic

signatures which offer a multitude of coordination geometries and mechanism of

cytotoxic action which is related to DNA binding affinity [25] and can also vary

accordingly as the biological activity is strongly dependent on structure–activity

relationship. Besides this, metal complexes also utilize or create open coordination

positions for DNA binding and hydrolysis generates reactive oxygen-containing

species or other radicals for DNA oxidation [26].

The interaction of transition metal complexes with DNA have been

extensively studied for their usage as probes for DNA structure and their potential

application in chemotherapy and are potent catalytic inhibitors of DNA gyrase

[27-29]. One of the important DNA related activity of the transition metal

complexes is that some of the complexes show the ability to cleave DNA.

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1.4.1. Transition metal complexes as anticancer agents

Metal complexes have been widely applied in clinics for centuries,

although their molecular mechanism has not yet been entirely understood [30, 31].

The great successes achieved with platinum-based antitumor agents, mainly

including cisplatin, carboplatin and oxaliplatin, have promoted the development of

metal-based drugs. However, all these Pt-based drugs are associated with severe

side effects and evolution of drug resistance during therapy processes, which has

stimulated inorganic chemists to seek and develop more effective, less toxic and

target-specific metal-based anticancer drugs [32].

1.4.2. Platinum based anticancer drugs

Platinum(II) complexes have been used as anticancer drugs. Among them,

cisplatin has proved to be a highly effective chemotherapeutic agent for treating

various types of cancers [33]. Cisplatin moves into the cell through diffusion and

active transport. Inside the cell it causes platination of DNA, which involves

interstrand and intrastrand cross-linking as well as formation of adducts, usually

through guanine, as it is the most electron rich site and hence, easily oxidized.

Formation of cisplatin DNA adducts causes distortion and results in inhibition of

DNA replication [34]. Cisplatin DNA adducts also serve as binding site for cellular

proteins such as repair enzymes, histones, transcription factors and HMG-domain

proteins [35,36]. The binding of HMG protein to cisplatin DNA adduct has been

suggested to enhance anticancer effect of the drug [37].

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1.4.3. Non-platinum anticancer agents

Platinum is not the only transition metal used in the treatment of cancer,

various other transition metals have been used in anticancer drugs [38]. Titanium

complexes such as Titanocene dichloride have been recognized as active

anticancer drug against breast and gastrointestinal carcinomas. Gold complexes

also show anticancer activity. These complexes act through a different mechanism

as compared to cisplatin [39].

The 2-[(dimethylamino) methyl] phenyl gold (III) complex has also been

proven to be anti-tumor agent against human cancers [40]. Gold nanoparticles

when used in combination with radio therapy or chemotherapy enhance DNA

damage and make the treatment target specific [41]. Copper as an essential element

for human with its bioessential activity and oxidative nature has attracted

numerous inorganic chemists to address Cu(II) complexes with the aim of medical

applications [42-45].

Ansari et al [46], studied some complexes of Mn(III) induce tumor

selective apoptosis of human cells. Many ruthenium complexes were studied

which showed anti-proliferative effects in human ovarian cancers. Ruthenium

complexes with oxidation state +2 or +3 show antitumor activity against

metastasis cancers. The relative binding of ruthenocene derivatives were very

high and even better than hydroxyl tamoxifen which is novel antagonist for

estrogen [47].

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1.4.4. Transition metal complexes as anti-infective agents

Transition metals like silver have been used as anti-microbial agents. Silver

has low toxicity as compared to other transition metals. Silver nitrate is still given to

the infants to prevent the development of opthalmia neonatorum. One of the most

commonly used compounds of silver is silver (I) sulfazine; it is used to treat severe

burns to prevent them from bacterial infection. Chlorohexidine- Silver Sulfadiazine is

an anti-infective metal complex against catheter infections in vivo [48, 49].

1.4.5. Transition metal complexes as anti-inflammatory agents

Transition metals have also been used as anti-inflammatory and anti-

arthritic agents. The transition metals complexes of Cu and Fe are capable of

catalyzing dismutation of the superoxide anion. A manganese(II) complex with bis

(cyclohexylpyridine)- substituted macrocyclic ligand has designed as a functional

mimic of the superoxide dismutase (SOD) enzymes that normally remove these

radicals [50]. Manganese complexes have also been used to treat cell and tissue

oxidative injuries by acting as superoxide anion scavenger [51]. Magnesium is

used for the treatment of asthma in children. Some Cu complexes are also active

against inflammation but their use is limited [52]. Cu (II) complexes tend to

dissociate and bind to natural ligands such as albumins [53]. Zinc has been proved

to be involved in the inhibition of pro-inflammatory cytokines [54].

1.5. Multinuclear Schiff base metal complexes

The chemistry of multinuclear Schiff base metal complexes, especially of

coupled systems is of special interest in various fields of science. The main reason

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probably is due to the phenomenon of interaction between metal centres lies at the

crossover point of two widely separated areas, namely the physics of the magnetic

materials and the role of polynuclear reaction sites in biological processes [55,56].

Trinuclear transition metal complexes bridged by polyatomic Schiff base ligands

have gained much attention in the recent years, towards synthesis and

characterization [57]. In particular, transition metal complexes have been the subject

for thorough investigation because of their extensive applications in wide ranging

areas from material science to biological sciences [58]. Metal complexes are well

known to accelerate drug action and the efficiency of a therapeutic agent can often

be enhanced upon coordination with a metal ion [59]. The pharmacological activity

has been found to be highly dependent on the nature of the metal ion and the donor

sequence of the ligands, with different ligands showing widely dissimilar biological

properties, although they may vary only slightly in their molecular structure.

1.6. Biological importance of Copper

The chemical nature of copper is very important in determining its

biological availability, both in the environment and in food. Some of the uses of

copper come from its ability to control the growth of organisms. This occurs when

copper is biologically available and at concentrations that are detrimental. As a

result, copper is used in a range of cidal agents. For example, copper has been

demonstrated to be an effective antibacterial, antiplaque agent in mouthwashes and

toothpastes. Copper also continues to be widely used for the control of unwanted

organisms in fish farming. Evidence in both fresh water and salt water indicates

that there were no hazardous effects on consumers of fish. Copper antifouling

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agents used on fish net pens have been considered a source of metal to the

sediments but there is little evidence that they provide an important source of

dissolved copper when there is adequate water exchange for fish farming.

Few dietary components are more misunderstood than copper. Although

copper is the third most abundant essential trace mineral in the body, after iron and

zinc, most people consider it unimportant. Even worse, many people have actually

taken steps to exclude it from their diets and dietary supplements, believing it to be

nothing more than a cause of free radical reactions. This is surprising, because

copper has been recognized as an essential nutrient since the 1920's [60]. In the

past seventy years, much has been learned about the important biological roles of

copper and the copper-dependent enzymes. In fact, copper is emerging as one of

the most important minerals in our diet. While unbound, free copper does generate

free radicals in vitro, the relevance of this in the body has been called more

imaginary than real [61]. In fact, copper has an entirely different role in the body,

being a component of two of our most important antioxidant enzymes, copper-zinc

superoxide dismutase and ceruloplasmin [62].

Unbound, free copper is not found in large quantities in the human body.

Instead, almost all of the copper in our bodies is bound to transport proteins

(ceruloplasmin and copper-albumin), storage proteins (metallothioneins), or copper

containing enzymes [63]. A substantial number of copper metalloenzymes have

been found in the human body. Copper is essential for the proper functioning of

these copper-dependent enzymes, including cytochrome C oxidase (energy

production), superoxide dismutase (antioxidant protection), tyrosinase

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(pigmentation), dopamine hydroxylase (catecholamine production), lysyl oxidase

(collagen and elastin formation), clotting factor V (blood clotting), and

ceruloplasmin (antioxidant protection, iron metabolism and copper transport) [64].

Most features of severe copper deficiency can be explained by a failure of one or

more of these copper-dependent enzymes. For instance, depigmentation can be

explained by a tyrosinase deficiency and the defects of collagen and elastin causing

abnormalities in the connective tissue and vascular system can be explained by a

lysyl oxidase deficiency.

Unfortunately, most research into copper deficiency has focused on acute,

severe deficiency. This is relatively rare in humans and animals on typical, varied

diets. Marginal, chronic deficiency, however, is much more common. The

determination of copper needs and marginal deficiency is complicated by the fact

that while copper deficiency doesn't necessarily lower the level of copper

dependent enzymes, it does significantly lower their activity [65]. As an example

in lysyl oxidase is one of the most important and best understood roles of copper in

the body. This is the main enzyme involved in the necessary cross-linking of

connective tissue. Optimal functioning of lysyl oxidase ensures the proper cross

linking of collagen and elastin, vital for the strength and flexibility of our

connective tissue. A reduction in lysyl oxidase activity affects the integrity of

numerous tissues, including our skin, bones, and blood vessels. In copper

deficiency the level of lysyl oxidase isn't altered, but the activity of the enzyme can

be reduced by more than fifty percent [66]. Not surprisingly, some of the hallmarks

of copper deficiency are connective tissue disorders, osteoporosis and blood vessel

damage.

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Although most research utilizing copper complexes has been to determine

anti-inflammatory activity, copper complexes have shown potential as a

physiological approach to the treatment of numerous chronic diseases. This

potential has been expanded to include, in addition to inflammatory diseases,

gastrointestinal ulcers, cancers, carcinogenesis and diabetes. In these conditions

much of the research interest has centered on finding that many copper complexes

demonstrate superoxide dismutase (SOD) activity. Because of this, many of these

compounds have been designated as SOD-mimetics. One of the better recent

reviews on this topic of copper complexes is a good example of the breadth of

research that has been published on this topic. Unfortunately, despite the

tremendous promise that copper complexes have in many varied diseases and

conditions, clinical interest in these compounds has been almost nonexistent.

While copper is slowly becoming less misunderstood, one can only hope that it

will eventually be properly utilized in its potential for maintaining health and

treating disease.

The polynuclear copper(II) complexes have aroused extensive interest due

to their importance in biological processes and in inorganic material science [67].

The metal ion dependent oxidative DNA cleavage by Cu(II) complexes is of

topical interest. In the presence of suitable chelating ligands, the d9 electronic state

of Cu(II) may easily accept an electron to produce stable d10 Cu(I) species. Such

redox-active systems may achieve the decomposition of H2O2 to reactive oxygen

species (ROS) which can oxidize DNA by multiple attacks at the sugars and

nucleobases [68]. The coordination geometry around the Cu(II) ion plays a major

role as well in the redox-mediated formation of reactive oxygen species. The

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activation of molecular oxygen by a mononuclear Cu(II) complex in the presence

of DNA is expected to lead to the abstraction of a proton/hydrogen from the sugar

backbone or from the bulk solvent.

1.7. Biological importance of Nickel

Although not recognized until the 1970s, nickel plays important roles in the

biology of microorganisms and plants [69]. Nickel compounds are present in the

active sites of urease and are used extensively in the design and construction of

new magnetic materials. The study of nickel compounds is of great importance in

various aspects of chemistry. The NiFe-hydrogenases contain nickel in addition to

iron-sulfur clusters. Such [NiFe]-hydrogenases characteristically oxidise H2.

A nickel-tetrapyrrole coenzyme, F430, is present in the methyl coenzyme

reductase which powers methanogenic archaea. One of the carbon monoxide

dehydrogenase enzymes consists of an Fe-Ni-S cluster [70]. Other nickel

containing enzymes include a class of superoxide dismutase [71] and a glyoxalase

[72].

In plants and microorganisms, the importance of nickel has been well

documented. In many cases, nickel is needed for the proper functioning of various

plant enzymes such as urease and hydrogenase. In the decreased presence of

urease, due to the lack of adequate nickel, urea accumulation leads to necrosis of

the plant [73]. In soyabeans, where hydrogenase activity was depressed due to

nickel-depletion, only low levels of nitrogen-fixation occurred, which resulted in

slow plant growth and decreased crop yields. Nickel depletion has also been linked

to necrosis of the leaves and stems of a variety of plants, lack of grain viability,

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and depressed vigor of seedlings [74]. Because of the broad distribution of plants

that exhibit a nickel requirement, the authors of the aforementioned studies suggest

that this element is an essential micronutrient for all higher plants.

Nickel complexes draw much attention due to the environmental toxicity

and carcinogenic nature of certain nickel compounds and the chemotherapentic

properties of other group VIII metal complexes [75]. Some researchers have shown

that bound proteins or synthetic ligands may increase the toxic effect of nickel

ions. However, up to now, the exact mechanism to involve carcinogenesis has not

been fully elucidated [76]. The elucidation of the mechanism is essential not only

for the risk assessment [77], but also for developing novel nickel complexes that

have potential applications in medicine and research, such as inhibitors of cancer

proliferation and useful DNA or RNA probes [78]. Therefore, further studies by

employing various ligands with different structures to evaluate and understand

those factors that can determine the DNA binding modes and cleavage mechanism

are necessary.

Nickel complexes in the presence of oxidants have been extensively used

for DNA cleavage reactions [79]. Nickel is an essential trace element for many

species. Chicks and rats raised on nickel deficient diets are found to have liver

problems. Enzymes known as hydrogenases in bacteria contain nickel. Nickel is

also an important additive agent in plant ureases. Nickel complexes in the presence

of oxidants have been extensively used for DNA cleavage reactions.

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1.8. Biological importance of Manganese

Manganese is an essential trace nutrient in all forms of life [80]. The

classes of enzymes that have manganese cofactors are very broad and include

oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, lectins and

integrins. The reverse transcriptases of many retroviruses (though not lentiviruses

such as HIV) contain manganese. The best known manganese containing

polypeptides may be arginase, the diphtheria toxin, and Mn-containing superoxide

dismutase (Mn-SOD).

Mn-SOD is the type of SOD present in eukaryotic mitochondria and also in

most bacteria (this fact is in keeping with the bacterial-origin theory of

mitochondria). The Mn-SOD enzyme is probably one of the most ancient, for

nearly all organisms living in the presence of oxygen use it to deal with the toxic

effects of superoxide, formed from the 1-electron reduction of dioxygen.

