Open Access Repository of Indian Theses

KADEPPAGARI, RAVI KUMAR

  
PREDICTION OF DIENE-DIENOPHILE REACTIVITY IN 
CYCLOADDITIONS AND PROBING THE NATURE OF SHORT C-C 
BONDS: A COMPUTATIONAL APPROACH  
 
A Synopsis of thesis submitted for the degree of 
DOCTOR OF PHILOSOPHY 
(IN CHEMISTRY) 
To 
OSMANIA UNIVERSITY 
 
 
By 
G. GAYATRI 
 
Under the guidance of 
Dr. G. NARAHARI SASTRY 
 
 
MOLECULAR MODELING GROUP 
INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY 
HYDERABAD-500 007, INDIA 
 
October 2008 
G. Gayatri 
 
Synopsis 
The thesis entitled “Prediction of Diene-Dienophile Reactivity in Cycloadditions and 
Probing the Nature of Short C-C Bonds: A Computational Approach” is divided into 
seven chapters. 
Chapter 1: Introduction 
Computational methods have become increasingly popular practical tools for chemists 
to predict a reaction’s outcome and the physical properties of the chemical systems. The 
development of ab initio and density functional theory based methods and their effective 
implementation in software packages has made a great impact on virtually all branches of 
chemistry, physics as well as in biology. Organic chemistry is perhaps one of the areas 
where the contribution from computations and their impact on experimental chemistry is 
outstanding. With the advent of a range of computational tools and software packages, and 
effective implementation of various theoretical methodologies, now computational 
chemistry has become an extremely important area of research in chemistry, and 
particularly in delineating organic chemical structures and mechanisms. Besides structural 
determinations, these methods have proved to be better in explaining the reaction barriers 
and electronic characteristics of the transition structures, especially in the case of pericyclic 
reactions, energies of the molecular systems, spectroscopic properties, etc.  
Pericyclic reactions were defined by R. B. Woodward and R. Hofmann as ‘reactions in 
which all the first-order changes in bonding relationships occur in a concert or closed 
curve’. This definition arose from the systematic study of the conservation of orbital 
symmetry in a series of reactions: electrocyclic reactions, cycloaddition reactions, 
sigmatropic shifts, cheletropic reactions and group transfer and elimination reactions.  
     Gayatri 
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The first insight of the pericyclic reactions was provided by Woodward and Hofmann in 
their monograph ‘The Conservation of Orbital Symmetry’. The basic idea is that the 
reactions occur readily ie thermally allowed, when there is congruence between the orbital 
symmetry characteristics of the reactants and products, while they occur with difficulty, ie 
thermally forbidden, when there is no congruence. In other words orbital symmetry is 
maintained in concerted reactions. This has been proved by Pearson by means of 
perturbation theory. 
Another important contribution was made by Fukui with his frontier orbital theory. 
According to this theory reactions should take place in the direction of maximum overlap of 
the HOMO and LUMO of the reacting molecules. The most important interaction involves 
the HOMO and LUMO with the smaller energy gap, while the maximum overlap is 
obtained by bonding the termini with larger coefficients. This has been very much 
successful in predicting the regioselectivity of the reactions. The regioselectivity can also be 
explained by DFT based descriptors such as Fukui function indices, softness, 
electrophilicity indices, triplet spin densities etc. 
Atoms in Molecules (AIM) and energy decomposition analysis (EDA) are proved to be 
reliable methods to understand the concepts of bonding. With the AIM approach, one can 
determine if there is or not a bond involved in a system by using criteria based on the 
electron density, which is a scalar field obtained by several quantum chemistry methods or 
by experimental techniques. EDA involves the electron-electron attractive and repulsive 
interactions. 
Chapter 2:  Methodology  
Chapter 2 deals with the various computational methodologies such as Hartree-Fock 
(HF), coupled cluster(CC) method, Møller-Plesset perturbation(MP) theory, density 
functional theory (DFT) and semiempirical methods that are employed in the thesis. This is 
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followed by a discussion on the appropriateness of the adopted methodologies on each of 
the problems tackled in the thesis. After dealing with the theoretical basis for the 
quantitative methodologies employed in the thesis, we turned to the qualitative models. 
Most of these models are derived from the quantum chemical principles and they include 
frontier molecular orbital theory, density functional theory descriptors such as chemical 
hardness, Fukui function indices, electrophilicity index, softness, electrochemical potential, 
Koopmans’ theorem, Hammond’s postulate, Valence Bond Configuration Mixing, etc. 
These qualitative models are used to rationalize the computed structural, stability and 
reactivity trends. 
Chapter 3: Bottlenecks in the Prediction of Regioselectivity of [4+2] Cycloaddition 
Reactions: An Assessment of Reactivity Descriptors 
Regioselectivity has been one of the most important aspects to keep in mind and 
understanding the factors controlling regioselectivity helps a great deal in synthetic 
strategies. A quick perusal of the combined experimental and computational studies reveal 
that prediction of regioselectivity in cycloaddition reactions is still a challenging task and no 
reliable criterion exists which explains all the expected observations involving 
regioselectivity. The present chapter deals with the regioselectivity of 128 distinct reaction 
channels of Diels–Alder reactions by taking all possible combinations from a series of 
dienes (1N-a, 1N-b, 2N, 1P-a, 1P-b, 2P, 1O, 1S) and dienophiles (NE, PE, OE, SE, AE, 
OHE, MeE, CNE) as in Scheme 1. B3LYP/6-31G(d) calculations were performed on all 
the reactants, transition states and products. The predictive ability of the values to gauge the 
regioselectivity of the putative [4+2] cycloaddition reactions is analyzed. No correlation is 
obtained between the reaction energies and activation energies. The extent of asynchronicity 
is measured based on the bond order analysis and all the reactions are found to be 
asynchronous. DFT-based descriptors such as local softness (sk+ and sk–), Fukui function 
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indices (fk+ and fk–), global electrophilicity index (ω) and local electrophilicity index (ωk) 
were found to be better than the conventional FMO predictions. These results are compared 
with that of the activation energies to measure performance of the DFT based descriptors. 
The success rate of regioselectivity prediction is found to be much higher with any of the 
DFT-based descriptors, which could explain the regioselectivity in about 38-43 cases, 
compared to conventional FMO-based descriptors, which predict the regioselectivity only in 
29 out of 64 cases. 
X
Y z
XY
z
XY z
 
