Open Access Repository of Indian Theses

R., SEETA

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ABSTRACT 
 
Singlet open-shell ground state molecules are rapidly gaining 
importance in the recent times due to the possible applications as organic 
field effect transistors (OFET) and in nonlinear optics (NLO).  They are usually 
characterized experimentally by small HOMO-LUMO gap (HLG) and long 
wavelength absorption in the UV/visible spectrum. Additionally SQUID 
measurements on the powdered sample can give us the singlet-triplet (ST) 
energy difference which would be very small. Theoretical studies using ab 
initio methods would require multideterminants for description hence the 
wavefunction shows spin contamination when single determinant methods are 
used. Calculated ST gap would also be less than 1 eV. One such set of 
molecules are the polycyclic aromatic hydrocarbons. They have the ability to 
show ambipolar transport in FET due to the small HLG. In the field of NLO 
they been shown to have large γ values both from experiment and theoretical 
calculations.  The major issue right now in these molecules which has to be 
overcome before applications in materials is the stability.  
 
Biradicals are molecules consisting of two nearly degenerate non 
bonding molecular orbitals (NBMO) occupied by two unpaired electrons. They 
could either have a triplet or Hunds rule violated singlet ground state. Due to 
this they are highly reactive and unstable. The type of molecules which can 
find applications in the field of materials is the conjugated biradical molecule. 
These can be represented by valence bond structures in resonance. 
Conjugated biradical systems can be broadly divided into two types – Kekule 
and non-Kekule systems. In some cases the degenerate NBMOs (HOMOs) 
due to substitutions would have a small energy gap and here the singlet 
would be more stable but with some biradical character (BRC) retained 
(partial occupation of LUMO). These types of molecules are called as 
biradicaloids. Though there is no clear threshold, one could imagine the 
closed shell molecule at one extreme and the biradical molecule at the other 
extreme and a biradicaloid anywhere in the middle. Molecules having a small 
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BRC are of advantage as they are stable and can also show the properties of 
the biradical described above. 
 
There are many molecules in literature which are derivatives of non-
Kekule type biradicals like tetramethylenemethane, tetramethyleneethane, 
tetramethylenebenzene etc.  Introduction of heteroatoms into the rings 
increases the zwitterionic character thus stabilizing the molecule but retains a 
small BRC. These molecules can be said to inherit the biradical nature from 
the parent molecule.  In fact the biradicaloid nature of many of these 
molecules can go unnoticed unless larger exact exchange is included into the 
density functional for the calculation. The thesis work was initiated by looking 
into such molecules from literature with two objectives, one to evaluate 
theoretically such molecules for material applications and the other for 
understanding the structure and stability to improve these molecules.  
 
This thesis consists of five chapters in which first chapter gives 
introduction biradicaloid molecules. Subsequent chapters are case studies on 
each set of molecules and their properties (mainly the linear optical, stability 
and semiconducting properties).    
 
Chapter 1 
      This chapter gives a general introduction to biradicaloid molecules. A 
brief out line of the theoretical/computational methods used for this work is 
also presented.  
 
Chapter 2 
Main factors responsible for the red to near infra red (NIR) absorption 
of the benzobis(1,2,5-thiadiazole) (BBT) derivatives have been investigated 
using high level computational studies. These molecules with NIR emission 
are of importance due to the recent reports of possible role in NIR Organic 
Light Emitting diodes (OLED) development. These molecules are having 
sulfur diimide bonding and are distinguished from many other compounds by 
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its non- classical structure. Calculations of these compounds therefore need 
more sophisticated quantum chemical methods. The electronic structure has 
been determined using closed-shell/open-shell DFT methods (B3LYP and 
BHandHLYP). Molecular geometries were calculated using B3LYP and 
BHandHLYP DFT methods. The obtained results are compared with 
experimental crystallographic data where available. The wavefunction 
stabilities of these BBT derivatives have been tested. It is noticed that using 
the BHandHLYP functional, the wave function becomes instable though large 
HLG are obtained. On the other hand a stable wavefunction is obtained using 
the B3LYP functional but the HLG is small. The B3LYP HLG is in good 
agreement with the available experimental data. Nevertheless results from 
both functionals indicate a possible LUMO occupation/ biradicaloid character 
(BRC).  Different computational methods are used to quantitatively determine 
the BRC of the BBT derivatives. It is found that at the DFT level the   S–T 
gaps calculated are less than 20 kcal/mol for the BBT derivatives. The 
computed CASSCF coefficients also point out to a BRC for the BBT 
derivatives.  It is observed that the BRC has a good correlation with the NIR 
absorption, smaller the excitation energy-larger the BRC. The transition 
energies of three known chromophores calculated at the SAC/SAC-CI level of 
theory were in excellent agreement with the experimental values. Charge 
transfer (CT) based on Mulliken charges of both ground and excited state are 
obtained from high level SAC/SAC-CI studies. From this data it is indicated 
that substantial CT from the side groups to the middle ring is taking place in 
the BBT derivatives. The excitation energies do not have a correlation to this 
CT. Ab initio valence bond studies are carried out for the unsubstituted BBT to 
predict the relative weights of the individual Lewis structures to the resonance 
picture. It is concluded that a more general description which include the 
zwitterionic and biradical structures are necessary for these molecules and 
not just simple donor-acceptor-donor(D-A-D) structures as suggested in the 
literature. 
 
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Chapter 3 
Recent literature reports indicate that derivatives of benzothiadiazole 
(BT) and benzobis(thiadiazole) (BBT), which differs from BT by an extra 
thiadiazole ring, exhibit good semiconducting properties like high electron 
mobility and low lying LUMO levels. In this chapter computational techniques 
like DFT, spin-flip DFT and valence bond methods are used to analyze the 
semiconducting properties of these molecules. Calculations at the B3LYP/cc-
pVTZ level reveal that all the BBT molecules, including the bare BBT ring, 
have lower lying LUMO energies (3.70 – 4.11 eV) compared to the BT 
derivatives (2.56 – 3.41 eV) with similar substitution.  The reorganization 
energies (λ+/λ-), obtained at this level of theory, of the BT derivatives are 
around (225-333)/(246-315) meV while BBT derivatives have much smaller 
reorganization energies and these are in the range (129-259)/(150-230) meV. 
It is observed here that the different behaviour of BBT is due to the inherited 
biradicaloid character from the parent molecule tetramethylenebenzene 
(TMB), a disjoint non Kekule biradical having NBMOs as the HOMO and the 
LUMO. The perturbation of the orbitals of the biradical TMB to obtain BBT 
also is the major cause for the BBT derivatives to have a larger EA and 
smaller HOMO-LUMO gap (HLG) when compared to BT derivatives.  
 