Exceptions include a few kinds of bacteria such as Lactobacillus plantarum and

related lactobacilli, which use a different non-enzymatic mechanism, involving

manganese (Mn2+) ions complexed with polyphosphate directly for this task,

indicating how this function possibly evolved in aerobic life. The human body

contains about 10 mg of manganese, which is stored mainly in the liver and

kidneys. In the human brain the manganese is bound to manganese metalloproteins

most notably glutamine synthetase in astrocytes [81].

It is also important in photosynthetic oxygen evolution in chloroplasts in

plants. The oxygen evolving complex (OEC) is a part of Photosystem II contained

in the thylakoid membranes of chloroplasts; it is responsible for the terminal

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photooxidation of water during the light reactions of photosynthesis and has a

metalloenzyme core containing four atoms of manganese [82]. For this reason,

most broad-spectrum plant fertilizers contain manganese.

The chemistry of manganese complexes is of interest as models for

biological redox systems involving manganese ions,such as the oxygen-evolving

center (OEC) of Photosystem II in green plants, superoxide dismutases (SOD) and

Mn-catalases (CAT). 4H2O + hν → 4H+ + O2 is considered the most simple written

and important reaction on the planet at this moment. This is because we owe our

life to the oxygen released from this reaction through photosynthesis. However, 2.7

billion years ago this reaction was the most polluting factor for the environment of

that day. The polluting factor was the oxygen. It was being discarded from the

prokaryotes biological systems in their attempt to consume as much H2O as

possible, to use as an energy source (food). This pollutant (O2) destroyed the

atmospheric environment consisting of CO2 and H2. As a result of this change, the

next step of evolution started, with the creation of higher and more complicated

biological systems, first plants, then animals and human beings. It has been written

that human beings are the highest and most perfect biological system on the planet.

I know that plants can survive without our presence, but we cannot survive without

the presence of plants, having a crucial defect, that of not having chloroplasts.

Coming to the chemical factors implicated in the above given reaction we can find

a metal, i.e. manganese, playing a key role. It catalyzes the cleavage of water in

oxygen evolving complexes (OECs). Besides that, manganese is an essential

element in many other biological processes. Concerning manganese, two

functional values can be distinguished; Mn(II) as a Lewis acid and in higher

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oxidation states [Mn(III), Mn(IV)] as an oxidative catalyst. In most Mn redox

enzymes [83], Mn exists in the oxidation states +2, +3 and +4. Mn(II), in the

manganese-containing ribonucleotide reductase [84-87]; a trimanganese center in

inorganic pyrophosphatase; a binuclear Mn(II) site in the Mn thiosulfate oxidase

[88]; a single Mn(III) center in the manganese SOD [89] catalyzing the

dismutasion of superoxide radicals to oxygen and hydrogen peroxide; Mn(III)

heme center in the manganese peroxidase (MnP) [90,91] capable for the oxidative

degradation of lignin; two Mn(III) single centers in the non heme manganese

catalase [92–95] and four Mn(II)–Mn(IV) in the OEC [96].

The coordination chemistry of manganese in the +2, +3 and +4 oxidation

states is receiving considerable attention due to the biological importance of these

ions [97]. The synthesis and characterization of manganese coordination

complexes to model the structure, reactivity and spectroscopy of manganese in its

various oxidation states, with various ligand types and nuclearities, has contributed

substantially to our understanding of the role and mechanism of manganese

enzyme.

The polynuclear manganese complexes are of great current interest due to

rich biochemistry [98] and remarkable magnetic properties [99]. In order to

understand the fundamental coordination chemistry in manganese systems that is

relevant to catalytic and biochemical functions, efforts have been centered on the

controlled preparation and characterization of manganese carboxylate complexes

with nitrogen donor ligands. A great deal of recent research in polynuclear mixed

valent manganese complexes has focussed on two main lines of enquiry. Firstly, in

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the bioinorganic area, many model complexes have been made to replicate the

structure and function of manganese containing proteins and enzymes including,

significantly, the water oxidation complex (WOC) in the Photosystem II (PSII) of

green plants and cyanobacteria, which is responsible for the catalysis of the light

driven oxidation of H2O to O2 [100, 101].

1.9. Biological importance of Vanadium

Vanadium has been recognized as a metal of biological importance. Its

importance has been recognized at the beginning of 20th Century. Vanadium plays

an important role in the biological system. Considerable effort has been made in

studying the function of vanadium in biological systems as well as its role in

catalytic and pharmaceutical applications [102, 103]. Salen type ligands, one of

the oldest classes of ligands in coordination chemistry, have been used extensively

to complex transition and main group metals [104]. Schiff base complexes

containing different metal ions such as Ni and Cu have been studied in great details

for their various crystallographic feature, structure/redox relationships and

enzymatic reactions, mesogenic characteristics and catalysis properties [105, 106].

Vanadium has a rich and varied chemistry which includes compounds in all

oxidation states from -1 to +5.

Among the transition elements, vanadium has been widely used in a

number of industrial system. Because of its extreme resistance to corrosive agents,

vanadium is extensively used in steel alloys [107]. Vanadium is an important

catalyst in oxidation reactions such as oxidation of naphthalene to phthalic acid

and conversion of toluene to benzaldehyde. V2O5 is an important catalyst in the

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manufacture of SO3 by the contact process. Recently vanadium has been widely

used in bioinorganic chemistry. Though the requirement of vanadium in mammals

is at nano to pico molar level, it is involved in promontory and inhibitory

biochemical processes [108, 109]. Vanadium compounds are also of major concern

because of their adverse effect on the hydro processing catalysis used in the

refining of crude oil [110]. There has been considerable interest in the coordination

chemistry of vanadium involving nitrogen and oxygen donor ligands due to the

increasing recognition of the role of this metal in biological systems. Compounds

in which vanadium is found in different oxidation states from -1 to +5, the higher

oxidation state is that corresponding to the total number of 3d and 4s electrons, that

is vanadium(V), which exists only in oxide species, such as V2O5 and is strong

oxidizing. Among these the most stable and generally important state is +4. which

exists only in oxo species such as (VO)2+

ie., oxovanadium (IV) ion. The

oxovanadium(IV) ion, VO2+, is considered to be the most stable oxocation of the

first row transition metal ion. It forms stable anionic, cationic and neutral

complexes with various types of ligands.

A square pyramidal geometry has been established or more frequently

assumed, for the five co-ordinate oxovanadium(IV) chelate complexes. The

electrolytic behaviour and polymerization tendency of the metal ion is well

documented and many polynuclear complexes of the metal ion have been isolated

and characterized. Because of the stability of the +4 state, compounds of vanadium

in the +3 and +5 states have a strong tendency to get oxidized/reduced to the +4

state.

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Biochemical role of vanadium has now become a widely chosen topic of

bioinorganic chemistry. Though, the requirement of vanadium in mammals is at

nano to picomolar level, its involvement in promontory and inhibitory biochemical

processes, viz., enzymes-nitrogens and halperoxidases, phosphates and ATPase

inhibitor, lowering of hyperglycemia and hyperlipidemida, etc. have enough

driving force for intensive research aimed at establishing its role as a micronutrient

in human beings.

The element has also been identified in the sun, stars and in meteorites. It

is found as a by-product in petrol refining process. Significant amounts are also

found in milk, cereals, seafood‟s and in vegetables. It has a natural affinity for fats

and oils. Food oils have high concentrations of vanadium. When excess amount

of vanadium are taken in the diet, the concentration of vanadium in the red cells

tends to increase. Parental administration increases levels in the lever and kidneys.

However, such increased amounts may only be transient. The lung tissues may

contain some vanadium, depending upon the exposure and route, but normally the

other organs contain its negligible amount. In a variety of plant and animal life, it

appears to play an important role.

1.10. DNA Interactions

The interest of the bioinorganic community in the field of metal / nucleic

acid interactions has burgeoned in the last decade. This interest and the resulting

progress have come about primarily because of the tremendous advances that have

occurred in nucleic acid technology. Bioinorganic chemistry has itself been

evolving from a field focused on delineating metal centers in biology to one that

23

includes also the application of inorganic chemistry to probe biological structures

and function. In the past decades it has become clear that nucleic acids,

structurally, functionally and even remarkably in terms of catalysis, play active and

diverse roles in nature. Transition metal chemistry, both in the cell and in the

chemist‟s test tube, provides a valuable tool both to accomplish and to explore

these processes.

There are also many practical motivations behind the study of how metal

ions and complexes interact with nucleic acids. Heavy metal toxicity in our

environment arises in part from the covalent interactions of heavy metal ions with

nucleic acids. In addition, these heavy metals interface with metalloregulatory

proteins and in so doing disrupt gene expression. We need to understand the

functioning of the natural metalloregulators of gene expression and we need to

design new metal specific ligands, which like the proteins themselves, capture

heavy metals before their damage is done. Heavy metal interactions with nucleic

acids indeed have provided the basis also for the successful application of cisplatin

and its derivates as anticancer chemotherapeutic agents. The design of new

pharmaceuticals like cisplatin requires a detailed understanding of how platinum

and other metal ions interact with nucleic acids and nucleic acid processing.

Furthermore, we are finding that metal complexes can be uniquely useful in

developing spectroscopic and reactive probes of nucleic acids, and hence may

become valuable in developing new diagnostic agents. Finally, Nature itself takes

advantage of metal / nucleic acid chemistry, from the biosynthesis of natural

products such as bleomycin, which chelates redox active metal ions to target and

damage foreign DNA, to the development of basic structural motifs for eukaryotic

24

regulatory proteins, the zinc finger proteins, which bind to DNA and regulate

transcription. In all these endeavors, we need first to develop an understanding of

how transition metal ions and complexes interact with nucleic acids and how this

chemistry may best be exploited.

Metal ions and complexes associate with DNA in a variety of ways. Both

strong covalent interactions and week non covalent complexes are observed. Each

may yield a significant perturbation in the nucleic acid and / or may be exploited to

obtain a site specific response. Clearly there are some general guidelines, based on

principles of coordination chemistry that may be helpful in sorting out these

interactions.

Most prevalent among covalent complexes with DNA are those involving

coordination between soft metal ions and nucleophilic positions on the bases. The

structure of cis – (NH3) 2Pt-dGpG is an example for its platinum center coordinates

to the N7 position of the guanine basis. In terms of interactions with the full

polynucleotide, it is likely that the cis – diammineplatinum center, with two

coordination sites available, would yield an intrastrand crosslink between

neighbouring guanine residues on a strand. Other nucleophilic sites targeted by

soft metal ions on the bases include the N7 position of adenine, the N3 position on

cytosine, and the deprotonated N3 position on thymine and uracil. Some additional

covalent binding to the N1 positions of the purines has also been observed.

Indeed, coordination by the metal to one site on the heterocyclic base lowers the

pKa and increases the metal binding affinity to secondary sites. It is noteworthy,

however, that in base paired double helical DNA only N7 positions on the purines

25

are easily accessible in the major groove of the helix. Base binding at the purine

N7 positions is, of course, not limited to soft metal ions such as Pt (II), and Ru(II).

Coordination at these sites has been evident also with first row transition metal

ions such as Cu (II) and Zn (II). For these, as is consistent with basic coordination

chemistry, the liability of complexes formed is higher.

1.10.1. DNA Cleavage Studies

Identifying molecules that intercalate into DNA helices has attracted

considerable interest over the last few decades. Intercalation was first proposed by

Lerman and is defined as insertion between base pairs [111]. The cleavage of

nucleic acids may be considered as an enzymatic reaction which comprises of

various biological processes as well as the biotechnological manipulation of

genetic material. The application of artificial DNA cleaving agents is manifold:

biotechnology, structural studies of nucleic acids, or development of new drug

[112]. Compounds showing the property of effective binding as well as cleaving

double stranded DNA under physiological conditions are of importance since these

could be used as diagnostic agents in medicinal and genomic research [113].

Noting the very sensitive nature of DNA towards oxidative cleavage, the majority

of the studies on artificial DNAs have been centered around molecules capable of

cleaving DNA with an oxidative mechanism. Several efficient cleaving agents

have been developed in course of time. These involve reactive oxygen species

(ROS) or free radicals that are able to induce an oxidative pathway. The antitumor

antibiotic leinamycin and its analogues have been shown to play the role of

26

chemical nuclease exhibiting reduction of molecular oxygen to form reactive

hydroxyl species [114].

Biochemistry also provides very sensitive techniques that have been

invaluable in characterizing interactions of metal complexes with nucleic acids.

First are simply gel electrophoresis experiments, which permits and assessment of

changes in the nucleic acid conformation, through its changes in gel mobility, as a

function of metal binding. A classic illustration is that of the unwinding of super

helical DNA as a function of intercalation. Closed circular DNA has much the

same topological constraints on it as does a rope or a telephone card; the DNA

helices can wind up in coils. We define the duplex turning in a double helix as

secondary helical turns and turns of the helices about one another as the super coils

or tertiary turns. As long as a DNA double helix is closed in a circle (Form I), the

total winding ie., the total number of secondary and tertiary turns, is fixed.

Molecules with differing extents of winding have different super helical densities.

In a circular molecule with one strand scission, what we call form II (nickel) DNA,

the topological constraints are relaxed and no super coils are apparent. The same,

by analogy, can be said telephone card off the phone receiver, which can turn

about itself to relax its many supercoils. Now let us consider a DNA unwinding

experiment, monitored by gel electrophoresis. Supercoil Form I DNA can be

distinguished from nicked DNA (Form II) in a agarose gel because of their

differing mobilities; the wound- up supercoiled molecule moves easily through the

gelatinous matrix to the positive pole, whereas the nicked species is more floppy

and thus is inhibited in its travels down the gel.

