Open Access Repository of Indian Theses

Vaidya, Nishtha

                       Synopsis
Soybean (Glycine max L.)  is one of the world’s leading oil seed crops.  It is one of the most important source of vegetable protein.  The defatted meal contain 45-50%  protein of good nutritional quality.  Among the many biologically active factors present in the soybean, the protease inhibitors have been shown to exert a negative impact on the nutritional quality of the protein.  

	Protease inhibitors are inactivated by heat treatment.  Heat treatment not only reduces trypsin inhibitor activity but also adversely affects the functional properties of the protein.  Therefore, in the process of heat treatment to soy products, it is essential to use optimum conditions of temperature, time, moisture and pressure such that destruction of trypsin inhibitor is maximum and at the same time the solubility of the proteins is not reduced considerably.

	 The surface properties of protein is a deciding factor in exhibiting the functional properties in solution, of particular interest being the foaming and emulsifying properties.    These properties and the manner in which proteins interact with other components, directly and/or indirectly affect their applications, food quality and ultimately the acceptance.   Any factor that disrupts the interactions between protein molecules and their confirmational structure will lead to changes in their physicochemical and functional properties.  For proteins to have the desired functional properties, several methods of protein modifications such as physical, chemical and enzymatic modifications are used. 

	 In the present thesis, an attempt is made to modify soy flour by physical and enzymatic means and to study the effect of these modifications on trypsin inhibitor inactivation, functional properties and physicochemical properties of the flour.  
	

	The present investigation entitled “ Enzymatic and physical modifications for the preparation and utilization of soy flour for better functional properties” is undertaken with the following work plan in view. 

1.	Functional and physicochemical properties of different soy flours.

2.	Physical modification of soy flour and its effect on functional and physicochemical properties of the flour. 

3.	Enzymatic modification of the physically modified soy flour and its effect on functional and physicochemical properties of the flour

4.	Incorporation of the modified flour in specific products for nutritional enhancement such as bakery products and related products. 

The results of these investigations are presented in the form of a thesis.  A brief outline of the important results obtained in the investigation is summarized below.  

	The thesis has been divided into Introduction, Scope and Objectives, Materials and Methods, Results and Discussion (in four chapters), Summary and Conclusions and References.  These are supplemented by tables with titles and figures with legends.  

	Chapter  I deals with the preparation of defatted soy flour and evaluation of its functional and physicochemical properties.  For comparison, two commercial varieties of soy flours were also evaluated for functional and physicochemical properties.   

 In this study, defatted soy flour was prepared in the laboratory by solvent extraction with less than 1% fat and with minimal heat treatment (flour 1).  Two other varieties of commercial soy flour  prepared under different conditions were also used for the comparative study (flour 2 and flour 3).  

	Protein content of the three flours were in the range of 49-50%.  The SDS-PAGE pattern showed seven major bands.  Gel filtration pattern of the three total proteins showed symmetrical  elution   profiles  with  four  peaks.    There  was  no  change  in the 
fluorescence properties of total proteins extracted from the different soy flours.  Scanning electron microscopic analysis showed that the particle size of the flours plays a major role.
	The functional properties of flours namely protein solubility, foaming capacity, foam stability, emulsion capacity and emulsion viscosity were all determined using standard procedures to compare the functional attributes under standard conditions of pH and salt concentration.  Protein solubility profile of three soybean flours showed increasing solubility with increasing and decreasing pH values, having minimum solubility (24%) at isoelectric point.  At pH 2, about 85% of the protein was soluble while more than 90% of the protein was soluble at pH 10.  Effect of sodium chloride on the solubility of soy protein was measured using  0.1, 0.25, 0.5 and 1 M sodium chloride.  In the presence of sodium chloride, solubility increases in the vicinity of the isoelectric point.  Extractability of protein was more at pH 2 and 10.  But with increasing concentration of sodium chloride there was a decrease in the protein extractability.  Out of the four dispersion media investigated, water solubilized the greatest percentage of nitrogen at pH 2 (85%).  At pH 10, nitrogen was soluble at 92% level in suspension prepared with water.  The results show that presence of salt enhances solubility of the protein at the isoelectric pH, which is an important characteristic for food formulations.

	The foaming capacities of the three flours were also compared.  Flour 1 showed the maximum foaming capacity of 70  3ml followed by flour 2 (62  3ml) and  flour 3 (40  2 ml).  Foam stability of   three flours were in the following order, flour 2 > flour 1 > flour 3.  Effect of sodium chloride on the foaming capacity and foam stability of soy flour showed that low salt concentration (upto 0.5 M) increased the foam stability.  At higher concentration of salt (1 M) destabilization of foam was observed.  Foaming capacity of soy flour as a function of pH showed a minimum foaming capacity of 32% at pH 4.0 and a maximum of 80% at pH 10.  Emulsion capacity of soy flour as a function of pH and sodium chloride was studied.  Both pH and salt concentration markedly affect the emulsion capacity of soy flour.  Emulsion capacities of soy flour dispersed in buffer,     0.1 M NaCl, 0.5 M NaCl and 1.0 M NaCl solutions are 184  2 ml,  178  3 ml,                  156  2 ml and 172  3 ml respectively.  A progressive decrease in emulsion capacity was observed with increase in salt concentrations upto 0.5 M  NaCl after which it increased upto 1.0 M NaCl.   The viscosity of the emulsion depended on NaCl concentration.  The viscosity significantly decreased with increase in NaCl concentration upto 0.65 M and then increased  upto 1.0  M concentration.  

The results indicate that though there is homogeneity among the total proteins extracted from the three different defatted soy flours, flour 3 might have undergone mild heat treatment during the processing period.  Flour 3 is having poor functional properties compared to flour 1 and flour 2.
	
The above results on the functional and physicochemical attributes of the three flours are presented in the form of  Tables and Figures for the purpose of discussion in the thesis. 

	Chapter II  describes physical modifications of soy flour and the effect of these modifications on the functional and physicochemical properties of the flour.  This chapter describes the effect of physical modifications on the trypsin inhibitor inactivation and digestibility of soy flour.  The highlights of the results are:

Dry roasting did not show any significant change in the trypsin inhibitor inactivation whereas 10 minutes of autoclaving reduced the trypsin inhibitor activity of the flour to a considerable level of 10.3 units from a control value of 65.8 units.  Almost 100% inactivation was achieved by 30 minutes of autoclaving.  

	Effect of heat treatment on the functional properties was studied.  Nitrogen solubility of control soy flour, roasted soy flour and autoclaved soy flour at different time intervals (2, 5, 10, 20 and 30 minutes) over a pH range of 2-10 was studied.  Dry roasting for 10 minutes did not reduce the solubility of the flour except with a slight reduction at the isoelectric pH range (pH 4-5).  30 minutes of dry roasting reduced the solubility of the flour in all the pH ranges.  Solubility of the flour decreased with increase in time of  autoclaving.  The nitrogen solubilities of control soy flour at pH 2, 4, 6, 8 and 10 were 85, 24,  53, 75 and 90% respectively while those of 30 minutes autoclaved flour were 43, 17, 17, 22 and 32% respectively.  The foaming capacity of roasted soy flour and autoclaved soy flour as a function of time was studied.  The roasted flour did not show any significant change in the foaming capacity from that of control flour.  The autoclaved flour did not form a stable emulsion.  

	Physicochemical properties of the heat processed flour was studied.  Total proteins were extracted from heat treated flour with 0.02  M phosphate buffer of  pH 7.9 and loaded on a polyacrylamide gel.  Under non-reducing conditions, the control soy protein showed 4 major electrophoretic bands.  There was no change in the electrophoretic pattern of the extracted protein from roasted and control flour.  Autoclaving  of flour causes a loss in the intensity and the disappearance of major protein band.  The gel filtration  studies of total proteins extracted from soy flours were carried out.   The elution profile showed 4 peaks.  Upto 20 minutes of dry roasting there was no change in the gel filtration profile compared to the control.  With increase in time of roasting there is a marginal decrease in the present area of peaks as compared to the control protein.  Autoclaving of the flour increases the concentration of last two peaks  (low molecular weight proteins and water soluble proteins).  

Scanning electron microscopic studies showed that heat processing under moist condition (autoclaved) causes aggregation of particles.   The fluorescence studies showed a decrease in the fluorescence emission intensity of autoclaved protein compared to the control protein.  The relative fluorescence intensities of control, 10 minutes autoclaved, 20 minutes autoclaved and 30 minutes autoclaved flour proteins were 342, 350, 355 and 357 nm respectively.  

The effect of heat treatment and trypsin inhibition inactivation on the trypsin digestibility of soy flour was studied.  The control flour was having a digestibility of     20-25%, whereas 30 minutes of autoclaving increased the digestibility to 90-95%.  

From the above results and discussion, it is evident that, physical modification of  soy flour reduced the trypsin inhibitor activity.  Solubility of the  flour  decreased with increase in time of heat treatment.  Foam capacity of the autoclaved flour was less compared to control flour.  Autoclaved flour did not form a stable emulsion.   Dry roasting did not have much effect on the functional properties of the flour.   Autoclaving increased the trypsin digestibility of the flour.
 	
Chapter III  describes the effect of enzymatic modification on the functional and physicochemical properties of soy flour.  A detailed investigation is carried out to study the effect of two proteolytic enzymes namely, papain and fungal protease on the functional and physicochemical properties of  heat processed soy flour. The combination of heat processing and enzymatic modification was carried out in two ways:                   (I) enzymatic modification followed by heat processing (II) heat processing followed by enzymatic modification. 

Enzymatic modification of autoclaved flour increased its acid solubility (pH 4-5) from 17% to 56% over a control value of 24%.  There was a concomitant increase in the solubility of enzyme modified flour and the flour autoclaved first and then enzyme modified.  The foaming capacity and foam stability of heat processed flour, enzyme modified flour and the flour, which was subjected to both heat processing and enzymatic modification with fungal protease and papain was studied.  In both the cases, enzyme modified flour had the least foam capacity and foam stability whereas those of the flour which was enzyme modified first and then autoclaved was more compared to the one which was first autoclaved and then enzyme modified. 

	Effect of autoclaving time on the emulsion capacity of fungal protease treated soy flour was studied.  10 minutes of autoclaving followed by 20 minutes of enzyme treatment gave maximum emulsion capacity of 174  5 ml/g flour.    The heat processed flour and fungal protease treated flour could not foam a stable emulsion.  The type of enzymes significantly influences the emulsion capacity.  

	The molecular weight distribution of autoclaved and enzyme modified proteins were studied by gel filtration chromatography.  After enzyme treatment there was a reduction in molecular size of the particles.  When the autoclaving was carried out prior to enzyme treatment, there is a substantial increase in the size of the peak corresponding to high molecular weight range whereas when the flour is autoclaved after enzymatic modification, there is a considerable decrease in the first peak and a substantial increase in the peaks corresponding to low molecular weight.  Subunit degradation occurring during proteolytic enzyme treatment of the autoclaved flour was investigated by SDS-PAGE.  Untreated control display six major bands and three minor bands.  Major protein bands were no longer observed in the enzyme treated flour.  The decrease in band intensities were apparent in the enzyme modified first and then autoclaved flour.  