or
Head-Head Head-Tail
X
Y z
X
Y
z
or+
 
X = CH2, NH, PH, O, S  Y = CH, N, P 
Z = CH2, NH, PH, O, S, CHCHO, CHOH, CHCH3, CHCN 
Scheme 1 
Chapter 4: Hetero Diels-Alder Reactions: A Testing Ground for the Predictive 
Performance of Qualitative and Quantitative Methods 
When more than one possible coupling route is available, the mechanism of transition 
state formation differs for different orientations of coupling, thereby preferring one over the 
other. The major task to be solved in such cases is regioselectivity, stereoselectivity and 
exo-endo selectivity of the products. Testing the reliability of any quantitative or qualitative 
approach requires the application of these methods on a large number of compounds. The 
purpose of the current study is to perform an exhaustive and systematic computational study 
on a large number of hetero Diels-Alder reactions. A total of 64 reactions which essentially 
lead to 247 distinct reactions channels by taking all possible combinations from a series of 
dienes (1, 1N-a, 1N-b, 2N, 1P-a, 1P-b, 2P, 1O, 1S) and dienophiles (E, NE, PE, OE, SE, 
AE, OHE, MeE, CNE) are considered. B3LYP/6-31G(d), HF/6-31G(d), CCSD(T)/6-
     Gayatri 
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31G(d) and MP2/6-31g(d) calculations are performed in order to look at the method 
dependency on the activation energies and the exothemicities of the series of reactions 
under study. Concerted and stepwise reactions for a set of reactions are evaluated which 
suggest that such reactions proceed through asynchronous and concerted mechanism. 
Singlet-triplet energy gaps are computed to understand the stability of the products. All the 
reactions studied are asynchronous with the asynchronicity ranging from 0.01 to 1.18 Å.  
N N
N
O
P
P
P
S
H
H
H
H
1                    1N-a                   1N-b                    2N                     1P-a
1P-b                  2P                     1O                            1S
Dienes
Dienophiles
NH PH O S
CHO OH CH3 CN
E        NE       PE OE SE AE OHE MeE        CNE  
Scheme 2 
In general head-to-head arrangement is observed to be less asynchronous compared to head-
to-tail arrangement in case of dienophiles NE, PE, OE, SE. However in the case of 
dienophiles AE, OHE, MeE, CNE head-to-head arrangement is observed to be more 
asynchronous. syn isomer is found to be more asynchronous than the anti isomer which can 
be attributed to lone pair-lone pair  and lone pair-bond pair interactions. However for the 
TSs where acroline is the dienophile favorable secondary orbital interactions operate to 
     Gayatri 
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facilitate the regioselectivity. To estimate how the activation energies can be predicted using 
simple measure, correlations are drawn with the singlet-triplet energy gaps of the dienes and 
the dienophiles, reaction energies and HOMOdiene-LUMOdienophile and LUMOdiene-
HOMOdienophile. The regioselectivity for the present set of compounds is explained based on 
the Fukui function indices and triplet spin densities. 
Chapter 5: Competitive Diels-Alder Reactions: Cyclopentadiene and Phospholes with 
Butadiene. 
Cycloaddition reactions of phospholes have been employed to access novel polycyclic 
organophosphorus compounds. Many of the phosphorus-containing cycloadducts have 
promising applications in the fields of homogeneous catalysis and molecular materials. The 
present chapter deals with the diene-dienophile competing Diels-Alder reaction pathways of 
cyclopentadiene, 1H-, 2H- and 3H-phospholes with butadiene (Scheme 3). The results are 
explored at B3LYP level using 6-31G(d) and 6-311+G(d,p) basis sets, and at CCSD(T)/6-
31G(d)//B3LYP/6-31G(d) level. Activation barriers show that cyclopentadiene acts as a 
diene rather than a dienophile. Pathways 1 and 2 (A and B) corresponding to butadiene as 
diene and dienophile respectively are predicted to be highly competitive in the case of 1H-
phospholes.  
In case of 2H- and 3H-phospholes the reactions require lower activation energy when 
the conversion of weak C=P to C-P occurs. Secondary orbital interactions and preferable 
bispericyclic nature of transition states are responsible for the stability of endo transition 
state. Some of the transition states are bispericyclic and most of them are highly 
asynchronous. The high stability of the products resulting via path 1 can be attributed to the 
less strain in the bicyclo[4.3.0]nonadiene skeleton compared to the norbornene derivatives 
obtained from path 2. Activation and reaction energy values for these Diels-Alder reaction 
     Gayatri 
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pathways are compared with the values reported for the [4+2] cyclodimerizations of each of 
the reactants to examine the likelihood of cyclodimerizations along these pathways. 
 
  
  
  
 
 
 
  
 
  
  
  
 
 
 
 
  
 
 
 
 
  
 
 
 
  
 
 
 
 
 
BD-Cp-xPr BD-Cp-nPr
Cp-tBD-nPr Cp-tBD-xPr
Cp-cBD-nPr Cp-cBD-xPr
a-1P-tBD-nPr a-1P-tBD-xPr
a-1P-cBD-nPr a-1P-cBD-xPr
s-1P-tBD-nPr s-1P-tBD-xPr
s-1P-cBD-nPr s-1P-cBD-xPr
A
B
a-BD-1P-xPr a-BD-1P-nPr
s-BD-1P-xPr s-BD-1P-nPr
A
B
Cp BD
1P BD
2P1-tBD-nPr 2P1-tBD-xPr
2P1-cBD-nPr 2P1-cBD-xPr
2P2-tBD-nPr 2P2-tBD-xPr
2P2-cBD-nPr 2P2-cBD-xPr
BD-2P1-xPr BD-2P1-nPr
BD-2P2-xPr BD-2P2-nPr
A
B
2P BD
3P1-tBD-nPr 3P1-tBD-xPr
3P1-cBD-nPr 3P1-cBD-xPr
3P2-tBD-nPr 3P2-tBD-xPr
3P2-cBD-nPr 3P2-cBD-xPr
BD-3P1-xPr BD-3P1-nPr
BD-3P2-xPr BD-3P2-nPr
A
B
3P BD
P
H
P
P
 
Scheme 3 
 
 
     Gayatri 
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Chapter 6: Estimating the Regio- and Stereo-selectivity in [4+2] Cycloadditions of 2-Vinyl 
and 3-Vinyl Cyclic Dienes with Maleic Anhydride 
Various Diels-Alder methodologies have been implemented for a long time for 
synthesizing natural products and also in pharmaceutical chemistry. One among these 
methodologies involves the use of alkenyl-substituted heteroaromatic systems. The present 
chapter is aimed to computationally explore the regio and stereo chemical preference of 
vinyl cyclopentadiene (1 and 2) and vinyl heterocyclic (1-N, 1-O, 1-S, 2-N, 2-O and 2-S) 
systems as dienes with maleic anhydride as dienophile. Stepwise and concerted pathways of 
pristine model systems 1 and 2 as dienes with ethylene as dienophile have been 
systematically explored and they are found to proceed through asynchronous and concerted 
pathway. The activation barriers and exothermicities of 1, 1-N, 1-O, 1-S, 2, 2-N, 2-O and 2-
S with maleic anhydride are evaluated. 3-vinyl systems are found to be more reactive 
compared to 2-vinyl systems. The regio- and stereo-selectivity preferences are evaluated 
based on activation energies, reaction energies, DFT based descriptors and Atoms in 
Molecules (AIM) analysis. Extra-annular cycloadducts are more feasible compared to the 
intra-annular cycloadducts. The formation of endo adduct is found to be more facile 
compared to exo adduct which may be traced to the favorable secondary orbital interactions. 
Computational results thus obtained are compared with earlier experimental observations 
that are available.  
 
 
     Gayatri 
 - 9 -  
Y
O
O
O
+
Y
O
O
O
Y
O
O
O
Y
O
O
O
Y
O
O
O
Extra-annular addition
Intra-annular addition
Endo-orientation
Endo-orientation
Exo-orientation
Exo-orientation
Y=CH2, O, S, NH
Y
O
O
O
+
Y
O
O
O
Y
O
O
O
Y
O
O
O
Y
O
O
O
Extra-annular addition
Intra-annular addition
Endo-orientation
Endo-orientation
Exo-orientation
Exo-orientation
Y=CH2, O, S, NH
 
Scheme 4 
Chapter 7: Further Shortening of the C-C Single Bond in Substituted Tetrahedranyl 
Tetrahedrane Systems  
Bond lengths and bond strengths are fundamental parameters in chemistry. It is 
generally assumed that bond lengths and bond strengths which are given by the bond 
dissociation energy (BDE) are inversely related to each other. However such a correlation is 
not always given and there are many cases known where a longer bond may be stronger 
than a shorter bond between the same atoms. The present chapter explores the electronic 
fine tuning of the exocyclic C-C single bond length in tetrahedranyl tetrahedrane (THTH) 
     Gayatri 
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as a function of various substituents. The factors which determine the bond lengths and 
bond strengths are examined by energy decomposition analysis (EDA) and Atoms in 
Molecules (AIM) analysis. The very short exocyclic C-C single bond in THTH-X where 
X=H can be further shortened when X is a halogen atom. The calculations predict that the 
C-C bond will become shorter by ~0.012 Å when X = F, Cl, Br compared to the silyl 
subsituted compound THTH-SiH3. The EDA results reveal that the equilibrium bond length 
of the exocyclic C-C single bond is determined by the interplay of attractive forces 
(electrostatic bonding and orbital interactions) and repulsive forces (Pauli repulsion) where 
it is the latter which prevents shorter bonds. Although the hyperconjugation in THTH-X is 
slightly stronger than in substituted ethanes X3C-CX3 the main reason for the very short 
exoskeletal C-C single bond in the former compound is the very high %s character of the 
bonding orbital at carbon. AIM analysis has been done on the systems to look at the 
molecular structures and the electronic charge densities of the eclipsed and staggered 
conformers which helped in the clear demarcation of the two conformers.  
Eclipsed Staggerred
X X
X
X
X
X
X
X
X
X
X
X
X= H, CH3, OH, OCH3, NH2, SiH3, F, Cl, Br, CN  
Scheme 5 


