Open Access Repository of Indian Theses

PALNITKAR, SANJAY S.

Abstract

This thesis reports the theoretical and experimental 
studies carried out to study the dissociation dynamics of 
multiply charged diatomic molecules (N2q+ 
and COq+) to gain insight into the electronic states of 
these molecular ions. On the theoretical front, ab initio 
calculations of potential energy curves for multiply charged 
N2q+ and COq+ (3≤q≤6) were performed to 
discuss the dissociation possibilities of these ions. Experimentally 
multiply charged N2 and CO molecular ions were generated in interaction 
of neutral parents with highly charged low energy Argon 
projectiles. The technique of time-of-flight 
mass spectrometry was used to study the interaction. The experimental data 
was obtained using time and position sensitive detectors in multi-hit 
coincidence mode. Kinetic energy release distributions were obtained for all 
the observed dissociation channels and were correlated to the existing and 
calculated potential energy curves of N2q+ and COq+ ions. 
In addition, this thesis reports the design and implementation of an 
electrostatic deceleration lens which would 
be used to decelerate and focus a beam of HCI having energy of few tens of 
keV/q so as to obtain a low energy (few tens of eV/q) 
well focused beam on the target. This setup is developed for carrying out 
ion-matter interaction studies with highly charged ions at much lower 
energies (~10 eV/q) and will be used to shed light on the role of the 
projectile's potential energy in ion-molecule interactions. 
With the new setup the ion-atom, ion-molecule interaction times will be of 
the order of few tens of femtoseconds. 
With these interaction times the initial as well the final states of the 
target atom or molecule are expected to be modified by the presence of the 
projectile. 
Quasi two body effects may appear in ion-atom interactions with 
the projectile and the atom being the two bodies, while ion-molecule 
collisions will offer a simple many body problem. 
A time-of-flight system using position sensitive detectors in multi-hit 
coincidence mode, coupled with the new setup will 
help us identify how the projectile modifies the states of the target. 

The multiple ionization and fragmentation of N2 induced by ion impact is studied using time and position sensitive detectors in multihit coincidence mode. With Ar9+ at a projectile velocity of 1 a.u. we observe a total of seven fragmentation channels originating from multiply charged transient molecular ions. The preference of symmetric charge breakup channels over the asymmetric ones is clearly observed. A signature of core excitation of the target followed by Auger emission in the N3+ - N3+ fragmentation channel. The kinetic energy release spectra of all the observed fragmentation pathways is explained on the basis of calculated ab initio (using multiconfiguration self-consistent-field method) potential energy curves. 

Ion-induced molecular fragmentation of CO has been studied using time-of-flight mass spectroscopy with position sensitive detectors in multihit coincidence mode. Ar8+ ions having a velocity of 1.1 a.u. were used as projectiles. The features observed in the kinetic energy release (KER) spectra for all the detected fragmentation channels are discussed in light of the existing and calculated ab initio potential energy curves. The preference of the symmetric breakup over the asymmetric one is clearly observed. For fragmentation channels originating from the same parent molecular ion, it is observed that the most probable KER value is higher for the dissociation channel having a higher charge on the oxygen ion. Occurrence for sharp peaks in KER spectra of some of the fragmentation channels hints towards the existence of shallow (possibly metastable) excited states of COq+ (q=4,5) molecular ions and calls for further theoretical investigations. 




English

MICROBIOLOGY AND BIOCHEMISTRY OF D-XYLOSE FERMENTATION

PRABHUNE, A

DESIGN, CHARACTERIZATION AND COMPARATIVE EVALUATION OF FOLATE MEDIATED TARGETING OF THERAPEUTICS







SYNOPSIS



CHANDRASEKAR D
M. Pharm.,


2006




Department of Pharmaceutics
Faculty of Pharmacy
Jamia Hamdard
New Delhi – 110 062