	The fluorescence spectrum of autoclaved and enzyme modified proteins were studied.  The max of control soy flour, 10 minutes autoclaved flour and fungal protease treated flour were 340, 348, 349, 354 and 346 nm respectively.   Scanning electron microscopic studies were utilized to study the ultra structural features of enzyme modified flour.   Surface hydrophobicity of autoclaved protein was less compared to that of control protein.   

The above results reveal that enzymatic modification of the heat processed soy flour increased its solubility and other functional properties.  The enzyme used and the degree of hydrolysis influenced the surface hydrophobicities and hence the functional properties of the flour.  The results suggested that partial hydrolysis might have initiated physicochemical changes of the flour.  The increased functionality of the soluble proteins appear to be related to changes in composition, hydrolysis and conformation resulting in exposure of unique hydrophobic domains.  The modified soy flour based on its functional and physicochemical properties should find application in many fabricated food systems, particularly in acidic beverages without affecting the  digestibility.      
      
	Chapter IV  describes the effect of incorporation of different levels of raw soy flour and modified soy flour on the quality attributes of products like saltine cracker and noodles.  The products were evaluated for physical characteristics such as texture, colour and spread ratio.  Electron microscopy was used to understand the effect of incorporation of different types of soy flours on the morphological characteristics of the products.  The effect of incorporation of different levels of raw soy flour and modified soy flour on the solubility profiles and hydrolysis profiles of the products were studied.  Cooking properties of the noodles, like leaching loss and swelling index were also studied.  

	Breaking strength of saltine crackers considerably increased from 1.4 to 2.3 kg by the incorporation of 30% raw soy flour whereas the value was decreased to 0.4 kg when 30% modified soy flour was incorporated.  The control sample had a protein content of 9.7%.  By the incorporation of either raw soy flour or modified soy flour in different levels, the protein content increased in the range of 12.5-17.7%.   Incorporation of raw soy flour increased the yellow index of saltine crackers.  

Incorporation of modified soy flour at different levels increased the protein solubility of saltine crackers.   In vitro digestibility of the saltine crackers was measured using 3 proteolytic enzymes namely, pepsin, trypsin and -chymotrypsin.  Incorporation of raw soy flour increased the rate of hydrolysis.  With 5 hours of trypsin hydrolysis the saltine cracker containing 10% soy flour showed 38% increase in hydrolysis compared to the control saltine cracker.  Though incorporation of modified flour increased the enzyme digestibility of the saltine crackers, with all the enzymes after 5 hours of hydrolysis there was no significant change in the rate of hydrolysis.  

	Incorporation of soy flour and modified soy flour on the quality characteristics of noodles were studied.  Addition of raw soy flour for more than 10% level increased the cooking loss of noodles.  There was a marginal decrease in cooking loss (8.85%) by the incorporation of 10% modified soy flour to that of control flour (9.65%).  At the level of 10% incorporation, there was no significant change in water absorption.  Incorporation of raw soy flour and modified soy flour affected the color  of noodles. Yellow index values increased with the addition of raw and modified soy flour.  Breaking strength of the noodles increased with the addition of 20 and 30% modified soy flour whereas higher concentration of raw soy flour made the noodles more softer compared to the control.  Increasing concentration of modified soy flour make the noodles  more stickier compared to that with control or raw soy flour.  

	Solubility of profile of noodle samples containing different levels of soy flour and modified soy flour was studied.  The farinograph characteristics of control (wheat flour) and wheat flour containing different levels of raw soy flour and modified soy flour were studied.  With the incorporation of 10, 20 and 30% defatted soy flour, water absorption reduced to 33.5%, 30.8% and 29.8% respectively.  When wheat flour and enzyme modified soy flour was used , there was not much difference in the water absorption values.  Incorporation of 10% defatted soy flour increased the dough development time drastically from 7  minutes to 27 minutes.  
	
The studies on product development can be concluded that an acceptable saltine crackers or noodles of better nutritional value can be prepared by supplementing wheat flour with 10% defatted soy flour.  Processing does not cause any important difficulties.  Slightly higher amount of water is required to develop the dough.  There was no significant change in the quality parameters of soy proteins supplemented saltine crackers and noodles with that of control, using only wheat, samples.

	Summary and conclusions gives the salient features of the present investigation in brief.  The literature cited in the complete text is arranged in alphabetical order under references, which gives all the relevant details including the title of the paper, chapters in books  and pagination. 





English

Circadian heart rate and blood pressure variability in apparently healthy subjects using ABPM

Kodru, Sireesha

Type II fatty acid synthesis (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials due to its intrinsic differences from Type I pathway operating in humans. β-Ketoacyl-acyl carrier protein reductase (FabG) is the only enzyme of this pathway, which has no isoforms, thus selective inhibitors can be developed for this player of the pathway. This thesis describes the successful cloning of FabG from Plasmodium falciparum, its expression in E. coli, single step affinity purification, kinetic characterization and its interaction with PfACP. The enzyme has a Km (app) value for the substrate acetoacetyl-CoA of 0.43 + 0.05 mM and Km (app) value for NADPH of 42.6 + 0.05 μM. The specific activity of the enzyme with acetoacetyl-CoA is 59.8 U mg-1 and the kcat is 259 + 25 s-1. The study was executed to gain insights into the structure and function of PfFabG to get better understanding of the interactions with its substrate and product analogs and acyl carrier protein (PfACP). Binding of the reduced co-factor, makes the enzyme catalytically efficient, as it increases the binding affinity of the substrate acetoacetyl-CoA, 16 fold. The binding affinity of PfACP to the enzyme also increases by 3 fold upon NADPH binding. Plasmodium FabG exhibits negative, homotropic co-operative binding for NADPH, which is enhanced in presence of acyl carrier protein. There are two NADPH and one PfACP binding sites in PfFabG in the absence of each other, whereas the fully active enzyme has two equivalent and inter-dependent NADPH and two PfACP binding sites. NADPH induced conformational changes were studied using the chaotropic denaturants. Differential denaturation of PfFabG was observed with urea and GdmCl in presence of NADPH, the active site organizer. Also, the residues of PfFabG important for the interaction with PfACP were successfully elucidated. It was found that the highly conserved α-II helix of ACP recognizes a positively charged hydrophobic patch on the FabG. Kinetic analysis and direct binding studies between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical for PfFabG-PfACP interaction.

Type II fatty acid synthesis (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials due to its intrinsic differences from type I pathway operating in humans. beta-Ketoacyl-acyl carrier protein reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this paper we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium acyl carrier protein (ACP) in solution, in presence and absence of its co-factor, NADPH, by monitoring the change in intrinsic fluorescence of PfFabG and by surface plasmon resonance. To address the issue of importance of the residues involved in a strong, specific and stoichiometric binding of PfFabG to PfACP, we mutated Arg-187, 190 and 230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinity of all the PfFabG mutants were reduced for acetoacetyl-ACP (the physiological substrate) as compared to wild type PfFabG to different extents, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and direct binding studies between PfFabG and PfACP confirmed the identification of Arg-187 and Arg-230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/ hydrophobic patch located adjacent to the active site cavities of the Plasmodium beta-ketoacyl-ACP reductase, for interactions with PfACP.

The urea and guanidine chloride induced unfolding of FabG, a β-ketoacyl-ACP reductase was examined in detail using intrinsic fluorescence of FabG, UV- circular dichroism (CD), spectrophotometric enzyme activity measurements, glutaraldehyde cross-linking  and size exclusion chromatography. The equilibrium unfolding of FabG by urea is a multistep process as compared to two state processes by guanidine chloride. FabG is fully unfolded at 6.0 M urea and 4.0 M guanidine chloride (GdmCl). Approximately 90% of the enzyme activity could be recovered on dialyzing the denaturants. We found two states in the reversible unfolding process of FabG in the presence of NADPH; one is an activity-enhanced state and the other, an inactive state in case or equilibrium unfolding with urea. On the contrary, in presence of NADPH, there is no stabilization of FabG in case of equilibrium unfolding with GdmCl. We hypothesize that the hydrogen-bonding network may be reorganized by the denaturant in the activity-enhanced state formed in the presence of 1.0 M urea, by interrupting the association between dimer-dimer interface and help in accommodating the larger substrate in the substrate binding tunnel, thus increase the activity. Furthermore, binding of the active site organizer, NADPH leads to compaction of the FabG in presence of urea, as evident by acrylamide quenching. We have shown here for the first time, the detailed inactivation kinetics of FabG, which have not been evaluated in the past from any of the FabG family of enzymes from any source. These findings provide impetus for exploring the influences of ligands on the structure-activity relationship of Plasmodium β-ketoacyl-ACP reductase.

The conformational stability of the homotetrameric Plasmodium falciparum β-ketoacyl-ACP reductase (FabG) was determined by both isothermal guanidinium chloride induced and thermal denaturation in the presence of GdmCl. The reversible unfolding transitions were monitored by intrinsic fluorescence, circular dichroism (CD) spectroscopy and by measuring the enzyme activity of FabG. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with unfolding of the protein. The data not only confirm to the simple   model of unfolding, based on the superimposability of CD and fluorescence transition and similar ∆G estimates for denaturation curves obtained at four different concentration of the FabG, but also well described by the linear extrapolation model for the nature of denaturant-protein interaction. In addition, both the conformational stability (∆Gs) and the ∆Cp for the protein unfolding is quite high, at about 22.68 kcal/mole and 5.83 kcal/ (mole K), respectively, which may be a reflection of the relatively larger size of the tetrameric molecule ( Mr 120, 000) and a consequent larger buried hydrophobic core in the folded protein. We have shown here for the first time, the detailed biophysical characterization of FabG, which have not been evaluated in the past from any of the FabG family of enzymes from any source. Our study can serve as prototype for conformational stability of other members of short-chain alcohol dehydrogenase/reductase super family.      

Type II fatty acid synthesis (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials due to its intrinsic differences from Type I pathway operating in humans. beta-Ketoacyl-acyl carrier protein reductase (FabG) is the only enzyme of this pathway, which has no isoforms, thus selective inhibitors can be developed for this player of the pathway. We report here intensive studies on the direct interactions of Plasmodium FabG with its co-factor NADPH, acyl carrier protein (ACP), acetoacetyl-CoA and other ligands in solution by monitoring the intrinsic fluorescence (λmax 334 nm) of the protein due to its lone tryptophan, as well as the fluorescence of NADPH (λmax 450 nm) upon its binding to the enzyme. Binding of the reduced co-factor, makes the enzyme catalytically efficient, as it increases the binding affinity of the substrate acetoacetyl-CoA, 16 fold. The binding affinity of ACP to the enzyme also increases ~3 fold upon NADPH binding. Plasmodium FabG exhibits negative, homotropic co-operative binding for NADPH, which is enhanced in presence of acyl carrier protein. ACP increases the accessibility of NADPH to FabG, as evident from the increase in the accessibility of the tryptophan of FabG to acrylamide from 81% to 98%. In presence of NADP+, the reaction proceeds in reverse direction (Ka=23.17 μM-1). These findings provide impetus for exploring the influence of ligands on the structure-activity relationship of Plasmodium β-ketoacyl-ACP reductase.