27

Figure.1.2. Relaxation of supercoiled circular form (Form I) of DNA into nicked

circular form (Form II) and linear form (Form III).

DNA strand scission can also be sensitively monitored and even more

importantly the specific nucleotide position cleaved can be pinpointed by

biochemical methods. This methodology has been applied successfully in

monitoring both the efficiency strand scission by metal complexes and specific site

cleaved and hence where the complexes are specifically bound on the helical

strand.

Relative extents of cleavage of DNA by different metal complexes can be

easily assayed in an experiment that is an extension of the unwinding experiment

described above. One simply measures the conversion of supercoiled (form I)

DNA to nicked (form II) species. One strand cleavage on the DNA circle releases

the topological constraints on the circular molecule and relaxes the supercoils. Two

cleavage events within twelve base points on opposite strands will convert the

DNA to a linear (form III) which also has a distinguishable gel mobility.

28

Figure.1.3. Metal complexes exhibit high ability of DNA cleavage through the

effective cooperation of metal with the nucleic acid.

1.10.2. Binding Interactions with DNA

The interaction of metal complexes containing N4 Schiff base ligands has

been thoroughly considered [115]. DNA binding is the critical step for DNA

activity. To design effective chemotherapeutic agents and better anticancer drugs,

it is essential to explore the interactions of metal complexes with DNA [116, 117].

Recently, there has been tremendous interest in studies related to the interaction of

transition metal ions with nucleic acid because of their relevance in the

development of new reagents for biotechnology and medicine [118]. These studies

are also important to understand the toxicity of drugs containing metal ions.

The transition metal complexes are octahedral, substitutionally insert

complexes and as a result of this coordinative saturation the complexes bind to

double helical DNA through a mixture of noncovalent interactions. Tris

(phenanthroline) metal complexes bind to the double helix both by intercalation in

the major groove and through hydrophobic association in the minor groove.

29

Intercalation and minor groove binding are, in fact, the two most common modes

of noncovalent association of small molecules with nucleic acids. In addition, as

with other small molecules, a non specific electrostatic interaction between the

cationic complexes and the DNA polyanion serves to stabilize association.

The extent of intercalative versus groove binding is seen to depend upon

environmental conditions, such as temperature and ionic strength, the charge of the

metal center and the DNA base sequence; groove binding is favored at AT – rich

sequences [119]. Second generation mixed ligand derivatives of the tris

(phenanthroline) series has been prepared and their interactions with DNA have

provided useful insight into the factors important for promoting either intercalation

or groove binding [120]. Aromatic heterocyclic ligands with increased surface

areas that are planar bind DNA with increasing avidity through intercalation,

irrespective of the charge on the metal center. Intercalative binding constants

greater than 107 M-1 can be easily achieved with planar heterocyclic ligands that

just out from the metal center. Not surprisingly, complexes containing ligands of

increasing hydrophobicity that are not planar, favor minor groove binding.

Figure.1.4. DNA binding ability of Metal complexes.

30

Transition metal complexes with aromatic ligand also generally associate

by minor groove binding or through the mix of intercalative and groove-bound

ineractions. Cu(Phen)2, a tetrahedral complex appears to favor minor groove

binding over intercalation. Perhaps the tetrahedral coordination does not permit

appreciable overlap of the phenanthroline ring with the bases in an intercalative

mode. Metalloporphyrins, despite their large expense and the presence commonly

of nonplanar substituents appear to bind to double- helical DNA both by

intercalation and by minor groove binding at AT-Rich sequences. Occupation of

the porphyrins by transition metal ions such as Cu(II), which bind axial ligands,

leads to the favoring of groove binding over intercalation.

1.11. Antimicrobial activity

An anti-microbial is a substance that kills or inhibits the growth of

microorganisms such as bacteria, fungi or protozoans. Antimicrobial drugs either

kill microbes or prevent the growth of microbes. Generally transition metal

complexes have higher antimicrobial activity. Such increased activity of the metal

chelates can be explained on the basis of chelation theory [121]. The transition

metal complexes also disturb the respiration process of the cell and thus block the

synthesis of proteins, which restricts further growth of the organism.

Organic compounds and their metal complexes are well known for their

antimicrobial activity. The metal complexes of ligands containing electron with

drawing group exhibit improved biological activity [122]. Schiff bases and their

metal complexes have been found to possess important biological [123] and

catalytic activity.

31

Azomethines bind to the metal ions through nitrogen, oxygen or sulphur

atoms so form an important class of biologically active ligands and provide models

for metal ligand binding sites in several enzymes [124]. These ligands and their

metal complexes are known to function as antimicrobial [125, 126], antimalarial

[127], antitumor [128] and antileukemic agents [129].

Metal complexes are toxic to most microorganisms at specific

concentrations and often cause serious upsets in biological processes. Some of the

metal complexes are essential for the growth of microorganisms at very low

concentrations and certain metal ions inhibit the growth of many microorganisms

at higher concentrations.

The toxicity of the metal complexes depends mainly upon the nature of

metal ion, ligands and their concentration. Other factor such as pH and

temperature are also reported to affect the toxicity of metals [130]. Though to a

lesser degree, the metal complexes of ligands containing electron-withdrawing

group exhibit improved biological activity.

Some biologically active compounds act via chelation, but for most of them

little is known about how metal coordination influences their activity. The

trinuclear metal complexes have respective controls produce different inhibition

zones against the tested bacterial strains.

32

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[126] R. K. Parashar, R. C. Sharma, A. Kumar and G. Mohan, Inorg. Chim. Acta.,

151 (1988) 201.

[127] M. G. Bhowon, H. L. K. Wah and R. Narain, Polyhedron., 18 (1998) 341.

[128] S. G. Clarkson and F. Basolo, Inorg. Chem., 12 (1975) 1528.

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135.

[130] M. Mohan, P. Sharma, M. Kumar and N. K. Jha, Inorg. Chim. Acta., 9

(1986) 125.

41

1.12. LITERATURE SURVEY

Kilic et al1

have reported the properties of the new Mn(II)–Co(II)–Mn(II)-type

hetero-trinuclear oxime metal complexes with N4 and N4O2 ligands. The metal

complexes were identified by combination of FT-IR spectra, UV–Vis spectra,

magnetic susceptibility measurements, mass spectra, X-ray powder diffraction

measurements, elemental analysis, molar conductivity measurements and their

morphology studied by SEM measurements. The aim of his study was to prepare

and characterize new different trinuclear metal–oxime complexes and then to

understand the structural properties of the coordination of different groups.

Zhang et al2 reported the synthesis, crystal structure and magnetic

characterization of a novel linear trinuclear copper(II) complex bridged by

phenoxy and benzyloxy oxygen atoms. The crystal structure of the complex

contains a linear trinuclear array of copper(II) ions in which the central copper(II)

ion is in an octahedron coordination sphere and lies on an inversion center of the

molecule, the terminal ones are in an identical square pyramid structure. Variable-

temperature magnetic data indicate that the complex displays a strong

antiferromagnetic coupling with J = −270(8) cm-1 between the metal ions.

42

Dede et al3

worked on the synthesis, characterization and extraction

studies of N,N‟‟-bis[1-biphenyl-2-hydroxyimino-2-(4-acetylanilino)-1-ethylidene]-

diamines and their homo-and heteronuclear copper(II) complexes. The homodi-,

homotrinuclear and heterodinuclear copper(II) perchlorate complexes of

tetradentate Schiff bases which possess N4 donor sets derived from the

condensation of 4-(arylaminoisonitrosoacetyl)biphenyl and diamine derivatives

were synthesized and characterized. Elemental analysis, FT–IR, ESR, molar

conductivity, magnetic moment measurements and thermal analyses studies were

utilized for the investigation of the complexes. Elemental analyses, stoichiometric

and spectroscopic data of the metal complexes indicated that the metal:ligand ratio

of dinuclear copper(II) complexes were found to be 2 : 1 while this ratio was 3 : 2

in trinuclear copper(II) complexes and the metal complexes indicated that the

metal ions are coordinated to the oxime and imine nitrogen atoms.

Saglam et al4 explored the oxidative cleavage of DNA by homo- and

heteronuclear Cu(II)-Mn(II) complexes of an oxime-type ligand. The novel

homodinuclear, heterodinuclear and homotrinuclear complexes with a novel

oxime-type ligand were prepared and their nucleolytic activities on pCYTEXP

43

were established by neutral agarose gel electrophoresis. The analyses of the

cleavage products obtained electrophoretically indicate that although the examined

complexes induces very similar conformational changes on supercoiled DNA by

converting supercoiled form to nicked form than linear form in a sequential

manner as the complex concentration or reaction period is increased.

Karabocek et al5 have reported the synthesis and structural studies of

(2E,3E)-3-[(6-{[(1E,2E)-2-(hydroxyimino)-1-methylpropylidene]amino}pyridin-

2yl)imino] butan-2-one oxime, ligand and its mono-, di- and trinuclear copper(II)

complexes. The dioxime ligand (H2Pymdo) and its copper(II) complexes were

characterized by 1H NMR, 13C NMR, elemental analyses, magnetic moments, IR

and mass spectral studies. The mononuclear copper(II) complex of H2Pymdo was

found to have a 1:1 metal:ligand ratio. Elemental analyses, stoichiometric and

spectroscopic data of the metal complexes indicated that the metal ions are

coordinated to the oxime and imine nitrogen atoms (C=N). In the dinuclear

complexes, in which the first Cu(II) ion was complexed with nitrogen atoms of the

oxime and imine groups, the second Cu(II) ion is ligated with dianionic oxygen

44

atoms of the oxime groups and are linked to the 1,10-phenanthroline nitrogen

atoms. The trinuclear copper(II) complex was formed by coordination of the third

Cu(II) ion with dianionic oxygen atoms of each of two molecules of the

mononuclear copper(II) complexes.

Yu et al6 identified the crystal structure of trinuclear nickel(II) complex

with Schiff base Ligand of N,N‟-Bis(Salicylidene)-1,3-Diiminopropane. All the

complexes were synthesized and characterized by elemental analysis,

thermogravimetric analysis, IR spectroscopy and X-ray diffraction techniques. All

the Ni(II) ions are coordinated in distorted octahedral geometry.

Deepalatha et al7 have reported the synthesis, physico-chemical and DNA

interaction studies of homo- and hetero-trinuclear complexes. Synthesis and

45

characterization of three new trinuclear metal complexes of type Cu3, Cu2Zn and

Cu2Ni was achieved by assembling simple mononuclear complexes, namely

2,2-bipyridyl 3,4-dihydroxo benzaldehyde copper(II) complex and

diethylenetriamine complexes of copper(II), nickel(II) and zinc(II) ions, through

the reaction of coordinated ligands. The FAB mass spectra for the complexes

showed fragmentation pattern in accordance with the molecular formula. The

frozen electron paramagnetic resonance spectrum of trinuclear copper complex

shows two sets of parallel lines with approximately 2:1 ratio. The simulation has

been carried out by considering dipolar interaction between the two types of

copper ions present in the complex. The trimetallic complexes, Cu3, Cu2Ni and

Cu2Zn show strong intercalation type of interaction with calf thymus DNA in

0.02 mol L-1

of phosphate buffer containing 60 mmol sodium chloride at pH 7.0 at

room temperature. The binding constant is found to be in the order Cu3

<Cu2Zn<Cu2Ni. The enhanced binding capability of Cu2Ni complex is attributed to

the increased symmetry in overall structure of the complex and the pronounced

binding character of positively charged Ni(II) ions with the purines.

46

3

Ray et al8 found three new mono-di-trinuclear cobalt complexes of

selectively and non-selectively condensed Schiff bases with N2O and N2O2 donor

sets: Syntheses, structural variations, EPR and DNA binding studies. In all the

three complexes psuedohalides (NCO− and N −) have been incorporated to

generate structural variation. Of the three Schiff bases, HL1 and HL2 were

obtained by selective condensation of two different 1,3-diamines with

2-hydroxyacetophenone and H2L3

resulted from non-selective condensation of

1,3-diamine with 2-hydroxyacetophenone. Only one –NH2 functionality of

1,3-diaminopropane and 1,3-diaminopentane was selectively condensed with

2-hydroxyacetophenone to generate Schiff bases HL1 and HL2, respectively. H2L3

was obtained by condensing both the amine functionality of 1,3-diaminopropane

with 2-hydroxyacetophenone. Therefore HL1

and HL2

behave as N2O donors,

whereas H2L3 provides a N2O2 donor coordination environment for the cobalt ions

in the respective complexes. The complexes have been characterised using IR, UV–

Vis spectroscopy and cyclic voltammetry. Structural aspects of the complexes have

been described by performing single crystal X-ray analysis. EPR analyses and DNA

binding abilities of all three cobalt complexes were studied in detail.

Jiang et al9 identified one trinuclear copper(II) complex derived from a new

Schiff base ligand based on the dianion of 4-chloro-6-(hydroxymethyl)-2-((3-

aminopropylimino)methyl)-phenol: Synthesis, structure, spectroscopic and

magnetic properties. A linear trinuclear copper(II) complex, prepared from a new

Schiff base ligand, namely the dianion of 4-chloro-6-(hydroxymethyl)-2-((3-

aminopropylimino)methyl)-phenol, was synthesized and characterized. The X-ray

structural study reveals that the geometry of the central Cu(II) ion is elongated

47

octahedral and that of the two side Cu(II) ions is distorted square pyramidal. The

magnetic susceptibility measurements from 2 to 300 K revealed a medium

antiferromagnetic interactions between the Cu(II) ions.

Bian et al10

have reported the synthesis, X-ray structure and magnetic

properties of trinuclear copper(II) tridentate Schiff base complexes containing a

partial cubane Cu3O4 core. Two copper(II) complexes with tridentate Schiff bases

AE and SE, which are condensed from N,N-dimethylethylenediamine and

acetylacetone or salicylaldehyde, respectively, was synthesized and characterized

by X-ray structural analysis, magnetic measurement, IR and UV spectra. Both of

the complexes contain a partial cubane Cu3O4 core consisting of [Cu(AE)] or

[Cu(SE)], nonbonded ClO4−

anions and water molecules.