The folate receptor (FR) is a 38 kDa glycosylphosphatidylinositol-anchored protein that binds to the vitamin folic acid with high affinity (KD < 1 nM). Following binding, rapid endocytosis delivers a fraction of the receptors into a low pH compartment where dissociation of the vitamin from its receptor is promoted. Interestingly, covalent conjugation of small molecules, proteins, and even liposomes to the γ-carboxyl moiety of folic acid does not alter its ability to bind the folate receptor and undergo endocytosis by receptor bearing cells. The FR-α isoform is present primarily in placenta, kidney, malignant tumors and various cancer cell lines. It is also expressed on the apical surfaces of some normal epithelia (e.g. bronchioalveolar cells of the lungs and choroid plexus of the brain), but the receptor at these epithelial sites is generally inaccessible to parenterally administered drugs. It has recently been discovered that activated (but not resting) synovial macrophages, taken from patients diagnosed with rheumatoid arthritis, possess a functionally active FR-β. The accumulated data also strongly demonstrate that activated macrophages can be selectively targeted with folate conjugates in cancer tissue and arthritic joints, a capability that opens new possibilities for the diagnosis and treatment of rheumatoid arthritis. The prevalence of FR overexpression among human tumors and activated macrophages makes it a good marker for targeted drug delivery to cancer and arthritis. Although FR-targeting has been found to enhance intracellular delivery of attached proteins, liposomes, viruses, antisense oligonucleotides, gene therapy vectors, polymeric drug carriers, imaging compounds, and neutron activation agents it has not yet been reported to improve net uptake of low molecular weight chemotherapeutic compounds, except in the case of certain antifolates. 
	The objective of this study was to utilize the folate-receptor overexpression in cancer cells and inflammatory macrophages for enhanced and site-specific delivery of anti-cancer and anti-rheumatic drugs to their respective sites. For this purpose folate was conjugated to different polymers such as PAMAM dendrimers, PLGA and loaded with drugs and evaluated for their targeting ability. Also folate was coupled to the drugs through a linker (pro-drug/polymer-drug conjugate concept) and determined for their enhanced site-specific targeting ability.
	The first part of the study deals with the folate mediated targeting of anti-arthritic drug indomethacin to inflammation. Indomethacin is a non-steroidal anti-inflammatory drug used for the management of rheumatoid arthritis, osteoarthritis and acute gout but has a limited efficacy coupled with gastric intestinal hemorrhage and other side effects such as renal dysfunction with conventional therapies. Site-specific delivery of indomethacin to the target site remains the best choice to overcome the side effects and to increase its efficacy.
	Poly(amidoamine) (PAMAM) dendrimers offer a defined branched chain structure capable of carrying multiple molecular entities that are either linked covalently to its surface or are encapsulated in its interior space. The controlled multivalency of dendrimers can be used to attach several drug molecules, targeting groups and other agents (solublizing/sensing) to the periphery of the dendrimers in a well defined manner. Dendrimers conjugates have been used for delivering drugs, DNA, radionucleides, MRI contrast agents and boron for neutron capture therapy.
	Full generation PAMAM dendrimers with amino terminal groups are suitable for direct coupling of folic acid and G4 with 64 surface amino groups was selected for the present study. Folic acid was coupled to the surface amino groups of G4-PAMAM dendrimer (G4D) via a carbodiimide reaction and loaded with indomethacin. Folate-PAMAM dendrimer conjugates with different degree of folate substitutions were synthesized and characterized by 1H-NMR and IR spectroscopy. Approximately 4, 13 and 21 folate molecules were conjugated to G4D when the molar ratio of folate was 8, 16 and 32 moles, respectively. The drug content and percent encapsulation efficiency increased with increasing folate content for the dendrimer conjugates. The in vitro release profile indicated a more controlled release of the drug with increasing folate content. Pharmacokinetic and tissue distribution studies in arthritic rats had shown preferential higher accumulation of indomethacin at the inflamed paw by the folate conjugates when compared with dendrimer at equivalent dose. The plasma concentration profile showed a biphasic curve indicating rapid distribution followed by slow elimination. The AUC0–∞, half-life and residence time of indomethacin in inflamed paw was higher for folate-dendrimer conjugates. The time-averaged relative drug exposure (re) of the drug in paw and overall drug targeting efficiency (Te) were higher for folate conjugate with 21 folate moieties (4.1 and 2.78, respectively) when compared with DNI (1.91 and 1.88, respectively). Among the conjugates, the formulation with 21 folate molecules (FN3) was found to possess good encapsulation efficiency, controlled release nature and highest targeting efficiency to the inflammatory region. This study demonstrated the superiority of active targeting over dendrimer mediated passive targeting and also for the first time, folate-mediated targeting of an anti-arthritic drug to the inflammatory tissues.
	The next formulation was prepared with anionic dendrimer (carboxylate terminated G3.5 PAMAM) as targeted drug delivery systems. This dendrimer has 64 surface carboxylate groups, which were conjugated to folate through a PEG spacer. Folate-PEG-PAMAM conjugates were synthesized by a two step reaction through a carbodiimide mediated coupling reaction. Conjugates with three different folate-PEG substitutions were prepared and loaded with indomethacin by continuous shaking method. Characterization of the conjugates revealed approximately 7, 11 and 20 folate-PEG moieties were attached to each molecule of G3.5 PAMAM dendrimer. Drug loading was significantly increased in the folate-PEG conjugates when compared with the non-conjugated dendrimer. The number of folate-PEG molecules influenced the percent drug loading into conjugates with maximum loading observed for the conjugate with 20 folate-PEG molecules attached. The in vitro release profile revealed the controlled release nature of the folate-PEG conjugates. The plasma indomethacin levels in arthritic rats showed a biphasic curve with rapid distribution phase followed by a slower elimination phase. The plasma pharmacokinetic parameters illustrated an increased AUC, half-life and mean residence time for the folate-PEG conjugates. The tissue distribution studies had shown high exposure (AUC) of the drug to stomach, heart and RES organs in the control group. The folate-PEG conjugates had shown reduced uptake by RES organs and in particular the exposure to stomach was nearly one-tenth of the non-conjugated dendrimer indicating the limited gastric-related side effects of indomethacin. Also the folate-PEG conjugates delivered significantly higher amount of drug to the inflamed paw than the control and dendrimer groups. Among the different folate-PEG conjugates, the conjugate with 7 folate-PEG molecules exhibited better overall drug targeting efficiency (3.44) and higher relative exposure of drug to the inflammatory region (2.37). This folate-PEG-PAMAM conjugate remains the ideal candidate for site-specific drug delivery with high efficiency to target the inflammatory tissues accompanied by reduced side effects.
	The polymeric prodrug conjugates increase aqueous solubility, enhance biodistribution and retain the inherent pharmacological properties of the drug intact. Utilizing this strategy, indomethacin nanoconjugates were synthesized by conjugating folic acid and indomethacin to α- and ω-terminal end group of poly(ethylene glycol)-bis-amine (FPI). FPI nanoconjugate micelles loaded with indomethacin were produced by both dialysis and emulsification method. The emulsification method yielded smaller particle size (179 nm) with maximum encapsulation efficiency at 25% drug loading. The in vitro release profile showed that the release of indomethacin from this micellar nanoconjugate could be controlled over 5 days. The plasma pharmacokinetics of the micellar nanoconjugate in arthritic rats had shown an increased circulation half-life of indomethacin. The tissue distribution studies revealed higher accumulation of the drug in inflammatory paw. The overall drug targeting efficiency (Te) value of micellar nanoconjugates for inflamed paw was 3.9 folds higher than the control and the time-averaged relative drug exposure (re) value was higher (23.86) indicating the potential of this micellar drug nanoconjugate for active targeting to inflammation in rheumatoid arthritis.
	In brief summary, all these folate targeted formulations exhibited enhanced site specific drug delivery to the inflammatory region of arthritis and serve as better carriers for indomethacin by reducing its side-effects and improving drug delivery at the target site.
	The second part of the study deals with the folate-mediated targeting of anti-cancer drug paclitaxel to tumor cells and their in vivo pharmacokinetics and biodistribution pattern in healthy rats. Paclitaxel is used for its antineoplastic action against malignant neoplasms of the breast and ovary, and in advanced non-small cell lung cancer, but has also been tried in other malignancies including tumors of the head and neck, prostate, and Kaposi’s sarcoma. Paclitaxel is also active against malignant gliomas and brain metastases. However, low to undetectable levels of the drug were found in the normal brains as well as in tumor brain tissue. Paclitaxel has been shown to be a substrate for the multidrug resistance 1 (MDR 1) expressed protein, p-glycoprotein (p-gp). Thus targeted delivery of paclitaxel to cancer cells and to brain remains the best choice to overcome the above mentioned barriers.
	Paclitaxel was conjugated to the carboxyl end groups of G3.5 PAMAM dendrimer and coupled with folate-PEG chains. Utilizing the pro-drug concept, the drug was also directly conjugated to folate-PEG-amine through a carbodiimide mediated coupling reaction after converting the hydroxyl group into a carbonate derivative. About 43 to 44 molecules of paclitaxel were found conjugated and the number of folate-PEG chains attached was 10. All these formulations exhibited controlled release over 12 days with less than 25% of drug released. When compared with paclitaxel-dendrimer conjugate, the release rate of folate conjugate was decreased in both PBS and rat plasma, which can be attributed to the more shielding effect provided by the surface folate-PEG chains. None of these conjugates were found to be hemolytic upto 5 mg/ml. The in vitro cytotoxicity of these conjugates confirmed the selective cytotoxicity towards the folate receptor positive (HeLa) cells. Also the in vitro uptake studies confirmed folate receptor mediated uptake of folate coupled conjugates. The in vivo plasma pharmacokinetics indicated increased bioavailability of all the conjugates with increased mean residence time and circulation half-life. Biodistribution studies revealed the brain targeting efficiency of the folate conjugates. Thus the folate conjugates of paclitaxel indicate a promising approach for overcoming the blood brain barrier and enhanced paclitaxel delivery to the brain.
	Finally, folate targeted copolymers comprising of PEG and PLGA (folate-PEG-PLGA, FPLG) was synthesized by a two step reaction utilizing the carbodiimide mediated coupling reaction. NMR and FTIR spectroscopy studies confirmed the conjugation of folate-PEG to PLGA. The DSC thermograms revealed the shift in Tg of FPLG confirming the formation of new copolymer. Nanoparticles with 0%, 50% and 100% FPLG (NP1, NP2 and NP3, respectively) were prepared by single emulsion method. Folate-PEG coupling increased the mean size of the nanoparticles from 258.7 nm to 290-295 nm. The shift in the zeta potential to positive charge and the XPS confirmation of the presence of nitrogen groups in N1s region prove the surface domination of nanoparticles by folate groups in nanoparticles prepared with FPLG. The encapsulation efficiency increased with increasing folate-PEG composition in the polymer matrix. The in vitro release profile indicated increased release rate for NP3 followed by NP2 in PBS and in rat plasma. Paclitaxel was released in controlled manner by all the nanoparticles with the release exceeding 12 days. Cytotoxicity studies revealed decreased IC50 for NP2 and NP3 in folate receptor positive cell lines (HeLa cells) and similar IC50 values as that of NP1 in folate receptor negative cell lines (A549 cells) indicating the selective cytotoxicity. The cell uptake studies confirmed the enhanced uptake of NP2 and NP3 by folate expressing cell lines through receptor mediated endocytosis which was inhibited in presence of folic acid due to competitive inhibition and saturation of the folate receptors. Thus these folate-PEG conjugated PLGA copolymers serve as a better carrier for paclitaxel with high encapsulation efficiency and selective targeting and cytotoxicity to folate receptor expressing cells.
	To summarize, the folate targeted paclitaxel conjugates and nanoparticles exhibited enhanced cytotoxicity to folate-receptor positive cells with enhanced uptake by these folate-receptor positive cells, thereby overcoming the side effects of paclitaxel. Also the folate conjugates were found to overcome the blood brain barrier and thereby increase the drug exposure to brain, which is of significant consequence in treating brain tumors.
	






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MICROBIAL ENZYMES: STUDIES ON PENICILLIN ACYLASE (EC 3.5.1.11) FROM Escherihia coli

RAM, SHASHI ANANTH

Abstract/Synopsis

Preparation and Characterization of Polymeric Materials for the Removal of Heavy Metal Ions from Water

This thesis embodies the results of laboratory studies on Cr(VI), Ni(II), Cu(II) and Pb(II) removal from water with a few selected polymeric sorbents. Based on these results the mechanism of metal sorption by these sorbents is established. The suitability of the sorbents for the development of metal removal technology is also discussed in detail.  The thesis is divided into four main chapters. In the first chapter a general introduction on metal toxicity and a review of various adsorbents studied for heavy metal removal so far are given in detail.  The second chapter deals with the experimental methods to analyze the water samples for various quality parameters and characterize the sorbents with standard reference and the procedure adopted to carry out the adsorption and kinetic studies that have been carried out. The third chapter presents the details of the results and discussion of the heavy metal removal studies with different sorbents and the results are presented under the following sub divisions.
III.1. Sorption studies of Cr(VI), Ni(II), Cu(II) and Pb(II) using commercial ion  
          exchange resins
III.2. Sorption studies of metal ions using modified chitosan beads
III.3. Sorption studies of metal ions using bio-inorganic polymeric composites
III.4. Sorption studies of metal ions using synthetic polymeric resins and 
III.5. Selectivity of metal ions and regeneration studies 
Suitable mechanism, isotherm and kinetic models for metal ions sorption onto the sorbents are established. The fourth chapter presents the summary and conclusion of the thesis which gives the details of the merits and demerits of the various sorbents studied based on their sorption capacity at various equilibrating conditions like time, dosage, pH, effect of co-ions, temperature, etc. 



SYNTHESIS AND CHARACTERIZATION OF THERMOTROPIC COPOLYESTERS

VYAS, ANURADHA

	STUDIES ON NARINGINASE, A DEBITTERING ENZYME, FROM PENICILLIUM SP.