A recombinant form of Plasmodium falciparum β-ketoacyl ACP reductase (PfFabG) was overexpressed in Escherichia coli BL-21 codon plus (DE3). The resulting insoluble inclusion bodies were separated from cellular debris by extensive washing with buffer containing 0.05% Tween-20 and solubilized by homogenization with 8 M urea. Attempts to refold PfFabG from solubilized inclusion bodies employing Rotofor (separation based on different pIs of proteins in a mixture) followed by Ni2+ or cation exchange chromatography were not successful either by bringing down the urea concentration instantaneously, stepwise, or by dialysis. Denatured PfFabG was therefore initially purified by cation exchange chromatography and was then correctly refolded at a final concentration of 100-200 μg/ml in a 20 mM Na-acetate buffer pH 5.3 with 300 mM NaCl, 10% glycerol and 0.05% Tween-20. The protein was found to be properly folded only in the presence of the cofactor NADPH and salt at a concentration < 300 mM by drop dilution method at 2-80C for 12 h. The purified final product was >98% pure by denaturing gel electrophoresis. The purified protein was biologically active in a standard enzymatic assay using acetoacetyl-CoA as a substrate. The enzyme was found to be stable up to fourth day of purification and glycerol was found to stabilize enzyme activity for several weeks, during storage. This effort paves the way for elucidation of the structure-function correlations for PfFabG as well as exploration of the enzyme for developing inhibitors against it for combating malaria.



Characterization of a DEAD-box RNA helicase mutant ΔcrhR in Synechocystis sp. PCC 6803

Khan, Shadab Ali

IV 
 
ABSTRACT 
 
Nanotechnology involves the synthesis and/or manipulation of materials at the 
nanometer scale either by scaling- up from single groups of atoms or by refining or 
reducing bulk materials. Comprehension of metal nanoparticles exotic 
physicochemical and optoelectronic properties, and organization of nanoscale 
structures into predefined superstructures promise to play an increasingly important 
role in innovative technologies. 
Recently, biological methodologies are considered highly promising for nanomaterial 
synthesis because of eco-friendly approaches alternative to the usual chemical 
methods. The biomimetic and biomineralization procedures, besides following the 
green chemical regulations for nanoscale material synthesis are supposed to yield 
novel and complex structural entities as compared to the conventional methods. Until 
now, researchers have been successful in obtaining a number of inorganic metal 
nanoparticles using microorganisms quite efficiently and to some extent started 
gaining access to design strategies to develop protocols to obtain nanomaterials of 
desired shape and size. Researchers also started deciphering the mechanistic aspect of 
formation of inorganic metal nanoparticles which are atleast easy to follow during 
their course of reaction with microorganisms such as gold nanoparticles. Despite all 
these efforts in the direction of biological synthesis of nanomaterials, one area which 
is still least explored or seldom encountered in literature is the green synthesis of 
oxide nanoparticles. Currently physico-chemical protocols for the synthesis of oxide 
nanomaterials involve high temperatures, toxic chemicals and harsh environmental 
condition, thus proving eco-unfriendly, whereas their biological counterparts have 
shown a great promise to overcome all these deficits. 
The present investigation details the study of such biosynthetic routes/protocols for 
the synthesis of natural protein capped, water dispersible and biomedically important 
oxide nanoparticles such as Gd2O3 and CeO2 using the fungus Humicola sp. Protein 
capped Gd2O3 nanoparticles were then functionalized and conjugated to the anticancer 
drug taxol and this nano-bioconjugate has shown an enhanced potency in killing 
cancer cells when incubated with THP-1 cell lines rather than taxol alone, thus 
creating new avenues in nanosized drug delivery applications. Another mesophilic 
fungus Fusarium oxysporum was then used to obtain silica (SiO2) nanoparticles 
through bioleaching of waste material such as fly-ash. Humicola sp. once again was 
V 
 
employed to obtain TiO2 nanoparticles using top-down approach. The fungus 
Humicola sp. not only showed a great potential in biomilling of bulk TiO2 to nanosize 
TiO2 but was also able to biotransform the shape and phase of TiO2 nanoparticles. 
Finally, in the last working section of the thesis an attempt has been made for the 
elucidation of mechanism of formation of gold nanoparticles. Sulphite reductase 
enzyme and organic capping molecule were purified to homogeneity from an 
actinomycete Thermomonospora sp. and employed for the in vitro synthesis of gold 
nanoparticles. Sulphite reducatse enzyme has been shown to reduce gold metal ions 
resulting in the subsequent formation of gold nanoparticles whereas the capping 
molecule has been shown to provide stability to gold nanoparticles without which 
nanoparticles tend to aggregate. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Biosynthesis of Oxides Nanoparticles using Microorganisms

Pandey, Amit Kumar

Abstract/Synopsis

Insulin resistance together with β-cell dysfunction leads to the appearance and gradual progression of type 2 diabetes. Type 2 diabetes, which accounts for almost 90% of the total diabetic population, stems from decreased responsiveness of the body to insulin, accompanied by the failure of pancreatic β-cells to secrete insulin to counteract this insulin-resistant state. Obesity-induced insulin resistance is thereby characterized by a loss of insulin sensitivity mediated by factors released from adipocytes, mainly free fatty acids and proteins, termed adipocytokines, which act to control various metabolic functions with well-described physiological effects. One such adipocytokine is tumour necrosis factor-α (TNFα), which has been identified as a significant contributor to insulin resistance, and its levels have been reported to be increased significantly in obese diabetic individuals and in several animal models of obesity. TNFα is believed to abrogate insulin action in target tissues, yet, the details of its role in hepatic gluconeogenesis in the insulin resistant state and the complex underlying mechanisms involved are not well studied. 
We have demonstrated an overall detailed analysis of the action of TNFα in HepG2 cells by encompassing the altered gene expression profile; the different GO functional terms these altered genes categories in and the consequent predicted signatures of conserved transcription factor binding sites in these sets of genes. Our results provide information towards identification of the underlying genes and transcription factors that determine among other significant liver phenotypes, hepatic steatosis and fatty liver that are critically associated with Diabetes. These results, add-on to the current knowledge of diabetic mechanisms and indicates that TNFα is one of the major contribution for cholesterol accumulation in the liver. Further we have demonstrated the role of TNFα in the regulation of gluconeogenesis and determined the underlying molecular mechanisms. Our results indicate that transcription factor, Foxa2, is responsible for the attenuating effect of TNFα on insulin action on PEPCK expression and glucose production in HepG2 cells. 

Although there are several reports of miRNAs playing important role in pathogenesis of many diseases, there role in the pathogenesis of diabetes is not completely understood. Therefore we studied the status of miRNAs in the diabetic db/db mice liver by microarray analysis and found that miR-29a levels are one of the most significantly elevated. Our finding reveals that miR-29a inhibits the PI3Kp85α proteins levels and through the PI3K-Akt pathway counteracts insulin inhibition of PEPCK gene expression. This presents a novel mode of regulation at this important point of metabolism. 





Study of the underlying molecular mechanism of insulin resistance and diabetes in liver

Gupta, Arvind Kumar

Cathepsin B inhibitory activities of phthalates isolated
from a marine Pseudomonas strain
Van L. T. Hoang,a Yong Lib,c and Se-Kwon Kima,b,*
aDepartment of Chemistry, Pukyong National University, 599-1 Daeyon 3-dong, Nam-gu, Busan 608-737, Republic of Korea
bMarine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea
cResource Institute, Academy of Sciences of Traditional Chinese Medicine of Jilin Province, Changchun 0431, China
Received 25 October 2007; revised 9 January 2008; accepted 24 January 2008
Available online 30 January 2008
Abstract—Two cathepsin B inhibitors were isolated from the culture supernatant of a marine Pseudomonas sp. PB01 (GenBank
Accession No. EU126129). Their structures were elucidated by spectroscopic analyses as dibutyl phthalate and di-(2-ethylhexyl)
phthalate. Both dibutyl phthalate and di-(2-ethylhexyl) phthalate showed dose-dependent cathepsin B inhibitions with IC50 of
0.42 and 0.38 mM, respectively. It is also observed from kinetic analyses that dibutyl phthalate and di-(2-ethylhexyl) phthalate acted
as noncompetitive inhibitors with Ki values of 0.64 and 0.42 mM, respectively. Furthermore, both of them caused inactivation of the
pericellular cathepsin B of murine melanoma cell with no acute cytotoxicity. The IC50 values were found to be 0.23 mM for dibutyl
phthalate and 0.14 mM for di-(2-ethylhexyl) phthalate, respectively, and were 50% compared to that of purified cathepsin B.
 2008 Elsevier Ltd. All rights reserved.
The lysosomal cysteine proteases cathepsin B (EC
3.4.22.1) is a unique cysteine member showing dual
roles as both endopeptidase and exopeptidase activi-
ties, due to the presence of the occluding loop in
structure.1 In normal conditions, cathepsin B plays a
role in intracellular protein catabolism; and is also in-
volved in hormone activation and processing of anti-
gens in immune response. Over-expression and
mislocation in cell membrane of cathepsin B, however,
have contributed to several pathological processes that
include cancer and neurogenerative disorders.2–4
Cathepsin B inhibitors have been reported to cause
changes in malignant tumors.5 The inhibition of
cathepsin B has also been proved to decrease the
severity of joint inflammation and to reduce the
destruction of particular tissues in the rat model of
antigen adjuvant-induced arthritis. Cathepsin B inhib-
itors are therefore expected to be useful for the treat-
ment of inflammatory joint disease, invasion of
cancer, and other diseases related to cathepsin B
disorder.
Inhibitors of cathepsin B include endogenous inhibi-
tors such as the cystatin superfamily, low molecular
weight natural, and chemical synthesis inhibitors, that
is, leupeptin, E-64, tokaramide A, CA030, and
CA074.6. Marine organisms, including microorganisms
represent as treasure sources for therapeutic natural
products. To explore new cathepsin B inhibitors, cul-
ture broths from more than 300 marine microorgan-
isms were investigated by enzyme inhibitory assay.
The most interesting microorganism Pseudomonas sp.
PB01 (GenBank Accession No. EU126129) was cho-
sen. In this study, we described the isolation, structure
elucidation, and enzyme inhibitory activities of pure
compounds from culture broth of strain PB01. More-
over, pericellular cathepsin B inhibition was studied in
order to assess in vivo potential of the isolated
compounds.
Cathepsin B and 3-(4,5-dimethylthiazol-2-yl)-2,5-diph-
enyltetrazoliumbromide (MTT) were purchased from
Sigma Chemical Co. (St. Louis, MO). Z-Arg-Arg-
MCA was purchased from Bachem AG (Switzer-
land). Murine melanoma cell, B16F10 (ATCC
CRL-6475) was obtained from American Type of
Culture Collection (Manassas, VA, USA). Cell cul-
ture medium and all the other materials required
for culturing were obtained from Gibco (Grand Is-
0960-894X/$ - see front matter  2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.097
Keywords: Phthalates; Cathepsin B inhibition; Pseudomonas species;
Fluorescent assay.
* Corresponding author. Tel.: +82 51 620 6375; fax: +82 51 628
8147; e-mail: sknkim@pknu.ac.kr
Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2083–2088