Asada et al11 have reported the preparations and structures of trinuclear

manganese(II) complexes with N-2-pyridiylmethylidene-2-hydroxy-5-substituted

phenylamine. In the molecular structures of these complexes, two terminal

manganese ions are coordinated with one oxygen and two nitrogen atoms of

ligand, two oxygen atoms of OAc− and a solvent molecule, to form a distorted

48

octahedral structure where the central manganese ion resides on a center of

symmetry and is surrounded by an O6 donor set of four oxygen atoms from four

bridging OAc− and two phenolic oxygen atoms of two ligands.

Liu et al12 studied the synthesis, structural characterization and magnetic

properties of new tripeptide Schiff base heterotrinuclear Cu(II)–M(II)–Cu(II)

(M = Ni and Mn) complexes. Two novel heterotrinuclear complexes,

[Ni(H2O)4(CuL)2]7H2O (1), [Mn(H2O)4(CuL)2] 8H2O (2), have been obtained by

the self-assembly of the mononuclear tripeptide Schiff base complexes [CuL] with

bivalent metal ion M2+, where H3L=N-salicylideneglycylglycylglycine. Single

crystal X-ray diffraction experiments indicate that the structures both 1 and 2, are

made up of centrosymmetric trinuclear units with the M atom lying on an inversion

center, formed the 1D supramolecular chain structures via hydrogen bondings and

M----O weak interactions. The magnetic susceptibility data (2–300 K) revealed

antiferromagnetic interactions between copper(II) ions and the central metal.

Neelakantan et al13 worked on the spectral characterization, cyclic

voltammetry, morphology, biological activities and DNA cleaving studies of

amino acid Schiff base metal(II) complexes. Metal complexes are synthesized with

Schiff bases derived from o-phthalaldehyde (opa) and amino acids viz., glycine,

l-alanine, l-phenylalanine. Metal ions coordinate in a tetradentate or hexadentate

manner with these N2O2 donor ligands, which are characterized by elemental

analysis, molar conductance, magnetic moments, IR, electronic, 1H NMR and EPR

spectral studies. The biological activity of the complexes has been tested on eight

bacteria and three fungi. Cu(II) and Ni(II) complexes showed an increased activity

49

in comparison to the controls. The metal complexes of opapal Schiff base were

evaluated for their DNA cleaving activities with calf-thymus DNA under aerobic

conditions. Cu(II) and VO(II) complexes show more pronounced activity in

presence of an oxidant.

Leelavathy et al14 have reported the synthesis and characterization of a

new series of an unsymmetrical macrocyclic binuclearvanadyl complexes:

Electrochemical, antimicrobial, DNA binding and cleavage studies. A new series

of an unsymmetrical macrocyclic binuclear bis-phenoxo bridged

oxidovanadium(IV) complexes have been synthesized and characterized by

elemental and spectral techniques. These complexes significantly promote the

oxidative cleavage of supercoiled plasmid DNA under physiological conditions in

the presence of H2O2. All the complexes show noticeable growth inhibition of

some plant pathogenic fungal species and human pathogenic bacterial species.

Karipcin et al15 synthesized, characterized and studied the properties of

heteronuclear ruthenium(III) complexes of N,N‟-o-bis[1-p-diphenylmethane-2-

(arylamine)-1-ethiliden]-phenylenediamine quadridentate ligands. In this study,

they synthesized the amine compounds (1-p-diphenylmethane-2-hydroxyimino-2-

(1-arylamino)-1-ethanones) [HL1 and HL2] as the starting material, and then

quadridentate tetraaza Schiff base ligands [H2L3 and H2L4] were prepared from the

reactions of the amine compounds with 1,2-phenylenediamine. A new series of

mononuclear Ru(III), heterodinuclear Ru(III)–Mn(II), Ru(III)–Ni(II), Ru(III)–

Cu(II) and heterotrinuclear Ru(III)–Cu(II)–Ru(III) complexes were synthesized.

50

The complexes were characterized by elemental analysis, molar conductivity, IR,

ESR, ICP-OES, magnetic moment measurements and thermal analyses studies.

Mathur et al16 reported new homodinuclear complexes of the type

[LM2IICl2], heterotrinuclear complexes of the type [LM2

II SnIV Cl6] where M= CuII,

MnII, CoII, NiII and CuII and NiII , respectively which were synthesized and

characterized by elemental analysis and various spectroscopic techniques. The

homodinuclear complexes possess two different environments (N2 and N2O2 donor

sets) for holding the metal ions. The metal ion in N2 set exhibits square planar

geometry with two chloride ions in the inner sphere but rhombic structure is found

in tetradentate N2O2 Schiff base cavity while in heterotrinuclear complexes SnIV

atom is in the octahedral environment. The interaction of complexes with calf

thymus DNA was carried out by absorption spectroscopy and cyclic voltammetry.

Fluorescence studies along with viscosity measurements were also checked to

authenticate the binding of metal complexes with DNA.

Sreedaran et al17 have reported the N-functionalized cyclam based

trinuclear copper(II) complexes: electrochemical, magnetic, catalytic and

antimicrobial studies. A series of trinuclear Cu(II) complexes have been prepared

by Schiff base condensation of 1,8-[bis-(3-formyl-2-hydroxy-5-methyl)benzyl]-

51

l,4,8,11- tetraazacyclotetradecane and 1,8-[bis(3-formyl-2-hydroxy-5-bromo)-

benzyl]-l,4,8,11-tetraazacyclotetradecane with aromatic and aliphatic diamines,

Cu(II) perchlorate and triethylamine. The complexes were characterized by

elemental and spectroscopic analysis. All the complexes were screened for

antifungal and antibacterial activity.

Durmus et al18 explored the thermal decomposition of some linear

trinuclear schiff base complexes with acetate bridges. Ni(II)–M(II)–Ni(II) nuclear

structured complexes were prepared from N,N‟-bis(salicylidene)-1,3-

propanediamine (LH2) and its derivatives N,N‟-bis(salicylidene)-2,2‟-dimethyl-

1,3-propanediamine (LDMH2) and N,N‟-bis(salicylidene)-2-hydroxy-1,3-

propanediamine (LOH3), where M represents one of the following metal ions;

Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II). Two different µ-bridges are found

between the metal nucleuses of the complexes. The phenolic oxygens and acetate

ions tend to form µ-bridges between the terminal Ni(II) ions and central metal(II)

ion. The coordinatively bonded DMF molecules, in the complexes, were observed

to abandon the structure between 160–180°C. Further heating resulted primarily in

the thermal decomposition of the complexes above 310°C, whereas metal oxide

residue mixtures were observed above 650°C.

52

Dulger et al19 studied the DNA cleavage by homo- and heterotetranuclear

Cu(II) and Mn(II) complexes with tetrathioether-tetrathiol moiety. Novel

homotetranuclear Cu(II) and heteronuclear Cu(II)-Mn(II) complexes with

tetrathioether-tetrathiol moiety has been prepared and their DNA relaxation

activities with plasmid pCYTEXP were electrophoretically established. The

cleavage products analyzed by neutral agarose gel electrophoresis indicated that

the interaction of the metal complexes with supercoiled plasmid DNA yielded

linear, nicked or degraded DNA. The relaxation activities of both homo- and

heterotetranuclear complexes are time and concentration dependent. His findings

suggest that a homo and heterotetranuclear complexes with potent nucleolytic

activity is a good nuclease substitute in the presence of cooxidant.

Mathur et al20 have reported a template synthesis of novel carboxamide

dinuclear copper(II) complex: spectral characterization and reactivity towards calf

thymus DNA. Dinuclear complexes Bis [aqua 1,8-(1,2-dicarboxamido benzene)

53

3,6-diazaoctane copper (II)/nickel (II)] tetrachloride (1 and 2) were synthesized by

a two component one-pot metal template condensation between phthalic anhydride

and 1,8-diamino 3,6-diazaoctane. Elemental analysis, molar conductance

measurements, electronic absorption, infra-red, electron paramagnetic resonance,

nuclear magnetic resonance, atomic absorption and electron spray mass spectral

studies have been performed to probe the nature and structure of the complexes.

The interaction of copper (II) complex with calf thymus has been studied by using

absorption, emission and circular dichoric spectral methods, viscometry and cyclic

voltammetry.

Prabahkara et al21 have studied the binding and photocleavage of DNA by

mixed ligand Co(III) and Ni(II) complexes of thiophene[2, 3-b] quinoline and

phenanthrolie/bipyridine. In order to systematically perform an experimental and

theoretical study on DNA binding and photocleavage properties of transition metal

complexes of the type [M(L)2(L1)](PF6)n xH2O (where M = Co(III) or Ni(II),

L = 1,10-phenanthroline or 2.2‟ bipryidine, L1 = Thiophene [2,3-b] quinoline (qt),

n = 3 or 2 and x = 5 or 2) have been synthesized and characterized by elemental

analysis, IR, 1H NMR, UV and magnetic susceptibility data. The DNA-binding

properties of these complexes have been investigated with UV-Vis, viscosity

measurements, thermal denaturation and cyclic voltametric studies. The

photocleavage studies with pUC19 DNA shows that all these complexes promoted

the conversion of SC form to NC form in absence of „inhibitors‟.

Liu et al22

have reported synthesis, DNA-binding and cleavage studies of

macrocyclic copper(II) complexes. Two macrocyclic copper(II) complexes have

54

been prepared and characterized by elemental analysis, UV–Vis., IR and mass

spectra. Absorption, fluorescence, circular dichroic spectra and viscosity

experiments have been carried out on the interaction of the two complexes with

calf thymus DNA. The results suggest that both complexes can bind to CT DNA

by intercalation via the aromatic moiety ring in the macrocycle into the base pairs

of DNA.

Mukherjee et al23

have reported three di-Schiff-base ligands, N,N‟-

bis(salicylidene)-1,3-propanediamine (H2Salpn), N,N‟-bis(salicylidene)-1,3-

pentanediamine (H2Salpen) and N,N‟-bis(salicylidine)-ethylenediamine (H2Salen)

react with Ni(SCN)2 4H2O in 2:3 molar ratios to form the complexes; mononuclear

[Ni(HSalpn)(NCS)(H2O)]H2O, trinuclear [{Ni(Salpen)}2Ni(NCS)2] and trinuclear

[{Ni(Salen)}2Ni(NCS)2] respectively. All the complexes have been characterized

by elemental analyses, IR and UV–VIS spectra, and room temperature magnetic

susceptibility measurements.

55

Maity et al24 have reported a novel trinuclear nickel(II) complex of an

unsymmetrical tetradentate ligand involving bridging oxime and acetylacetone

functions. The trinuclear nickel(II) complex, [Ni3(L)2(H2O)2](ClO4)2, where L is a

bridging unsymmetrical tetradentate ligand, involving o-phenylenediamine,

diacetyl monoxime and acetylacetone (H2L = 4-[2-(3-hydroxy-1-methyl-but-2-

enylideneamino)-phenylimino]-pentan-2-one oxime) has been synthesized and

characterized structurally. In the complex, an octahedral Ni(II) centre is held in the

middle by two square planar units with the aid of oxime and ketonic bridges.

Refat et al25 have reported Synthesis, infrared spectra and thermal

studies of Zn(II), Cd(II) and Hg(II) complexes with 2-aminobenzaldehyde

phenylhydrazone “nitrin” ligand. The nitrin, 2-aminobenzaldehyde

phenylhydrazone was synthesized by refluxing 2-nitrobenzaldehyde with

phenylhydrazine in ethanolic solvent. Three transition metal (II) complexes have

been prepared. Elemental analysis, molar conductivity, IR, UV, 1H NMR and mass

spectra, as well as TG/DTG have been used to characterize these complexes. The

ligand and its complexes have been studied for their possible biological activity

including antibacterial and antifungal activity.

56

Kwiatkowski et al26 have reported Nickel(II) and palladium(II) complexes

with singly condensed diprimary triamines and 2-aminobenzaldehyde. Six new

nickel(II) and one palladium(II) complexes are obtained by complexation of

unsymmetrical Schiff bases from 1:1 condensation of 2-aminobenzaldehyde with

1,5-diamino-3-azapentane, 1,7-diamino-4-azaheptane and 1,7-diamino-4-methyl-4-

azaheptane.

Dragancea et al27

have reported a Bis(o-aminobenzaldehyde)

thiocarbohydrazone (HL) forms with copper(II) nitrate a tetranuclear complex

[Cu2(L)(NO3)3]2 2H2O, in which two dinuclear units are joined by nitrate bridges.

The dihydrazone ligand behaves ditopically, providing NNS and NNN binding

sites, with the four coppers essentially in a squarepyramidal geometry. The

tetranuclear molecule displays intramolecular magnetic interactions, with the

antiferromagnetic exchange between the copper(II) ions within each dinuclear

moiety dominant over weak interdimer ferromagnetic coupling.

Abdallah et al28 have reported spectroscopic study of molecular structures

of novel Schiff base derived from o-phthaldehyde and 2-aminophenol and its

coordination compounds together with their biological activity. New Schiff base

(H2L) ligand is prepared via condensation of o-phthaldehyde and 2-aminophenol.

The metal complexes of Cr(III), Mn(II), Fe(II), Fe(III), Co(II), Ni(II), Cu(II) and

Zn(II) with the ligand are prepared in good yield from the reaction of the ligand

with the corresponding metal salts. They are characterized based on elemental

analyses, IR, solid reflectance, magnetic moment, electron spin resonance (ESR),

molar conductance, 1H NMR and thermal analysis (TGA).