The flavonoid glycoside, naringin(4', 5, 7-trihydroxyflavanone-7-rhamnoglucoside), chiefly contributes to the bitterness in citrus fruits thereby affecting its keeping quality. Enzymatically naringin is cleaved by naringinase, an enzyme complex with an α-L-rhamnosidase (E.C. 3.2.1.40) and a flavonoid dependent β-D-glucosidase (B.C. 3.2.1.21) activity. The two components act sequentially cleaving the parent naringin to give naringenin, a tasteless compound. A fungal isolate identified as Penicillhim citrinum was selected for the present study. Naringinase from this isolate was not inhibited by the various components of the juice, i.e. sugars and citric acid, a problem faced by enzymes reported so far. The routine assay performed for enzyme activity was a spectrophotometric method using DNSA to estimate reducing sugars. However, the enzyme was also assayed by HPLC to account for the activity in terms of total naringinase as well as the individual enzyme components. Shake flask level experiments for media optimisation resulted in a yield enhancement from a basal level of 20 U/L to 600 U/L. Naringinase was found to be an extracellular enzyme produced in the late log phase. Media optimisation with respect to components of media, their concentration and physiological conditions, like pH and temperature was carried out. Naringinase production was higher in organic media and addition of glucose further enhanced enzyme production. Growth patterns were similar in the pH range from 5.5 to 7. However, maximum enzyme production occurred at pH 6.5. A temperature of 30⁰C was found most suitable for growth and enzyme production. Naringin acted as an inducer for enzyme production and its addition after growth phase proved most beneficial. In the fermenter a similar pattern of enzyme production was obtained. The enzyme was purified to homogeneity by a series of chromatographic steps involving IEC on DEAE-methacrylate, HIC on methylmethacrylate and GPC through Sephacryl S-200. A fold purification of 1397 with a specific activity of 47.5 U/mg was obtained. The enzyme had a molecular weight of 105 kD as deduced by SDS-PAGE. Naringinase had a pH optimum between pH 3.5 and 4 and a temperature optimum between 40 and 45⁰C. Moreover, in the pH range between 4 to 12 the enzyme retained more than 90% activity at 4⁰C and more than 65% activity at 40⁰C for a period of 24 h. The Km and Vmax values for naringinase as determined by DNSA assay were 2.45 mM and 145.5 U/mg protein and that as determined by HPLC were 2.9 mM and 138 U/mg protein respectively. The enzyme was not inhibited by glucose, fructose, sucrose or citric acid and retained greater than 80% activity in the presence of 2% w/v rhamnose. The enzyme was immobilized on Matrex PAE silica 1000L by adsorption and stabilized by 1.5% glutaraldehyde treated for 1 h. The immobilized enzyme had a pH optimum between 3 and 3.5 and temperature optimum between 50 and 55⁰C. In the pH range between 4 and 8.5 the enzyme retained greater than 70% activity at 4⁰C for a period of 24 h. The kinetic constants Km and Vmax as determined by DNSA assay were 1.12 mM and 0.82 U/g of matrix and by HPLC were 1.42 mM and 0.34 U/g of matrix, respectively. The immobilized enzyme was also not inhibited by glucose, fructose, sucrose and furthermore even by rhamnose at low and high concentrations. This property is a report, first of its kind. The debittering studies on naringin and simulated juice were carried out using a fluidized bed reactor. For 30 mL of naringin solution or juice processed, 90% naringin was removed in 2 h at 40⁰C at a flow rate of 2 mL/min. The enzyme retained 100% activity for 7 runs in 7 days. 



CELL ENGINEERING BY PROTOPLAST FUSION: TRANSFER AND EXPRESSION OF CELULLASE AND XYLANASE GENES IN STABLE HYBRIDS BETWEEN CELLULOMONAS SP. AND ZYMOMONAS MOBILTS ATCC 10988

RANE, VILAS

 
 
i 
 
SYNOPSIS 
Cells are continuously challenged by conditions which cause stress. In order to adapt 
to environmental challenges and ensure survival, cells respond by a rapid and transient 
reprogramming of cellular activities called stress response. In response to such critical stress 
situations, cells employ a variety of mechanisms, and this involves increasing the abundance 
of an important group of biomolecules in the cells, namely the heat shock proteins (HSPs) or 
stress proteins. Heat shock proteins were first identified in response to heat stress but were 
later found to be constitutively present and up-regulated under various stress conditions. 
HSPs function as molecular chaperones and are involved in a plethora of functions including 
facilitating the synthesis and folding of proteins throughout the cell. 
Heat shock proteins are classified, based on their molecular weights, into six major 
families i.e. Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and small heat shock proteins (sHsps). 
Chapter I describes role of HSPs in stress response with particular emphasis on sHsps. The 
sHsps form a ubiquitous family of molecular chaperones, of which the monomer size typically 
ranges between 12 and 43 kDa. They are characterized by a conserved stretch of 80-100 
amino acid residues, called the α-crystallin domain, a more variable N-terminal sequence, and 
in most cases, a short and variable C-terminal extension. Notably, many of these sHsps are 
shown to exhibit chaperone-like activity and enhance stress tolerance. The human genome 
codes for ten sHsps, namely, HspB1 (Hsp27), HspB2, HspB3, HspB4 (αA-crystallin), HspB5 (αB-
crystallin), HspB6 (Hsp20), HspB7 (cvHsp), HspB8 (Hsp22), HspB9 and HspB10 (ODF1).  
Chapter I provide a contextual review of small heat shock proteins (sHsps). Three 
sHsps have been crystallized namely Hsp16.5 of Methanococcus janaschii (thermophillic 
methanogenic archae), Hsp16.9 of wheat and Tsp36 of Taenia saginata (tapeworm); these 
provide insights into the structural and functional organization of sHsps. However, no small 
heat shock proteins of mammalian origin have been crystallized so far. sHsps exhibit ATP-
independent chaperone-like activity. Moreover, role of sHsps in cytoskeletal rearrangements, 
their crosstalk with extracellular environment, interaction with apoptotic cascade and metal 
 
 
ii 
 
ions have been discussed. sHsps provide protective role in several pathological conditions. 
Chapter I also describes the role of sHsps in pathological conditions. Several mutations in 
sHsps have been linked to cataract, myopathies and neuropathies. The work reported in this 
thesis is related to the structure and function of human small heat shock protein HspB3. This 
chapter reviews the literature available on HspB3 and provides the scope of this work. 
 Chapter II describes the cloning, over-expression and purification of the recombinant 
human HspB3. HspB3 was purified to homogeneity and characterized for its structural and 
functional properties by various biophysical methods. Circular dichroism (CD) studies revealed 
that HspB3 has significant β-sheet content, similar to other well characterized members of the 
sHsp family. Quaternary structure study shows that HspB3 predominantly exists as a trimer. 
Our studies show that HspB3 efficiently prevents the thermal aggregation of alcohol 
dehydrogenase (ADH), moderately prevents the thermal aggregation of citrate synthase (CS) 
and fails to prevent the DTT-induced aggregation of insulin. Thus our study shows that HspB3 
exhibits target protein-dependent chaperone-like activity. 
 HspB3 is a unique member of the small heat shock protein family, it has a very small C-
terminal extension (PVGTK). The C-terminal extension of sHsps, have been predicted to play a 
role in solubilization of target-chaperone complex. In order to investigate the role of C-
terminal extension in sHsps, we made a chimeric protein containing the N-terminal domain 
and the α-crystallin domain of HspB3 fused to the C-terminal extension of HspB5 (αB-
crystallin). Chapter II describes our studies on the structure and chaperone properties of the 
chimeric protein. Our study indicates that the C-terminal extension modulates the 
oligomerization status and chaperone activity of the protein. The chimeric protein shows a 
major dimeric population with a significant population of higher oligomeric species. The 
chapter further shows that the chimeric protein exhibits protection against insulin aggregation 
in addition to that of ADH and CS. Our results suggest that the C-terminal extension in sHsps 
may play a role in imparting wide range chaperone-like activity.  
 Several human sHsps have been associated with neurodegenerative disorders such as 
Alzheimer’s and Parkinson disease.  Cu2+ has been reported to accumulate in extracellular 
 