Molecular Studies on Gut Associated Microbiota in Housefly and Fleshfly

Jalasutram, Vanajakshi

SCOPE AND OBJECTIVES
Proteins from fish muscle are considered as very nutritious owing to its easy digestibility and its association with polyunsaturated fatty acids in fish muscle. Fish muscle proteins are mainly contributed by actin and myosin. In comparison to other muscle proteins, fish proteins are less stable for processing and storage. Because of this fact a good amount of this high value proteins ends up in fishmeal plants or go as manure. Preparation of shelf stable products from under utilized species and its quality improvements are very important. Preparation of surimi and its storage life enhancement will helps an efficient utilization of this unexplored resources. Surimi is the stabilized fish mince, frozen stored and used as a raw material for the production of various convenient products.
Stabilization of proteins under various storage conditions attracted the attention of protein chemists. Since fish proteins are of less stable in character, stabilization of the same required a very deep understanding of the structure and function. Various additives like low molecular weight carbohydrates, sugars and sugar alcohols were used to prevent the denaturation of protein during frozen storage. The extent of protection induced by these compounds to fish muscle proteins is the limitation in keeping the mince for a considerable time. 
The commonly used cryoprotectants to surimi are sucrose and sorbitol which impart a sweet taste to the products. Mannitol is a polyhydric alcohol whose potentials to protect the proteins from various denaturing conditions are not studied in detail. Being a low molecular weight carbohydrate and with structural similarity to sorbitol, this can act as a good cryoprotectant. Mannitol is being produced from microbial sources and can be used at low cost. Various metal ions influence the structure - activity of enzymes and proteins. Sometimes metals act as cofactors for these proteins or it can be inhibitory too. Various divalent cations can be helpful in inducing better storage stability to fish proteins. Calcium can be a useful additive to synergies the protective effect. Addition of calcium compounds also helps to restore the nutritional quality of mince prepared from fish muscle. Specific effect of each these compounds and its optimum concentration are very important for stabilization in the system. Divalent metal ions such as zinc also are of importance in the structural integration of proteins. Zinc is considered to be an important micronutrient. The specific alteration in the structure and properties of fish actomyosin in presence of zinc ions is an important phenomenon to be looked in. 
Actin and myosin are the major functional component of fish muscle. The quality and functionality of fish muscle based products mainly based on these two structural proteins from muscle tissue. The specific interaction of polyhydric alcohols to these proteins will be useful to understand the mechanism of action of these compounds with actomyosin complex. The different concentrations of the above compounds and actomyosin help to derive a clear idea of this interaction. The enzymatic, structural, biochemical and biophysical approaches are proved to be good tools for the extensive understanding of this phenomenon. 
The various additives and treatment to extend the storage stability and improve the quality of proteins requires a thorough understanding of structural details of proteins and its effect on functionality. The specific nature of interaction between these compounds and proteins helps to formulate a convenient formulation for the enhancement of shelf life of product with a clear mandate on quality attributes.  This also helps us to find out the mechanism of interaction of these small molecules with selected protein that can lead to a better understanding of interaction of multimeric proteins and ligand molecules. The specific objectives can be briefly summarized as follows:
Detailed analysis of the proteins from Indian oil sardine (Sardinella longiceps) in terms of its physicochemical and functional attributes will be studied to characterize the system. Isolation and characterization of major protein fraction the actin myosin complex to understand the exact mechanism of action of various additives to system will be undertaken.
Understanding the mechanism by in vitro effects of selected polyhydric alcohols and specific metal ions on the properties of isolated actomyosin for application from a biotechnological angle on protein- protein stabilization and protein-protein interactions are also the objectives of this study.
Pretreatment using selected polyhydric alcohol/cryoprotectant such as mannitol on frozen storage of Sardinella longiceps from the point of view of stabilization of macromolecules and measurement of altered physical, chemical and enzymatic properties of the proteins are investigated. Effect of selected salts at several concentrations on the biochemical, biophysical and enzymatic properties of the proteins for utilization of the results in biotechnological processes such as stabilization and destabilization of the proteins/enzymes during prolonged low temperature storage.
The mechanism of extension of proteins quality during processing and storage by the addition of various compounds perhaps would provide a clear understanding of the nature of interaction and range of utilization of these techniques for various biotechnological applications helps to improve products. The use of alternative additives helps to reduce the cost of production and make it more cost effective process for storage and utilization. Mannitol can be a good alternative cryoprotectant, which can protect the proteins during low temperature storage and also the use of divalent metal ions like calcium help to prevent deterioration of proteins thus enhancing the quality. This synergistic approach could be of immense application in quality.
The specific interaction of various polyols with actomyosin may clearly lead us to find out the mechanism of interaction of these with the major protein fraction of muscle. Polyols like sorbitol and mannitol can have a very specific interaction pattern on the actomyosin molecules and helps for its further utilization. Various metal ions like zinc can helps to impart the changes in structure and activity of actomyosin from fish muscle and also the role of zinc as a micronutrient needs to be revealed in such studies. The interaction of this divalent cation with actomyosin will help to conclude the use of low levels of metal ions as additive to various muscle systems for enhancing the quality of the meat.




Studies on production of 1,3-propanediol from glycerol by microbial fermentation

Chhabra, Ravindresh

  2
STUDIES ON SUPPORTED VANADIUM CONTAINING 12-
MOLYBDOPHOSPHORICACID AND ITS AMMONIUM SALT FOR 
AMMOXIDATION FUNCTIONALITY 
 
ABSTRACT  
 
 
Heteropoly compounds as green catalysts: 
 
The fine chemical industry has experienced a tremendous growth over the past few 
years due to the high demand for products like pharmaceuticals, pesticides, fragrances and 
flavoring, and feed additives. The production of these products that require stringent 
specifications is definitely non-trivial. The stoichiometric organic synthesis, currently being 
followed, leaves large quantities of inorganic salts as effluents whose disposal is a serious 
problem due to the heightened environmental awareness and the tighter regulations. 
Furthermore, increased competition in industry has pushed the research and development 
towards finding more cost-effective catalytic   routes.   One specific area that can have a 
large impact on the environmental issues is the utilization of solid heteropoly compounds 
(HPC) as substitutes for conventionally used reagents such as HF, H2SO4 etc.  Even though 
many developments like incorporation of heteropolyacids into the channels of mesoporous 
materials have been adopted for improving product selectivity there are still many areas for 
which new methods have to be explored.  
 
Problems associated with the application of heteropolyacids as solid catalysts: 
 
The application of heteropolyacids (HPA) as catalysts has now been extended to the 
synthesis of fine chemicals like drugs and drug intermediates. Preparation of 2-
cyanopyrazine (CP) by the ammoxidation of 2-methylpyrazine (MP) is one such example, 
wherein enhancing the redox property of the catalyst could derive higher CP selectivity.  
  3
Cyanopyrazine is an intermediate in the production of 2-amidopyrazine, an effective anti-
TB drug. The conventional multi-step process is unacceptable both economically as well as 
environmentally.  In the catalytic route the vanadium based catalysts, though found to be 
active, operate at high reaction temperatures. For an exothermic reaction, like that of the 
ammoxidation, high reaction temperatures substantially decrease product selectivity 
besides posing the danger of reactor runaway. So there is an ample scope for design and 
development of new catalysts, which are active at low temperatures and offer high nitrile 
selectivity.   
The HPAs are emerging as green catalysts in the fine chemical industry. Bulk 
heteropolyacids have low thermal stability and low surface area. The development of new 
solid catalysts with enhanced characteristics of strength, surface area, porosity etc. has been 
a challenging task. Replacing the protons of heteropolyacids with ammonium ions 
preparing ammonium salts of HPA and dispersing these compounds on supports like TiO2, 
ZrO2, CeO2, SiO2 and AlF3 can enhance the thermal stability and surface area.  
Increasing the redox property can be achieved by vanadium substitution for 
molybdenum in molybdophosphoric acid (MPA), which has been the subject of latest 
interest. A few reports are available on the synthesis of bulk vanadium substituted MPA 
and its ammonium salt (AMPA). Identification of various methods to increase the surface 
area and the thermal stability of vanadium substituted MPA and AMPA by supporting them 
on metal oxides. It has been observed that the Keggin structure can also be preserved on the 
support up to a higher temperature than that of the unsupported one. 
 
 
 


Study of miRNAs as critical regulators of cell death and tumorigenesis

Verma, Pramod Kumar



Synopsis



The thesis entitled “Towards The Total Synthesis of Phorboxazole A, Synthesis And Biological Evaluation of Coumarin Analogues As DNA Gyrase Inhibitors And Development of New Synthetic Methodologies” is divided into three chapters.
CHAPTER I: This chapter further divided into two sections.
SECTION A: This section describes biological importance of some antitumor    molecules     	and earlier synthetic approaches to C20-C32 fragment of the    	phorboxazoles.
SECTION B: This section comprises present work, the stereoconvergent synthesis of C20-C32 fragment of phorboxazoles.
Phorboxazole A and B (Scheme 1) rare marine macrolides isolated from the Indian ocean sponge Phorbas sp. by Searle and Molinski.1 Both C13 epimers comprised of a 21-membered macrolide ring that embodies three oxane rings, one oxazole and subtends a side chain that contains a second oxazole and a hemiketal oxane ring. These are among the most cytostatic natural products known, inhibiting the growth of tumor cells at nanomolar concentrations with mean GI50 = 1.58 x 10-9 M against NCI panel 60 tumor cell lines. These scarce natural products arrest cancer cell growth in S phase at low to subnanomolar concentrations, making them promising candidates for therapeutic development. The enormous biological activity and the fascinating molecule structure have stimulated synthetic efforts by a number of research groups and seven elegant total syntheses have been reported so far. The remarkable biological activities associated with the phorboxazoles make these compounds important leads for biotherapeutic development.2
We have depicted our strategy in the Scheme 1, which reveals the disconnection approach. The convergent synthesis of C20-C32 oxazole-oxane 1 achieved by coupling two subunits, pentasubstituted oxane 2 and oxazole sulfone 3 via modified Julia olefination. The oxane moiety 2 was synthesized from the bicyclic ketone 4.