57

Singh et al29 have reported synthesis and spectroscopic studies of

biologically active compounds derived from oxalyldihydrazide and benzil, and

their Cr(III), Fe(III) and Mn(III) complexes. A new series of complexes have been

synthesized by template condensation of oxalyldihydrazide and benzil in

methanolic medium in the presence of trivalent chromium, manganese and iron

salts forming complexes of the type [M(C32H24N8O4)X]X2 where M = Cr(III),

Mn(III), Fe(III) and X = Cl−1

, NO3 −1, CH3COO−1. The complexes have been

characterized with the help of elemental analyses, conductance measurements,

magnetic susceptibility measurements, electronic, NMR, infrared and far infrared

spectral studies. On the basis of these studies, a five coordinate square pyramidal

geometry has been proposed for all these complexes. The biological activities of

the metal complexes have been tested in vitro against a number of pathogenic

bacteria to assess their inhibiting potential. Some of these complexes have been

found to exhibit remarkable antibacterial activities.

Shebl et al30 have reported synthesis, spectroscopic characterization and

antimicrobial activity of mono-, bi- and tri-nuclear metal complexes of a new

Schiff base ligand. Condensation of o-acetoacetylphenol and 1,2-diaminopropane

in 1:1 molar ratio under condition of high dilution yielded the mono-condensed

dibasic Schiff base ligand with a N2O2 donors. The mono-condensed ligand has

been used for further condensation with 2-hydroxy-5-nitrobenzaldehyde to obtain

the new asymmetrical dicompartmental Schiff base ligand, H3L, with N2O3 donors.

The structure of the ligand was elucidated by analytical and spectroscopic tools

(IR, 1H and 13C NMR spectra) which indicated that the coordinating sites are

oxygen atoms of the phenolic OH groups, nitrogen atoms of the azomethine groups

58

and the oxygen atom of the ketonic group. Reactions of the ligand with metal salts

yielded mono- and homo-bi-nuclear complexes. The Schiff base and its metal

complexes were evaluated for antimicrobial activity against Gram positive bacteria

(Staphylococcus aureus), Gram negative bacteria (Escherichia coli) and fungi

(Candida albicans and Aspergillus flavus). The ligand and some of its complexes

were found to be biologically active.

Yang et al31 have reported synthesis, characterization and DNA

interaction of copper (II) complexes with Schiff base ligands derived from

2-pyridinecarboxaldehyde and polyamines. A series of copper (II) complexes

with Schiff bases ligands derived from the condensation reactions between

2-pyridinecarboxaldehyde and different polyamines (ethylenediamine,

diethylenetriamine, triethylenetetramine and tetraethylenepentamine) were

synthesized and characterized by elemental analysis, FT-IR spectroscopy, HRMS,

molar conductance and molecular modeling studies. The interactions of the copper

59

complexes with DNA were investigated by the UV spectra, viscosity

measurements and gel electrophoresis under physiological conditions. The

experimental results indicated that the complexes could bind to DNA via an

intercalative mode and showed a different DNA cleavage activity.

Chaviara et al32 have exhibited DNA interaction studies and evaluation of

biological activity of homo- and hetero-trihalide mononuclear Cu(II) Schiff

base complexes. Quantitative structure – activity relationships. A new series of

mixed-ligand mono- or hetero-trihalide Cu(II) complexes of the type

[Cu(dienXX)Y(YZ2)], where dienXX = Schiff dibase of diethylenetriamine with

2-thiophene-carboxaldehyde (dienSS), 2-furaldehyde (dienOO) or 2-pyrrole-2-

carboxaldehyde (dienNN), Y = Cl, Br and Z = Br, I was synthesized by the

reaction of the precursors of the type [Cu(dienXX)Y]Y with iodine or bromine in

1:1 molar ratio. The distorted square pyramidal configuration of the new homo-

and hetero-trihalide Cu(II) mononuclear complexes was identified by C, H, N, Cu

analysis, spectroscopic methods (IR, UV–visible), molar conductivity and

magnetic measurements. The effect of the new compounds on the single stranded

(ss), double stranded (ds) and pDNA led either to the formation of a DNA-complex

cationic adduct, or to its degradation, evidenced by DNA electrophoretic mobility

and DNA interaction spectroscopic titration studies. Moreover, the antimicrobial

activity of Cu(II) complexes against Gram positive and Gram negative bacteria can

be attributed to the synergistic action of the dissociation species, namely the

cationic [Cu(dienXX)Y]+ and anionic [YZ2]− ones.

60

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[19] S. Dulger, N. Saglam, A. O. Belduz, S. Guner and S. Karabocek,

Biometals., 13 (2000) 261.

[20] S. Mathur and S. Tabassum, Biometals., 21 (2008) 299.

[21] M. C. Prabahkara and H. S. B. Naik, Biometals., 21 (2008) 675.

[22] J. Liu, T. B. Lu, H. Deng and L. N. Ji, Transition. Met. Chem., 28 (2003)

116.

[23] P. Mukherjee, C. Biswas, M. G. B. Drew and A. Ghosh, Polyhedron., 26

(2007) 3121.

[24] D. Maity, P. Mukherjee, A. Ghosh, M. G. B. Drew and G. Mukhopadhyay ,

Inorg. Chim. Acta., 361 (2008) 1515.

[25] M. S. Refat and A. A. Ibrahim, Spectrochim. Acta. A., 70 (2008) 234.

[26] E. Kwiatkowski, G. Romanowski and K. Suwinska, Polyhedron., 21 (2002)

2071.

62

[27] D. Dragancea, A. W. Addison, M. Zeller, M. E. Foster, M. J. Prushan,

L. K. Thompson, M. D. Revenco and A. D. Hunter, Inorg. Chim. Acta.,

363 (2010) 2065.

[28] S. M. Abdallaha, G. G. Mohamed, M.A. Zayedb and M. S. Abou El-Ela,

Spectrochim. Acta. Part. A., 73 (2009) 833.

[29] D. P. Singh, R. Kumar and J. Singh, Eur. J. Med. Chem., 44 (2009) 1731.

[30] M. Shebl, S. M. E. Khalil, S. A. Ahmed and H. A. A. Medien, J. Mol.

Struct., 980 (2010) 39.

[31] X. B. Yang, Y. Huang, J. S. Zhang, S. K. Yuan and R. Q. Zeng, Inorg.

Chem. Commun., 13 (2010) 1421.

[32] A. T. Chaviara, E. E. Kioseoglou, A. A. Pantazaki, A. C. Tsipis,

P. A. Karipidis, D. A. Kyriakidis and C. A. Bolos, J. Inorg. Biochem., 102

(2008) 1749.

63

1.13. SCOPE AND OBJECTIVES OF THE WORK

The chemistry of multinuclear Schiff base metal complexes, especially of

coupled systems is of special interest in various fields of science. The main reason

probably is due to the phenomenon of interaction between metal centres lies at the

crossover point of two widely separated areas, namely the physics of the magnetic

materials and the role of polynuclear reaction sites in biological processes.

Trinuclear transition metal complexes bridged by polyatomic Schiff base ligands

have gained much attention in the recent years, towards synthesis and

characterization.

The multinuclear copper(II) complexes have aroused extensive interest due

to their importance in biological processes and in inorganic material science. The

metal ion dependent oxidative DNA cleavage by Cu(II) complexes is of topical

interest. Nickel complexes in the presence of oxidants have been extensively used

for DNA cleavage reactions. Manganese plays an essential and versatile role in the

biochemistry of many organisms. The chemistry of manganese in various

oxidation states is currently receiving much attention owing to its importance in

many enzymatic systems such as superoxide dismutase and catalase. The vanadium

ions can play a role in biology as counter ions for protein, DNA, RNA and in

various biological organelles.

Hence the present work described in the thesis concerns with the reactions

of some of the Cu(II), Ni(II), VO(II) and Mn(II) complexes with p-phenylenediamine,

o-phthalaldehyde/2,3-pentanedione/benzil and 2-aminobenzaldehyde. The new homo

64

and heterotrinuclear Schiff base complexes formed with N4 donor sets. The structures

of these complexes have been investigated by using various physico chemical

methods.

The synthesised complexes have been effectively used as DNA cleavage

studies. The cleavage of nucleic acids may be considered as an enzymatic reaction

which comprises of various biological processes as well as the biotechnological

manipulation of genetic material. The application of artificial DNA cleaving agents

is manifold: biotechnology, structural studies of nucleic acids or development of

new drug.

Some of the complexes are used for DNA binding studies. These complexes

have effectively bind to CT DNA. DNA binding is the critical step for DNA

activity. To design effective chemotherapeutic agents and better anticancer drugs,

it is essential to explore the interactions of metal complexes with DNA. Recently,

it has been tremendous interest in studies related to the interaction of transition

metal ions with nucleic acid because of their relevance in the development of new

reagents for biotechnology and medicine. The antibacterial properties of the

complexes have also been screened against some pathogenic bacteria.

Objectives of the study

1. Template Synthesis of mononuclear Cu(II), Ni(II) and Mn(II) Schiff base

complexes derived from p-phenylenediamine and o-phthalaldehyde (or)

2,3-pentanedione (or) benzil.

2. Synthesis of homo and heterotrinuclear Schiff base metal complexes by

conventional methodology as given in the literature. The mononuclear

65

complex has been used as a ligand for further condensation with

2-aminobenzaldehyde and also react with metal salts to obtain the homo

and heterotrinuclear Schiff base metal complexes.

3. To characterize the homo and heterotrinuclear Schiff base metal complexes

using various analytical and spectral techniques such as elemental analysis,

molar conductance, FT-IR, UV-Visible spectroscopy, electrochemical,

thermal, magnetic and EPR studies. Several attempts to isolate crystals

suitable for X-ray diffraction were unsuccessful.

4. DNA binding Studies

The interaction of the homo and heterotrinuclear Schiff base metal

complexes with calf thymus DNA (CT-DNA) were studied using

absorption spectra, cyclic voltammetry and viscosity measurements.

5. DNA Cleavage studies

The cleavage study was monitored by gel electrophoresis method.

6. Antimicrobial activity

The in vitro biological screening effects of the synthesised compounds

were tested against the some Gram positive and Gram negative bacteria by

the well diffusion method.

66

1.14. EXPERIM ENTAL METHODS AND ANALYTICAL TECHNIQUES

This present chapter deals with the chemicals used, purification of the

solvents, synthesis of the compounds and their analytical and instrumental

techniques used in the determination of the stoichiometry of the complexes

prepared.

1.14.1. Analytical Reagents

The main reagents used are summarized in Table 1.1.

Table.1.1. Reagents used in this thesis

S.No

Product

Provider

1 CuCl2. 2H2O (A.R Loba)

2 NiCl2 6H2O (A.R Loba)

3 Cu(ClO4)2.6H2O (A.R Aldrich)

4 Ni(ClO4)2.6H2O (A.R Loba)

5 VOSO4. H2O (A.R Aldrich)

6 MnCl2. 4H2O (A.R Loba)

7 o-phthalaldehyde (A.R Aldrich)

8 2,3-pentanedione (A.R Aldrich)

9 Benzil (A.R Aldrich)

10 p-phenylenediamine (A.R Loba)

11 2-nitrobenzaldehyde (A.R Loba)

12 Ammonia (A.R Qualigens)

13 Ferrous sulphate heptahydrate (A.R Merck)

14 Sodium hydroxide (A.R Loba)

15 Triethylamine (L.R Qualigens)

67

16 Sodium hydroxide (A.R Loba)

17 Oxalic acid (A.R Loba)

18 Nitric acid (A.R Loba)

19 EDTA (A.R Merck)

20 Eriochrome Black-T (A.R Merck)

21 Ammonium Chloride (L.R SDS)

22 Ammonium Acetate (L.R SDS)

23 Tetrabutylammonium perchlorate (A.R Aldrich)

24 Hydrochloric acid (L.R Loba)

25 Potassium sulphate (L.R Qualigens)

26 Calcium sulphate (L.R Qualigens)

27 Diethyl dithiocarbamate (L.R Qualigens)

28 Bromine water (L.R Loba)

29 Nickelammonium sulphate (L.R Qualigens)

30 Hydroxylamine hydrochloride (A.R Loba)

1.14.2. Solvents

Solvents used in this thesis are gathered in Table 1.2

Table.1.2. Solvents used in this thesis

S.No Solvents Provider

1 Ethanol (A.R Loba)

2 Methanol (A.R Loba)

3 Dimethyl formamide (A.R SDS)

4

Dimethyl sulfoxide

(A.R Merck)

5

Acetonitrile

(A.R Merck)

6

Acetone

(A.R Merck)

68

1.14.2.1. Purification of solvents

Common solvents such as methanol, ethanol and DMF, all of commercial

grade were purified as usual [1]. Ethanol and methanol were purified by the

following procedures [2].

1.14.2.2. Ethanol

Commercial ethanol was distilled, collecting the middle fraction (80%). It

was then refluxed over ignited quicklime for 4 hours and again distilled at

atmospheric pressure, when 98% ethanol was obtained.

1.14.2.3. Methanol

The commercial grade methanol was refluxed for 4 hours with quicklime

and distilled. The middle fraction was collected and used.

1.14.3. Synthesis of 2-aminobenzaldehyde

Figure.1.5. Synthesis route of 2-aminobenzaldehyde

1.14.3.1. Procedure

A three necked flask is employed as a reaction vessel from which the

product can be steam-distilled immediately after completion of the reaction. It is

convenient to arrange the apparatus for the reaction and that for the steam

distillation on the same steam bath, with provision for the rapid connection of the

69

flask to the distillation assembly at the desired time. For use as a reaction vessel,

the flask is mounted on a steam bath and fitted with a mechanical stirrer and a

reflux condenser, third neck is closed by a cork.

In a steam-distillation assembly of the small necks of the flask is fitted with

a steam-inlet tube, connected through a water trap to a steam line, the other small

neck is closed by a cork. The central neck is fitted to Kjeldahl trap leading to a 50

cm. Allihn condenser set downward and connected in series to a 50 cm Liebig

condenser. The second condenser leads to a 500 ml three necked flask used as the

receiver. The received flask is immersed in an ice bath and fitted with an Allihn

reflux condenser.