 
iii 
 
spaces of the patient suffering from neurodegenerative disorders. Cu2+ deposition could cause 
neurodegeneration via oxidative damage. Cu2+ is known to accelerate amyloid fibrillation of 
proteins such as α-synuclein. Chapter III describes our investigation on the interaction of 
HspB3 with Cu2+. We found that HspB3 binds to Cu2+ with picomolar affinity, sequesters Cu2+ 
and prevents Cu2+-ascorbate-mediated ROS generation. This binding also confers 
cytoprotection from Cu2+-ascorbate-mediated oxidative damage. Further, our study shows 
that Cu2+ binding also prevents Cu2+-mediated α-synuclein fibrillation, by extracting the Cu2+-
bound to α-synuclein. Our study shows that another sHsp, Hsp27, which is associated with 
neurodegenerative disorders, also exhibits Cu2+-binding property, prevents ROS generation, 
confers cytoprotection and inhibits α-synuclein fibrillation. 
 R7S mutation in HspB3 has been correlated with axonal motor neuropathy. Chapter IV 
describes the cloning, bacterial expression and purification of R7S HspB3 and the effect of R7S 
mutation on the properties of HspB3. We find that the mutation leads to a small change in the 
secondary structure and a significant loss in the tertiary structure of the mutant protein. The 
R7S mutant of HspB3 also shows slight reduction in the chaperone-like activity. Our 
immunolocalization study on COS-1cells transiently transfected with wild type and mutant 
HspB3 shows that under normal, unstressed conditions both predominantly localize to the 
cytoplasm. Further, our study shows that under heat shock conditions wild type HspB3 
translocates into the nucleus, whereas R7S mutant fails to do so and remains in the 
cytoplasm.  
 Chapter V summarizes our findings and conclusions. The thesis reports the cloning, 
over expression, purification, structural and functional characterization of the human small 
heat shock protein HspB3. It also sheds light on the role of C-terminal extension in sHsps. This 
study reports on the Cu2+-binding property of HspB3 and its role in preventing Cu2+-mediated 
oxidative damage and amyloidogenesis. Work reported in the thesis characterizes the axonal 
motor neuropathy-causing R7S mutant of HspB3, and its mis-localization under heat shock 
conditions. Further studies may help in elucidating the molecular basis of this disorder. 
  


PREPARATION, CHARACTERIZATION AND EVALUATION OF BASIC SOLID CATALYSTS FOR DIRECT CONVERSION OF METHANE TO HIGHER HYDROCARBONS

MENON, SHAMAL

Abstract of the thesis

Synthesis, characterization and catalytic activity of ordered Sn, Al-SBA-15 and immobilization of Ntn-hydrolase family enzymes on SBA-15 mesoporous molecular sieves

The thesis is divided into six chapters with proper references at the end of each chapter. 

Chapter 1: Introduction and literature survey
                  Chapter 1 provides a brief and general introduction to the importance of environmentally friendly solid acid catalysts, such as those based on MCM-41 and SBA-15. It also summarizes the available literature on mesoporous molecular sieves. It describes the synthesis strategies, the role of templating surfactants and the mechanisms proposed for their synthesis. It also elucidates the physical principles of the characterization techniques used to study mesoporous materials described in brief. It mainly describes the published literature on the structure, preparation and catalytic aspects of mesoporous molecular sieves. This Chapter is mainly a review of the literature on the above said topics.

Chapter 2: Synthesis, characterization and catalytic activity of Sn-SBA-15 mesoporous molecular sieves
	Chapter 2 describes the preparation and characterization of Sn-SBA-15 with different metal loadings synthesized by post grafting as well as by direct hydrothermal synthesis methods. The direct hydrothermal synthesis appears to be not feasible since this mesoporous phase is synthesized in strongly acidic medium. Hence, post-synthesis method is overwhelmingly used. Mesoporous silica SBA-15 molecular sieves have been incorporated with various amounts of Sn by repeated post grafting technique using two different metal precursor viz., Sn acetate and Sn chloride. Adding dilute HCl to parent SBA-15 in the acetate precursor is likely to have increased the number of silanol groups, thereby facilitating the incorporation of higher amounts of Sn. There are probably two types of Sn species formed in SBA-15 during impregnation techniques, the ratio of which depends on Sn content. The first one is stabilized atomically (well dispersed, Si-O-Sn-O-Si- groups) on the walls, which form Sn2+ upon reduction treatment (easily reducible from Sn4+ to Sn2+). The second types are large site entities of Sn4+ (SnO2 clusters) distributed in the external pore structure which is seen as large (45 nm) particles in TEM.   
Highly acidic conditions of preparation of Si-SBA-15 normally prohibit the incorporation of Sn due to the high solubility of their precursors. It is still a challenge to find a one-step route of metal incorporation into SBA-15 in order to increase the acidity without changing its structural order or increasing the complexity of the synthesis. The second chapter also reports an optimized procedure for the synthesis of Sn-SBA-15 materials under milder acidic conditions made through an adjustment in the molar H2O/HCl ratio. The samples prepared under three different acidic conditions have been characterized by various techniques. Incorporation of Sn in SBA-15 framework is successfully achieved by adjusting the molar ratio of nH2O/nHCl to 796 of the gel. These Sn-SBA-15 samples are different from the samples prepared either at lower ratios of nH2O/nHCl (276) or without any addition of HCl to the synthesis gel. By adjusting the H2O/HCl molar ratio to 796, Sn gets incorporated into the lattice of SBA-15 at a low Sn concentration, which is evidenced by XRD, TEM, UV, 29Si MAS NMR and Sn-Mössbauer spectroscopic data. 29Si MAS NMR spectra of samples prepared under an intermediate acid condition show the presence of Si in (2Si, 2Sn) i.e., Q2 environment may point to the incorporation of considerable Sn4+ ions in tetrahedral positions. Sn-Mössbauer spectroscopic studies reveal that Sn2+ species form upon reductive treatments and can probably be stabilized in the pore wall upon reoxidation. This to some extent is an indication of the formation and stabilization of Si-O-Sn-O-Si linkages in Sn-SBA-15. 
The studies reveal that the method of preparation, nH2O/nHCl ratio and the nSi/nSn ratio (concentration of SnCl4) of the gel significantly influence the type of tin species in the resulting Sn-SBA-15 samples. In the present study, we have examined the catalytic properties of these Sn-SBA-15 samples in three industrially important reactions, viz., trans-esterification of diethyl malonate, synthesis of nopol by Prins condensation of β-pinene with para-formaldehyde and the epoxidation of norbornene. In each of this reaction, while demonstrating the catalytic role of Sn species, we show that requirement of the type and the environment of Sn species is different for these reactions. The effect of various reaction parameters such as Si/Sn ratio, amount of the catalyst, temperature and reaction time on the influence of reaction is also studied. Sn-B-40 is more efficient for Prins condensation and epoxidation reaction than Sn-A-40, Sn-C-40 and other Sn impregnated samples. In other words, support influences the catalytic activity. The activities of Sn-B-40 catalysts are lower for transesterification of diethylmalonate. The structure of the catalyst, amount and type of acidity and the method of preparation influenced the catalytic activity.

Chapter 3: Synthesis, characterization and catalytic activity of mesoporous Al-SBA-15 molecular sieves 
Chapter 3 describes the synthesis and characterization of Al-SBA-15 by post grafting as well as by direct hydrothermal synthesis method. Physico-chemical characterization was done by various spectroscopic methods to understand the chemical nature of incorporated Al. In case of impregnated samples, the presence of silanol OH group of the wall of SBA-15 sample is being used up to react with Al precursors to form Si-O-Al-O linkages, which ultimately develop into thin films on the walls of SBA-15. Highly acidic conditions of preparation of SBA-15 normally prohibit the incorporation of Al due to the high solubility of their precursors. We report here an optimized procedure for the synthesis of Al-SBA-15 under milder acidic conditions were made through an adjusting the molar H2O/HCl ratio which indicate the formation of Si-O-Al linkages that lead to isomorphous substitution of Si4+ by some Al3+ ions. By adjusting the H2O/HCl molar ratio, Al gets incorporated into the lattice of SBA-15, which is evidenced by XRD, TEM, 29Si and 27Al MAS NMR spectroscopic data. Aluminium incorporated SBA-15 samples retained both structural and textural properties of SBA-15. The 27Al MAS NMR confirms that a large proportion of the Al is inserted into tetrahedral positions within the framework. The Al3+ ions could assume a tetrahedral coordination and be part of the hexagonal structure of silica in Al-SBA-15. In the present study, we have examined the effectiveness of Al-SBA-15, as an acid catalyst for the esterification reaction and in benzylation of anisole. The effect of various reaction parameters such as Si/Al ratio, amount of the catalyst, temperature and reaction time on the influence the reaction is also studied. The high activity of Al-20 sample in esterification reaction has been attributed to isolated, generally tetrahedrally coordinated, framework Al species. Larger Al2O3 particles are known to inhibit esterification reactions by decreasing the number of accessible catalytic sites. While in benzylation of anisole, the best result was achieved with Al-5 catalyst, which has a larger number of acid sites. The Al content increases the conversion and also yield due to an increase in the number of active centers. The selectivity for benzylanisole increases and that for dibenzylether decreases with increase in the Si/Al ratio of Al-SBA-15.