Chapter 4 
Keeping in view the possible applications of singlet open-shell 
molecules as semiconductors, non-classical derivatives of the heterocyclic 
rings benzobis(thiadiazole) (BBT) and its positional isomer 
thiadiazolothienopyrazine (TTP) have been characterized using DFT 
methodologies. M06-2X, B3LYP and BHandHLYP functionals have been used 
to optimize the geometries and estimate the vertical transition energies. It is 
observed that unlike BHandHLYP functional (50% exchange)  which gives 
rise to spin contaminated solutions for all molecules in the series, M06-2X 
(54% exchange) for most of the molecules affords wavefunction either with no 
instability or negligible instability. The results have been compared with the 
earlier reported experimental data and those obtained in this work using spin 
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flip (SF)-5050 method. It is found that B3LYP does not fare well while on the 
other hand the M06-2X and SF-50-50 are in good agreement with the 
experimental results. It is seen that M06-2X TD-DFT for the molecules can be 
carried out without major spin contamination and also the more time 
consuming CI can be avoided for calculation of transition energies.  Biradical 
nature of the molecules are estimated by singlet-triplet gap. Intramolecular 
charge transfer is calculated. It is found that the ring  substituents donate 
charge in the ground state creating a zwitterionic structure. Thus the 
substituents play an interesting dual role, decreasing the stability of the 
molecule by increasing the biradical character (small HOMO-LUMO gap), and 
stabilization of this ground state by intramolecular charge transfer. 
Chapter 5 
Squarines and croconine dyes have oxyallyl biradical substructure and 
hence inherit the character of the parent biradical (Biradicaloids). Earlier 
TDDFT studies with different DFT functionals fail to predict the right excitation 
energies in these molecules. The croconate dyes, however, have larger 
deviation from the experimentally observed values in all the functionals 
studied even after inclusion of the solvent effects. The deviations are also not 
systematic. Biradicaloids with high BRC in general may require multi-
reference description to obtain the first vertical singlet-singlet excitation 
energy.  In this chapter detailed electronic structure for a series of croconate 
molecules with varying BRC is analyzed.  It is observed that at the single 
determinant/RB3LYP level the HLG match the experimental absorption for 
molecules with high BRC. The stable broken symmetry (BS)-UDFT solutions, 
which are only slightly lower in total energy yield overestimated HLG due to 
spin contamination. For medium and small BRC the RB3LYP has stable 
solutions but the HLG deviates, the deviation being quite large in the latter 
case.  The Spin Flip (SF)-TDDFT/TDA method is used which is known to 
cover non-dynamical electron correlation in biradicals. Excitation energies 
calculated using single reference SF-TDDFT methods are in good agreement 
with the experiment for the molecules with medium-small BRC. 
 


English

ENZYMATIC HYDROLYSIS OF CELLULOSE

B., SEETARAMARAO

Abstract
The thesis describes the isolation and structural determination of bioactive constituents from well known medicinal plant Cedrus deodara used in Indian traditional system of medicine. Series of analogues of (-)-carinol were prepared and their biological activities were discussed and quantitative estimation of chrysophanol as marker compound in the rhizomes of Rheum emodi using HPTLC has been studied. In addition, some new synthetic methodologies have been developed using Lewis acid catalysts.
The first chapter deals with the chemical examination of the bark of Cedrus deodara which resulted in the isolation of eleven compounds designated as A, B, C, D, E, F, G, H, I, J, and K. Compounds A, B, C, D, E, F, G, H, I, and K were identified as pinocembrine, matairesinol, divanillyltetrahydrofuran, (-)-nortrachelogenin, dibenzylbutyrolactol, chrysin, cedeodarin, pinobanksin, a new lignan 8′-hydroxydivanillyltetrahydrofuran, secoisolarciresinol and a new dimeric lignan respectively. Among the compounds compound C, F, G and H are isolated for the first time from the genus Cedrus.
Compound I was obtained as an amorphous powder, m.p. 69-71 0C, [α]25D –12.330 and analyzed for C20H24O6 (M+ 360).  The IR spectrum has shown absorption band at 3300 cm-1 suggesting the presence of an hydroxyl group and general nature of an aromatic compound. The 300 MHz 1H NMR spectrum in CDCl3 has shown the general features of a tetrahydrofuranoid lignan. It has shown a multiplet integrating for six protons at δ 6.60-6.76, which were accounted for six aromatic protons of a 1,3,4- tri substituted aromatic system. Two broad singlets at δ 5.56 and 5.65 are typical of hydroxyl groups. A sharp singlet at δ 3.79 integrating for six protons is indicative of two aromatic methoxy groups. A multiplet integrating for two protons at δ 3.90-3.95, a double doublet at δ 3.70-3.78 (J 14 Hz, 13.5) and a doublet at δ 3.60 (J 14 Hz) each integrating for one proton were characteristic of furanoid protons (H-9, 9′). A doublet at δ 2.88 (J 14 Hz) integrating for one proton, a multiplet at δ 2.70-2.81 integrating for two protons and a double doublet at δ 2.50-2.61 (J 14, 11.5 Hz) integrating for one proton indicate the presence of two benzylic methylenes (H-7, 7′). A multiplet at δ 2.28 - 2.40 integrating for one proton are assigned for H-8. The above data upon comparison with carinol showed that, no profound change has not been found at aromatic region and benzylic methylenes, remarkable downfield shift was observed for the protons present at H-8, 9 and 9′. The 13C NMR spectrum of compound I in CDCl3 showed the presence of 20 carbons. The notable feature of the 13C NMR spectrum was the down field shift of C-9 and C-9′ carbons when compared to Carinol. This downfield shift is due to the presence of furanoid ring, which was further confirmed by comparing the values with similar type of lignans.  The DEPT experiment has revealed the presence of four methylenes, seven methines, two methyls and seven quaternary carbons. The negative ion ESIMS spectrum of the compound gave (M+-1) at 359. In view of above considerations along with its mass spectrum, compound I was identified as a new lignan, is arrived by 8′-hydroxy divanillyltetrahydrofuran (9). Further confirmation of the structure is arrived by its synthesis from (-)-nortrachelogenin. 
All the known 8′-hydroxy lignans are essentially lactones. However the present new compound is an 8′-hydroxy furanoid lignan. Hence its occurence is biogenetically significant.