English

Characterisation of a maltogenic alpha amylase of Aspergillus niger and studies on the enzyme overproducing mutant

P.C, SRINIVASA

PAGE  




i
Synopsis		


Plastics have become part and parcel of our everyday life and the plastic industry has emerged as a rapidly expanding industry in the past several decades. Approximately 40,000,000 tones of plastic packaging is used annually world wide, and a majority of this is put to one time use and is discarded later. This contributes to an appreciable amount of total waste stream (around 20% volume world wide) and in India its contribution is approximately 3 million tones. The treatment of waste plastics has become a serious problem because of the difficulty of ensuring reclaimed land and burning by incineration. The industry is now facing ecological and legislative issues for handling plastic raw materials and finished products. Their total non-biodegradability as well as an increased environmental consciousness by the consumers and Government bodies has paved the way to look for alternate approaches. Also due care is necessary not to deteriorate the environment by using non-biodegradable and non-recyclable materials. This development has for the best part led to focusing on alternative packaging films derived from natural biopolymers which are replenishable and completely biodegradable under a variety of ecological systems. Biopolymer films are generally prepared by using biological materials such as polysaccharides, proteins and their derivatives, which are naturally and abundantly available. Natural biopolymeric films have the advantage over synthetic biopolymers since they are totally biodegradable and are derived from renewable raw materials. They can be used effectively as an alternative to synthetic plastics. Biopolymeric films have also desirable overall mechanical and barrier properties.
 
Food packaging is an important discipline of food technology concerned with the protection and preservation of all types of foods from oxidative and microbial spoilage. The petroleum based synthetic thermoplastic materials, currently being used extensively, may gradually loose importance as packaging materials because of waste disposal and non-biodegradable problems, and as a consequence threat to the environment. As an alternative, interest in the study of biodegradable packaging films has increased steadily during the past decade. Although it is not feasible to entirely replace synthetic plastic packaging films, the biodegradable films do have potential to reduce and replace plastic packaging films in some specific applications. A clean pollution free environment is the need of the day.

	Polysaccharides such as cellulose and starch (derived from agricultural resources), chitin/chitosan (derived from marine food processing wastes) and pullulan (from microbial sources), either in their native or modified forms, as well as their blends have the ability to form films. Bioplastic consisting of synthetic monomeric or polymeric materials, graft copolymerized with natural biomolecule are also shown to be useful as biodegradable packaging materials. Use of certain additives such as plasticizers, antioxidants and antimicrobials will enhance their functional value to a great extent. Use of chitosan and its derivatives in such applications has the additional advantages of being biocompatible and antimicrobial.      
  
        Chitin, a naturally occurring and abundantly available polysaccharide obtained form crustacean wastes, consists mainly of  (1-4)-linked-2- acetamido-2-deoxy-D-glucose units. Chitosan is obtained from chitin by N-deacetylation using strong alkali. The cationic property of chitosan offers an opportunity to take advantage of its electrostatic interaction properties. Chitosan films are used in the separation of ethanol from water by evaporation, water purification, and controlled release of pharmaceuticals, but has been reported to have limited application as far as packaging film is concerned.  Therefore, it was felt desirable that a study be initiated to evaluate the properties of chitosan film prepared under different drying conditions and to modify the films by incorporating various additives and to look for their application to storage studies of fruits, vegetables, dairy and bakery products, and also to study their antimicrobial properties. With these objectives in mind, work was carried out and the results obtained are consolidated in the form of a thesis having the following layout.
  Chapter 1

This chapter provides a General introduction of the subject matter, with reference to problems due to non-biodegradable plastics, different ways of handling these plastics and different   sources of biodegradable plastics. The main focus is on occurrence and distribution of chitin/chitosan, their chemical structure, physicochemical properties, and application in various fields including food, medicine, agriculture and industry. Emphasis is given on the preparation of chitosan-based films and their application to shelflife extension of fruits, vegetables and other products. Finally, the Aim and Scope of the present investigation are indicated.

Chapter 2

This section includes a brief introduction on the preparation of chitosan films, with a detailed account of characterization of chitosan samples from two different sources. Chitosan sample (CH1) is of molecular weight 1,00,000 Da with Degree of deacetylation (DD) of 83%, whereas chitosan sample (CH2) is of molecular weight 2,00,000 Da with DD >90%. Chitosan films were wet casted on different base materials such as glass, Teflon, aluminium sheet, stainless steel sheet and polyester sheet for easy peeling off from the base, of all the polyester base gave the best quality of film. Chitosan films were prepared by using different (solvent) acids such as acetic acid, lactic acid, formic acid and propionic acid and their properties studied. Formic acid cast film had a higher tensile strength (48.34 ± 4.28 MPa) and lactic acid cast films had the lowest value (21.9 ± 4.2 MPa). On the other hand, chitosan films prepared using acetic acid showed easy handling and good film properties. Chitosan films were prepared under different drying conditions, such as ambient drying, oven drying and infrared drying. The results showed IR drying to be faster and superior in preserving desirable functional characteristics of chitosan films. Though subtle variations in the crystallinity pattern were observed between differently dried chitosan films, no significant differences were observed in their mechanical and barrier properties. Typical drying curves were been obtained for films dried at different drying conditions, and their kinetic constants determined. The sorption curves of the films showed a sigmoid shape and WVTR of the films showed an increase with higher relative humidity. The tensile strength (TS), percent elongation (%) and modulus of elasticity (ME) of films were studied at different RH, temperature, and storage period using response surface methodology.  Simultaneous optimization by desirability approach resulted in an overall desirability score of 0.8429, where in TS, % and ME values were 35.79 MPa, 19.86% and 896.73 MPa and these values were obtained when the independent variables such as temperature, RH, and storage days were 20.1°C, 40% and ~7 days, respectively. Lastly an attempt was made to fabricate a prototype model for continuous preparation of chitosan film under infrared drying condition.

Chapter 3

This chapter describes blending of chitosan with polyols (glycerol, sorbitol and PEG), fatty acids (stearic acid and palmitic acid) and a water-soluble polymer, PVA, before casting the films. The optical properties (colour density), mechanical properties (tensile strength, %, ME, tearing strength, burst strength and impact strength), barrier properties (WVP and OTR) were all determined. The chemical nature of these films was studied by FTIR, heat flow by DSC and changes in crystallinity by X-ray diffractogram.  The sorption isotherms of all blend films were studied and observed for validity of different models.   

 The result indicated that yellowness of the film increased with the addition of plasticizers. The opacity of film increased with the addition of PEG but no differences seen in glycerol and sorbitol added films. TS decreased to 6.08 and 6.24 MPa in glycerol and sorbitol containing films respectively, whereas in PEG both decreasing and increasing trends were noticed. The ME also showed a decreasing trend. The Impact strength was increased with the addition of polyols. Burst strength of the film decreased with addition of glycerol and sorbitol, but in PEG an increasing trend with a value of (190 kPa) was observed.  The WVP of native chitosan film was 0.01322 g.m/m2.day.kPa. With the addition of glycerol the WVP decreased to 0.008 and in sorbitol it increased to 0.0163 g.m/m2.day.kPa. In PEG films it was 0.019 g.m/m2.day.kPa. The OTR values decreased with the addition of PEG, whereas with glycerol and sorbitol it increased to 98.01 and 141.14 x 10-6 cc.m/m2. day. kPa, respectively.  