When all the apparatus has been set up and tested the flask is connected to

the reaction assembly and 175 ml of water, 105 g of ferrous sulphate heptahydrate,

0.5 ml of concentrated hydrochloric acid and 6 g of o-nitrobenzaldehyde are

introduced in the order given. The stirrer is then started and the flask is heated by

means of the steam bath. When the temperature of the mixture reaches 90oC, 25 ml

of aq.NH3 is added to one portion and at 2-minute intervals, three 10 ml portions of

ammonium hydroxide are added. Stirring and heating are continued throughout.

The total reaction time is 8 minutes.

Immediately after the addition of last portion of ammonium hydroxide, the

reflux condenser and stirrer are removed and the flask is connected to the steam

distillation assembly. The mixture is steam-distilled as rapidly as possible and two

250 ml fractions of distillate are collected during a period of 12 minutes. The first

fraction is saturated with sodium chloride and the solution is stirred until

70

precipitation appears to be complete. The solid is collected on a Buchner funnel

and derived in the air. The product weights 2.5 g. (60 %) and metals at 38oC. The

second fraction of the distillate is saturated with sodium chloride and combined

with the filtrate remaining from the first fraction. The combined solution is

extracted with two 45 ml portions of ether. The combined ether extract is filtered,

dried over anhydrous sodium sulphate and concentrated by distillation, finally

under reduced pressure. The residue solidifies on cooling and weights 1.0 g, it can

be purified by steam distillation from 40 ml of saturated sodium chloride solution

until 100 ml of distillate is collected, saturated the distillate with sodium chloride,

cooled and filtered. The pure product so obtained weights 0.87 g. The total yield is

3.0 g (70 %). The submitters have obtained yields of about 70% in runs eight times

as large as that described. This amino aldehyde undergoes self-condensation on

standing, especially in desiccators and so the product should be used immediately.

1.14.4. Synthesis of trinuclear Schiff base complexes derived from

o-phthalaldehyde

Synthesis of homo and heterotrinuclear Schiff base metal complexes from

p-phenylenediamine, o-phthalaldehyde and 2-aminobenzaldehyde were carried

out in two stage processes. In the first stage mononuclear Cu(II) and Ni(II)

complexes were prepared. In second stage, the mononuclear complex has been

used as a ligand for further condensation with 2-aminobenzaldehyde and also react

with metal salts to obtain the homo and heterotrinuclear Schiff base metal

complexes.

71

Stage I

Template synthesis of mononuclear Schiff base complexes derived from

p-phenylenediamine and o-phthalaldehyde. The mononuclear complexes were

synthesized by the following general procedure [3]. The p-phenylenediamine (2 mmol,

0.216 g) and o-phthalaldehyde (1 mmol, 0.134 g) in ethanol were refluxed for

2 hours. Metal salt (0.5 mmol) in ethanol was added to the resulting solution and again

refluxed for one hour. The resulting solution was evaporated and dried in air. The solid

product was collected. Synthesis pathway is shown in Figure 1.6.

Figure.1.6. Synthesis approaches for mononuclear Schiff base metal complexes

derived from o-phthalaldehyde.

72

Stage II

The bis-(p-phenylenediaminephthalaldehyde)metal(II) complexes (1 mmol)

and 2-aminobenzaldehyde (4 mmol) in hot methanol were refluxed for 2 hours and

metal salts in methanol were then added drop-wise to the resulting solution. The

contents were again refluxed for one hour. It was cooled to room temperature. The

product was obtained. It was filtered and dried over in decicator. Synthesis

pathway is shown in Figure 1.7.

Figure.1.7. Synthesis route of trinuclear Schiff base metal complexes derived from

mononuclear complexes and 2-aminobenzaldehyde.

73

Table.1.3. Physical data of trinuclear Schiff base complexes derived from o-

phthalaldehyde

Compounds Molecular Formula Color

Yield

(%)

Melting

Point

(°C)

[Cu3(L1)Cl2]4ClO4 [(C68H56N12)Cu3] 4ClO4 Brown 75 >250

[Ni3(L1)Cl2]4ClO4 [(C68H56N12)Ni3] 4ClO4 Pale 70 >250

Brown

[NiCu2(L1)Cl2]4ClO4 [C68H56N12)CuNi2] 4ClO4 Black 65 >250

[CuNi2(L1)Cl2]4ClO4 [C68H56N12)NiCu2] 4ClO4 Black 70 >250

[Cu(VO)2(L1)Cl2]2SO4 [C68H56N12)Cu(VO)2] 2SO4 Brown 73 >250

1.14.5. Synthesis of trinuclear Schiff base complexes derived from

2,3-pentanedione

Synthesis of homo and heterotrinuclear Schiff base metal complexes

derived from p-phenylenediamine, 2,3-pentanedione and 2-aminobenzaldehyde

were carried out in two stage processes. In the first stage mononuclear Cu(II),

Ni(II) and Mn(II) complexes were prepared. In second stage, the mononuclear

complex has been used as a ligand for further condensation with

2-aminobenzaldehyde and also react with metal salts to obtain the homo and

heterotrinuclear Schiff base metal complexes.

Stage I

The ethanolic solution (20 ml) of p-phenylenediamine (2 mmol) and

ethanolic solution (20 ml) of 2,3- pentanedione (1 mmol) were mixed slowly with

constant stirring. This mixture was refluxed at 90°C for 2 hours. Metal chloride

74

(0.5 mmol) in ethanol (20 ml) were then added to the resulting solution and again

refluxed for one hour as shown in Figure 1.8. When it was cooled to room

temperature, the solid product was obtained.

Figure.1.8. Synthesis approaches for mononuclear Schiff base metal complexes

derived from 2,3-pentanedione.

Stage II

An ethanolic solution (20 ml) of bis-(p-phenylenediamine,

2,3-pentanedione) metal(II) complexes in hot condition (1 mmol) and

2-aminobenzaldehyde (4 mmol) were refluxed for 2 hours and metal chloride

(2 mmol) in ethanol(20 ml) were then added drop wise to the resulting solution.

75

The contents were again refluxed for one hour as shown in Figure 1.9. It was

cooled to room temperature. The precipitate was obtained. It was filtered and dried

over fused CaCl2.

Figure.1.9. Synthesis route of trinuclear Schiff base metal complexes derived from

mononuclear complexes and 2-aminobenzaldehyde.

76

Table.1.4. Physical data of trinuclear Schiff base complexes derived from

2,3-pentanedione

Compounds Molecular Formula Color

Yield

(%)

Melting

Point

(°C)

[Cu3(L2)Cl6] [(C62H60N12Cl6)Cu3] Black 81 >250

[Ni3(L2)Cl6] [C62H60N12Cl6)Ni3] Dark

brown

78 >250

[Mn3(L2)Cl6] [C62H60N12Cl6)Mn3] Brown 75 >250

[CuNi2(L2)Cl6] [C62H60N12Cl6)CuNi2] Black 74 >250

[NiCu2(L2)Cl6] [C62H60N12Cl6)NiCu2] Black 72 >250

1.14.6. Synthesis of trinuclear Schiff base complexes derived from benzil

Synthesis of homo and heterotrinuclear Schiff base metal complexes

derived from p-phenylenediamine, benzil and 2-aminobenzaldehyde were carried

out in two stage processes. In the first stage mononuclear Cu(II), Ni(II) and Mn(II)

complexes were prepared. In second stage, the mononuclear complex has been

used as a ligand for further condensation with 2-aminobenzaldehyde and also react

with metal salts to obtain the homo and heterotrinuclear Schiff base metal

complexes.

Stage I

An equimolar amount of the ethanolic (20 ml) solution of benzil and

p-phenylenediamine was refluxed at 90°C for 2 hours. CuCl2 (1 mmol) in ethanol

(20 ml) were then added to the resulting solution and again refluxed for one hour

as shown in Figure 1.10. The reaction mixture was cooled to room temperature,

filtered and filtrate on slow evaporation yielded greenish brown coloured solid was

obtained. It was filtered and dried over fused CaCl2.

77

obtained. The same procedure was adopted to the synthesis of mononuclear Ni(II)

and Mn(II) complexes.

Figure.1.10. Synthesis approaches for mononuclear Schiff base metal complexes

derived from benzil.

Stage II

A hot ethanolic solution (20 ml) of bis-(p-phenylenediamine, benzil)

metal(II) complexes (1 mmol) and 2-aminobenzaldehyde (4 mmol) in 1:4 molar

ratio were refluxed for 2 hours and metal chloride (2 mmol) in ethanol (20 ml)

were then added drop wise to the resulting solution. The contents were again

refluxed for one hour. It was cooled to room temperature. The precipitate was

from mononuclear complexes and 2-aminobenzaldehyde.

78

Figure.1.11. Synthesis routes of trinuclear Schiff base metal complexes derived

79

Table.1.5. Physical data of trinuclear Schiff base complexes derived from benzil

Compounds Molecular Formula Color Yield (%)

Melting Point

(°C)

[Cu3(L3)Cl6] [(C80H64N12Cl6)Cu3] Black 78 >250

[Ni3(L3)Cl6] [C80H64N12Cl6)Ni3] Brown 75 >250

[Mn3(L3)Cl6] [C80H64N12Cl6)Mn3] Brown 75 >250

[CuNi2(L3)Cl6]

[C80H64N12Cl6)CuNi2]

Black

72

0250

[NiCu2(L3)Cl6]

[C80H64N12Cl6)NiCu2]

Black

74

>250

1.14.7. Apparatus and Experimental conditions

1.14.7.1. Solubility

Solubility of the complexes were checked in hot and cold water,

dimethylsulfoxide (DMSO), dimethylforamide (DMF) and other organic solvents

by shaking a small amount of the complexes in a test tube.

1.14.7.2. Melting points

The melting points were determined by placing a fine powdered sample in

glass capillary and heated by using Technico Melting point apparatus.

1.14.7.3. Eleme ntal analysis

The elemental analyses (Carbon, Hydrogen and Nitrogen) were performed

using a Carlo Eraba 1106 instrument.

1.14.7.4. Molar conductivity

Molar conductances of the complexes in DMF solution were measured with

ELICO CM 185 conductivity Bridge. Conductance or conductivity can also be

explained as the power of electrolytes to conduct electrical current. Conductance is

the reciprocal of resistance. Its units are Mho or Ohm-1.

80

Where,

Λ = 1/R

Λ = conductance

R = Resistance

Conductance is affected by different factors including

(i) Concentration

(ii) Solvent

(ii) Temperature

Generally, conductance is decreased by increasing concentration and vice

versa. Concentration increases by dilution because on dilution, the ionic mobility

increases and interionic attractions decreased. The conductance of a solution of an

electrolyte generally increases with rise in temperature (2-3% of for one-degree

rise in temperature). The increase in conductance with rise in temperature is due to

the speed of ions with which they move towards the electrodes. With rise in

temperature velocity of the solvent decreases which makes the ions to move freely

towards the electrodes.

The molar conductivity of an electrolyte in a particular solvent depends on

various factors like viscosity, polarity and donor property of the solvent. Geary [4]

has given the ranges of molar conductivity of various types of electrolytes in

different solvents, which is given in Table.1.6.

81

Table.1.6. Molar conductance (S cm2 Mol-1) of various electrolytes in different

solvents

Electrolyte type

Solvents 1:1 1:2 1:3 1:4

Nitrobenzene

Acetone

20-30

100 – 140

50-60

160 – 200

70-82

260

90-100

270

Acetonitrile 120 – 160 220 – 300 340 – 420

500

DMF 65 – 90 130 – 170 200 – 240

300

Methanol

80 – 115

160 – 220

290 – 350

450

Ethanol 35 – 45 70 – 90

120

160

1.14.7.5. Infrared spectroscopy

Fourier Transform Infrared Spectroscopy (FT-IR) is a spectroscopic

technique that deals with the middle infrared region of the electromagnetic

spectrum (400 − 4000 cm−1

). Each covalent bond of organic functions and

inorganic species exhibits a characteristic frequency of vibration in FT-IR

spectroscopy which can be used to identify its component. The vibration forms of

molecules are primary two types: 1) stretching vibration (ν), 2) bending vibration

(δ). Generally, stretching vibration modes are observed at higher frequency than

that of bending vibrations for a same covalent bond.

The infrared spectra were recorded on the Perkin Elmer FT-IR-8300 model

spectrometer using KBr disc in the region 4000–400 cm−1. Infrared spectroscopy is

an extremely powerful analytical technique for both qualitative and quantitative

82

analysis. The infrared spectrum of any given substance is interpreted by the use of

known group frequencies. It is one of the most widely used tools for the detection

of functional group in pure compounds and mixtures, for compound comparison

and for the identification of the substances.

Infrared spectroscopy involves twisting, bending, rotational and vibrational

motion of the atomic groups in a molecule. Infrared radiation does not possess

sufficient energy to cause the excitation of electrons however, it causes atoms and

groups of atoms to vibrate faster about covalent bond or bond, which connect them.

The compounds absorb infrared energy in the particular region of spectrum. A highly

complex absorption spectrum is obtained which is characteristic of the functional

group comprising the molecule and overall configuration of the atom as well.

The infrared region constitutes three parts:

i. Near Infrared region: In the near infrared region, which meets the visible

region at about 12,500 cm−1

and extends to about 4000 cm−1

are found in

many absorption bands resulting from harmonic overtones of fundamentals

bands and combination bands often associated with hydrogen atoms.

Among these, the first overtones of the O-H and N-H stretching vibrations

are near 7140 cm−1 and 6667 cm−1 respectively, combination bands

resulting from C-H stretching and deformation vibration of alkyl groups at

4548 cm−1 and 3850 cm−1.

ii. Mid Infrared region: Middle infrared region is divided into the “group

frequency” region, 4000−1300 cm−1 and the finger print region, 300-650

cm−1.