Compound K was isolated as white solid, hygroscopic in nature m.p. 75-77 0C,  [α]25D -5.150 and analyzed for C38H40O14 (M+-1, 719). The IR spectrum has shown a broad band at 3388.60 cm-1 indicating the presence of hydroxyl groups, a sharp band at 1759 cm-1 suggesting the presence of a lactone carbonyl and indicating the general features of an aromatic compound. The 200 MHz 1H NMR spectrum in acetone-d6  has shown the general features of a lignan, the spectrum showed a multiplet at δ 6.78-6.52 integrating for twelve protons indicating the presence of 12 aromatic protons, a four proton multiplet at δ 4.01-3.87 assigned for two sets of oxymethylene protons (H-9, 9′). Two sharp singlets at δ 3.85, 3.79 each integrating for three protons were characteristic of two aromatic methoxyl groups. A multiplet at δ3.18-3.03 integrating for two protons, a four proton multiplet at 2.90-2.75 and a multiplet at δ2.58-2.41 integrating for four protons were attributed to  benzylic and aliphatic methine protons respectively.
	The 13C NMR spectrum in acetone-d6 displayed the presence of 38 carbons and the 13C NMR DEPT experiment has revealed the presence of 14 methine carbons, 6 methylenes, 14 quaternary carbons and 2 oxymethyls. All the signals were assigned based on DEPT experiment in conjunction with HSQC spectra and comparison with (-)-nortrachelogenin. A close look at 13C NMR spectrum shows the presence of two oxymethylene carbons at δ 70.84 and 70.79. All the values in 1H and 13C NMR spectra were comparable to those in (-)-nortrachelogenin except in the case of protons present on C-9 and C-9″. In the HMBC spectrum no correlations were observed between protons present on C-9 and C-4″′. Further, when compound K is converted to acid chloride followed by esterification, it has not resulted in the formation of any ester. Hence it was concluded that linkage between the two monomers is through C-9′. The downfield shift of C-9 carbon in 13C spectrum to δ 70.84 is attributed to the fact that the hydroxyl present at C-9 may be in hydrogen bonding with the hydroxyl group present on the aromatic ring. 
Based on the above facts and taking into consideration the data obtained from 1H, 13C, 1D and 2D NMR, Mass spectral data, the structure of compound K is concluded as a lignan dimer. Positions of the two methoxyl groups were assigned based on their 2D spectral correlations. The earlier literature reveals that the carbons present in A and C rings occur at downfield when compared to B and D rings hence the position of methoxyls were assigned at C-3 of A and C-3 of C rings. The low resolution negative ion mass spectrum indicated the mass as 719 which corresponds to M+-1 and suggestive of a dimeric lignan. The earlier literature reveals that the carbons present in A and C rings occurs downfield when compared to B and D rings hence the position of methoxyls were assigned at C-3 of A and C-3 of C rings. DEPT experiment showed the presence of 12 aromatic methine carbons ruling out the possibility of a phenyl-phenyl linkage of the two monomers. The other possible linkage is through phenolic hydroxy to phenolic hydroxyl, which is also ruled out based on the mass. Hence, the only possibility is to have the linkge through lactone carbons. This is possible when the lactone is hydrolyzed to acid and alcohol. In such case there should be two possibilities one is through acid and the other is through oxymethylene, out of these the linkage through alcohol is ruled out on the basis of chemical evidence and its 2D spectral features. Thus, compound K is identified as new dimeric lignan; obviously formed through lactone ring opening of the monomeric lignan and further condensation with phenolic –OH of another unit of monomeric lignan. This is the first report of the occurrence of a dimeric lignan formed through the condensation of both the monomers leading to an ester linkage. Hence its occurrence is biogenetically singnificant. A cold acetone extract of C. deodara wood powder also showed the presence of the new dimeric lignan on TLC. This clearly shows that the new compound is not an artifact.
	The second chapter describes the synthesis of (-)-carinol analogues as free radical scavengers and enzyme inhibitors. The aliphatic C-9, 9′-O-acyl esters of (-)-carinol were prepared by condensing the corresponding aliphatic acids with (-)-4, 4′-di-O-benzyl carinol in the presence of dicyclohexylcarbodiimide (DCC) in anhydrous methylene chloride followed by debenzylation using palladium on carbon under hydrogen atmosphere to yield the target compounds. The aromatic esters of (-)-carinol were obtained by treating the (-)-4, 4′-di-O-benzyl carinol in methylene chloride with the corresponding aromatic acid in the presence of dicyclohexylcarbodiimide (DCC) and catalytic amount of DMAP followed by debenzylation to yield the target compounds. 
	(-)- Carinol and its 9,9′-O-acyl esters were screened for their free-radical scavenging activity. (-)-Carinol 9, 9′-O-acyl esters have showed moderate level of DPPH radical scavenging activity than parent compound (-)-carinol. It is evident from the present study that the antioxidant activity is due to the presence of free hydroxyl groups in the molecule present at C-9, 9′ of (-)-carinol. The introduction of an acyl group (aliphatic or aromatic) at C-9, 9′ positions of (-)-carinol has not affected the free radical scavenging property. Introduction of an aromatic acyl group at C-9, 9′ position of (-)-carinol had a moderate effect on free radical scavenging inhibitory potential. 
	(-)-Carinol and its acyl esters were tested for their xanthine oxidase enzyme inhibitory activity. Parent compound and its aliphatic acyl esters have shown moderate level of xanthine oxidase inhibition. It has been observed that interesting results have been found in the case of aromatic acyl esters. Thus the introduction of different functional groups on the aromatic ring at C-9, 9′ position increases the enzyme inhibitory property of the lignans. 
It may be concluded from the present study, that the presence of free hydroxyl groups at C-9, 9′ positions in (-)-carinol reduces the xanthine oxidase inhibition to a greater extent. The introduction of an aromatic acyl group at these positions showed better inhibitory pattern. The trends observed in the experiments could be explained on the basis of lipophilicity of the substrates. Substituted aromatic acyl esters showed better inhibitory potential. The effect of various functionalities on the aromatic ring can be well understood from the IC50 values of the selected aromatic esters. An interesting observation is that, in the case of 4m ((-)-C-9, 9′-O-vanilloyl carinol ) the presence of hydroxyl at position para and methoxyl at meta position showed a better inhibitory potential in the case of xanthine oxidase as well as free radical scavenging property. Hence, among all the aromatic esters 4m could be a better therapeutic candidate.
In summary, the incorporation and increase in xanthine oxidase inhibitory properties along with increase in lipophilicity of aromatic esters of (-)-carinol, may offer exciting opportunity for their development as therapeutic agents in disorders where increase in free radical generation and xanthine oxidase levels play an important role in pathogenesis.     
The third chapter deals with standardization of the medicinal plant Rheum emodi, a widely used medicinal plant. Quality evaluation of herbal drugs is absolutely necessary for authenticity, purity, safety and efficacy. Of all the protocols the marker compound based quality evaluation using HPTLC/HPLC is highly reliable. The latest development of quality evaluation is the use of biological standardization. In an effort to isolate and identify the antioxidant principles from Indian medicinal plants, chemical examination of a well known medicinal plant Rheum emodi has yeilded chrysophanol as the major constituent and it is quantitatively estimated using HPTLC. Over the years HPTLC has emerged as an important analytical tool for the evaluation of herbal formulations on a phytochemical basis.  In the process of estimation, chrysophanol (> 99.0% purity) thus isolated is used as an external standard. As part of standardization of indigenous medicinal plants, a simple, specific and efficient method for the estimation of chrysophanol present in the rhizomes of Rheum emodi has been developed using HPTLC.
The fourth chapter deals with the development of new synthetic methodologies using Lewis acids as catalyst. Efficient and chemoselective methods have been developed for the synthesis of chromanoquinolines, selective deprotection of prenyl ethers in presence of prenyl esters and regeneration of carbonyl compounds from their oximes have been discussed using three different Lewis acid catalysts. 
	Cycloaddition reactions are important synthetic routes for the fast assembly of polycyclic structures. The [4+2] Diels-Alder reaction between N-aryl amines and electron rich dienophiles is a powerful synthetic strategy for N-containing six membered heterocyclic compounds and in the synthesis of natural products. In particular intra molecular hetero Diels-Alder reactions provide multiple opportunities for the stereo selective construction of tetrahydroquinolines and their derivatives having wide range of biological activities. In conclusion a simple and highly efficient one-pot syntheses of tetrahydrochromano [4,3-b]quinolines from anilines and O-prenyl derivatives of salicylaldehydes via intramolecular cyclisation of imines in the presence of catalytic amount Bi (OTf)3 has been demonstrated. The advantage of this procedure is the operational simplicity, mild reaction conditions. The method offers several advantages including high yields and short reaction times. We believe that this method presents a better and more practical alternative to the existing methodologies for the synthesis of tetrahydrochromano [4,3-b] quinolines. It was thus demonstrated for the first time that Bi (OTf)3 is an efficient catalyst for the intramolecular[4+2] cycloaddition reactions.
	Protection of phenolic hydroxyls and subsequent deprotection is very useful for multi-step transformation and synthesis of several bioactive naturally occurring molecules. Phenols are frequently protected as ethers due to ease of their preparations and stability. Among these unsaturated ethers prenyl (2-methyl-2-butenyl) ethers are routinely prepared and utilized for protection of phenols. In conclusion, p-TSA was demonstrated to be a simple and convenient deprotecting agent for prenyl group. The advantage of the method is inexpensive nature of the reagent, co-reagents not needed, extremely simple experimental procedures (anhydrous, inert (or) dry atmosphere conditions are not required), high selectivity towards the deprotection of prenyl ethers and high yields. These advantages are very useful in complex multistep synthesis which require sequential protection and deprotection of various functionalities.
Oximes are also useful as protecting groups especially in the case of carbonyl compounds. So regeneration of carbonyl compounds from oximes is an important reaction in organic chemistry. The utility of BF3.Et2O/NaI at ambient temperatures for the regeneration of carbonyl compounds in high yields. Nineteen (1-19) were prepared from their corresponding aldehydes and ketones according to the standard oximation method. In the first steps of analysis the treatment of 3-methoxy-4-hydroxy benzaldoxime with BF3.Et2O/NaI at ambient temperature results the 3-methoxy-4-hydroxy benzaldehyde in more than 90% yield. In view of the above observed selectivity, mildness, less reaction times, it is believed that the scope and generality of the reaction has considerably improved with respect to deprotection of various oximes. A mechanism is also proposed for the deprotection of oximes. 