DSC thermogram showed a difference in the ΔH values for various polyol containing films, ΔH values increased as glycerol concentration increased in the blend films.  Water capacities of the blend films showed different characteristics, glycerol showed a early evaporation at around 125 C with high ΔH values, compared to other two plasticizers.  X-ray diffraction of polyol blend films showed no significant differences.

With the addition of fatty acid the density of blend film decreased from 1.4024 to 1.2692 g/ml in palmitic acid and 1.4024 to 1.2585 g/ml in stearic acid blend film. TS of blend film decreased with the addition of fatty acid and no significant variation was observed in % elongation and modulus of elasticity.  The WVP results showed no significant differences. The FTIR showed hydrogen bonds between hydroxyl groups and water molecules to remain intact. The methyl and methylene stretching appeared at around 2918 and 2850 cm-1, which were attributed to amide stretching. The palmitic and stearic acid blend chitosan films showed melting peaks at around 63 C and 56 C, respectively, and noticeable water content were found in these films.    
  
The optical properties and TS of the chitosan-PVA blend films increased while %elongation decreased with increase in chitosan concentration and a blend ratio of 60-40 was found to be the best which had a value of 41.24 MPa. The burst and impact strength of the blend films increased with the addition of PVA. The increase in the impact strength was attributed to the chain flexibilities of the blend films. The WVP of films decreased to 0.006 g.m/m2.day.kPa. FTIR spectrum showed a characteristic peak shifting to a lower frequency range due to hydrogen bonding between -OH of PVA and –OH or NH2 of chitosan. The blending ratio showed a regression coefficient of 0.94.  DSC thermogram of chitosan-PVA showed endotherm around 140-160 C and 215 C. The exotherm peak of chitosan at around 300 C was diminished as PVA concentration increased due to overlapping of PVA endotherm.  X-ray diffraction patterns showed 2 peaks 11.92, 21.28 and 23.28, the latter was due to drying of chitosan acetate salts. The intensity of peak around 19 increased as the concentration of PVA increased. Moisture sorption isotherms showed sigmoid pattern, indicating the influence of polyols/fatty acid/PVA on the blend film. Sorption data were useful in choosing suitable packaging material having a desirable water vapour barrier property. The GAB model showed a better fit compared to other models and was applicable to a wide range of water activity values.  Chitosan blend films with polyols and fatty acids showed complete bio-degradation. 

Chapter 4

This chapter describes storage studies of fruits and vegetables, dairy and bakery products using chitosan film.  Shelflife extension of mango fruits was studied and compared with the sensory profile.  Control fruits were found spoiled in 10 days, whereas chitosan covered fruits showed better sensory quality.  The latter were observed with high levels of carotenoids, sugars, free from off-flavour and fungal growth compared to LDPE covered fruits. In PCA plot the chitosan-covered fruits showed several desirable quality attributes. At the end of storage period (20 days), the chitosan covered fruits showed better sensory quality than LDPE covered fruits. 

Tomatoes stored in chitosan covered cartons showed uniform colour development, free from off –flavour and retention of sugar level for more than 30 days of storage, compared to control fruits which had 15 days of storage, while LDPE packed tomatoes showed non-uniform colour development. Similarly, chitosan and LDPE packed bell pepper pods showed a shelflife of 16 days. The textural studies data showed that modified atmosphere packaged conditions can extend the shelflife of tomato and bell pepper, which are beneficial for sustainable fluctuating market availability associated with limited and seasonal availability.  No differences were observed in headspace gas levels during the storage period, while greater changes were observed in colour development and its retention. Changes in chemical parameters of stored fruits were very marginal. Sensory profiling indicated that synthetic film packaged fruits exhibited loss of typical aroma, while chitosan packaged fruits retained it.  Firmness and development of red colour and retention of green colour are the major factors in deciding the price and market value of tomatoes and bell pepper, respectively. The unpackaged fruits showed decaying symptoms at an early stage than the packaged samples, which indicated a beneficial role of chitosan films for extending the shelflife of bell pepper. 

  Dairy product (peda) stored in chitosan coated butter paper gave considerably extend storage. A similar trend was seen in the package of bakery product (bread). Further, incorporation of chitosan into the dough gave an additional 30 days of shelflife extension.


Chapter 5

This chapter deals with antimicrobial characteristics of chitosan film. Our results conclusively demonstrated the antimicrobial efficiency of chitosan film even at very low concentrations in liquid medium. Although there was lesser diffusion of chitosan on the agar surface, there was no visible microbial growth, which makes it a potential packaging film for use in food preservation. Growth curve of microorganisms showed effectiveness of chitosan film in inhibiting growth of microbes. SEM studies revealed the effectiveness of chitosan film as an antimicrobial agent. A chitosan-based film with a broad spectrum of antimicrobial activity will have a higher potential as food packaging material. 

 	The presentation is finally concluded with a note on the Highlights of Significant Findings from this investigation, followed by Bibliographic listing of the literature referred to in preparing this thesis.



         		
P.C.SRINIVASA            		                 R.N.THARANATHAN    
  (Candidate)                               		        (Guide)








Process development of biodegradable chitosan-based films and their suitability for food packaging

R., CHETANA

Abstract
The thesis entitled “Synthesis of C-Linked Carbo-3, 4-Amino Acids, Peptides and Studies Towards the Synthesis of Marinomycin A” is divided into three chapters. Chapter I is dealt with the synthesis of new mixed -peptides from the -Caa monomers having D-xylose, D-lyxose, D-ribose and D-arabinose side chains, to understand the impact of the side chains. Chapter II is divided into two sections, wherein Section A is dealt with the synthesis of new -amino acid and its conversion into /-peptides to realize 12/10-helices, while, the Section B is dealt with the synthesis of new bifunctional -amino acid and its conversion into mixed -peptides to realize 9-helix. Chapter III is dealt with the attempted synthesis of marinomycin A. 

Chapter I: Synthesis of Mixed -Peptides from C-Linked Carbo--Amino Acids (-Caas) and Study on the Impact of Carbohydrate Side chain.
	Carbohydrates as well as peptides and proteins are essential biopolymers of life. To perform biological functions, correct folding of biopolymers which creates active site is crucial1. β-Amino acids are much less frequent in nature than α-amino acids and have also been found incorporated into naturally occurring peptides with important pharmacological properties2. Seebach et al.3 and Gellman et al.4 were the first to report β-peptides with novel helical structures.5 Based on the structures of nikkomycins6, our group reported the synthesis of C-linked carbo-β-amino acids7 (β-Caa), with carbohydrate moieties as side chains and converted them into β-peptides8 with 12/10-helical patterns. 
  This chapter is dealt with the synthesis of new mixed carbo--peptides from -Caas having D-xylose, D-mannose, D-ribose and D-arabinose side chains. The main idea behind the study is to understand the sterochemical as well as other structural features of the carbohydrate side chains that are essential for minimum compatibility to adopt well-defined helices in the mixed peptides.
       Thus, the requisite -Caa monomers 1-6 were prepared by aza-Michael addition with benzyl amine on the esters derived from the corresponding aldehdyes obtained from D-glucose, D-mannose, D-ribose and D-arabinose (Scheme 1). Thus, reaction of the esters 7 with benzyl amine gave the esters 8 and 9 as separable mixtures of diastereoisomers. Hydrogenolysis of 8 and 9 followed by reaction of the respective amines with Boc2O and Et3N in THF afforded the -Caa monomers 1-6.   
        