Chapter 4: Non ambient powder X-ray diffraction studies on mesoporous SBA-15, Sn-SBA-15 and Al-SBA-15 molecular sieves
Chapter 4 includes two parts which describes the thermal stability of SBA-15, Sn-SBA-15 and Al-SBA-15 material and the effect of isomorphous substitution of Sn and Al on the thermal expansion behavior investigated by in-situ HTXRD analysis for the first time in literature. The study of structural and spectral aspects of a material is of great importance to understand its chemistry. As there is no study reported on the thermal expansion behavior of SBA-15 in literature, we have made an attempt to study the structural changes that occur in SBA-15, Sn and Al-SBA-15 as a function of temperature by in-situ HTXRD studies. The Chapter includes two parts. 
Part-1: HTXRD studies of SBA-15 and Sn-SBA-15 samples
Part I describes the in-situ HTXRD studies in the temperature range 298-1573 K carried out on SBA-15 and Sn-SBA-15 mesoporous molecular sieves (with Si/Sn=80, 60, 40 and 10) prepared through direct synthesis route. SBA-15 is found to be thermally highly stable upto 1473 K and appearance of α-cristobalite was observed at 1573 K also supported by the thermogravimetric (TG) data. A strong negative thermal expansion was observed on heating from 298 to 1573 K (αa=-4.3 x 10-6 K-1). Sn containing samples (Si/Sn = 80 and 60) showed a positive thermal expansion (6.745 x 10-6 K-1 and 9.944 x 10-6 K-1 respectively). On the other hand, the samples with Si/Sn = 40 and 10 having higher Sn content, showed a strong negative thermal expansion (-4.12 x 10-6 K-1 and -7.64x 10-6 K-1) similar to SBA-15. The linear thermal expansion coefficient varied in the order: Si/Sn= 60 > 80 > 40 > 0 > 10.  This variation in αa with Sn concentration is due to the presence of higher concentration of Sn4+ ions in the SBA-15 lattice framework in Sn-60 and Sn-80 as compared to Sn-40 and Sn-10 samples. Sn4+ ions exhibit both tetrahedral and octahedral coordination depending upon the location of these ions either on the walls of the silica (in samples with Si/Sn = 80 and 60) or in the corona region of the structure (in samples with Si/Sn = 40 and 10) respectively. 

Part-2: HTXRD studies of Al-SBA-15 samples 
Part II describes the in-situ HTXRD studies in the temperature range 298-1573 K carried out on Al-SBA-15 mesoporous molecular sieves (with Si/Al = 80, 60, 40, 20, 10 and 5) prepared through direct synthesis route. Al-SBA-15 sample shows the positive linear thermal expansion coefficient for Al-80 sample and negative for Al-60 and Al-40 samples from 298-1473 K. On cooling the sample from 1473 K to RT, the only positive thermal expansion coefficient was observed and the values are 5.59x10-6 K-1, 10.02x10-6 K-1 and 16.01x10-6 K-1 for Al-80, Al-60 and Al-40 samples respectively. The magnitude of the coefficient of thermal expansion increases in this order, Al-80 > Al-60 > Al-40. For high Al containing samples (Al-20, Al-10 and Al-5), the linear thermal expansion coefficient is negative both during heating in the temperature range 298-1473 K and on cooling from 1473-373 K. On cooling, the values of the linear thermal expansion coefficient from 1473-373 K are –19.84 x10-6 K-1 and –27.33x10-6 K-1 and –47.48x10-6 K-1 for Al-20, Al-10 and Al-5 samples respectively. The very strong NTE coefficients observed for these samples is likely due to the transverse vibration of the bridging oxygen in two-fold coordination between two polyhedrons causing a shortening of Si–Si non-bonding distance. The magnitude of the coefficient of thermal expansion increases in this order, Al-20 > Al-10 > Al-5. There is a recognizable correlation between the Si/Al ratio and the thermal expansion behavior of the material which is dependent on the concentration of Al. The thermal decomposition of the samples (TG data) is correlated to the thermal expansion behavior (HTXRD data). TG-DTA data reveal a shift in the decomposition temperature of template to higher value with increasing Sn or Al concentration. This indicates that the template ions located within the pores is strongly influenced by the presence of metal ions (Sn or Al) in the framework and a ‘soft’ interaction probably exists between the template ions and the Sn or Al sites.

Chapter 5: Structural features of immobilized Penicillin G acylase and Bile salt hydrolase adsorption on APTES functionalized SBA-15
Chapter 5 describes the immobilization of Ntn hydrolase family enzymes e.g., Penicillin G acylase (PGA) and Bile salt hydrolase (BSH) on aptes functionalized SBA-15. This chapter is also divided into two parts:

Part-1: Immobilization of Penicillin G acylase enzyme 
Part 1 describes the effect of the immobilization of PGA on the amino (APTES)-functionalized SBA-15 materials and the effect of silica as host matrix on enzyme kinetics. Physico chemical characterization was done by nitrogen adsorption, powder XRD and TEM methods to understand the nature of immobilized PGA enzyme. XRD and nitrogen adsorption data reveals that these samples retain both structural and textural properties of mesoporous SBA-15. The maximum loading of the enzyme was observed at pH 7.8, slightly below the isoelectric point of the enzyme. The trapped enzyme is more stable than the soluble form, both to temperature and to pH environments and retained 73% of its activity after immobilization. This enhanced stability is attributed to the protective nature of the cage itself and to the rigidity of the SiO2 matrix, which reduces the freedom of peptide-chain refolding of molecular motions that occur in denaturation processes. It is important that the immobilized enzyme retains as much catalytic activity as possible after ten cycles of reusability. 

Part-2: Immobilization of Bile salt hydrolase enzyme 
Part 2 describes that the encapsulated BSH enzyme grafted through glutaraldehyde groups inside the channels of APTES functionalized SBA-15 imparts higher stability by the formation of covalent bonds with functionalized surface which effectively prevent the loss of activity. BSH is considered as an important enzyme which catalyzes the hydrolysis of glycine- and taurine-conjugated bile salts into amino acid residues and free bile acids. It reduces serum cholesterol level with its bile salt deconjugation property. Physico- chemical characterization was done by nitrogen adsorption, powder X-ray diffraction, TEM and 29Si CP-MAS NMR methods to understand the nature of immobilized BSH enzyme. The pH and temperature stability of Bile salt hydrolase (BSH) enzyme was also enhanced by the physical entrapment in SBA-15. The adsorption of BSH on SBA-15 from buffered solutions with different pH values has been studied as a model protein adsorption system. We examined the dry and wet storage of the BSH immobilized SBA-15 sample at room temperature over six months resulted in marginal loss of the initial activity, demonstrating their working stability in aqueous and low-water media. We have also carried out experiment to check the activity of immobilized enzyme towards co-precipitation of cholesterol. The experiment indicates that immobilized BSH enzyme may be the promising catalyst beneficial for bile deconjugation activity in vivo. 

Chapter 6: Summary and conclusions 
The Chapter 6 present a brief summary of the work described in previous chapters and general conclusions arrived from the work. Thus the thesis includes new investigations on different aspects such as synthesis procedures, synthesis mechanisms, heteroatom insertion, catalytic applications, thermal stability, physico-chemical characterization and enzyme immobilization.  



N-ACRYLOYL Y-AMINOBUTYRIC ACIDAND ITS POLYMERS

RATH, ASOKE

Abstract:

The free hydroxyl groups of the cellulose chains within lignocellulosic fibers (LCFs) tend to attract water molecules in moist environment causing the fibers to swell and causing the cellulose chains lose integrity because of hydrolysis and oxidation because of the actions of biogenic enzymes or because of chemical factors such as acidity, alkalinity, and salinity or UV irradiation.  
In this research a non-hazardous and inexpensive chemical process has been developed for treating LCFs, e.g., jute and sisal using an alkaline water-based emulsion of vegetable oil (neem oil, rice bran oil) and plant based phenolic resin to enhance their short-term tensile strength and degradation resistance without potential toxic leachate generation.  The treatment involves part transesterification of cellulose hydroxyl groups of fibers with vegetable oil or blocking of hydroxyl groups with phenolic resin via hydrogen bond formation.  Treatment with alkaline neem oil-phenolic resin emulsion was found to be most effective for enhancing tensile strength as well as degradation resistance of LCFs.  Yarns were spun manually or mechanically from treated jute fibers.  Geotextiles were also fabricated in a commercial jute mill from the treated fibers to establish the industrial adaptability of the process.  Additionally, treatment was applied on commercial grade geotextiles manufactured from untreated jute fibers.  The treatment did not adversely affect flexibility and filtration properties of the geotextiles.      
The treatment caused the short-term fiber tensile strength to increase by 75 % for jute and 62 % for sisal, the water absorption to decrease from 255 % to 79 % (in terms of dry fiber weight) for jute, and from 185 % to 62 % for sisal.  Treated jute and sisal fibers retained, on an average, 80 % and 75 % of their initial tensile strengths, respectively, after 120-d biodegradation.  The corresponding tensile strength retention for untreated jute and sisal fibers were only 10 % and 15 %, respectively.  After 120-day immersion into a 3 % saline environment, treated jute and sisal fibers retained 72 % and 68 % of their initial tensile strengths, while untreated jute and sisal fibers retained only 6 % and 7 %.  Further the treated fibers and geotextiles were found to be stable upon being exposed to pH ranging between 4 and 9.  The biodegradation and UV-degradation related half-lives for geotextiles after treatment found to exceed 3 to 4 times compared to untreated samples.