MICROBIAL ENZYMES: IMMOBILIZED WHOLE CELL SYSTEMS IN FERMENTATION

SUTAR, IBRAHIM

STUDIES ON PROTEOLYTIC ENZYME

LAGU, MEENA

MOLECULAR ANALYSIS OF PLANT GENOMES WITH SPECIAL REFERENCE TO THREE CUCURBITACEAE SPECIES

PESHWAI, SUNANDA RAMARAO

Abstract
The present work with Salvadora persica was carried out with three main aims : ( I ) . To develop a procedure for micropropagation, using seedling as well as mature tree explants as the starting material (II) to study the response of Salvadora shoot cultures to different concentrations of sodium chloride and sea water and (III) to study the variation in total fatty acid or oil contents among seeds from different trees and also within an individual tree. These aspects have been divided into three main Chapters. The results obtained in Chapters III, IV and V have been summarised as follows: 
Chapter - III : Micropropagation This Chapter has been divided into two sections: Sectionl : Covers studies on clonal . propagation using both seedling and mature tree explants. 1. A high percentage of seed germination (70-80%) was obtained on 1/2 strength MS (1962) basal medium, within 10 days. 2. The conditions for maximum shoot multiplication both with seedlings and mature tree explants were fairly similar. 3. The best explants for seedlings were cotyledonary nodes and nodal segments and from mature trees the axillary buds. 4. A modified Murashige and Skoog's (1962) medium supplemented with cytokinins, kinetin (0.3 mg/1) or benzyl amino pur?ne (0.5 mg/1) gave better results (4-5) healthy shoots as compared to zeatin, diphenylurea or N6 (2-Isopentyl adenine). Zeatin and 2ip produced less number of shoot multiples (2-3), whereas DPU produced rhizogen?sis. 5. Six to eight healthy uniform shoots were produced on a modified MS (19,62) medium to which a combination of kinetin (0.3 mg/1) and benzylamino purine 0.5 mg/1 were added. 6. Addition of adenine (10 mg/1) to MS medium containing kinetin (0.3 mg/1) and benzyl aminopurine (0.5 mg/1) increased shoot multiplication (9-12 shoots). Other purines and pyrimidines tested were less effective. 7. At adenine concentrations above 10 mg/1 few shoots were produced with an increase in callusing, indicating that on increased concentrations of adenine shoot growth is inhibited. 8. None of the auxins tested viz. IAA, IBA, IPA, NAA or 2,4-D, enhanced shoot multiplication. 2,4-D inhibited shoot growth and NAA produced rhizogenesis and callusing. 9. Sucrose at 2% was the best carbohydrate. 10. Among the physical factors tested, a temperature of 28-30?C with an illumination of 800-1000 lux light intensity for 18 h proved optimum. Rooting and transfer to field : The rooting response for shoots produced from seedling and mature trees differed only in respect of the auxin concentration. 1. Seedling explants rooted after treatment with NAA (10 mg/1) whereas a concentration of NAA (5 mg/1) was sufficient for shoots raised from mature tree explants. This is the reverse of the observations made for other tree species like tamarind ( 273 ), and teak ( 278 ) where a higher auxin concentration was essential for rooting of mature tree cultures, as compared to seedling. 2. It was essential to transfer the auxin treated shoots to Jg strength MS semi solid media containing 0.25% activated charcoal after a 48 h auxin treatment (auxin added aseptically in Jg strength MS liquid medium) for obtaining 80-90% rooting. 3. Healthy rooted plantlets were gradually hardened and transferred to field, where a high survival was obtained. Section - II : Embryogenesis This section includes experiments carried out for inducing embryogenesis. 1. Cotyledons from mature seeds were used as inoculum. 2. Direct regeneration of embryoid like structures were obtained from cotyledon tissues of mature Salvadora seeds. 3. Of the two auxins tested 2,4-D (0.5 - 3.0 mg/1) was the best auxin for induction of embryogenesis, while NAA produced slow growing callus and rhizogenesis. 4. Coconut milk inhibited embryogenesis. Sections taken of the regenerative tissue showed ?arly stages of the embryogenic pathway. Chapter IV : Studies on the response of Salvadora shoots to various concentrations of sea water and sodium chloride: Inoculum raised from mature trees was used for these studies. Two sets of experiments were conducted. In the first set, the multiplication medium MS basal medium + kinetin (0.3 mg/1) + benzyl-amino purine (0.5 mg/1) + adenine (10 mg/1) was prepared with 50% sea water or as 100% sea water containing MB ( MS vitamins) + kinetin (0.3 mg/1) + benzyl-amino purine ( 0 . 5 mg/1) and adenine (10 mg/1) prepared in sea water, at two pH values (6.3 ? 1 and 5.0). Since 6.3 ? 1 was found to be pH of sea water and pH 5.0 was optimal for shoot multiplication. In the second set, various concentrations of NaCl from 0.5% to 10% were tested by adding to the shoot multiplication medium at pH 5.0. In media containing 50% sea water and pH 5.0 the growth of the 'shoot was better as compared to pH 6.3. In 100% sea water shoot growth was inhibited. A multiplication of 7-8 shoots occured on media containing 0.5% NaCl after the thi rd transfer. On media containing 1, 2 and 3% NaCl the multiplication rate was lower (2-3 shoots). Shoots grown with a higher level (5% and above) of NaCl turned white and did not survive. The shoots in both 50% sea water and 0.5% NaCl were stunted and succulent. Successful rooting of shoots grown both in 50% sea water and 0.5% NaCl has been obtained. The shoots elongated when they were rooted. These rooted plants are now in field and are being water with 50% sea water or 0.5% NaCl respectively. 
Chapter V : Studies on oil analysis : This Chapter includes experiments conducted to study the variability in content of total fatty acids and lauric and myristic acids of seeds from 3 different trees and also within an individual tree. Seeds which contained comparatively high lauric and myristic acids were identified from 95 seed samples analysed. Seeds were suface sterilized, dissected aseptically and the embryos inoculated into 1/2 strength MS medium and cotyledons labelled correspondingly. The cotyledons were extracted and analysed for total fatty acids and also lauric and myristic acid. Standard procedures were used for extraction and estimation. From the results of the analysis it can be concluded that a variation in oil and fatty acids contents exists between different and within individual trees. 