Scheme 1

Mixed -peptides from (R)-β-Caa(x) and (S)-β-Caa(l) with D-xylose and D-lyxose side chains
      	 Based on the molecular mechanics calculations of Wu and Wang,9 and from the earlier studies of our group, in the present study on the synthesis of mixed-β-peptides from β-Caa(x) 2 and β-Caa(l) 3, it was planned to adopt the same stratergy of alternating chirality. Both the side chains are furanoside in nature and in D-form, while, the acetonide groups are in 1,2- and 2,3-postions and are in α-and β-face in 2 and 3 respectively. 
Synthesis of mixed-β-peptides is based on the 1:1 alternating use of (R)-β-Caa(x) 2 and (S)-β-Caa(l) 3 (Scheme 2). The peptides were prepared using standard peptide coupling conditions in the presence of EDCI and HOBt, with DIPEA as a base in dry CH2Cl2. Accordingly, the dipeptide 10, tetrapeptide 11 and hexapeptide 12 were prepared by adopting segment condensation method. NMR, CD and MD studies clearly indicated the presence of novel right-handed 10/12-mixed helical pattern in the mixed--peptides 11 and 12 (Scheme 2).
Mixed-β-peptides from (R)-β-Caa(x) and (S)-β-Caa(r) with D-xylose and D-ribose side chains
 	In a further study to understand the impact of the carbohydrate side chains, a new design of a mixed peptide was proposed wherin the β-Caa(x) with D- xylose as side chain will remain the same, while, -Caa(l) with D-lyxose side chain is replaced with -Caa(r) with D-ribose as side 
Scheme 2

chain, wherein, the sterocentres at C-1, C-2 and C-3 are enantiomeric to -Caa(l). Accordingly, the peptides 13-15 were prepared from (R)-β-Caa(x) 2 and (S)-β-Caa(r) 5 in the presence of EDCI and HOBt, with DIPEA as a base in dry CH2Cl2 (Scheme 3). The NMR, CD and MD studies of peptides 14 and 15 clearly indicated the presence of robust 12/10-mixed helix (Scheme 3).
Scheme 3

Mixed-β-peptides from (R)-β-Caa(l) and (S)-β-Caa(Da) with D-lyxose and D-arabinose side chains
	From the above two designs using β-Caa(x) having a D-xylose side chain with two different β-Caas having D-lyxose and D-ribose side chains, which are enantiomeric at the C-1, C-2 and C-3 positions, the mixed peptides have shown the presence of 12/10-mixed helices. Thus, it indicated that in these systems the sugar side chain has no impact on helix formation. Hence, in a new design, it was proposed to use a different β-Caa side chain in place of D-xylose moiety to understand more about the side chain impact. In the new design, β-Caa(Da), having a D-arabinose side chain, was used in place of β-Caa(x) monomer. The stereochemistry of the 1,2-O-isopropylidene group in β-Caa(Da) 6 is now inverted (in β-face) unlike in β-Caa(x) 1. Accordingly, the peptides 16-18 were prepared from (R)-β-Caa(l) 4 and (S)-β-Caa(Da) 6 in the presence of  EDCI, HOBt and DIPEA in CH2Cl2 (Scheme 4).
Scheme 4

Thus, even though we were successful in the synthesis of peptides having alternating β-Caa(l) 4 and β-Caa(Da) 6, the spectral analysis revealed that they exist as rotamers in CDCl3 solutions. Due to the presence of rotamers always, the secondary structures could not be understood well in the NMR studies on these peptides. Thus, for the first time it has become evident that the compatibility of the side chain with appropriate sterochemistries is equally important for a robust helix formation in this class of β-peptides.
Mixed-β-peptides from (R)-β-Caa(l) and (S)-β-Caa(r) with D-lyxose and D-ribose side chains
From the above study it was well understood that the compatibility of the side chain is also an essential and important aspect in defining a proper secondary helical structure. To further understand the side chain impact, mixed β-peptides were proposed from alternating use of (R)-β-Caa(l) 4 and (S)-β-Caa(r) 5 with D-lyxose and D-ribose side chains, wherein, though both the side chains have 2,3-O-isopropylidene groups, the stereocentres at the C-1, C-2 and C-3 in both these  monomers are enantiomeric to each other. Accordingly, the peptides 19-21 were prepared from (R)-β-Caa(l) 4 and (S)-β-Caa(r) 5 in the presence of  EDCI, HOBt and DIPEA in CH2Cl2 (Scheme 5).
Scheme 5
      
 In 1H NMR of 20 and 21, characteristic information on a well definded secondary structure was not observed. Further, solvent titration studies did not show their involvement in H-bonding.
Thus, mixed -peptides were prepared utilizing the concept of ‘alternating chirality’ from -Caas 1-6 having D-xylose, D-lyxose, D-ribose and D-arabinose side chains. The spectral and analytical studies amply indicated that, all the side chains are not compatible in giving novel secondary structures. The structural features and enantiomeric nature of the carbohydrate side chain plays a role in defining the conformations in such peptides. 

Chapter II: Synthesis of New C-Linked Carbo-γ4-Amino Acids and Their use in the Synthesis of Peptides with Novel Helical Structures
Section A: Synthesis of New C-Linked Carbo-γ4-Amino Acids (γ4-Caas) and their Conversion into /-Hybrid Peptides
Most of the unnatural foldamers reported so far have homogeneous backbones i.e., they are constructed from single class of amino acids. Heterogeneous backbones composed of two or more residue types also display well-defined folding behavior, and such an exploration is important because mixing monomer classes leads to an exponential increase in the range of potential foldamers.
Based on our earlier findings on /-hybrid peptides10 derived from (S)--Caa and L-Ala repeats with unprecedented 11/9-mixed helices and recent theoretical studies by Hofmann et al.11, we envisaged that /- hybrid peptides from (S)--Caa and L-Ala would adopt novel 10/12- or 12/10-mixed helical structures in such peptides. Inspired by these findings, (S)--Caa(l), a C-linked carbo--amino acid 22 from D-mannose, was prepared by homologation method from the known -amino acid (Scheme 6).  The new design on /-hybrid peptides from alternating L-Ala and (S)--Caa, would thus be the first motif devoid of β-amino acid for a 12/10-mixed helix. 
Scheme 6

Accordingly, the di-, tetra- and hexapeptides 26-28 were prepared from alternating (S)-γ-Caa 22 and L-Ala in the presence of  EDCI, HOBt and DIPEA in CH2Cl2 (Scheme 7). NMR, CD and MD studies clearly indicated the presence of novel right-handed 12/10-mixed helical pattern in the γ/-hybrid peptides 27 and 28 (Scheme 7). 
Scheme 7

Similarly, new /γ-peptides 29-31 (Scheme 8) with L-Ala at the N-terminus were also prepared from L-Ala and (S)-γ-Caa 22 under standard peptide coupling conditions (EDCI, HOBt and DIPEA) in CH2Cl2. NMR, CD and MD studies evidently proved the presence of a right-handed 12/10-mixed helical pattern in /γ-hybrid peptides 29-31.
Scheme 8


Section B: Synthesis of New Bifunctional C-Linked Carbo-γ4-Amino Acid and Conversion into Mixed -Peptides Using Alternating -Caa and GABA
Based on our earlier findings8b on mixed β-peptides and -peptides derived from alternating (S)-β-Caa/β-hGly and -Caa(x)/GABA, resulting in left-handed 12/10-mixed helices 9-helix,12 respectively, a new bifunctional (S)--Caa(l) 32 was proposed. The new (S)--Caa(l) 32 was prepared by homologation of the bifunctional -amino acid prepared from D-mannose (Scheme 9).  The new (S)--Caa(l) 32 would not only result in the 9-helix in the mixed peptides with GABA, but also would help in the solubility in water, that would enhance the possibility for biological evaluation.
Using -(S)-Caa(l) 32, previously reported 4-(S)–Caa and GABA, di-, tetra- and hexapeptides 36-38 under standard peptide coupling conditions (EDCI, HOBt and DIPEA) in CH2Cl2 were prepared. NMR and CD studies confirmed the presence a left-handed 9-helix in tetrapeptide 37. Though the presence of some conformation is indicated in CD of 38, it could not be deducted from NMR due to severe overlapping of the signals (Scheme 10). 
Scheme 9