Scheme 1
	The bycyclic alcohol 6 was prepared from the bycyclic ketone 4 in three step sequence using (-) Ipc2BH asymmetric hydroboration3 as a key step. It was converted to keto compound 7 by PCC oxidation in DCM in 85% yield (Scheme 2). Next task was to open the bicyclic ketone 7. It was thought that the best way of opening the bicyclic ketone was by making the corresponding ester 8 and hydrolyzing it. Thus a Baeyer – Villiger oxidation of the ketone 7 using m-CPBA and NaHCO3 in DCM yielded the ester 8 in 90% yield (Scheme 2). 

Scheme 2
Methanolysis of lactone 8 using catalytic amount of sulfuric acid in MeOH afforded the acetal ester 9 in 85% yield with a minor amount of the -isomer (at C-1 center). This ester consists of three stereocenters out of five in the central pyran moiety 1.  Next we turned our attention to obtain the stereocenters at C1 and C2 carbons of the pentasubstituted pyran ring 1. In that direction ester functionality of 9 was reduced with LAH in THF at ambient temparature to the corresponding alcohol 10 in 90% yield.  Then benzylation of the resulting primary alcohol with BnBr, NaH, and TBAI (catalytic) in THF furnished dibenzyl acetal 11 in 95% yield (Scheme 3).

Scheme 3
	Next the dibenzyl methyl acetal 11 was converted to lactol 12 under acidic conditions AcOH/H2O (2:1) at 55-60 oC for 12 h with 55% yield. Then the lactol 12 was oxidized to lactone 13 using PCC, CH3COONa, celite in CH2Cl2 for 3 h with 90% yield (Scheme 4).




Scheme 4
	Having made flat form to obtain the stereocenters at C1 and C2 of pentasubstituted pyran moiety 1, we thought to epimerize the α-methyl at C2 of the lactone 13. For this purpose we made experiments using MeONa/MeOH and refluxing KOH/EtOH which were resulted in ring opening of the lactone to corresponding hydroxy ester and hydroxy acid respectively. And acid catalyzed cyclization of hydroxy acid resulted  in epimerized lactone 14, and base catalyzed cyclization of hydroxy ester also led to the same 14. To avoid ring opening of lactone it was thought that to use mild base such as DBU. Epimerization was successfully carried out with DBU in THF for 12 h with 85% yield with 100% conversion (Scheme 5).4 Epimerization was confirmed by 1H NMR and NOE studies.

Scheme 5
	Accordingly, nucleophilic addition of lithiumtrimethylsilyl acetylide which was generated in situ from acetylenetrimethylsilane and n-BuLi at -78 oC to the resulting epi-lactone 14 at -78 oC in THF provided inseparable anomeric mixture of hemiketals 15 in 90% yield. Reduction of hemiketal to pyran 16 was carried out with excess Et3SiH/BF3.Et2O in CH2Cl2/CH3CN (1:1) at -40 oC.5 The reaction was proceeded to completion within 1 h, furnishing exclusively pentasubstituted pyarn moiety 16 in which ether linkage between C1 and C5 is in syn configuration (Scheme 6). The stereochemistry of 16 was confirmed by 1H NMR and NOE experiments.

Scheme 6
	Desilylation and hydrolysis of the triple bond via oxymercuration in Markownikoff’s fashion was effected in a single transformation using yellow HgO and aq.H2SO4 in refluxing acetone for 2 h with 70% yield to give keto oxane 2 (Scheme 6).6

Scheme 7
	Many attempts went in vain to make the desired oxazole 5. Finally we followed the Conforth’s synthesis of oxazoline 19, which was prepared by employing ethyl acetimidate hydrochloride 17 and DL-serine methyl ester hydrochloride 18 in CH2Cl2 in presence of Et3N for 12 h. The resulting oxazoline 19 was oxidized to oxazole 5 by using CuBr2, HMTA and DBU in CH2Cl2 for 2 h at ambient temparature in 75% yield. Further the ester functionality in oxazole was reduced to alcohol 20 by employing DIBAL-H.7 Then the alcohol was made into thioether 21 under Mitsnobu reaction conditions using mercaptobenzothiazole, DEAD and TPP in THF for 4 h. Thioether was oxidized to oxazole sulfone 3 by oxone in THF:H2O:MeOH (2:1:1) for 12 h in 90 % yield (Scheme 7).
Scheme 8
Coupling of oxazole sulfone and the ketone under the modified Julia olefination reaction conditions gave the C20-C32 fragment as separable E:Z isomers in a ratio of 9:1 in 50% yield (40% conversion) based on recovered starting material (Scheme 8).
	In conclusion, the practical synthesis of the penta substituted pyran C20-C32 fragment achieved from bycyclic ketone is described. Modified Julia olefination reaction between sulfone and the ketone was also achieved. 

CHAPTER II : This chapter again divided into two sections.
SECTION A: This section deals with introduction to coumarin and quinolone antibiotics and dual action drugs.
SECTION B: This section deals with the present work, synthesis and biological                                              		    evaluation of coumarin linked fluoroquinolones, phthalimides and naph-		                thalimides as potential DNA gyrase Inhibitors. 
	Over the past decade, bacterial DNA gyrase has drawn much attention as a selected target for finding potent antibacterial agents. Accordingly, a number of synthetic quinoline antibacterial agents have been developed and are now widely used for treatment of bacterial infectious diseases. Quinolones inhibit DNA gyrase and topoisomerase  IV, and  cause  bacterial cell death. Besides the quinolones, naturally occurring antibacterial agents, such as novobiocin  (NB; Fig. 1) have also been identified as bacterial DNA gyrase inhibitors.8  Novobiocin  inhibits ATPase activity of DNA gyrase by competing  with ATP for binding to the gyrase subunit B. Based on this background it was considered of interest to prepare new type of dual-acting coumarin-linked hybrids possessing phthalimide, naphthalimide and quinolone moieties to unravel their antibacterial activity and DNA gyrase inhibition potential.
	A hybrid molecule synthesis strategy was employed to develop new molecules with potential antibacterial activity. Three types of coumarin-linked hybrids have been synthesized. The coumarin ring system has been linked to phthalimido, naphthalimido and quinolone moieties at 4-position through piperazino alkane spacers. These compounds have been evaluated for their antibacterial activity. Some of these new hybrids particularly phthalimido and naphthalimido ones have exhibited DNA gyrase inhibition activity.

Figure. 1
Chemistry
	The coumarin precursors 4-(1-piperazinyl)-2H-1-benzopyran-2-one 25a and 25b were synthesized9 from the starting materials o-hydroxy acetophenones 23a, 23b in two step sequence. Compounds 23a, 23b were treated with diethylcarbonate in the presence of Na to reflux for 4 h to obtain  substituted 4-hydroxy coumarins 24a, 24b which 
inturn heated with piperazine at 160 oC for 1h to obtain the precursors 25a and 25b (Scheme11). 
Scheme 9

	 Quinolone precursors were obtained from substited anilines 26a and 26b by reacting with diethylethoxymethylenemalonate at 110 oC for 1.5 h to give 2-(3,4-dihaloanilino-methylene)melonates 27a, 27b which upon heating with diphenylether at 250 oC for 1h furnished  28a, 28b (Scheme 10).

Scheme 10
	N-alkylbromoquinolones 32a-g were synthesized by reacting 28a, 28b with dibromoalkanes 29a-d in acetone in the presence of potassium carbonate. The precursors N-alkylbromophthalimides 33a,b and N-alkylbromonaphthalimides 34a,b were prepared by reacting phthalimide 30 and naphthalimide 31 with corresponding dibromoalkanes 29a-d (Scheme 11).

Scheme 11

	The new cross linked hybrids of coumarins with quinolones, phthalimides and naphthalimides have been synthesized by coupling 4-(1-piperazinyl) coumarin 25a or 25b with N-alkylbromo-6,7-dihaloquinolones 32a-g, N-alkylbromophthalimide 33a,b and N-alkylbromonaphthalimides 34a,b respectively. The reaction was carried out in the presence of anhydrous K2CO3 at reflux in acetonitrile for 24 h to afford the coumarin-quinolone hybrids 35a-k, the coumarin-phthalimide analogues 36a-d and coumarin-naphthalimide analogues 37a-d (Scheme 12) in good yields. 







Scheme 12

Antibacterial activity
	The in-vitro antibacterial activity of the compounds was assessed along with ciprofloxacin by Kirby-Bauer method on both Gram negative and Gram-positive bacteria. The minimum inhibitory concentration of the compounds was determined by inoculating the test organisms (E. coli MTCC 443, B. subtilis MTCC 736) on Mueller-Hinton agar plates with different concentrations of compound (4 mg/mL, DMSO) followed by incubation at 37 oC for 16-18 hrs. To ensure that the solvent had no effect on bacterial growth, a control test with DMSO was also performed. Some of the compounds exhibited mild activity against E. coli. (Table1). However, these compounds were almost inactive against B. subtilis (MIC >60 g/mL). 

Inhibition of DNA supercoiling
	The ability of the coumarin linked hybrids to inhibit the supercoiling activity of DNA gyrase was tested. For this purpose very well studied E. coli DNA gyrase was employed. Ciprofloxacin was used as positive control in these assays as it has been shown to be a potent inhibitor of DNA supercoiling activity of the enzyme. Many of the compounds tested showed dose-dependent inhibition of enzyme activity. From the data (Table 1) it is clear that some of the compounds inhibit the DNA gyrase activity, particularly the phthalimido and naphthalimido linked coumarin hybrids (36a-d, 37a-d). One of the molecules from the quinolone linked coumarin series (35d) also exhibited promising DNA gyrase inhibition, which could be considered as dual activity coumarin. Although compounds 36b,d and 37b-d  had good inhibitory activity against DNA gyrase, it did not show any antibacterial activity, presumably because of low permeability through bacterial membrane. Compounds 35d, 36a, 36c and 37a had potent DNA gyrase inhibitory activity and showed moderate antibacterial activity (Table 1). It is interesting to note that precursors 25a,b, 30 and 31 did not exhibit any significant inhibition of DNA-gyrase up to 50ug/mL. The precursors 28a,b could not be evaluated because of the insolubility in DMSO. Therefore, it appears that there is no intrinsic activity for the precursor fragment in these hybrid molecules.













Table 1. Minimal inhibitory concentrations (MIC) of coumarin hybrids against DNA gyrase and bacteria

Sl. No	Compound	R	X	Y	n	MIC against E. coli (g/mL)	MIC against DNA gyrase (g/mL)
  1	35a	CH3	F	Cl	2	>60	a
  2	35b	CH3	F	Cl	3	>60	a
  3	35c	CH3	F	Cl	4	>60	50
  4	35d	CH3	F	Cl	6	  53	30
  5	35e	C2H5	F	Cl	2	>60	a
  6	35f	C2H5	F	Cl	3	>60	50
  7	35g	CH3	F	F	2	>60	50
  8	35h	CH3	F	F	3	>60	a
  9	35i	CH3	F	F	4	>60	50
 10	35j	C2H5	F	F	2	>60	50
 11	35k	C2H5	F	F	3	>60	a
 12	36a	CH3	-	-	2	  35	30
 13	36b	CH3	-	-	3	>60	30
 14	36c	C2H5	-	-	2	  42	30
 15	36d	C2H5	-	-	3	>60	30
 16	37a	CH3	-	-	2	  40	30
 17	37b	CH3	-	-	3	>60	30
 18	37c	C2H5	-	-	2	>60	30
 19	37d	C2H5	-	-	3	>60	30
 	Ciprofloxacin					            5	10

aNo inhibition was observed.