83

iii. Far infrared region: Far infrared region between 667 cm−1 and

10 cm−1

contains the bending vibrations of carbon, nitrogen, oxygen and

fluorine with atoms heavier than mass 19 and additional motion in cyclic or

unsaturated systems.

The infrared spectrum can give a perfect picture of the structural formula

without a chemical investigation.

1.14.7.6. Electronic spectra

Electronic absorption spectra in the ultraviolet (UV) – Visible range were

recorded on Perkin Elmer Lambda-25 between 200 and 800 nm by using DMF as

the solvent. The spectra of transition metal complexes depend on the transition of

unpaired electrons from the ground state to an excited state. Most of the transition

metal complexes are colored; the color is observed due to a d-d transition in the

visible region. Transitions between a given set of p or d orbitals, where d-d

transitions are forbidden (e.g. in octahedral complexes), render such complexes

colorless.

The electronic structure of coordination compounds is mainly described by

the molecular orbital theory. The atomic overlap in metal-ligand bonds allows d-

electrons to penetrate from the central atom to the ligand. The transitions are

affected by the effect of ligands on the energies of the d orbital of the metal ions.

Since octahedral, square-planar and tetrahedral fields cause splitting of d orbitals in

different ways, the geometry will have a pronounced effect on the d-d transitions in

a metal ion complex. Thus, spectral data of these transitions provide useful

information about the structure of complexes.

84

Cu(II) complexes are usually blue or green, exception to this, is generally

caused by strong ultraviolet and charge transfer bands tailing off into the blue and

visible spectrum thus causing the substances to appear red or brown. The blue or

green colors are due to the presence of Cu d-d absorption bands in 500-800 nm

regions of the spectrum. The envelopes of these bands are generally unsymmetrical

seeming to encompass several overlapping transitions, but definite resolution into

the proper number of sub-bands with correct location is difficult.

1.14.7.7. Electron paramagnetic resonance (EPR) Spectroscopy

ESR spectra were recorded on a Varian JEOL-JES-TE100 ESR

spectrophotometer at X-band microwave frequencies for powdered samples at

room temperature. EPR is a valuable technique, both because of its high sensitivity

and its general capability to identify unambiguously paramagnetic species which

contain unpaired electrons. It can reveal the oxidation state, electronic

configuration and coordination number of a paramagnetic ion. This technique has

been used in catalytic chemistry, biological systems food production. Here, we will

focus on this technique and its characterization on homo-and heterotrinuclear

Schiff base metal complexes.

Principle

EPR is an absorption spectroscopy in which radiation having frequency in

microwave region is absorbed by paramagnetic species to induce transition

between magnetic energy level of electrons with unpaired spin. Each electron

processes a magnetic moment and a spin quantum number S = 1/2, with magnetic

components ms = +1/2 and ms = −1/2. In the presence of an external magnetic field

85

with strength B0, the electron‟s magnetic moment aligns itself either parallel

(ms = +1/2) or antiparallel (ms = −1/2) to the field. The separation between

ms = +1/2 and ms = -1/2 state is ∆E =geµBB0, where ge is the electron‟s so-called g-

factor, B0 is the magnetic field, µB is the Bohr magneton. An unpaired electron can

move between energy levels by either absorbing or emitting electromagnetic

radiation of energy ε = hν such that the resonance condition, ε = ∆E, is obeyed.

Therefore, the fundamental equation of EPR spectroscopy is obtained:

hν = geµBB0, In practice, the frequency (ν) of radiant energy is usually fixed to

approximately 9.5 GHz in X-band EPR and the external magnetic field (B0) is

varied until that the gap between the ms = +1/2 and ms = −1/2 energy states

matches the energy of microwaves.

Figure.1.12. Energy-level diagram for S = ½ and no zero field splitting.

By increasing an external magnetic field, the gap between the ms = +1/2

and ms = −1/2 energy states is widened until it matches the energy of the

microwaves, as represented by the double arrow in the diagram above. At this

point the unpaired electrons can move between their two spin states. Since there

are typically more electrons in the lower state, due to the Maxwell Boltzmann

86

distribution, there is a net absorption of energy and it is this absorption which is

monitored and converted into a spectrum.

Figure.1.13. An example for EPR spectra

As an example of how hν = geµBB0 can be used, consider the case of a free

electron, which has ge = 2.0023, and the simulated spectrum shown at the right in

two different forms. For the microwave frequency of 9388.2 MHz, the predicted

resonance position is a magnetic field of about B0 = hν/geµB = 0.3350 tesla = 3350

gauss, as shown. Note that while two forms of the same spectrum are presented

in the figure, most EPR spectra are recorded and published only as first

derivatives. Because of electron-nuclear mass differences, the magnetic moment of

an electron is substantially larger than the corresponding quantity for any nucleus,

so that a much higher electromagnetic frequency is needed to bring about a spin

resonance with an electron than with a nucleus, at identical magnetic field

strengths.

87

1.14.7.8. Magnetic susceptibility measurements

Magnetic susceptibility data were collected on powdered sample of the

compounds at room temperature with PAR 155 vibrating sample magnetometer.

Experimental methods to determine susceptibility

Volume magnetic susceptibility is measured by the force change felt upon

the application of a magnetic field gradient [5]. Early measurements were made

using the Gouy balance where a sample is hung between the poles of an

electromagnet. The change in weight when the electromagnet is turned on is

proportional to the susceptibility.

The behavior of a compound in a magnetic field can be classified as being

diamagnetic, paramagnetic, ferromagnetic or antiferromagnetic. We will be

concerned with the first two types of behavior. A diamagnetic substance is weakly

repelled by a magnet, whereas a paramagnetic substance is attracted by a magnet.

If the substance is diamagnetic the mass of the magnet as measured by the

balance will appear to increase, because the magnet is repelled by the sample. On

the other hand, if the substance is paramagnetic, the mass of the magnet will

appear to decrease, because the magnet is attracted to the sample. The force of

attraction between the sample and the magnet is given by

( H 2 − H 2 ) g∆m = µ o χ m nm

1 2

2l

(1)

where g is the acceleration due to gravity, ∆m is the apparent mass change

of the magnet, µo is the permeability of a vacuum, χm is the molar magnetic

88

susceptibility (its units are cm3mol-1), H1 and H2 are the magnetic field strengths at

the opposite ends of the sample, nm is the moles of sample and l is the height of the

sample in the sample tube.

The magnetic behavior of a substance is related to the electronic structure

of its atoms. A moving charged particle generates a magnetic field. Thus, the

electrons within atoms generate magnetic fields due to their orbital motion around

the nucleus and to their “spin”. Atoms with unpaired electrons are paramagnetic

since the unpaired electrons behave as tiny magnets and in the presence of an

external magnetic field these magnets become aligned with the external field and

cause the sample to be attracted to the external magnet. In this experiment it will

be concerned with the magnetic properties of compounds containing transition

metal ions. Many of these compounds are paramagnetic due to the presence of

unpaired electrons in the transition metal ion. In such compounds the electrons‟

orbital motion does not contribute to the paramagnetism and the permanent

magnetic moment of the compound, µ, is given by the “spin only” formula:

µ S = µ B n( n + 2 ) (2)

where, µS, is the spin contribution to the magnetic moment of an ion having n

unpaired electrons. µB, is the Bohr magneton, the fundamental unit of magnetic

moment.

Diamagnetism arise from the fact that in an external magnetic field, the

moving electrons in an atom experience an additional force which, in turn, results

in an induced magnetic field that opposes the external field. This causes the

89

2 1 m m

sample to be repelled by the external field. All atoms will exhibit diamagnetism

regardless of whether or not they also possess permanent magnetic moments.

A general expression for χm is

χ m = χ diamag + Nµ 2

3kT

(3)

where µ is the permanent magnetic moment per atom, N is Avogadro‟s

number, T is the absolute temperature, k is the Boltzmann constant and χdiamag is

the diamagnetic contribution to the molar susceptibility. In paramagnetic

substances, the first term is equation (3) is negligible compared to the second term,

and we have:

µ = 3kTχ m

N

= 2.84 µ B

Tχ m

(4)

As we have seen, for transition metal compounds, the paramagnetism is

due to the presence of unpaired electrons in the transition metal ion and we would

expect µ to have the “spin only” value given by equation (2). Thus, equations (1),

(2) and (4) provide us with an experimental method for determining the number of

unpaired electrons in a transition metal ion.

In practice, equation (1) is written as

χ m = 2 lg ∆m

= β ∆m

µo ( H 2 − H 2 ) n n (5)

Where β is a calibration constant that depends upon g, µo, the magnetic

field strength, the magnetic field gradient and the dimensions of the sample. Note

90

m

that in equations (1) and (2), ∆m and χm can be either positive or negative,

depending on whether the substance is diamagnetic or paramagnetic, respectively.

The value of β is determined experimentally, by measuring ∆m and nm for a

reference substance, then

χ mref

β = (∆m n

)ref

(6)

where the subscript “ref” refers to the reference substance. The reference

substance to be used is Mohr‟s salt, iron(II) ammonium sulfate hexahydrate,

FeSO4(NH4)2SO4.H2O. It‟s molar magnetic susceptibility is given by

χ m = 3.7278

T + 1 (7)

1.14.7.9. Thermal Studies

Thermal studies were carried out in the 100−900oC range using an

SETSYS Evolution -1750 model thermal analyzer. Thermal methods of analysis

may be defined as those techniques in which changes in physical and / or chemical

properties of a substance are measured as a function of temperature. Methods that

involve changes in weight, dimensions or changes in energy come within this

definition.

Thermogravimetry (TG) a technique in which a change in the weight of a

substance is recorded as a function of temperature or time.

Differential thermal analysis (DTA) a technique in which the temperature

difference between a substance and a reference material is measured as a function

91

of temperature whilst the substance and reference are subjected to a controlled

temperature programme.

The basic instrumental requirement for thermogravimetry is a prescision

balance with a furnace programmed for a linear rise of temperature with time. The

results may be presented as a thermogravimetric (TG) curve, in which the weight

change is recorded as a function of temperature or time, or as a derivative

thermogravimetric (DTG) curve, where the first derivative of the TG curve is

plotted with respect to either temperature or time.

1.14.7.10. DNA binding experiments

A solution of CT DNA in 0.5 mM NaCl/5 mM Tris–HCl (pH 7.0) gave a

ratio of UV absorbance at 260 and 280 nm of 1.8:1.9, indicating that the DNA was

sufficiently free of proteins [6]. A concentrated stock solution of DNA was

prepared in 5 mM Tris–HCl/50 mM

NaCl in water at pH 7.0, and the concentration of CT DNA was determined per

nucleotide by taking the absorption coefficient (6600 mol−1 cm−1) at 260 nm [7].

Absorption titration experiments were performed by varying the concentration of

the DNA with the complexes.

1.14.7.12. Viscosity experiments

Viscosity measurements were carried out from observed flow time of CT

DNA containing solution (t > 100 seconds) corrected for the flow time of buffer

alone (t0), using Ostwald‟s viscometer at 30 ± 0.1°C. Flow time was measured with

a digital stopwatch. Each complex was measured three times and an average flow

92

time was calculated. Data were presented as (η/η0) versus binding ratio

([complex]/[DNA]), where η is a viscosity of DNA in the presence of complex and

η0 is the viscosity of DNA alone. Viscosity values were calculated from the

η = t – t0 [8].

1.14.7.12. Gel electrophoresis

The cleavage of pUC18 DNA was determined by agarose gel

electrophoresis [9]. The gel electrophoresis experiments were performed by

incubation of the samples containing 40 µM pUC18 DNA, 50 µM metal complexes

and 50 µM H2O2 in Tris-HCl buffer (pH 7.2) at 37°C for 2 hours. After incubation,

the samples were electrophoresed for 2 hours at 50 V on 1% agarose gel using

Tris–acetic acid–EDTA buffer (pH 7.2). The gel was then stained using 1 µg/cm3

ethidium bromide and photographed under ultraviolet light at 360 nm. All the

experiments were performed at room temperature.

1.14.7.13. Cyclic voltammetry

All voltammetric experiments were performed with a CHI 760

electrochemical analyzer, in single compartmental cells using tetrabutylammonium

perchlorate as a supporting electrolyte. The redox behavior of the complexes has

been examined in absence and in presence of CT DNA at a scan rate of 0.1 Vs−1 in

the potential range +2.0 to –2.0 V. A three-electrode configuration was used,

comprising a glassy carbon electrode as the working electrode, a Pt-wire as the

auxiliary electrode and an Ag/AgCl electrode as the reference electrode. The

electrochemical data such as cathodic peak potential (Epc) and anodic peak

potential (Epa) were measured.

93

1.14.7.14. Antimicrobial activity

The in vitro antibacterial activity of the ligand and the complexes were

tested against the bacteria Klebsiella pneumoniae, Escherichia coli and

Staphylococcus aureus by well diffusion method using nutrient agar as the medium

[10]. Streptomycin was used as standard component. The stock solution (10−1 mol

L−1

) was prepared by dissolving the compound in DMF, and the solution was

serially diluted to find minimum inhibitory concentration (MIC) values. In a

typical procedure, a well was made on the agar medium with microorganisms in a

petri plate. The well was filled with the test solution and the plate was incubated

for 24 h for bacteria at 35°C. During the period, the test solution diffused and the

growth of the inoculated micro-organisms was affected. The inhibition zone was

developed, at which the concentration was noted.

Glasswares

Glasswares used in the present investigation were thoroughly washed, dried

and were sterilized at 121oC for fifteen minutes in autoclave.

Grams staining

In the gram, staining test was performed and the morphology cell shape and

arrangement were observed.

Gram stain reagents

Crystal violet - 1.0 gm

5% Na2CO3 - 1.0 ml

Distilled water - 99 ml

94

Crystal violet was added to sodium bicarbonate into a mortar. Using a

pestle grind to a good paste. Distilled water was added and mixed well. It was

then filtered and stored in brown bottles.