Scheme 10     


Chapter III. Attempts Directed Towards the Synthesis of Marinomycin A

	Marinomycin A 39, a macrodiolide, isolated from a novel marine Actinomycete13, (Marinispora strain CNQ-140), exhibited potent antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF), with MIC90 values ranging from 0.1 to 0.6 µM. Marinomycin A 39 is a C2 symmetric14 system and arose great interest to the organic chemists.15a,b 
The retro analysis of 39 indicates that it can be prepared from the dimerisation of 40, which in turn could be envisaged from the fragments 41-43 (Scheme 11). 
Scheme 11      

	Synthesis of fragment 41 is achieved as shown in Scheme 12. The chiral epoxide 44,on reaction with 45, using n-BuLi, at -78 oC gave the alcohol 46 in 64% yield, which was protected as MOM ether using MOMCl and DIPEA in CH2Cl2 at 0 oC to offord 47 in 93% yield. Compound 47 was selectively deproteced using cat. PTSA in MeOH to give primary alcohol 48 in 87% yield, which on readuction with Red-Al in THF at 0 oC afforded trans allylic alcohol 49 in 91% yield. To generate the next chiral centre, this allylic alcohol was subjected to epoxidation under Sharpless epoxidation conditions using (+)-DIPT, Ti(i-OPr)4 and cumene hydroperoxide at -20 oC to obtain the chiral epoxide 50 in 77% yield. Epoxide 50 was then selectively opened as 1,3-diol using Red-Al in THF in 85% yield, which was subsequently protected using PMB acetal and cat. PTSA as the benzylidene derivative 52 in 67% yield. The acetal 52 was regioselectively opened into primary alcohol 53 with DIBAL-H in dry CH2Cl2, which on oxidation with Dess-Martin periodinane at 0 oC gave aldehyde 54 in 96% yield. Witting olefination of 54 gave the
Scheme12
  

terminal olefin 55. in 72% yield. The olefin on epoxidation with m-CPBA in dry CH2Cl2 furnished the racemic epoxide 56 in 85% yield, which on Jacobsen Kinetic resolution using (R, R)-Jacobsen catalyst afforded the chiral epoxide 41 (C1-C7 fragment with three out of five stereocentres) in 43% yield.
	
Synthesis of 42 was initiated from propargyl alcohol (Scheme 13). The allylated product  58 was  subjected to epoxidation using m-CPBA in CH2Cl2 to afford the racemic epoxide 59 in 92% yield, which was resolved by Jacobsen Kinetic resolution using (S,S) Jacobsen catalyst to 
Scheme13

afford the chiral epoxide 60 in 44% yield. The epoxide 60 was treated with ethyl propiolate in presence of n-BuLi at -78 oC to give the ester 61 in 84% yield, which on reaction with MOMCl and DIPEA in CH2Cl2 at 0 oC furnished 62 (C8-C16 framework with one stereocentre) in 92% yield. Several attempts to functionalize 62 met with failure.




Studies on the use of sucrose alternatives in traditional sweetmeats

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Strain and process improvement for polygalacturonase production by aspergillus carbonarius

De, Jaysankar

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ABSTRACT 
 
 
 
 
 
 
 
 
 
Abstract                                                                                                                                                             i                                                                                                                                              
 MIXED OXIDES AND METAL SUPPORTED APATITE CATALYSTS 
FOR ORGANIC TRANSFORMATIONS  
 
The thesis mainly deals with the development of heterogeneous solid catalysts and 
their applications in organic transformations particularly C-C bond formation reactions. 
Chapter I highlights the importance of heterogeneous catalysis and discuss various options 
for the synthesis of heterogeneous catalysts. The need and development of cleaner and 
greener alternative technologies using truly heterogeneous catalytic system in the synthesis 
of pharmaceuticals and fine chemicals is also described. Chapter II describes the 1,4-
addition of phenylboronic acids to α,β-unsaturated carbonyl compounds by using rhodium 
fluorapaptite (RhFAP) catalyst. Chapter III deals with the conversion of azides to N-(tert-
butoxycarbonyl)amines and the synthesis of 1,3-butadienes by using palladium fluorapatite 
(PdFAP) catalyst. Chapter IV deals with the synthesis of tetrazoles and propargylamines 
by using zinc based catalysts. Chapter V mainly deals with the synthesis of α-diazo-β-
hydroxy and α-diazo-β-amino carbonyl compounds using Mg/La mixed oxide catalyst and 
β-keto esters by using and Pd0/Mg-La mixed oxide catalyst. 
 
Chapter-I: Introduction 
 
This chapter describes the various catalyst / process options available for an 
industrial chemist to effect different organic transformations. It includes a brief 
introduction of homogeneous, heterogeneous catalysis and basic concepts commonly 
encountered in catalysis such as selectivity, turnover number, atom economy etc. This 
chapter also describes the structure, properties and applications of basic supports such as 
apatites, hydrotalcites, mixed oxides and their importance as catalysts in organic reactions. 
Abstract                                                                                                                                                             ii                                                                                                                                              
The need for the design and development of environmentally cleaner catalytic 
methodologies using supported solid catalysts is also presented in this chapter. 
Chapter-II: Rhodium fluorapatite catalyzed conjugative addition of phenylboronic 
acids to ,-unsaturated carbonyl compounds  
Rhodium catalyzed carbon-carbon bond formation via cross-coupling of 
organoboron compounds and organic electrophiles is one of the most important reactions 
in organic synthesis. Rhodium catalyzed conjugate addition of arylboronic acids to ,-
unsaturated carbonyl compounds has received considerable attention in the field of organic 
synthesis, ever since it was reported by Miyaura et al. in 1997. The discovery of such and 
important reaction has triggered much research activity in the area of carbon-carbon bond 
formation catalyzed rhodium complexes. Despite the advantages of homogeneous metal 
complex catalysts, difficulties in recovering the expensive catalyst metals and ligands from 
the reaction mixture severely limit their industrial applications. Moreover the use of toxic 
and expensive phosphine ligands is undesirable in viewpoint of environment. 
Heterogeneous catalysis is particularly attractive as it allows production and ready 
separation of large quantities of products with the use of a small amount of catalyst. 
The present chapter describes the conjugate addition of phenylboronic acids to ,-
unsaturated carbonyl compounds to yield conjugate addition products (Scheme 1). 
R1B(OH)2
RhFAP
   Methanol,
1,5-cod, N2,  65 °C
+
O
R2
O
R2R
1
 
Scheme 1. Conjugate addition of arylboronic acids to α,β-unsaturated carbonyl compounds 
catalyzed by RhFAP 
Abstract                                                                                                                                                             iii                                                                                                                                              
Chapter-III: One pot approach for the synthesis of N-(tert-butoxycarbonyl)amines 
and 1,3-butadiene derivatives using highly efficient palladium fluorapatite catalyst 
This chapter is divided into two sections. 
Section I: This section deals with the chemoselective reduction of azides to N (t-
butoxycarbonyl)amines by using PdFAP catalyst. 
Section II: This section deals with the three component coupling of aryl iodides, alkynes 
and alkenes by using PdFAP catalyst. 
Section I: Palladium fluorapatite catalyzed chemoselective reduction of azides to N-
(tert-butoxycarbonyl)amines 
 Organic azides are an important class of compounds, particularly for producing 
primary amines via reductive transformation, and several reducing agents have been 
developed to date. In a closely related field, protection of amines with appropriate 
protecting agents also plays an important role in multistep synthesis. Among the various 
amine-protecting group, the tert-butoxycarbonyl (Boc) group is frequently used due to its 
chemical stability to acids, bases, and hydrogenation. Based on this background, it is 
reasonable to consider that the combination of azide reduction and N-Boc-protection 
should offer an efficient approach to the manipulation of organic amino compounds. 
 The present chapter describes one pot method for the transformation of azide to N-
Boc-protected amines by using palladium fluorapatite (PdFAP). This catalyst is recovered 
and recycled for several times without loss of activity (Scheme 2). 
 