STUDIES ON TISSUE CULTURES OF SALVADORA PERSICA L.

PATANKAR, SHUBHADA

CONDENSED CHROMATIN IN HIGHER PLANTS

SANE, MUKUND

                                                                                                                Summary 
182 
 
The drug discovery and development process has undergone dramatic 
changes particularly in the last decade. Progress in drug discovery has been 
fuelled by improvements in methodologies and technologies including 
automated high performance liquid chromatography (HPLC), fast HPLC, 
automated method development, LC-MS and high-throughput purification 
methods. During the drug discovery and development process, rugged 
analytical methods are highly desirable to evaluate early drug targets, drug 
metabolism, pharmacokinetics, process research, preformulation and 
formulations. In view of the continuous demand for stability indicating 
methods for maintaining the quality and safety of drugs and bioanalytical 
methods in clinical and pre-clinical studies during drug discovery, the present 
work has been proposed.  
The present thesis entitled “DEVELOPMENT OF LIQUID 
CHROMATOGRAPHY-MASS SPECTROMETRIC METHODS FOR 
ANALYTICAL AND BIOANALYTICAL APPLICATIONS” has been divided into 
six chapters.  
Chapter 1 deals with a brief introduction on role of liquid chromatography and 
mass spectrometry in drug discovery & development. 
Chapter 2 deals with LC-ESI-MS/MS studies on forced degradation of abacavir 
sulphate an anti HIV drug. 
Chapter 3 deals with LC-ESI-MS/MS studies on forced degradation of 
indinavir suphate an anti HIV drug. 
Chapter 4 deals with development of an on-line two dimensional liquid 
chromatography combined with tandem mass spectrometric method for the 
determination of an antibiotic drug rifaximin in rat serum. 
Chapter 5 describes development and validation of a LC-ESI-MS method for 
determination of an antibiotic drug rifaximin on dried blood spots using 
monolithic column.  
                                                                                                                Summary 
183 
 
Chapter 6 describes the development of an ionic liquid based dispersive liquid-
liquid micro extraction procedure for the extraction and determination of 
rifaximin in rat serum by HPLC.  
Chapter 1: Role of liquid chromatography and mass spectrometry in drug 
discovery & development. 
This chapter gives a brief introduction to various roles of liquid 
chromatography in pharmaceutical analysis and bioanalysis. In modern 
pharmaceutical industry, high-performance liquid chromatography (HPLC) is 
the major and integral analytical tool applied at all stages of drug discovery, 
development, and production. At each phase analyses of various samples are 
performed to adequately control and monitor the quality of the prospective 
drug candidates, excipients, and final products, which require bioanalytical as 
well as stability indicating methods. 
Effective, eco-friendly and rapid method development is of paramount 
importance throughout the drug development life cycle. This requires a 
thorough understanding of HPLC principles and theory which lay a solid 
foundation for appreciating the many variables that are optimized during fast 
and effective HPLC method development and optimization. HPLC basics, types 
of chromatography, detectors, method development and validation are 
discussed briefly in this chapter. Hyphenated techniques such as LC-MS and 
LC-MS/MS were also discussed.  
In lieu of continuous demand for stability indicating and bioanalytical 
methods the work has been divided into two parts. Part A deals with 
development of stability indicating methods and part B with development of 
bioanalytical methods. The model drugs rifaximin, abacavir sulphate and 
indinavir sulphate were chosen and following objectives had been set:  
 
                                                                                                                Summary 
184 
 
Part A: Development of stability indicating methods 
i. LC-MS/MS studies on forced degradation of abacavir sulphate. 
ii. LC-MS/MS studies on forced degradation of indinavir sulphate. 
Part B: Development of Bioanalytical methods 
iii. On-line 2D-LC-ESI-MS/MS determination of rifaximin in rat serum 
iv. LC-ESI-MS determination of rifaximin on dried blood spots 
v. Ionic liquid based dispersive liquid-liquid microextraction and 
determination of rifaximin in rat serum 
Part A: Development of stability indicating methods 
Chapter 2: LC–MS/MS studies on forced degradation of abacavir sulphate. 
Abacavir sulphate (ABC) was subjected to forced degradation under the 
conditions of hydrolysis (acid, alkali and neutral), oxidation, photolysis and 
thermal stress. It resulted to eight degradation products. In case of acid 
hydrolysis drug was degraded completely. Hence its kinetic behaviour at 
temperatures 50 and 65 oC was also studied. Rate constant, half-life and t90 of 
the drug were also calculated. The rate constant was increased from 0.0474 to 
0.0064 h-1 as temperature increase from 50 to 65 oC. Abacavir sulphate and its 
degradation products separation was accomplished on Waters XTerra C18 (250 
mm ×4.6 mm, 5µm) column using 20 mM ammonium acetate: acetonitrile as a 
mobile phase in gradient elution mode. Based on the LC-MS/MS data the 
structure of degradation products and their fragmentation pathways were 
proposed. The proposed structures (Fig. 1) and fragmentation pathway was 
confirmed by HRMS data which provide accurate mass measurements. 
 