Conclusion
	The present studies demonstrate the synthesis of these hybrid molecules and evaluate their antibacterial activity as well as their ability to inhibit DNA gyrase. These new coumarin based hybrids exhibit moderate antibacterial activity against E. coli. with promising DNA gyrase inhibition activity and as such warrant further investigation by modulation of linker spacers and functional groups in the coumarin ring system. It is anticipated that this structure activity based approach could lead to the development of much improved inhibitors of DNA gyrase with better in-vivo efficacy. 



CHAPTER III: This chapter describes new methodologies for the synthesis of 2-				    deoxyglycosides and 2,3-unsaturated glycosides. This chapter again 			    divided into two sections.
SECTION A: This section describes the synthesis of 2-deoxyglycosides from D-glycals using CeCl3.7H2O-NaI as a novel reagent.

	Deoxyglycosides are well-known structural components of several biologically active compounds, especially antitumor antibiotics such as anthracyclines, aureolic acids, orthosamycins, angucyclins, and enediynes. In particular, 2-deoxy-α-glycosides are present in many bioactive natural products including compactin, olivomycin, mithramycin, daunomycin, calicheamicin and many others.10 In this context, several methods have been developed for the preparation of 2-deoxy sugars in a multi-step sequence. Of these, the acid-catalyzed addition of an alcohol to acetylated glycals appears to be the most direct method for the synthesis of 2-deoxy pyranosides. However, only a few methods have been reported so far for the direct synthesis of 2-deoxy- α -glycosides from glycals. Thus the development of a neutral alternative such as the CeCl3.7H2O– NaI reagent system would extend the scope of this transformation. 


Scheme 13
	This is a mild and efficient method for the synthesis of 2-deoxyglycosides from glycals and alcohols using the CeCl3·7H2O–NaI reagent system. Thus, treatment of 3, 4, 6-tri-O-acetyl-D-glucal with alcohols in the presence of cerium(III) chloride heptahydrate–sodium iodide in acetonitrile afforded the corresponding 2-deoxyglycopyranosides in good yields with high α-selectivity (Scheme 13). The α -anomer was obtained exclusively, the structure of which was characterized by spectroscopic data (Table 2.). 



	However, in the absence of NaI, the glycals underwent Ferrier rearrangement under the influence of CeCl3·7H2O in refluxing acetonitrile to afford the corresponding 2,3-unsaturated hexopyranosides in good yields (Scheme 14). The products were obtained as a mixture of - α and β-anomers, favoring the α-anomer. The ratios of products were determined by the examination of the 1H NMR spectra of the crude products. 


Scheme 14

SECTION B: This section describes the synthesis of 2,3-unsaturated C and O and S-       			glycosides from D-glycals using phosphomolybdic acid supported on 			silica gel as an efficient catalyst.

	The acid-catalyzed allylic rearrangement of glycals in the presence of nucleophiles such as alcohols, thiols etc., and known as Ferrier rearrangement11 is widely employed to obtain 2, 3-unsaturated glycosides. Allyl C-glycosides are attractive synthons due to the presence of terminal double bond that is amenable to easy functionalization, for instance, by hydroxylation, hydrogenation, epoxidation and amino hydroxylation.  These glycosides or pseudoglycals represent versatile chiral intermediates towards the synthesis of modified carbohydrates and nucleosides with important pharmacological properties. This class of compounds can be transformed in to 2-deoxy or 2, 3-dideoxy sugars, which are building blocks for the total synthesis of many antibiotics.12 Due to its great significance in the area of carbohydrate chemistry, there has been a growing interest in the improvement of the Ferrier reaction by using a variety of catalysts.  
	Phosphomolybdic acid (PMA) belongs to the class of heteropoly acids (HPA). Catalysis using HPAs and related polyoxometalate compounds is a field of growing importance.13 HPAs are commercially cheap and environmentally friendly catalysts. They exhibit high activities and selectivities and allow cleaner processes over conventional catalysts. HPAs are promising solid acids, redox and bifunctional catalysts in homogeneous as well as in heterogeneous conditions. HPAs have a very strong, approaching the superacid region, Bronsted acidity greatly exceeding that of ordinary mineral acids and solid acid catalysts.  


Scheme 15
	In a typical experiment, a solution of triacetyl glucal and allyl trimethylsilane in acetonitrile was stirred with 1 mol% PMA-SiO2 at room temperature. The reaction was complete within 10 minutes to produce exclusively corresponding -4,6-di-O-acetyl-2,3-unsaturated C-allyl glycoside with high yield (Table 3., Scheme 15). After the reaction the catalyst was filtered and reused 5 times without any appreciable loss in catalytic activity and yields.


Scheme 16
	 2,3-unsaturated glycopyranosides were successfully prepared with nucleophiles such as alcohols and thiols. A mixture of two anomers of 2,3-unsaturated glycopyranosides ( and ) were obtained in the case of glucal and galactal when alcohol was the nucleophile, while exclusively -anomers were obtained with thiols (Table 3., Scheme 16). The predominant formation of this -anomer may arise from thermodynamic anomeric effect. 

























References
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xix




Ultrafast Spectroscopic Studies on Chemically and Biologically Relevant Aqueous/Non-Aqueous Environments

Khattri, Arun





PAGE  



	Synopsis	iii



Synopsis of the thesis submitted for the award of Ph. D. degree (Biochemistry) of the University of Mysore, Mysore, India

Title of the thesis	‘Studies on water extractable feruloyl polysaccharides from native and germinated rice (Oryza sativa) and ragi (Eleusine coracana)’

Candidate		R. Shyama Prasad Rao


	Carbohydrates are the principal components/macronutrients of food, and apart from providing bulk of the caloric intake they play a variety of functions in human food and nutrition. In particular, arabinoxylans have attracted the attention of many researchers because of the complexity of their structure-function relationships. They are the major non-starch polysaccharides in cereals and represent bulk of the soluble and insoluble dietary fiber (unavailable/un-digestible carbohydrates) intake in human food. Nutritionally, dietary fibers/non-starch polysaccharides are known to exert many physiological/metabolic effects in reducing the risks of diseases (known as diseases of lifestyle/civilization) such as diabetes, obesity, atherosclerosis, hypertension, constipation, diverticulosis, colorectal cancer and so on. Due to their various physicochemical properties, water soluble non-starch polysaccharides (NSP), mainly arabinoxylans, are also known to have many functional roles in human food.
	Although general structure of arabinoxylans is known from many cereals, detailed investigations pertaining to the water soluble arabinoxylans/feruloyl arabinoxylans (feraxans) are sparse. Moreover, large variations in the fine structure of arabinoxylans isolated from various cereals are observed and they in turn may have influence on their physicochemical/functional roles. Thus for better understanding of fine structure of arabinoxylans and for their utilization in human food and nutrition with precise functional effects, arabinoxylans are characterized from diverse sources and conditions.
	The present study is taken up as there was no detailed investigation on the water soluble feraxans from rice and ragi, the major cereal and millet respectively. Structural and functional characterizations of water soluble feraxans from these two cereal grains are investigated with the following objectives:
(a) Isolation and preliminary characterization of water extractable feruloyl polysaccharides from native and germinated rice and ragi,
(b) Fractionation and purification of water extractable feruloyl polysaccharides,
(c) Structural characterization of purified polysaccharides using methylation, GLC-MS analysis, Smith degradation and 13C-NMR and
(d) Functional properties of water soluble feruloyl polysaccharides i.e., viscosity, gelling and foam stabilization and effect on dough characteristics, determination of their antioxidant and prebiotic activity in vitro.

The research work carried out towards achieving these objectives forms the subject matter of the thesis. The thesis is divided into 5 chapters:

Chapter 1: Introduction
	This chapter begins with the general account on carbohydrates, their classification, digestibility and importance in human nutrition. A brief account of the various methods employed in the structural characterization of carbohydrates/polysaccharides is given. Cereal feruloyl arabinoxylans, their biosynthesis and degradation, fine structures and physicochemical/functional roles in food and nutrition are discussed. Finally, with a brief account on rice and ragi, the chapter highlights the aims and scopes of the present study.

Chapter 2: Material and Methods
	This chapter starts with the information on the general procedures, and various chemicals and instruments used in the present study. Various colorimetric estimation methods employed for the analysis of feraxans and enzyme assay methods are discussed. Isolation, fractionation and purification procedures employed for water soluble feraxans are described. Structural characterization procedures included chemical methods such as methylation analysis, periodate oxidation and Smith degradation, and spectroscopic methods such as GLC-MS, NMR, IR and UV studies.
	Methods employed for the functional characterization of feraxans included various antioxidant assays; viscosity, gelation and foam stabilization experimental protocols; and farinograph, extensograph, amylograph and bread making procedures. A list of bacteria used for in vitro fermentation experiments, their media and growth conditions, and in vitro fermentation procedures are also described.

The following three chapters present the findings of the investigation in the form of results and discussion.