 
 
Scheme 2. Chemoselective conversion of azide to Boc-protected amine using PdFAP 
R
NH-Cbz
R
NH-CbzPdFAP
H2 atm, Boc2O
EtOAc, r.t.
R=Phenyl
NH-BocN3
Abstract                                                                                                                                                             iv                                                                                                                                              
Section II: An efficient palladium fluorapatite catalyzed three component coupling of 
aryl iodides, alkynes and alkenes 
One of the most attractive synthetic strategies favored by organic chemists is the 
multi-component domino coupling reactions for constructing polyfunctional molecules by 
linking several organic moieties through carbon-carbon bond formation in one step. In 
particular, the transition-metal catalyzed coupling reactions are recognized as versatile 
tools for constructing complex molecules from simple starting materials. Among such 
reactions, palladium-catalyzed carbon-carbon bond formation reactions are the most 
versatile by virtue of their generality and ability to tolerate a wide range of important 
organic functional groups. In the present chapter, a three-component coupling of aryl 
iodides, alkynes, and alkenes to generate 1,3-butadiene derivatives using palladium 
fluorapatite (PdFAP) catalyst is described (Scheme 3).  
DMF/H2O (9:1)
+ +
Yield: up to 71 %
+
130 oC
PdFAP
R1I
R1
R4
R4R2 R3
R2
R1
R3
R4
 
Scheme 3. Three-component coupling of iodobenzene, diphenylacetylene and butyl 
acrylate by using PdFAP catalyst. 
Chapter-IV: Synthesis of 5-substituted 1H-tetrazoles and propargylamines by using 
zinc based catalysts 
This chapter is divided into two sections.  
Section I: This section deals with the synthesis 5-substituted 1H-tetrazoles by using zinc-
hydroxyapatite catalyst 
Section II: This section deals with the synthesis of propargylamines using zinc dust. 
Section I: Zinc hydroxyapatite catalyzed efficient synthesis of 5-substituted 1H-
tetrazoles 
Abstract                                                                                                                                                             v                                                                                                                                              
Tetrazoles, a class of heterocycles, received considerable attention because of their 
wide range of applications in pharmaceuticals as lipophilic spacers, in speciality 
explosives, photography, and information recording systems. They are important ligands 
for many useful transformations and also precursors for a variety of nitrogen containing 
heterocycles. The most convenient route for the synthesis 5-substituted 1H-tetrazoles is the 
addition of azide ion to nitriles. The earlier reports for this transformation fall in to three 
main categories: i) the use of tin or silicon azides, ii) the use strong Lewis acids, and iii) 
running the reaction in acidic media. The drawbacks in the earlier methods are, use of 
expensive and toxic metals, severe water sensitivity, and the presence of hydrazoic acid, 
which is highly toxic and explosive as well as volatile. It is also known that acid-catalyzed 
cycloaddition between hydrazoic acid and isocyanides is one of the main routes to 5-
substituted 1H tetrazoles. However, this procedure needs the direct addition of large excess 
amounts of dangerous and harmful hydrazoic acid. Therefore, it is desirable to develop a 
more efficient and convenient method for the synthesis of 5-substituted 1H- tetrazoles. 
In the present chapter, the synthesis of 5-substituted 1H- tetrazoles by addition of 
sodium azide with organic nitriles using zinc hydroxyapatite is described. This catalyst is 
recovered and recycled for several times without loss of activity (Scheme 4). 
R
CN
NaN3
R N N
N
H
N
DMF, 120-130 oC
+
ZnHAP
 
Scheme 4. Synthesis of 5-substituted 1H-tetrazoles using ZnHAP 
Section II: Efficient one-pot synthesis of propargylamines using zinc dust 
 One pot multi-component coupling reaction (MCR) by coupling of several organic 
moieties in one step is an attractive synthetic strategy. Propargylamines are important 
Abstract                                                                                                                                                             vi                                                                                                                                              
synthetic intermediates for biologically active compounds such as β-lactums, 
conformationally restricted peptide, isosteres, natural products and therapeutic drug 
molecules. The standard practice for synthesis of propargylamines is carried out by 
amination of propargylic halides, propargylic phosphates or propargylic triflates.  
The present chapter deals with the three-component coupling of aldehydes, amines and 
alkynes to generate propargylamines using zinc dust without using any co-catalyst or 
additive (Scheme 5). 
 Easy separation of the catalytic by simple centrifugation. 
 No other co-catalyst or additive is required. 
 No pre-activation of Zn dust is required. 
 
Scheme 5. Synthesis of propargylamines by using zinc dust 
Chapter-V: Synthesis of α-diazo-β-hydroxy and α-diazo-β-amino carbonyl 
compounds using Mg/La mixed oxide catalyst and β-keto esters by using Pd0/Mg-La 
mixed oxide catalyst 
This chapter is divided into two sections. 
Section I: This section deals with the synthesis of α-diazo-β-hydroxy and α-diazo-β-amino 
carbonyl compounds using highly basic Mg/La mixed oxide catalyst. 
Section II:  This section deals with the arylation of Baylis-Hillman adducts with iodo- and 
bromoarenes using Pd0-Mg/La mixed oxide 
Section I: Synthesis of α-diazo-β-hydroxy and α-diazo-β-amino ester compounds via 
aldol type reaction of aldehydes and imines with ethyl diazoacetate using basic Mg/La 
mixed oxide catalyst 
R1-CHO + R2R3NH +
Zn dust
reflux
NR2R3
R1
R4
HR4 CH3CN
Abstract                                                                                                                                                             vii                                                                                                                                              
 α-Diazo carbonyl compounds are potential source of amino alcohols and acids. α-
Diazo carbonyl compounds are generally prepared by the azido transfer reaction of 
carbonyl compounds. Condensation of acyldiazomethane to aldehydes and imines yields α-
diazo carbonyl compounds but requires deprotonation of the acyldiazomethane. Some of 
the methods reported require low temperature and absolutely anhydrous conditions; 
moreover the use of strong bases may not be compatible with functional groups in the 
substrates. In recent years, there has been increasing emphasis on the use and design of 
environment-friendly solid catalysts to reduce the amount of toxic waste. The use of a solid 
catalyst allows easy separation of catalyst from the reaction mixture in large-scale 
production to conform to the concept of green technology.  
The present chapter addresses the synthesis of β-hydroxy-α-diazo carbonyl 
compounds and β-amino-α-diazo carbonyl compounds by condensation of a wide variety 
of aldehydes and imines with ethyl diazoacetate using the Mg/La mixed oxide catalyst in 
water (Scheme 6). The noticeable merits of Mg/La mixed oxide catalyst in synthesis of β-
hydroxy-α-diazo carbonyl compounds and β-amino-α-diazo carbonyl compounds are:  
1) The reaction is performed at room temperature.  
2) Low catalytic loading with good reusability. 
3) Water is used as a solvent in the reaction, which is safer, cheaper and more 
environmentally friendly.  
 