                                                                                                                Summary 
185 
 
 
Fig. 1 The chemical structures of proposed degradation products of ABC. 
Chapter 3: LC–MS/MS studies on forced degradation of indinavir sulphate. 
Indinavir sulphate (IDV) was subjected to acid hydrolysis, base 
hydrolysis, neutral hydrolysis, oxidation in presence of hydrogen peroxide and 
AIBN, thermal degradation and photo degradation. Total six degradation 
products were formed. Among the six degradation products three were from 
acid hydrolysis, two from oxidation and one of the degradation products 
obtained from neutral hydrolysis also. Indinavir sulphate and its degradation 
products were well separated on Waters XTerra C18 column. Based on the LC-
MS/MS data the structure of degradation products and their fragmentation 
pathways were proposed. The proposed structures (Fig. 2) and fragmentation 
pathways were confirmed by HRMS data. The oxidation degradation products 
obtained in this study are the important metabolites of indinavir sulphate 
formed in human body. 
                                                                                                                Summary 
186 
 
N
N
OHN
N
OO
N
N
O
HN
N
N
H
OOH
OH
NH2
OHN
N
O
HN
N
HO
O
OH
DP1IND DP3DP2
N
N
O
HN
N
N
H
O
OH
OH
O
O
N
N
O
HN
N
N
H
O
OH
OH
O
DP4 DP5
 
Fig. 2 The chemical structures of proposed degradation products of IDV. 
Part B: Development of Bioanalytical methods  
Chapter 4: On-line 2D-LC-ESI-MS/MS determination of rifaximin in rat 
serum. 
This chapter describes the  development of a direct injection method for 
the determination of rifaximin in rat serum. To facilitate this method two 
dimensional liquid chromatography was employed. The 2D-LC-ESI/MS/MS 
system consisted of a restricted access media column for trapping proteins as 
the first dimension and a Waters C18 column as second dimension using 0.1% 
aqueous acetic acid: acetonitrile as mobile phase in a gradient elution mode. 
2D-LC could be performed in either on-line or off-line modes. The on-line 
approach narrows the choice of LC mode because of the mobile phase 
compatibility for direct transfer to the second dimension. This approach 
minimizes sample losses, which is an advantage for sensitivity compared with 
the off-line mode. 
 
                                                                                                                Summary 
187 
 
Chapter 5: LC-ESI-MS determination of rifaximin on dried blood spots. 
This chapter describes the suitability of dried blood spot (DBS) technique 
was evaluated for sample collection. A patient can collect blood samples from 
finger prick on a blood spot card without much special training to a clinic for 
analysis. A high-throughput liquid chromatography–electrospray ionization 
mass spectrometric (LC–ESI-MS) method for screening of rifaximin on DBS was 
developed and validated. It involves solvent extraction of a punch of DBS (Fig. 
3) followed by reversed-phase LC on a relatively new monolithic column 
consisting of a silica rod with bimodal pore structure and detection by ESI-MS. 
 
Fig. 3 Optimized method for processing and extraction of rifaximin from DBS.  
 
Chapter 6: Ionic liquid based dispersive liquid-liquid microextraction and 
determination of rifaximin in rat serum. 
An efficient and environmental friendly ionic liquid based dispersive 
liquid-liquid micro extraction followed by RP-HPLC method for determination of 
rifaximin in rat serum was developed (Fig. 4). A water immiscible 1-butyl-3-
methylimidazolium hexafluorophosphate (BmimPF6), was found to be an 
effective extraction solvent among the five ionic liquids tried in the present 
                                                                                                                Summary 
188 
 
investigation. The effects of ionic liquid, dispersive solvent, 
extractant/disperser ratio and salt concentration on sample recovery and 
enrichment were studied. Using BmimPF6 and methanol as extraction and 
dispersive solvents, the recovery was found to be more than 98% at an 
extractant/disperser ratio of 0.43. The recovery was further enhanced to 99.5% 
by the addition of 5.0% NaCl solution. Rifaximin extracted in to BmimPF6 was 
spiked with rifampicin used as an internal standard and directly injected on to 
a Waters XTerra® C18 (5 μm, 250 x 4.6 mm) column, using 0.1% aqueous acetic 
acid: acetonitrile (45:55, v/v) as a mobile phase in an isocratic mode of elution 
and detected at 239 nm using photo diode array detector by reverse phase high 
performance liquid chromatography. A 3-fold enhancement in detection limit 
was achieved compared to the previously reported extraction method using 
protein precipitation.  
 
 
 
 
 
 
 
 
 
Fig. 4 Schematic representation of optimized IL based DLLME of rifaximin from 
rat serum. 


STUDIES ON CATALYTIC HYDROGENATION OF o-NITROPHENOL TO o-AMINOPHENOL IN THREE PHASE SLURRY REACTOR

RANADE, SHIRISH ANAND

Abstract

Expression of Insecticidal Toxin Coded by Modified Full length and Truncated Bt-cry1Ac Gene in Transgenic Tomato for Assessment of their Stability and Efficacy Against Target Insects

In spite of several modifications reported to improve expression of the cry1Ac gene in plants achieving high expression of Cry1Ac toxin does not seems to be a routine affair and reflect complexcities for differential expression of insecticidal toxin in plants from full-length and truncated version of the gene (Diehn et al., 1996; Rocher et al., 1998; Burma et al., 2011). In a recent report it has been demonstrated in tobacco and cotton that the expression of Cry1Ac endotoxin has detrimental effect on both in vitro and in vivo growth and development of transgenic plants (Burma et al., 2011). These observations have inspired us to give another dimension to our research objective. Tomato expressing Cry1Ab insecticidal protein from Bt, giving complete protection against H. armigera damage in laboratory, green house and field has been reported earlier (Kumar and Kumar 2004; Srivastava S Ph.D thesis ). The truncated cry1Ab gene bears 80% homology with truncated cry1Ac gene and the results which would be generated regarding truncated and full length cry1Ac transgenic tomato will be compared with that of cry1Ab transgenic tomato. The outcome of this comparative study will reveal the reliability and toxicity of both the truncated variants of the cry1A genes, in tomato. In view of these facts the present problem is identified with following objectives.  
	Development of transgenic population of tomato with modified full-length and truncated version of cry1Ac gene and cry1Ab.
	To assess the stability and efficacy of insecticidal toxin coded by full-length cry1Ac gene in plants compared to 3’ truncated version.
	 Recovery of promising transgenic event in tomato with full-length cry1Ac gene and cry1Ab.
	Molecular characterization of insecticidal toxins coded by two forms of the gene in transgenic tomato plants.
	The stability, efficacy and inheritance of cry1Ac gene shall be compared to that of cry1Ab gene in transgenic tomato. 