Chapter 3: Isolation, fractionation and purification of water soluble feraxans
Results on the characterization of water soluble non-starch polysaccharides (NSP), phenolic acids, variations in feraxanases during malting and fractionation and purification results are presented.
Water extractable non-starch polysaccharides (WEP) represent a small proportion (0.6 – 2.2%) of the total flour and their content increased by 2 to 3 folds upon malting (96 h controlled germination). Their water un-extractable counterparts (WUP) are present in higher proportions (7.5 – 20.3%). The WEP and WUP have high amount (2.8 – 11.0%) of uronic acid, which is slightly higher in malts, probably due to the faster degradation of mixed glucans than arabinoxylans as indicated by pentose to hexose ratio.
	Ferulic acid is the major bound phenolic acid ester-linked both in WEP and WUP, and over 90% of the total ferulic acid are bound to the latter. Malting resulted decrease in the bound ferulic acid content, due to the action of induced ferulic acid esterase. p-Coumaric acid is also found as bound phenolic acid mainly in WUP, in addition to ferulic acid.
	Protocatachuic acid is the major free phenolic acid with small amounts of gallic, caffeic and ferulic acids and their overall contents decreased upon malting. Presence of very low amount of free ferulic acid suggested that the bound ferulic acid hydrolyzed during malting would be quickly degraded in the system.
	All the major feraxanases were detected in both rice and ragi flours with many folds higher activity in malts indicating their induction during malting. In specific, xylanase activity increased by 2 to 3 folds and ferulic acid esterase activity increased by 50 to 100 folds upon malting. Arabinofuranosidase and xylopyranosidase, two key enzymes in the feraxanase system also induced during malting. These xylanolytic enzymes, acting together, are responsible for the loosening/degradation of cell wall matrix during germination and in turn increasing the content of WEP.
	WEP is sparingly soluble in water and its content (water soluble non-starch polysaccharides – NSP) increased by 3 to 5 folds up on malting. The major portion of water soluble NSP is arabinoxylan type of polysaccharide as indicated by sugar composition and it contained high amount of uronic (2.6 – 6.1%) and ferulic (492.5 – 528.0 g/g) acids.
	Water soluble NSP was fractionated on DEAE-cellulose into 5 fractions by eluting with water, 0.1 and 0.2 molar ammonium carbonate (AC) and 0.1 and 0.2 molar NaOH. The major (0.1 molar AC eluted) fraction is arabinoxylan type of polysaccharide with high amount of ester-linked ferulic acid as indicated by its strong UV absorption and HPLC analysis, and thus was designated as water soluble feruloyl arabinoxylans (feraxans). Interestingly, ferulic acid content of malt feraxans is around 12 and 7 folds higher than native (un-germinated) feraxans for rice and ragi respectively. On the contrary, ferulic acid content of 0.2 molar AC eluted fractions was higher in native polysaccharides compared to malts. This indicated possible mobilization of feruloyl arabinoxylans during malting due to the action of xylanolytic enzymes.
	Sephacryl S-300 gel permeation chromatography yielded two peaks each for native and malted rice and ragi water soluble feraxans. They were further purified on Sephacryl S-300 and their homogeneity was ascertained by HPSEC, capillary and cellulose-acetate paper electrophoresis.

Chapter 4: Structural characterization of water soluble feraxans
This chapter highlights the results regarding structural features of water soluble feraxans from rice and ragi.
The molecular weight of purified feraxans ranged between 15,400 to 2,31,500. Molecular weight of feraxans decreased upon malting and the yield of high molecular weight peaks also decreased. This is due to the action of xylanolytic enzymes, in turn leading to the better extractability/solubility of degraded polysaccharides in water.
	Purified feraxans have high arabinose to xylose (Ara/Xyl) ratio and are rich in uronic (8.0 – 13.4%) and ferulic (54.0 – 1471.6 g/g) acids, which are higher in malt feraxans. The presence of high amount of galactose seems to be the characteristic feature of rice and ragi water soluble feraxans.
	Methylation analysis of the carboxyl reduced feraxans showed very high amount of 2,3,5-Me3-arabinose indicating that majority of arabinose residues are terminally linked. Detection of di-methylated arabinose residues indicated the presence of branching site provision for arabinose and ester-linked ferulic acid. Presence of terminally linked galactose and glucuronic acid (4-O-Me) are confirmed by their tetra methyl derivatives. Di and mono-methylated xylose residues are in almost equal amounts and un-methylated xylose is found in good amount indicating high branching.
Periodate oxidation and Smith degradation studies showed that about 60% of sugar residues have adjacent free hydroxyl groups, which is in close agreement with the methylation and PMR data.
The low negative optical rotation values (-0.3 to -7.4) indicated the polymer primarily to be β-linked. Signals corresponding to -L-arabinofuranoside (δ ~110 ppm) and β-D-xylopyranoside (δ ~104 ppm) are detected in the 13C-NMR spectra of water soluble feraxans. Glucuronic acid is found to be in 4-O-Me form as indicated by 13C-NMR spectral signals at ~178 ppm (for >C=O), ~98.8 and ~72.1 ppm (for C-1 and C-3 of -D-glucuronic acid) and ~59.5 and ~18.0 ppm (for -O-CH3). It is also confirmed by GLC-MS analysis.
	Proton magnetic resonance (PMR) spectra of feraxans showed almost equal distribution of di, mono (2/3) and un-substituted xylose residues as quantified by the integration of the anomeric signals arising from the arabinose residues. Interestingly, the amount of di-substituted xylose increased in malt feraxans with concomitant decrease in the content of mono-substituted residues. On the other hand, amount of un-substituted residues remained almost equal in both native and malt feraxans. Similar trend is observed both in rice and ragi feraxans.
With their higher Ara/Xyl ratio and lower molecular weight, malt feraxans have higher di-substituted xylose residues. The substitution pattern of xylose residues is correlated with Ara/Xyl ratio and molecular weight of feraxans. There is a trend in the xylose substitution pattern. As the Ara/Xyl ratio increases and/or molecular weight decreases, content of di-substituted xylose residues increases while the un-substituted residues remain overall same. A trend of decrease in the Ara/Xyl ratio with increasing molecular weight is also observed.
The PMR spectra showed the signals corresponding to ferulic acid bound to the water soluble feraxans. Infrared spectra of feraxans showed signals typical to arabinoxylans with uronic/ferulic acid >C=O signal at ~1730 cm-1.
	With this information in hand, a structural model has been proposed for rice and ragi water soluble feraxans. They have a β-linked xylose backbone with -linked arabinose residues as side branches, similar to other cereal arabinoxylans. However, they differed in many other respects. They are of small molecular weight and have high Ara/Xyl ratio and hence highly branched, with almost equal amount of di, mono and un-substituted xylose residues. They are particularly rich in O-2 substituted xylose residues unlike many other cereal arabinoxylans especially from wheat. Presence of high amounts of galactose, glucuronic (4-O-Me) and ferulic acids are the characteristic features of water soluble feraxans.
	In spite of their positions in the widely separated clades, water soluble feraxans from rice and ragi are essentially similar, and structurally resembled highly branched regions of rye and maize arabinoxylans than to wheat arabinoxylans. Water soluble feraxans from malts are of low molecular weight with higher Ara/Xyl ratio and higher content of ferulic acid. This is probably due to the action of xylanolytic enzymes induced during malting which preferentially acted upon the less substituted region of large molecular (native) feraxans.

Chapter 5: Functional characterization of water soluble feraxans
	This chapter presents the findings of functional characterization of water soluble feraxans.
	Water soluble NSP/feraxans showed many functional characteristics. With their high amount of bound ferulic acid, water soluble NSP/feraxans exhibited very high antioxidant activity. The activity pattern observed for different fractions could well be correlated with their bound ferulic acid content. However, antioxidant activity of feraxans is several folds higher than the expected activity due to their bound ferulic acid content. This is, in part, related to the molecular weight/chain length of the polysaccharides. Possible antioxidant effect of negatively charged sugar residues is also shown.
	Water soluble NSP/feraxans exhibited very low viscosity except for ragi malt NSP. This property may make them ideal to be incorporated in fibre/antioxidant depleted/deprived foods/drinks requiring low viscosity. Changes in the viscosity in relation to concentration, temperature and pH are also shown. Interestingly, due to the bound ferulic acid, feraxans showed different trends in viscosity with respect to pH in different buffers. The presence of NaOH in the alkaline pH hydrolyses hydrophobic bound ferulic acid and increases the viscosity of feraxans due to freed –OH groups and increased hydrophilic interactions.
	Despite considerable amount of bound ferulic acid, water soluble NSP/feraxans showed no gelling ability. However, they showed good foam stabilization property. Water soluble NSP has higher foam stabilization effects compared to purified feraxans possibly due to the cumulative effect of several polysaccharide populations in NSP.
	Incorporation of water soluble NSP into wheat dough resulted in overall positive effects. Farinograph values indicated higher water absorption and lower dough development time with slightly lower dough stability. Both extensibility and resistance to extension are increased upon the addition of water soluble NSP, the effect is similar to that of dough improvers. Amylograph studies showed increased viscosity of wheat dough upon the addition of NSP.
	Test baking indicated improved bread characteristics with the addition of water soluble NSP. Weight, loaf volume and specific volume are increased, while firmness of bread decreased. Thus addition of water soluble NSP/feraxans has overall positive functional effects on dough compared to the negative effect exerted by their insoluble counterparts.
	The in vitro fermentation characteristics/prebiotic activity of water soluble NSP/feraxans are studied with probiotic cultures of lactic acid bacteria. In general, feraxans are only partly fermented by few lactic acid bacteria, which are able to utilize arabinose or xylose. Feraxan non-fermenters could not utilize constituent sugars – especially xylose. Degradation/fermentation of feraxans is constrained by the xylanolytic enzymes especially lack of xylanase in the probiotic bacteria.
	Utilization of feraxans by lactic acid bacteria resulted in increased OD, dry cell mass and viable cell counts, and concomitant decrease in the pH, which is related to the production of SCFA. Acetate is the chief SCFA produced. Arabinofuranosidase, the key enzyme in the feraxans’ degradation is shown to be induced in cells by the presence of pentose sugars/feraxans in the culture medium. Rat cecal/faecal mixed cultures completely degraded feraxans, which is related to their high xylanase activity. Pre-hydrolysis of feraxans with xylanase facilitated their fermentation by lactic acid bacteria. Pure cultures of lactic acid bacteria, thus have limited ability to ferment feraxans and their complete fermentation might require consortium of bacteria like in mixed cultures.
	Although many food borne pathogenic bacteria are able to ferment constituent sugars, they are unable to utilize feraxans. The culture broth of lactic acid bacteria grown on feraxans showed antimicrobial/bacterio-static activity towards these pathogenic bacteria. The water soluble feraxans with their ability to support the growth of probiotic lactic acid bacteria are shown to have prebiotic activity. The malt feraxans showed slightly better functionality compared to the native ones.
	Overall, a comparative investigation is made on the structural and functional characteristics of water soluble feraxans from rice and ragi, and their changes upon germination.

	A summary and conclusions are given at the end of results and discussion section.

	The thesis ends with a list of references arranged in alphabetical order.


(Dr. G. MURALIKRISHNA)			        (R. SHYAMA PRASAD RAO)
Guide									Applicant













Identification and Characterization of Novel Genes involved in Human Spermatogenesis

Aboobacker, V.M.

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i



ABSTRACT


	The  thesis entitled “Approaches Towards Synthesis of Enantiomerically Pure Ethyl (R)-2-Hydroxy-4-Phenyl Butyrate (HPB) Ester and (R)-3-Amino-3-Phenyl-1-Propanol Through Biocatalysis" has been divided into four chapters .

Chapter I deals with  a brief introduction to biocatalysis . 

Chapter II deals  with lipase mediated synthesis of enantiomerically pure (R)-and(S)-enantiomers of 2-acetoxy-4-phenyl-(E)-but-3-enenitrile. It has been divided into four sections. 

Section I describes the determination of  enantiomeric excess of 2-acetoxy-4-phenyl-(E)-but-3-enenitrile  and 2-hydroxy-4-phenyl-(E)-but -3-enenitrile by chiral HPLC.
 
Section II deals  with preparation and lipase mediated resolution of the racemic mixture via enantioselective alcoholysis on a laboratory scale. 