THERMOTROPIC LIQUID CRYSTALLINE COPOLYESTERS

PETHE, VAIJAYANTI VASANT

iii
ABSTRACT
Protease, also known as peptidyl-peptide hydrolase, an important
industrial enzyme, is responsible for approximately 60% of all enzyme sales,
projected as 1.8 billion USD in the year 2015. Proteases are utilized
extensively in leather industry for de-hairing. Until recently, most proteases
used in enzymatic de-hairing studies were bacterial in origin. These proteases
are not stable over a wide range of temperature and pH. Fungal expression
systems are capable of producing quantities of enzymes larger than by
bacterial expression systems and are stable over a wide range of
environmental conditions (pH and temperature). This is seen as advantageous
for use in the leather processing industry. The focus of this thesis is on
alkaline protease production by Aspergillus tamarii in a Real Time Biological
Reaction Calorimeter (BioRTCal) for bio thermokinetic data generation.
Calorimetry was found well suited for the online monitoring of
bioprocesses, i.e. studying the growth process of microbial cultures or for the
detection of biological key components. Since heat is a global parameter for
all living species, analysis of their metabolic activity through employment of
calorimetry for in-line monitoring and control of bioprocesses is gaining
importance since the last decade.
iv
Calorimetry was employed to correlate enzymatic activity with heat
flow profile as it was an inherent parameter of metabolism. This study
revealed that both growth and non-growth related reactions involved in this
cell culture metabolism could be monitored efficiently by calorimeter and the
heat yield values used for better design of fermentor and scale up. So far, no
report on biocalorimetric studies on the production of an alkaline protease
from Aspergillus tamarii is available. Attempts were made now to enhance
the yield of alkaline protease by optimizing process conditions like
temperature, pH, inoculum size, stirrer speed and aeration. It was aimed to
observe the correlation of oxygen uptake rate to enzyme activity, biomass
growth and heat production rate at lag, exponential and stationary growth
phases and compare the metabolic heat profile with fungal growth and
substrate depletion. This should provide an insight to real-time metabolic
process and behavior of the fungal strain employed for the production of
alkaline protease.
Optimization studies for growth and enhanced production of
alkaline protease by Aspergillus tamarii were carried out in a batch stirred
tank bioreactor. The four bioprocess variables (pH, temperature, inoculum
size and agitation) were critically examined using the thee-level four factor
CCD. Using Minitab (v15), regression equations were developed for enzyme
production using experimental data and solved. It was observed that model
predicted was in good agreement with experimental observations (R2 = 0.969)
vand protease production was significantly influenced by the listed bioprocess
parameters.
Fungal growth in pellet form was monitored calorimetrically and
reliable information on the growth dynamics of the organism achieved. The
heat yields estimated due to oxygen uptake and biomass growth, helped to
understand the energetics of the organism under study. Comparison of heat
release pattern to morphology revealed that under lag and exponential phase,
the growth process involved, germination, small pellet formation followed by
fully mature dense hairy pellets. For microbial cultures exact determination of
heat of combustion could be found experimentally in a bomb calorimeter.
Using Hess’s law the relationship between the online metabolic heat variable
and the offline bioprocess variables, i.e. biomass generation, substrate
consumption were explored and validated.
Bioenergetics of Aspergillus tamarii during protease production
using agricultural by-products was attempted in a Real Time Bio-reaction
Calorimeter. Catabolite repression was predominant in enzyme production by
A. tamarii at glucose concentration of 10 g/L; the enzyme activity was
reduced to 45-50 % in comparison to that of the glucose concentration at
5 g/L, indicating that an optimal concentration of the carbon source was vital
for maximum enzyme production. Production of alkaline protease by
A. tamarii was accomplished using agricultural by-products such as cotton
vi
seed meal and skimmed milk within a lower incubation period compared with
what was reportedly obtained with wheat bran and soya flour. Macro-
morphology and broth rheology studies coupled with BioRTCal studies
revealed that pellet formation resulted in low broth viscosity and high enzyme
production. Also towards the end of fermentation, the fungal pellets existed as
small, and meagerly branched with free hypal elements. A maximum enzyme
activity of 1511 U/mL was achieved at optimized conditions in cotton seed
meal supplemented with glucose as carbon source for growth.
 Alternative methods of oxygen supply are important, especially in
viscous fermentations and shear-sensitive fermentations. Liquid phase (need-
based) oxygen supply through H2O2 for alkaline protease production in
BioRTCal by A. tamarii fungal cells was successfully attempted. This
methodology helped to eliminate the gas–liquid transport resistance. In
BioRTcal studies, the optimal H2O2 concentration was 0.5 mM, and was
added in pulses to maintain the required DO level. Higher protease yield was
obtained in H2O2 based cultivation in comparison with conventional aeration
method.


A MOLECULAR APPROACH FOR THE ASSESSMENT OF VARIABILITY IN INDICA RICES

PILLUTLA, RAJANI

1 
 
Abstract 
 
Name of Scholar : Mr. Uttam Paliwal 
Name of Supervisor 
(Designation and Address) 
: 
: 
Dr. K.B. Joshi 
Associate Professor 
Computational Materials Science Lab 
Department of Physics 
University College of Science 
M.L. Sukhadia University, Udaipur 
 
 
 
Title of Thesis : Study of Structural and Electronic Properties of 
Some Beryllium and Cadmium Based Materials 
 
: csjhyh;e ,oa dSMfe;e vk/kkfjr dqN inkFkksZa ds 
lajpukRed ,oa bysDVªkWfud xq.kkas dk v/;;u             
 
Date of Registration : 07-02-2009 
Date of Submission : 20-04-2012 
 
In this thesis a theoretical study of structural and electronic properties of some 
beryllium and cadmium based semiconducting compounds, ternary and quaternary 
alloys is presented. The beryllium based materials Be3N2, Be3P2, BexZn1-xTe; and 
cadmium based materials CdO, Cd3P2, CdSxSe1-x and ZnxCd1-xSySe1-y; are studied. The 
structural properties like lattice parameters, atomic positions, bulk modulus and its 
pressure derivative, elastic constants, X-ray structure factors and cohesive energy are 
evaluated. Among electronic properties the electronic band structure, bandgap and its 
nature, branch point energy and other related quantities such as refractive index and 
dielectric constants are computed. The pressure-induced structural phase transitions 
among various polymorphs of Be3N2, Be3P2, CdO, and Cd3P2 are examined. The effect 
of pressure on bandgap is also studied and deformation potential and pressure 
coefficients of bandgap are computed in some cases. The bonding properties are studied 
in terms of the ionicity factors.  
Two methodologies are adopted to compute the structural and electronic 
properties. For compounds (Be3N2, Be3P2, CdO, Cd3P2), the first-principles calculations 
based on density functional theory (DFT) together with Hartree-Fock (HF) theory using 
linear combination of atomic orbitals (LCAO) method are performed implementing the 
2 
 
CRYSTAL code. The hybrid scheme is also applied in some cases. For alloys i.e. 
BexZn1-xTe, CdSxSe1-x ZnxCd1-xSySe1-y, the empirical pseudopotential method (EPM) is 
applied considering modified virtual crystal approximation (MVCA). The results 
obtained from EPM for BexZn1-xTe, and ZnxCd1-xSySe1-y alloys are compared with DFT 
calculations.   
A procedure to fit the Murnaghan equation of state (EOS) to the computed E(V) 
data is invoked. The EOS of four polymorphs of Be3N2 and two polymorphs of Be3P2, 
CdO and Cd3P2 each, are studied using DFT. The pressure dependent structural phase 
transitions and electronic properties are explored. The results on structural and 
electronic properties are compared with the experimental data. The study suggests new 
pressure-induced structural phase transitions in some cases. These results call for   
experimental verification. A critical analysis of the results on bandgaps obtained from 
DFT and hybrid schemes is also carried out on CdO. The EPM calculations are 
performed to study structural and electronic properties for BexZn1-xTe, CdSxSe1-x 
ZnxCd1-xSySe1-y alloys. Out of several compositions of BexZn1-xTe alloys, we have 
focused on the alloys showing lattice matching with InAs, InP and ZnSe. A comparison 
of the results obtained from DFT and EPM is made for a few compositions of BexZn1-
xTe and ZnxCd1-xSySe1-y alloys.  
The thesis is divided into 6 Chapters containing 38 Figures and 22 Tables. The first 
Chapter is about the introduction and review of literature. In the second Chapter detailed 
description about the two methodologies is presented. The work carried out on four 
compounds and three alloys is presented in Chapters 3,4 and 5. The overall findings are 
summarized in Chapter 6. The work presented in the thesis and the subsidiary work 
done in the course of Ph.D. degree is published in the form of 11 research papers in 
peer-reviewed journals. Partly the work is presented in 8 conference proceedings also.  
 