MOLECULAR STUDIES IN PLANT GENOMES WITH SPECIAL REFERENCE TO COWPEA AND PIGEONPEA

SINHA, SANJAI

ABSTRACT 
 
Rice (Oryza sativa), a member of Gramineae family, is a major crop plant that feeds almost 
half of the global human population. In recent years, this plant has emerged as a model 
system for functional genomics studies among the monocot plants. The availability of its 
complete genome sequence, easy transformation protocols and various mutant lines have 
been instrumental in unravelling the genetic and epigenetic molecular mechanisms that 
regulate gene expressions and various developmental pathways including reproductive 
development in rice. However, despite these advances, one of the important epigenetic 
regulators, the process of DNA methylation, has largely remained unexplored partly 
because of increased complexity in higher plant organization. With the view to get a clearer 
picture of the role of this regulatory process and to be able to understand the roles of its 
molecular components in modulating gene expression patterns, we selected an early land 
plant Physcomitrella patens as the second model organism for the present study. P. patens 
is a simple moss plant with fully sequenced genome. This study in the two model 
organisms has provided significant insight into the conservation and diversity among the 
methylation machinery components and their evolutionary relationships in land plants.  
The objective of this Ph.D. work was to first establish conclusively the role of DNA 
methylation in regulating growth, development and differentiation in both, Physcomitrella 
and O. sativa and then to identify and characterize the core component of the DNA 
methylation machinery, cytosine DNA methyltransferases, in these plants. As a first step in 
this direction, the effect of global hypomethylation induced by cytidine analog, zebularine, 
on growth and development of O. sativa and Physcomitrella was examined. Thereafter, a 
genome-wide in silico study was undertaken to identify the genes encoding cytosine 
methyltransferases in these plants. A comprehensive structural and phylogenetic analysis of 
rice and moss methyltransferase gene families was then carried out. This was followed by 
expression analysis of genes during different developmental stages and in response to 
abiotic stress conditions. Thereafter, putative functions of the genes were speculated by 
studying in vivo subcellular localization patterns of methyltransferase-GFP fusion proteins 
in onion epidermal cells and moss protonema cells. Finally, functional characterization of 
selected rice methyltransferase genes (OsCMT2, OsMET1-2, OsDRM2, OsDRM3 and 
OsDNMT2) was undertaken using gene silencing and overexpression strategies.  
The concise review of literature presented at the beginning of the thesis is aimed to discuss 
and highlight the evolutionary relatedness among the methyltransferase genes in eukaryotes 
and the pivotal roles played by cytosine DNA methylation in plant development. The 
experimental procedures used in this study and the results thus obtained have been 
summarized in separate sections. The new and recent findings and their relevance to the 
current research scenario has been discussed and significant conclusions have been drawn. 
References cited throughout this study and the research publications earned through this 
work have been compiled at the end. 
This research not only enhances our understanding of the evolution and functional 
conservation/diversification of DNA methylation and components of the methylation 
machinery, but it also provides evidence for the role of methyltransferases in modulating 
various developmental pathways regulating growth and development in both, O. sativa and 
Physcomitrella patens.  
  
Abstract 


ELECTRON SPECTROSCOPIC STUDIES OF SOME HYDROGEN IMPLANTED METALS

KUNTE, SUNITA S.

Abstract



CHAPTER I
SYNTHESIS AND ANTITUMOR EVALUATION OF NITROVINYL BIPHENYLS: ANTICANCER AGENTS BASED ON ALLOCOLCHICINES
               In the present study, considering the structures of colchicine and allocolchicines, a new class of compounds, nitrovinyl biphenyls were designed, synthesized and demonstrated as inhibitors of tubulin polymerisation and cause mitotic arrest in cells. The majority of compounds were found to possess potent anticancer properties with IC50 values in the range of 0.06-7 M and they are equally potent as colchicine against HeLa and MCF7 cells. Compounds 26e and 26f inhibited tubulin assembly greater than 60% and flow cytometry studies indicated growth arrest of cells at the G2/M phase of the cell cycle in a concentration dependent manner.  Treatment of cells with 26f up-regulated Cyclin B1 and Aurora Kinase B mRNA levels suggesting that the mode of action is by virtue of growth arrest at G2/M phase of the cell cycle.




Scheme 1. Synthesis of Nitrovinyl Biphenyls using Palladium catalyzed Suzuki cross coupling.
A majority of these compounds, except, 26c, g and k demonstrated significant anti-proliferative activity against a majority of cell lines tested.  The compounds 26e and 26f possessed anti-tubulin activity both in vitro as well as within cells as demonstrated by the soluble: polymerized tubulin ratio. Cell cycle arrest at G2/M phase and up-regulation of Cyclin B1 and Aurora Kinase B substantiates the anti-mitotic effects of 26e and 26f.  Overall, the potent anticancer compounds were found to have the ‘oxygen’ substitution at the 4th position of the ring C or the presence of bulky tert-butyl group. 



Table 1. Invitro Cytotoxic Effects of Compounds 26a-k and Colchicine on HELA, MCF7, HEPG2, A549 and SKNSH Human Cancer cells.


























Cell lines were treated with different concentration of compounds for 48 h as mentioned in “materials and methods section. Cell viability was measured employing SRB assay. IC50 values are indicated as mean ± SD (standard deviation) of three independent experiments.
CHAPTER II
Cu-CATALYZED AZIDE-ALKYNE 1,3-DIPOLAR CYCLOADDITION: SYNTHESIS OF 1,4-DISUBSTITUTED 1,2,3-TRIAZOLES

This chapter is divided into three sections:
Section I deals with the Regioselective synthesis of 1,4-disubstitited 1,2,3-triazoles via three-component coupling of secondary alcohols, TMSN3 and alkynes in the presence of catalytic amount of a Cu(OTf)2 and Cu powder. 
Section II employs an efficient approach to the one-pot synthesis of -ketotriazoles by the successive treatment of secondary alcohols with [hydroxy(tosyloxy)iodo]benzene (HTIB), NaN3 followed by CuI catalyzed 1,3- dipolar cycloaddition with terminal alkynes.
Section III deals with the hydroazidation of homoallyl alcohols to the corresponding allylic azides with migration of the double bond using PdCl2 as a catalyst and TMSN3 as an azide source. The formed allylic azides were converted to 1,4-disubstituted 1,2,3-triazoles using copper iodide. 