Section III describes a novel porous vessel bioreactor for the synthesis of (R)-2-acetoxy-4-phenyl-(E)-but-3-enenitrile with lipase immobilized in gelatin matrix (gelozymes) on a large scale (100 g). 

Section IV deals with the conversion of nitrile to (E)-2-hydroxy-4-phenylbut-3-enoic acid ethyl ester and its enzymatic resolution  with Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PSL).
 
Chapter III has been divided into two sections. 
Section I describes a survey of recent literature in the application of baker’s yeast in organic synthesis. 
Section II deals with the enantio- and the regiospecific reduction of ethyl 4-phenyl-2,4-dioxobutyrate with baker’s yeast leading to the synthesis of ethyl (R)-2-hydroxy-4-phenyl butyrate (HPB) ester. 

Chapter IV is divided into three sections.
Section I deals with the known methods for the synthesis of chiral 1,3 amino alcohols. 

Section II deals with the biotechnological applications of penicillin G acylase in organic synthesis.

Section III deals with the synthesis of  3-amino-3-phenyl-1-propanol from 3-oxo-3- phenyl-1-propanol and its resolution with penicillin G acylase.

Chapter I    : Biotransformations In Organic Synthesis  An Overview.
	Enzyme biocatalysts are finding increasing applications because of the growing demand for biotransformations with a high chemo- and stereospecificity in the fine chemicals industry such as pharmaceuticals, agrochemicals, and health care products. Incorporation of biotransformation steps, using microorganisms or isolated enzymes, is increasingly being exploited both in industry and academic synthesis laboratories. The primary consideration for incorporation of a biotransformation in a synthetic sequence is the regio- and stereo-control that can be achieved using an enzyme-catalyzed reaction. Present chapter describes a few aspects in the area of enzyme-catalyzed organic synthesis.   







Chapter II   : Approaches Towards Synthesis Of Enantiomerically Pure Ethyl (R)-2-Hydroxy-4-Phenyl Butyrate (HPB) Ester

Section I   : Determination of Enantiomeric Excess of 2-Acetoxy-4-Phenyl-(E)-But -3-Enenitrile by  Chiral HPLC and 2-Hydroxy-4-Phenyl-(E)-But-3-Enenitrile.
	2-Acetoxy-4-phenyl-(E)-but-3-enenitrile 1 is an important intermediate in the synthesis of ethyl (R)-2-hydroxy-4-phenyl butyrate (HPB) ester. Determination of its enantiomeric purity is based on chiral HPLC analysis on a chiral stationary phase. The reaction mixture on enzymatic reaction of racemic ester 1 consists of the (R)- and (S)-acetates 2 & 3, and the (R)- and (S)-cyanohydrins 4 & 5 accompanied by the formation of cinnamaldehyde 6 as the byproduct (Fig-1).	

Fig-1: Components of reaction mixture

The reaction mixture after proper dilution can be directly injected on the column for the analysis where the peaks for the (R)- and the (S)- cyanohydrins are well resolved. 

The optimized conditions for chiral HPLC analysis are as follows: column Chiralcel OD, 5x250 mm (Daicel Chemical Industries, Japan);  mobile phase 10% isopropanol in hexane;  flow rate of 0.7 ml/min.  and  detection wavelength  255 nm. Under these conditions retention times are:  aldehyde 6, 11.05;  (S)-ester 3, 14.56; (R)-ester 2, 15.63; R-cyanohydrin 4,  24.72; (S)-cyanohydrin 5, 27.50 min.

Section  II  :  Lipase Mediated Synthesis of Both (R)- and (S)-Enantiomers of 2-Acetoxy-4-Phenyl-(E)-But -3-Enenitrile
	Present chapter describes the preparation of racemic title compounds prepared from cinnamaldehyde, acetic anhydride and sodium cyanide under phase transfer catalysis and its resolution via lipase catalyzed alcoholysis. The methodology is summarized in Fig-2.



Figure-2: Preparation of (R)- and (S)-enantiomers of 2-acetoxy-4-phenyl-(E)-but-3-enenitrile by lipase catalyzed alcoholysis. 

Lipase from Pseudomonas sp. shows selectivity towards (S)-cyanohydrin acetate while the lipase from Candida rugosa  shows selectivity towards the (R)-acetate. Among all the lipases, Amano Ps lipase showed highest reactivity towards 1. The enzymes were immobilized in gelatin matrix by sol-gel technique (gelozymes). 

Section III : A Novel Porous Vessel Bioreactor for Reactions With Gelozymes
	In this section we describe a scale up (100 g scale) of the enantiomerically pure cyanohydrin ester by enantioselective alcoholysis of the racemic ester using an immobilized lipase (Gelozyme) using a novel porous bioreactor. Conventional bioreactors  such as the continuous stirred tank reactors (CSTR) and packed  bed reactors were tried for the scale up but  during our studies with the lipases immobilized in gelatin matrix (gelozyme) we encountered several problems. In the present section we describe the design of a novel porous vessel bioreactor (Fig-3) for the reactions with immobilized enzymes. The porous vessel bioreactor described in the present invention eliminates  the step of filtration and overcomes the problem of enzyme loss through attrition and handling and the  biocatalyst can be used in any of the immobilized forms such as beads, flakes, pellets, granules, and extrudes; and recycled several times, thus making the process economical. 


Figure-3: A Novel Porous Vessel Bioreactor for Reactions with Immobilized Biocatalysts

Section IV : Enzymatic Resolution Of (E)-2-Hydroxy-4-Phenylbut-3-Enoic Acid Ethyl Ester 
	The synthesis of enantiomerically pure multifunctional intermediates such as (E)-2-hydroxy-4-phenylbut-3-enoic acid  is of considerable interest since these can be used as versatile intermediates for several reactions such as asymmetric epoxidation, dihydroxylation, and halogen addition. The (R)-enantiomer 9a can also be easily converted to ethyl (R)-2-hydroxy-4-phenylbutyrate (HPB ester) 8 which has gained significant importance as a raw material for production of a variety of Angiotensin converting enzyme (ACE) inhibitors such as Enalapril, Lisinopril and Benazepril.
	A direct conversion of cyanohyrin acetate 2 to 7 with ethanolic  HCl, gave the required ester in 60% yield, but the reaction was accompanied by partial racemization and it was necessary to remove the unwanted (S)-enantiomer. It is known that the stereochemical preference for the esterification at the hydroxyl function of the hydroxy acid/ester for Pseudomonas lipase is (S). Thus it was expected that the hydrolysis of the racemic hydroxy acid by the same lipase would produce the (S)-acid leaving the (R)-enantiomer unreacted. However, we found that the stereochemical preference is reversed during hydrolysis at the carboxylic acid ester function and the acid produced is (R). Similarly, Candida rugosa lipase hydrolyzes the (S)-ester preferentially and produces the (S)-acid (Fig-4). 

Figure.-4 : Resolution of racemic (E)-2-hydroxy-4-phenylbut-3-enoic acid ethyl ester with lipases

Although it was possible to prepare 9a and 9b in enantiomerically pure forms through lipase mediated enantioselective hydrolysis, the reaction with Pseudomonas cepacia lipase (Amano Ps) was severely inhibited by the product while the enantioselectivity E for CRL catalyzed reaction was only 2.5 and hence synthetically uninteresting. A new route to HPB ester was hence developed.

Chapter 3 : Baker’s Yeast Mediated Ketone Reduction
Section  I : Importance of Baker’s Yeast (Saccharyomyces cerevisiae) in ketone Reduction Gives an introduction to the application of baker’s yeast in ketone reduction.   

Section II : Enantio- and Regiospecific Reduction of Ethyl 4-Phenyl-2,4-Dioxobutyrate with Baker’s Yeast: Preparation of Ethyl    (R)-2-Hydroxy-4-Phenylbutyrate (HPB Ester)
	The synthesis of enantiomerically pure (-)-ethyl (R)-2-hydroxy-4-phenylbutyrate (HPB ester) 8 is of great interest since it is a versatile key intermediate for the synthesis of a variety of angiotensin converting enzyme (ACE) inhibitors such as Cilazapril., Benazepril, Enalapril etc.  and several strategies have been devised for its synthesis. In this section we describe enantioselective reduction of the2,4-dioxo ester 12b prepared by Claisen condensation of acetophenone with diethyl oxalate in presence of sodium ethoxide, by baker’s yeast (Saccharyomyces cerevisiae), to give (-)-ethyl (R)-2-hydroxy-4-oxo-4-phenylbutyrate 13b  (e.e. 98%) in 80 % isolated yield which is then hydrogenated over Pd-C to obtain (-)-ethyl (R)-2-hydroxy-4-phenylbutyrate 8 without loss of enantiomeric purity in quantitative yield (Fig-5). Another interesting product, the β-keto alcohol 15, was formed in 90% yield when the yeast reaction was allowed to continue for a longer time. The β-keto alcohol 15 is an important intermediate for synthesis of 1-phenyl-1,3-propanediol, an important intermediate to drugs such as Fluoxetine.
               

Fig.5: Preparation of HPB-ester via baker’s yeast reduction in key step

Chapter 4: Preparation of  3-Amino-3-Phenyl-1-Propanol from 3-Oxo-3-Phenyl-1-Propanol  and its Resolution with Immobilized Penicillin G Acylase

Section 1: Gives  a brief introduction on  the known methods to the synthesis of chiral (R)-3-amino-3-phenyl-1-propanol 


Section II: Biotechnological Applications of  Penicillin Acylases
This section gives a brief survey of usefulness of Penicillin acylase in organic synthesis

Section III : Chemo-Enzymatic Synthesis of Enantiomerically Pure 3-Amino-3-Phenyl-1-Propanol
	A number of approaches to enantiomerically pure 1,3-amino alcohols have been reported. However, these approaches require costly reagents, drastic conditions, laborious work-ups and purification. Since chirality can be introduced via an enzyme catalyzed reaction, it was considered prudent to develop a methodology based on enzyme assisted synthesis. A careful survey of literature showed that no such methodology exists for the synthesis of (R)- or (S)-3-amino-3-phenyl-1-propanol 16.
In the present section we describe an alternate, efficient and convenient  chemo-enzymatic synthesis of both (R)- and (S)-3-amino-3-phenyl-1-propanol 16 using immobilized penicillin G acylase (Fig.-6).


Fig.-6: Preparation of enantiomerically pure 3-amino-3-phenyl-1-propanol
	Reductive amination of 15 with sodium cyanoborohydride in presence of ammonium acetate gave the racemic amino alcohol 16 in  65% yield. The amino group was protected with phenylacetyl chloride under Schotten-Baumann conditions to get racemic N-phenylacetyl derivative 17 in 95% yield and  resolved with penicillin G acylase (immobilized on epoxy resin Eupergit C) to obtain the (S)-amide 17a and (R)-amino alcohol 16a  in high enantiomeric purity (e.e.>99%) and quantitative yields. The reaction was found to be completely enantioselective and only the (R)-enantiomer is hydrolyzed by the enzyme.



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