Ethyl Alcohol

Ethyl alcohol (100%) 95 ml

Distilled water 5 ml

Saffranin

Saffranin 1 gm

Distilled water 100 ml

Grams iodine

Iodine crystals 2 g

NaOH 10 ml

Distilled water 90 ml

Sodium hydroxide was added to the iodine crystals kept in a mortar and

grind to a good paste. Distilled water was added and mixed well. It was then

filtered and stored in brown bottles.

1. A thin smear of sample is made on glass slide.

2. Sear is allowed to dry and heat fix it.

3. The slides are holded using slide rack.

4. Each smear is covered with crystal violet for 30 sec.

5. Each slide is then washed with distilled water.

6. Slides are covered with iodine solution.

95

7. Iodine solution is washed off by 95% ethyl alcohol.

8. Slides are washed by distilled water and drained.

9. Saffranin was flooded on the smear for 30 min.

10. Washed with distilled water and blot dried with absorbent paper.

11. The slides are allowed to dry.

12. Slides are observed under microscopically using oil immersion

objectives.

Motility test

Motility test was performed using hanging drop method. The motile and

non-motile organisms were differentiated.

1. Take two cavities slides and washed carefully.

2. Then cover slips are washed.

3. Dropped a small amount of culture on these cover slips each culture on

one cover slip.

4. Then applied Vaseline on the four corners of both the cover slips.

5. Then attached the cover slip by inverting it on the cavity slide to

scaffold the drop of culture inside the cavity.

6. Then the slide was examined under the microscope.

Catalase test

Some organism posses catalases and splits hydrogen peroxide into oxygen

and water.

1. Nutrient agar was prepared melted and pored into the test tube.

2. The test tubes were sterilized and then allowed in a stand position to

prepared slants.

96

3. After solidification, the organisms were streaked and stand position to

incubate at 370C for about 12 to 24 hours.

4. After incubation 3%, hydrogen peroxide is added to slant.

Oxidase test

The enzyme oxidase that forms the part of the electron transport system is

possessed by some bacteria. The enzyme oxidizes the reagent N-N tetramethylene

paraphenylene diamine dihydrochloride to a coloured product into phenol.

Sugar fermentation test

Some organisms produce small amount of acid either by fermentation or by

oxidation. These small amounts of acids are neutralized by large amount of

alkaline products released during protein metabolism and hence acid production is

not noticed. Thus, carbohydrate fermentation reaction can be tested in minimum

protein.

Indole test

Tryptophan present in peptones of the culture media is act upon by the enzyme

tryptophanase and converted into indole, skatol and induce acetic acid. Indole reacts

with aldehydes to produce a red coloured product. Therefore, organisms are grown in

tryptophan rich medium and tested for the presence of indole.

Tryptone broth

Tryptone 1.0 g

NaCl 0.5 g

Distilled water 100 ml

97

Kovacs reagent

Paradimethylamino benzaldehyde 5 g

Amyl alcohol 75 g

pH 7.4

Conc.HCl 25.5 ml

Dissolved the aldehyde in alcohol and added slowly. Stored in brown bottles.

Methyl Red Voges-proskauer Test

Enterobacteriaceae ferment glucose via pyruvate and produce mixed acids

and other products. Among them, one group produces mixed acids such as acetic,

lactic, succinic, formic acid, ethanol, CO2 and H2.

Because of the abundant acid production, the final pH of the broth drops to

less than 4.5, which can be detected by pH indicators.

Other group of organisms can produce the butylenes glycol and acetone,

which are more neutral in nature and not much drop in pH noticed. The end

products are detected by reagent.

MR-VB broth preparation

Peptone 7.0 g

Glucose 5.0 g

K2PO4 5.0 g

Distilled water 1000 ml

Final pH 6.9

98

MR-Reagents

Methyl red 0.1 g

Ethyl alcohol 300 ml

Distilled water 200 ml

Dissolved methyl red in alcohol and then add distilled water. Stored in brown

bottles at 40C.

VP reagent preparation

VP-A

Alpha napthol 5.0 g

Ethyl alcohol 100 ml

Dissolve alpha-naphthol in small amount of alcohol first and then add the

remaining alcohol to 100ml. Store in brown bottles at 40C.

VP-B

KOH 40.0 g

Distilled water 100 ml

Cooled the cylinder in cold-water bath with 80 ml of water and add KOH crystals,

dissolved and made upto 100 ml. Stored in polythene bottles at 40C.

Citrate utilization test

Certain organisms can utilize as sole carbon source and grow. During the

growth acetate and other alkaline carbonated are produced. This reaction is shown

by the change in colour of the indicator.

99

Simmon‟s citrate agar media

NaCl 5.0 g

MgSO4 0.2 g

NH4H2PO4 1.0 g

K2PO4 1.0 g

Sodium citrate 2.0 g

Distilled water 1000 ml

pH 6.9

TSI Test

Some bacteria liberate sulfur from sulfur containing amino acid or other

sulfur containing compounds. The sulfur is used as final hydrogen acceptor

leading to the formation of H2S. When the following conditions are present, H2S

can be detected by the test system.

Yellow - A (acid)

Yellow gas - AG+

Pink - AK (Alkali)

Black precipitation - H2S

TSI Medium

Beef extract 0.3

Yeast extract

0.3

Peptone

2.0

NaCl

0.5

Glucose

0.1

100

Lactose 1.0

Sucrose 1.0

Fe2(NH4)SO4 0.2

NaHSO4 2.5

Casein digests animal tissue 10.0 g

Phenol red 2.5

Distilled water 100 ml

Agar 20 g

Final pH 7.4

Urease test

Urea is diamide of carbonic acid. Urease the enzyme possessed by the

bacteria hydrolyses urea and release ammonia and carbon dioxide. NH4 reacts in

solution to form ammonium carbonate, which is alkaline leading to the increase in

the pH of phenol red which is incorporated in the medium changes its colour from

yellow to red in alkaline pH, thus indicates the presence of urease activity.

Christensons Urea agar

Peptone 0.1 g

Glucose 0.1 g

NaCl 0.5 g

K2PO4 0.2 g

Phenol red 1.0 g

Agar 2.0 g

Distilled water 1000 ml

pH 6.8

101

Primary culture media

Nutrient agar and Mac Conkey was used as the primary culture media.

Enriched media

Blood agar and citramide agar are used for isolation and identification of

the pathogenic microorganisms during the study.

Nutrient agar

Peptone 0.5 g

NaCl 0.5 g

Beef extract 0.3 g

Yeast extract 0.3 g

Agar 2 g

Distilled Water 100 ml

pH 7.2

Blood agar

Peptone 0.5 g

NaCl 0.5 g

Beef extract 0.5 g

Agar 2 g

Distilled water 100 ml

Sheep blood 5 ml

pH 7.4

102

Mac Conkey agar

Peptone 20 g

Lactose 10 g

Bile Salt 1.5 g

Neutral red 0.03 g

Crystal violet 0.001 g

Agar 15 g

Distilled water 1000 ml

pH 7.4

1.14.8. Metal Estimations

1.14.8.1. Estimation of copper [11]

The amount of copper in the complexes was estimated

spectrophotometrically by the following method. The addition of an aqueous

solution diethyldithiocarbamate to a slightly acidic or an ammoniacal solution of

the copper salt produces a brown precipitate of a sparingly soluble copper

derivative. With minute amounts of copper, a golden brown coloration is obtained.

The copper derivative is soluble in organic solvents such as CCl4 and the optical

density of the yellow-brown colored solution is compared with that of similar

standard solution.

Procedure

The complex was decomposed by adding 10 ml of concentrated sulphuric

acid and few drops of perchloric acid and the mixture was digested in a kjeldahl‟s

flask until the solution become colorless. The solution was cooled and made up to

103

50 ml. Dilution was effected by taking 5 ml of the solution in a 50 ml volumetric

flask and making up to the mark by addition of water. An aliquot of 2 ml of the

solution was transferred into a beaker and rendered alkaline using a dilute solution

of ammonia. The excess ammonia was boiled off. The pH of the solution was

adjusted to 8.5 using very dilute solutions of ammonia and sulphuric acid. 15 ml of

4% solution of EDTA (disodium salt) was then added and the solution was allowed

to cool to room temperature. The solution was transferred to a separatory funnel

and treated with 10 ml of 0.2% aqueous solution of diethyl dithiocarbamate. The

solution was thoroughly mixed by shaking for 45 seconds during which a yellow-

brown color developed in the solution, was immediately extracted with 5 ml of

CCl4. The lower organic layer was collected in a dry vessel. Extraction was

repeated until the entire complex was removed.

The extracted organic layer was washed by shaking with 20 ml of 5%

solution of sulphuric acid for 15 seconds in a reparatory funnel. After allowing

enough time for the two layers to separate, the organic layer was separated and

made up to a total volume of 25 ml. The copper content was then determined by

measuring the optical density of the made up solution at 435 nm and compared

with the standard solutions of copper treated similarly. The standard solution was

prepared by dissolving 0.0623 g of AR grade copper (II) sulphate pentahydrate in

100 ml of water in a standard flask. A calibration curve was obtained by measuring

the optical density of solutions prepared as above using different volumes of the

standard solution.

104

1.14.8.2. Estimation of nickel [12]

The nickel content in the complex was estimated colorimetrically using

dimethylglyoxime after the complex had been decomposed completely by

dissolving in 10 ml of warm 1:1 nitric acid, boiling the solution and was then

cooled and made upto 250 ml with distilled water in a volumetric flask.

An aliquot of 5 ml of the solution was taken in a 50 ml volumetric flask. To

that was added, with mixing after each addition, 5 mlof the 10% citric acid

solution, 2 ml of saturated bromine water, 2 ml of 1:1 ammonium hydroxide

solution and finally 1 ml of the 1% solution of dimethylglyoxime in rectified spirit.

Nickel content was then determined by measuring the optical density of the made

up solution at 530 nm and compared with the O.D of standard solution of nickel

similarly treated. The standard solution was prepared by dissolving 0.0673 g of

nickel ammonium sulphate in 100 ml of water. A calibration curve was obtained

by measuring the O.D of the solution prepared as above, using different volumes of

the standard solutions.

1.14.8.3. Estimation of manganese [13]

About 0.075 g of the complex was taken and dissolved in dilute nitric acid

(1:3) in a 250 ml conical flask. The solution was boiled for 5 minutes and cooled.

Then 0.5 g of A.R sodium bismuthate was added again and again boiled for

5minutes. A pink color was produced due to the formation of permanganic acid.

Then a concentrated solution of freshly prepared sodium sulphite was added drop

wise until the solution became clear. The solution was boiled to expell all oxides of

nitrogen and cooled to about 15ºC.

105

To the cooled solution, A.R Sodium bismuthate was added until no further

appreciable alteration in color was noticed. Then, excess of 0.5 g of sodium

bismuthate was added and the mixture was stirred for 2-3 minutes. To this, 50 ml

of the prepared 3% nitric acid was added and filtered through a sintered crucible

into a 350 ml conical flask. The residue was washed well with about 100 ml of the

3% nitric acid until it is free from permanganic acid.

Then the pink colored solution was added slowly with 0.003N FAS in

slight excess as indicated by the disappearance of pink color. This solution was

titrated immediately with a standard solution of (0.03) KMnO4 to the first faint

pink color.

The FAS solution was standardized by running a blank. In a conical flask,

50 ml of 1:3 nitric acid was taken, then a little of sodium bismuthate was added

and diluted with 10 ml of 3% nitric acid. To this an equal volume of the FAS was

added and titrated against the standard KMnO4. The difference between the

titrations gives the amount of permanganate equivalent to that of permanganic acid

formed in the determination.

1ml of KMnO4 = 0.0109 of Mn

1.14.8.4. Estimation of vanadium [14]

The amount of vanadium in the complexes was estimated volumetrically by

the following method. About 0.055g of the complex was weighed out and

dissolved in 2N H2SO4 acid. Approximately 0.1 N KMnO4 solutions was prepared

and standardized by titrating against standard solution of oxalic acid. The complex

106

solution was titrated against KMnO4 solution. The amount of vanadium was

calculated by the following formula.

Percentage of Vanadium = Titre value × N/100 ×51 × 100/W

Where

N – Strength of KMnO4

W – Weight of the vanadium complex

107

References

[1] A. Weissberger, E. S. Proskaver, J. A. Riddick and E. E. Toops, “Organic

solvents in techniques of Organic Chemistry”, 3rd Edn., Interscience., 7 (1970).

[2] A. I. Vogel “A Text book of practical organic analysis”, Longmans (1971).

[3] A. K. Panda, D. C. Dash, P. Mishra and H. Mohanty, Ind. J. Chem., 35

(1996) 848.

[4] W. J. Geary, Coord. Chem. Rev., 7 (1971) 81.

[5] L. N. Mulay, A. Weissberger and B. W. Rossiter, Techniques of Chemistry.

4. Wiley-Interscience: New York. p. 431, (1972).

[6] J. Marmur, J. Mol. Biol., 3 (1961) 208.

[7] M. E. Reicmann, S. A. Rice, C. A. Thomas and P. Doty, J. Am. Chem. Soc.,

76 (1954) 3047.

[8] S. Mathur and S. Tabassum, Biometal., 21 (2008) 299.

[9] N. Raman, J. Raja and A. Sakthivel, J. Chem. Sci., 119 (2007) 303.

[10] G. G. Mohamed, M. M. Omar and A. A. Ibrahim, Spectrochim. Acta. A., 75

(2010) 678.

[11] A. I.Vogel, A Text Book of Quantitative Inorganic analysis, ELBS 3rd

Edn,

794 (1975).

[12] A. I.Vogel, A Text book of Quantitative Inorganic analysis, ELBS 3rd Edn,

796 (1975).

[13] A. I.Vogel, A Text Book of Quantitative Inorganic analysis, ELBS 4th Edn,

160 (1979).

[14] A. I.Vogel, A Text book of Quantitative Inorganic analysis, ELBS 3rd Edn,

(1975).