SECTION I
REGIOSELECTIVE SYNTHESIS OF 1,4-DISUBSTITITED 1,2,3-TRIAZOLES VIA THREE-COMPONENT COUPLING OF SECONDARY ALCOHOLS , TMSN3 AND ALKYNES
In this section, the reactivity of alcohols in nucleophilic substitution reaction was studied. Whereas, it has been shown that nucleophilic substitution of alcohols with alkali azides generally requires activation of the hydroxyl group of the alcohols by formation of corresponding tosylates. herein, a one-pot two-step reaction between secondary alcohols, TMSN3 and terminal alkynes in the presence of catalytic amount of a Cu(OTf)2 and Cu powder to give 1,4-disubstituted 1,2,3-triazoles was presented (Scheme 2). The products contain various substituents on triazole ring and alkyl side chains on N-1 of the triazole. 
This three-component coupling reaction proceeds via nucleophilic substitution of the secondary alcohols with TMSN3 to afford the corresponding azides in the presence of Cu(OTf)2 without activation of the hydroxyl group. The in situ generated azide on Cu(I) catalyzed reaction with terminal alkynes gave 1,4-disubstituted 1,2,3-triazoles in excellent yields. The reaction conditions were optimized by varying the catalyst or the solvent.



Scheme 2.


SECTION II

TANDEM OXIDATIVE Α-TOSYLOXYLATION OF ALCOHOLS/NUCLEOPHILIC ADDITION OF AZIDE/COPPER-CATALYZED 1,3-DIPOLAR CYCLOADDITION
an efficient method for the synthesis of 1,4-disubstituted 1,2,3-triazoles from a variety of secondary alcohols, sodium azide and terminal alkynes has been developed via a simple one-pot, three-step procedure involving the oxidative α-tosyloxylation of alcohols, followed by azidation of the formed α-tosyloxyketones and copper catalyzed 1,3-dipolar cycloaddition (Scheme 3).

Scheme 3.


SECTION III
EFFICIENT SYNTHESIS OF ALLYLIC AZIDES AND ONE-POT REGIOSELECTIVE SYNTHESIS OF 1, 4-DISUBSTITUTED 1,2,3-TRIAZOLES FROM HOMOALLYL ALCOHOLS
In this section the hydroazidation of homoallyl alcohols  to the corresponding  allylic azides with migration of the double bond using PdCl2 as a catalyst and TMSN3 as an azide source has been developed. the formed allylic azides were subsequently converted to 1,4-disubstituted 1,2,3-triazoles through CuI catalyzed 1,3-dipolar cycloaddition with terminal alkynes without isolation and purification of azides (Scheme 4).

Scheme 4.
This conceptually new approach provides a straightforward and efficient access to allylic azides. In addition, the reaction can be coupled to copper catalyzed 1,3-dipolar cycloaddition to generate 1,4-disubstituted 1,2,3-triazoles in one-pot without isolating the azide intermediate and should prove to be useful for generating multivalent structures.

CHAPTER III
CATALYST AND BASE FREE WATER PROMOTED SNAR REACTION OF HETEROARYL HALIDES WITH THIOLS
Heterocycles make up an exceedingly important class of compounds. In fact more than half of all known organic compounds are heterocycles. Many natural drugs such as quinine, papaverine, emetine, theophylline, atropine, procaine, codeine, morphine and reserpine are heterocycles. Almost all the compounds we know as synthetic drugs such as diazepam, chlorpromazine, isoniazid, metronidazole, azidothymidine, barbiturates, antipyrine, captopril and methotrexate are also heterocycles. Some dyes (e.g. mauveine), luminophores, (e.g. acridine orange), pesticides (e.g. diazinon) and herbicides (e.g. paraquat) are also heterocyclic in nature.
A simple and efficient route for the synthesis of biaryl sulfides have been developed in aqueous medium under base and catalyst-free conditions (Scheme 5). A wide variety of heteroaryl halides and thiols underwent SNAr reaction to provide diaryl sulfides in good to excellent yields. The remarkable key features of the reaction include the use of water as an inexpensive and environmentally benign reaction medium, absence of any additional reagent/catalyst and easy isolation of the products.

Scheme 5

CHAPTER IV
AgOTf CATALYZED DIRECT AMINATION OF BENZYL ALCOHOLS WITH SULFONAMIDES
Catalytic amination reactions have received significant interest. Substituted amines are of considerable importance in the pharmaceutical, agrochemical and fine chemical industries. Numerous biologically active molecules such as alkaloids, amino acids and nucleotides contain the amine group.
Nucleophilic substitution of the hydroxy group in alcohols by amines generally requires preactivation of the alcohols by transformation in to good leaving groups such as halides, carboxylates and carbonates (Scheme 6).

Scheme 6. Substitution of benzyl alcohol by (a) preactivation   (b) catalytic hydrogen transfer, and (c) direct catalytic substitution.
We have developed an efficient procedure to obtain benzylic amines from the corresponding primary alcohols and sulfonamides in the presence of silver triflate as catalyst. The methodology is straightforward and environmentally benign with the formation of water as the only by-product (Scheme 7).

Scheme 7.



CHAPTER V
TITANIA SUPPORTED PALLADIUM (0) NANOPARTICLES FOR SUZUKI-MIYAURA CROSS-COUPLING OF ARYL AND HETEROARYL HALIDES 
The foundation of synthetic organic chemistry rests on the ability to form and manipulate carbon-carbon bonds. The increasing demand of coupled products in chemical and pharmaceutical industries has prompted the development of several transition metal catalysts, which aim to exert the highest turnover number and turnover frequency.
The biaryl moiety is an important structural motif in a great number of biologically active compounds and functional molecules. Which includes the natural products Biphenomycin, the pharmaceuticals Valsartan, Telmisartan, Felbinac, Losartan, Imatinib, the agrochemical Boscalid, liquid crystals for LCD screens, chiral ligands for catalysis and organic functional materials.
In this chapter, nanocrystalline TiO2-Supported Pd(0) Catalyst was prepared as outlined in the scheme 8 and used for the Suzuki cross coupling reaction of various aryl and heteroaryl halides with aryl/heteroaryl boronic acids at room temperature under air (Scheme 9). The conversion and selectivity results obtained for many substrates were excellent and similar to those provided by more active or even homogeneous catalysts. 

Scheme 8. Preparation of TiO2 supported palladium catalyst.
This methodology is similarly effective using 2-bromo, 3,4,5 trimethoxy benzaldehyde as the coupling partner and gave products in good yield. Further it has been shown that it is useful for the synthesis of terphenyl and tetraphenyls. The catalyst is quantitatively recovered from the reaction by simple filtration and reused for a number of cycles without significant loss of activity. The operational simplicity and the mild reaction conditions add to the value of this method as a practical alternative to the Suzuki coupling. 

Scheme 9. Suzuki coupling of aryl and heteroaryl halides with aryl boronic acids. 


















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