 
 
 
(Mr. Uttam Paliwal) 
Research Scholar 
Senior Research Fellow- CSIR 
Department of Physics 
University College of Science 
M.L.S. University, Udaipur 
 


MATHEMATICAL MODELING OF BIOSYSTEMS

PHADATARE, SANGITA

PAGE  



IV
Synopsis


Synopsis

	The thesis entitled “Synthetic efforts towards FR901483, Microsclerodermin A&B and development of new methodologies in poly(ethylene glycol)” has been divided into three chapters. 
Chapter I: Chapter I deals with the brief introduction to immunosuppressants and synthetic efforts on FR901483.
Chapter II: Chapter II describes the stereoselective synthesis of the C1-C20 segment of microsclerodermin A&B. 
Chapter III: This chapter deals with introduction to alternative solvents and poly (ethylene glycol) (PEG) as a reusable solvent for organic reactions. This chapter is further subdivided into two sections.
Section A:  Section A deals with poly(ethylene glycol) (PEG) as reusable solvent for the Heck reaction.
Section B:  This section describes poly(ethylene glycol) (PEG) as rapid and reusable solvent for OsO4 catalyzed dihydroxylation reactions.
Chapter I: This chapter deals with the brief introduction to immunosuppressants and synthetic efforts on FR901483.

INTRODUCTION:

Organ transplant recipients can attribute survival, in part, to the discovery of the immunosuppressive agents cyclosporin A and FK-506 (tacrolimus). However, these compounds are toxic at high doses and, consequently, the identification of additional immunosuppressants which function by mechanisms of actions different from these two drugs remains an ongoing concern. To that end, the Fujisawa Company screened a number of microbial culture broths for the inhibition of 12-O-tetradecanoylphorbol 13-acetate stimulated T-cell proliferation in the presence of exogenous IL-2, conditions which suppress the antiproliferative activity of tacrolimus. An attractive compound, FR901483 (1), was isolated from the fermentation broth of Cladobotrym sp. No. 11231 that was retrieved from litter collected at Iwaki, Japan. 

Experimental results suggest that FR901483 is likely to operate through an antimetabolite effect on immunocompetent cells by interfering with the enzymes adenylosuccinate synthetase and/or adenylosuccinase. Further information concerning the biological activity of this compound has not been forthcoming. However, one can speculate that the activity may result from FR901483 serving as a competitive inhibitor for inositol monophosphate (IMP) or an analogue of IMP. It was further demonstrated that FR901483 significantly prolongs graft survival time in the rat skin allograft model, and moreover, evidence was obtained which is suggestive of a different mechanism of action, namely, inhibition of purine nucleotide biosynthesis. The promising biological activity and structural novelty have elicited substantial interest in 1 at synthetic chemical level.







Retrosynthetically, FR901483 (1) can be disconnected into two subunits azabicyclic amine 3 and -bromo ester 6. We envisioned that the azabicyclic amine 3 could be accessed via ‘Robinson annulation’ type reaction of the functionalized pyrrolidine carboxaldehyde 4, which in turn would be synthesized from commercially available trans-4-hydroxy-L-proline 5. The other subunit -bromo ester 6 would be synthesized from the Wittig product of anisaldehyde 8, after the Sharpless asymmetric dihydroxylation and regioselective deoxygenation. The retrosynthetic analysis is represented in scheme 1.





























Our first goal was to devise an efficient route to azabicyclic amine 3. Accordingly initial modification of trans-4-hydroxy-L-proline 5 was done by sequential esterification and protection of amine as its tert-butylcarbamate to give 9 in 95% yield. The next step of the synthesis was the transformation of the hydroxyl group in 9 to the corresponding azide 10. For this purpose, a two-step sequence was used. The mesylation of the hydroxy function of 9, followed by azidation furnished the azido compound 10 in 90% yield (scheme 2). 





The obtained azide 10 was subjected to ‘Staudinger reaction’ using TPP in THF/H2O at room temperature to afford the corresponding amine 11 in 86% yield. The tosylation of amine with TsCl, DIPEA followed by methylation using CH3I, K2CO3 furnished the tosyl protected methylamino compound 13 in 88% yield.  The construction of the pyrrolidine carboxaldehyde 4 was now investigated from 13. After reduction of the carboxylic ester in 13 with LiAlH4 in diehyl ether, the resulting primary alcohol 14 was oxidized to the requisite aldehyde 4 utilizing CrO3 in quantitative yield (scheme 3). 














The azaspiro-ring system could be assembled by Michael addition followed by aldol condensation. Accordingly, the conjugate addition of pyrrolidine carboxaldehyde 4 to methyl vinyl ketone in the presence of K2CO3, and catalytic amount of tetra-n-butyl ammonium bromide provided 15 as a diastereometic mixture which was subjected to the intramolecular aldol condensation induced under the alkaline condition using catalytic amount of alcoholic KOH yielded enone 16 in 78% yield along with small amount of hydroxy ketone 16a. Mesylation of 16a with methanesulfonyl chloride, TEA followed by column chromatography on silica gel, gave enone 16 after elimination (scheme 4).



















The resulting enone 16 was subjected to catalytic hydrogenation using 20% Pd(OH)2/C in benzene to afford the ketone 17 in 96% yield. Subsequent ketalization of 17 with ethylene glycol under acid catalyzed process by the azeotropic removal of water provided the compound 18 in 85% yield. In the final transformation, deprotection of the N-Boc group of 18 was accomplished by treatment with TFA:CH2Cl2(1:1) leading to free amine 3 (scheme 5).













At this juncture, we were positioned to synthesize -bromo ester 6 that commenced with the preparation of methyl cinnamate 19, which was easily obtained as a single isomer in 95% yield via Wittig reaction of aldehyde 8 with a stabilized Wittig reagent (MeO2CCH=PPh3). Next, Sharpless asymmetric dihydroxylation using AD-mix- was explored to incorporate the vicinal dihydroxy group followed by selective hydrogenolytic removal of the benzylic hydroxy group furnished the -hydroxy ester 7 in 82% yield.  The hydroxy functional group of compound 7 was converted to bromo ester 6 in two steps. Accordingly, 7 was treated with MsCl and DIPEA to furnish mesylate. Displacement of mesylate with LiBr proceeded with complete inversion to provide the desired compound 6 in 90% yield (scheme 6). 
 
















Now the two fully functionalized fragments, azabicyclic amine 3 and -bromo ester 6 are ready to couple to get the tricyclic core of the target molecule 1 via 21 which is being pursued in our research group (scheme 7). 







Chapter II: Chapter II describes the stereoselective synthesis of the C1-C20 segment of Microsclerodermin A&B.
Studies of sponges of the order Lithistida have provided many bioactive metabolites, most of which are peptides that contain unusual amino acids.  Detailed studies of the metabolites of different populations of lithistid sponges are valuable in both the structure activity and chemotaxonomic arenas. The microsclerodermins are a growing family of cyclic peptides. Faulkner et al., reported the isolation and structural elucidation of two new cyclic peptides microsclerodermin A and B from a deep-water sponge and commented on the nature of the symbiotic filamentous bacterium associated with the sponge.  The crude aqueous extract showed antifungal activity against Botrytis cinerea, Candida albicans, Fusarium oxysporum, Helminthosporium sativum, and Pyricularia oryzae. 
	The basic structural motif of the microsclerodermins consists of a 23-membered ring constructed from six amino acid residues three of which namely glycine, N-methyl glycine and (3R)-4-amino-3-hydroxybutyric acid (GABOB), are common to all members of the family. The variable units are a modifieded tryptophan residue, an unusual 3-aminopyrrolidone-4-acetic acid moiety and various ω-aromatic 3-amino-2,4,5-trihydroxyacids.
As a continuation of our studies on the synthesis of structurally novel and challenging natural products, we have attempted the total synthesis of these unique cyclic peptides. This chapter describes an efficient synthesis of the C1-C20 fragment of this potent natural product. 












		
	Our retrosynthetic analysis envisioned the late installment of the four contiguous stereocenters of 23 through the application of an iterative asymmetric dihydroxylation on diene ester 26. This inturn could be obtained by deoxygenative  rearrangrment of alkynol 27 which was further synthesized from commercially available S-(-)-citronellol 28 (scheme 8).































We initially investigated the synthesis of diene ester 26 from S-citronellol 28. The S-(-)-citronellol 28 was protected as its tetrahydropyranyl ether 29. The compound 29 was subjected to ozonolysis to furnish aldehyde, which was elaborated to unsaturated ester 30 by Wittig olefination (scheme 9). 





The stepwise reduction of 30 with Mg-MeOH caused the selective reduction of olefin to give saturated ester 31 followed by ester reduction to primary alcohol 32 was achieved using LiAlH4. The primary alcohol group in 32 was protected as its benzyl ether 33 by treatment with NaH and benzyl bromide in 90% yield. The selective release of one of the primary alcohols was achieved by using p-TSA in MeOH to realize 34 (scheme 10). This allowed us to extend the right side of the fragment.







	





The oxidation of 34 using IBX furnished the aldehyde 35, which was immediately exposed to lithiated ethylpropiolate to realize the formation of hydroxy alkynoate 27 as a diastereomeric mixture, which was unseparated. The critical diene ester intermediate 26 has been obtained from hydroxy alkynoate 27 by triphenylphosphine mediated deoxygenative rearrangement via allene. This rearrangement allowed us to obtain the diene ester ready for the incorporation of stereoselective hydroxy groups via iterative Sharpless asymmetric dihydroxylation (scheme 11).





				
				


At a first glance Sharpless asymmetric dihydroxylation followed by  Sharpless asymmetric aminohydroxylation reaction seemed to be an apparent option to establish the required four stereogenic centers at C2-3, C4-5 in 23. The enantio and regioselective Sharpless asymmetric dihydroxylation of diene ester 26 with AD mix- in tBuOH:H2O (1:1) provided the diol 36 which was masked as its acetonide 37 using 2,2-dimethoxy propane and catalytic camphorsulfonic acid (CSA) in 85% yield (for two steps) with 96% de. The major isomer was separated by column chromatography (scheme 12).




 

	The regioselective incorporation of cis-aminohydroxyl group was planned in next step by Sharpless asymmetric aminohydroxylation. However this method was unsuccessful. Thus we envisaged that, this issue could be well addressed by another asymmetric dihydroxylation-double inversion (at C-3) sequence. Thus we changed the strategy to a second Sharpless asymmetric dihydroxylation on 37, with a diastereomerically matched chiral reagent AD mix- in tBuOH-H2O at 0 oC to afford the diol 38 in 87% yield with excellent diastereoselectivity (10:1) (scheme 13). Efforts to introduce the amine functionality at C-3 always ended up with the elimination product.






 	To avoid the competitive elimination reaction, the dihydroxy ester 38 was reduced to triol 39 with LiAlH4 in THF. Then, the triol 39 was subjected to regioselective 1,2-acetonide protection using 2,2-DMP in DCM under acid catalyst p-TSA to give 40 in 85% yield. Treatment of the diacetonide 40 with Tf2O in the presence of pyridine gave triflate 41. The sequential displacement of the triflate 41 by bromide ion (Bu4NBr) followed by azide ion (NaN3) provided the nitrogen function with retention of configuration at C-3. The required five contiguous asymmetric centers at C2-C6 for the key building block 23 are now been established by the synthesis of azido compound 43 (scheme 14). 
















	

	At this point a one-pot deprotection-reduction-protection strategy was utilized. The azide 43 was subjected to Pd(OH)​2/C catalyzed hydrogenation  in presence of (Boc)2O effected the benzyl ether deprotection, azide reduction and Boc-protection of the resulting amine to afford the compound 24 in 92% overall yield.The alcohol 24 was oxidized with IBX to furnish the desired aldehyde, which was subjected to Wittig olefination with (4-methoxy-phenylmethylene) triphenylphosphorane in THF was used to provide 44 as a mixture of E/Z isomers in a ratio of 3:2 in 70% yield. Treatment of the mixture of E/Z isomers with catalytic amount of Pd(CH3CN)2Cl2 in 0.5 M DCM at room temperature caused the isomerization of the double bond and afforded the isomerically pure E-isomer 45 in  92% yield (scheme 15). 










	     The terminal acetonide in 45 was selectively deprotected under mildly acidic conditions (PPTS in MeOH) to give the diol 46. The two liberated hydroxyl groups were protected selectively. The TBS group was introduced regioselectively at the primary hydroxyl group to yield 47. The introduction of methoxy methyl protection at the remaining free hydroxyl group was achieved by the reaction of 47 with MOMCl and diisopropylethylamine in DCM to obtain 48 in 90% yield. The conditions for the regioselective removal of protecting group from 48 were investigated next. It was possible to remove the TBS protecting group utilizing TBAF in THF to provide the key fragment 23, which could be oxidized at a later stage (scheme 16). All these transformations completed the stereoselective synthesis of C1-C20 segment of microsclerodermins A and B.  













































Chapter III: Introduction to alternative solvents

Solvents are widely used in commercial manufacturing and service industries. Despite abundant precaution, they have inevitably contaminated our air, land and water because they are difficult to contain and recycle. Researchers have therefore focused on reducing solvent use through the development “solvent alternatives” like, supercritical fluids, ionic liquids or fluorous phases. However, these approaches have their limitations. Environmental advantage alone probably will not enable alternative solvents to achieve widespread applicability. The pollution prevention technologies must include not only environmental or “green” advantages, but also advantages related to performance, health and cost. To addresses some of these issues, we initiated a new program to identify any liquid polymer or low melting polymer can be used as reusable reaction medium and we have found that poly (ethylene glycol) (PEG) is an efficient reusable reaction medium for organic reactions.
Poly (ethylene glycol) (PEG) was used as solvent medium for very few reactions in the literature, but not extensively studied. The complete toxicological profiles are available for a range of PEG molecular weights. Their low-toxicity, low volatility, and biodegradability represent important environmentally benign characteristics, which are particularly attractive when combined with their relatively low cost as a bulk commodity chemical. This material is widely used in food and beverages and medical purposes, which is a testament to its benign character. In addition, aqueous PEG solutions may often substitute for expensive and often toxic PTCs. The developed state of knowledge with regard to the toxicological properties of PEG is of considerable current advantage compared to the paucity of knowledge for many other potential alternative solvent systems.  
Considering all these attractive “green” advantages, we explored the PEG as a potential alternative solvent medium and here we described the Heck reaction and dihydroxylation reactions.



Section A:  Section A deals with a poly(ethylene glycol) (PEG) as reusable solvent for the Heck reaction.
Palladium catalyzed arylation and vinylation of olefins called as the Heck reaction. Heck reaction is normally carried out in the presence of phosphine ligand and base under inert atmosphere. However, the relatively high price of the palladium complex has greatly limited the industrial application of the Heck reaction, and some of the phosphine ligands are sensitive to air and moisture.
In view of the economy of the reaction, the recovery as well as recycling of the expensive palladium catalyst is required. In addition, phosphine-freed reaction conditions are highly desired due to the environmental effects associated with phosphine.    Here, we report poly(ethylene glycol) (PEG) is an efficient reusable reaction medium for the Heck reaction (scheme 17). We have noticed the following advantages.
The Heck reaction proceeded without phosphine ligands, which are expensive, toxic, and contaminates the products. The stereo and regioselectivities are also different from those with conventional solvents and ionic liquids. The reaction works very well for electron-deficient and electron-rich olefins, with equal ease and with high regio and stereoselectivities. One major advantage of the present protocol is that the catalyst is easily reusable up to five runs without loss activity even after fifth run we could obtain product in 70% yield. Benign character of PEG as well as easy operation makes the present Heck reaction attractive.













Section B:  This section describes poly(ethylene glycol) (PEG) as rapid and reusable solvent for OsO4 catalyzed dihydroxylation reactions.
Undoubtedly, the dihydroxylation of olefins using catalytic amounts of OsO4 is the most sought after method for the preparation of vicinal diols. The importance of this reaction has been enhanced substantially through the use of cinchona alkaloids, which result in chiral vicinal diols as reported by Sharpless et al. Even though the products of dihydroxylation have a prominent role in pharmaceuticals and fine chemicals, the cost of osmium and chiral ligands as well as the high toxicity and volatility of the osmium component has restricted its use in industry. To addresses this issue several groups investigated this reaction in reusable solvents. We intended to apply our procedure to Os-catalysis (scheme 18) to attempt all the above-mentioned issues and found very interesting observations.   

OsO4 in PEG not only as a recoverable and reusable system for dihydroxylation but also as a medium where reaction is rapid (2h) and high yielding at very low concentrations (0.5 mol%) of OsO4. All the reported protocols require 1–5 mol% catalyst and 12–48 hours time for completion for a similar result. Reusability of the solvent and the catalyst have been achieved up to five runs without loss of activity even after fifth run we could isolate diol in 90% yield.





 
Encouraged by these results, then we attempted the asymmetric dihydroxylation of olefins according to the Sharpless procedure and we have observed very interesting results (scheme 19). Conventionally, this reaction required longer reaction times (up to 24 h) and addition of 1 equivalent of methanesulfonamide is required for dihydroxylation of internal olefin. In our method the reaction is quite rapid (2 h) and no additional reagent like metanesulfonamide is required.





	
Reusability: To check the reusability of OsO4 and possibly ligand as well as trans-stilbene was subjected to asymmetric dihydroxylation (table 3). After 2 h, the product was separated by ether extraction and fresh trans-stilbene was added. To our great surprise, in the second run diol was obtained with more than 80% ee. Further addition of 0.5-mol% ligand helped us to obtain diol with more than 90% ee (Table 2).








	
































	

















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