Scheme 6. Synthesis of diazo carbonyl compounds by using Mg/La mixed oxide. 
R
X
H
N2
CO2Et
R
N2
CO2Et
XH
Water, rt
+
Mg/La mixed oxide
X= O, N-TS
Abstract                                                                                                                                                             viii                                                                                                                                              
Section II: Synthesis of β-keto esters via arylation of Baylis-Hillman adducts with 
iodo- and bromo- arenes catalyzed by Pd0/Mg-La mixed oxide catalyst 
Baylis-Hillman adducts are interesting class of disubstituted olefins possessing both 
allylic alcohol and α,-unsaturated ester moieties in which carbon-carbon double bond is 
an integral part of both allyl alcohol and α,-unsaturated ester moieties. Such a highly 
desirable olefin arylation is an important area in the synthesis of β-keto esters products, an 
important and useful class of synthons. Few reports on such coupling are reported in 
literature using homogeneous palladium salts. Despite the synthetic elegance and high 
turnover numbers, the non-reusability of the precious palladium precludes wide synthetic 
applications in the pharmaceutical industry. Heterogeneous catalysts have been proven to 
offer similar levels of activity to their homogeneous counterparts whilst eliminating the 
need for an extensive work-up and preventing the metal from being found in the product, 
which restricts its use in industry.  
In the present chapter, the arylation of di-substituted (Baylis-Hillman adducts) 
olefins with iodo- and bromoarenes to afford -keto ester products in good yields via -
hydride elimination in a single pot by using Pd0/Mg-La mixed oxide catalyst (Pd0/Mg-La 
MO) (Scheme 7) is described.  
 
 
 
Scheme 7. Arylation of Baylis-Hillman adduct with iodo- and bromobenzenes using 
Pd(0)/MgLa MO catalyst 
R2
CO2CH3
OH
R1
X
Pd0/Mg-La MO
DMF, Base
R2
CO2CH3
O
R1X =  Br , I
90o C
R2
CO2CH3
OH
R1
+
R2= Aromatic,
     Aliphatic


Mercury-Resistant Marine Bacteria and their Role in Bioremediation of Certain Toxicants

SHARMA, DINESH KUMAR

PAGE  



		209
                                                                                                                                          




                                Abstract
The thesis describes isolation of Enzyme inhibitory constituents from three Ayurvedic medicinal plants are Stereospermum personatum, Hydnocarpus wightiana and Piper nigrum.
Chapter I describes the isolation and charecterisation of compounds A-L from the petroleum ether extract of the stem bark of Stereospermum personatum (Bignoniaceae). Compounds A, B, D, E, H, I, K and L are identified as known compounds from the same plant. Compound-C was isolated as white semi solid and analyzed for C26H42O3  (M+ 402). The IR spectrum revealed the presence of a hydroxyl group at 3406 cm-1, and ester carbonyl at 1733 cm-1 and indicated the general nature of an aromatic compound. The 200 MHz 1H NMR spectrum in CDCl3 has shown two doublets each integrating for two protons at δ 7.20 (J = 8.5 Hz) and δ 7.0 (J = 8.5 Hz) suggesting the presence of an AB substituted aromatic ring. Multiplet at δ 5.3 (J = 4 Hz) integrating for two protons are attributed to isolated cis double bond protons. Two triplets at δ 4.28 and δ 2.92 each integrating for two protons the former is attributed to oxy-methylene group of an ester and the latter is attributed for a benzylic methylene. The above data clearly suggested the presence of a 4-hydroxy phenyl ethyl group. 
The 1H NMR spectrum has further showed a multiplet at δ 0.90 integrating for three protons characteristic of a terminal methyl group of an aliphatic chain. A triplet integrating for two protons at δ 2.25 was characteristic of a methylene adjacent to a carbonyl group, two multiplets at δ 2.30 and δ 2.50 integrating for four protons are attributed to allylic methylenes. Broad singlets at δ 1.30 integrating for twenty protons are attributed to methylene protons of aliphatic chain. The multiplet at δ 1.60 integrating for two protons was accounted for the methylene adjacent C-2" carbon. The 13C NMR   spectrum in CDCl3 has shown ester carbonyl at δ 173.2 and assignments were made by comparing  with that of bongrardol, an octacosanoic acid ester of  4-hydroxy phenyl ethanol. From the above data and its ESIMS spectrum the structure of compound-C was established as an oleic acid ester of 4-hydroxy phenyl ethanol. Compound-C is thus identified as a new compound and its natural occurrence is reported for the first time.
Compound-F was confirmed as sterequnthal-B from all its physical, spectral data and further evidence from X-Ray studies.
 	Compound-G was isolated as pale yellow semi solid and analysed for C20H18O4 by HREMS (M+) (m/z 322.119, calcd 322.120). Its IR spectrum has indicated the presence of carbonyl groups at 1713 and 1665cm-1. The 200 MHz 1H NMR spectrum in CDCl3 and 13C NMR have shown the general features of a substituted anthraquinone system. The 1H NMR has shown two doublets at  8.20 (1H, J = 8.5 Hz) and at  7.68 (1H, J = 2.5 Hz) each integrating for one proton, a double double at  7.22 (1H, J = 1 and 8 Hz) integrating for one proton suggesting the presence of a 1, 2, 4-trisubstituted aromatic ring. Two doublets at  8.18 (1H, J = 8.0 Hz) and at  7.55 (1H, J= 8.0 Hz) each integrating for one proton was indicating two orthocoupled aromatic protons. Two triplets at  3.10 (2H, J= 7.2 Hz) and at  2.78 (2H, J= 7.2 Hz) both integrating for two protons accounted for two adjacent metylene groups. The former is attributed to the one present adjacent to carbonyl group and latter is attributed to benzyllic methylene. A sharp singlet at  3.98 integrating for three protons (3H), was attributed to a methoxy group. Two sharp singlets at  2.75 (3H) and at  2.20 (3H) are due to two methyl groups, the former is attributed to an aromatic methyl, the latter is characteristic of methyl of acetyl group. The down field shift of aromatic meythyl group may be attributed to its proximity to one of the carbonyl group. 13C NMR spectrum in CDCl3 indicates the presence of 20 carbons are present and the assignments were made on the basis of DEPT experiment. 13C NMR DEPT experiment has suggested the presence of three methyl carbons, two methylenes, five methine carbons, seven quaternary and   three carbonyl carbons. The 1H NMR and 13C NMR data has shown close resemblance to that of anthraqunthone except for the aromatic region. The postion of methoxy group and aromatic methyl was established based on HMBC NMR experiment. In HMBC C-7 shown correlation with H-3 and H-5 and C-2 has shown correlation with H-6 only. Based on all the above data, compound-G confirmed as a new 4- methoxy anthraqunthone named as sterequinone-G isolated first time from nature. 
Compound-J was obtained as pale yellow solid m.p. 84-86C and analysed for C20H1804 (M+ 322). It is further supported by HR FABMS (M+H+) m/z 323.129 calcd 323.128. The IR spectrum has shown two bands at 1717 cm-1 and 1664 cm-1 indicating the presence of two carbonyl groups. The 300 MHz 1H NMR spectrum of compound in CDCl3 displayed two doublets at at  8.18 (J = 8.6 Hz),  7.20 (J = 2.6 Hz) and a double doublet at  7.20 (J = 8.6 and 2.6 Hz) each integrating for one proton suggesting a typical 1, 2, 4-trisubstituted aromatic ring, a sharp singlet at  8.13 integrating for one proton was accounted for an oliefinic proton with no α-hydrogens, a sharp singlet at  3.98 integrating for three protons was accounted for an aromatic methoxy group. A double multiplet at  3.02 integrating for two protons, and a multiplet at  2.25 integrating for two protons indicated the presence of two adjacent methylenes. Two sharp singlets at  2.70 and  1.65 each integrating for three protons were accounted for the presence of two-olifeinic methyl groups. The downfield shift of the former could be explained due to its close proximity to one of the carbonyl groups. The above 1H NMR data has close resemblance to that of kiglinol isolated from Kiglia pinnata, which is also a member of Bignonaceace family. The 13C NMR Spectrum in CDCl3 has revealed the presence of 20 carbons, and the assignments were made on the basis of DEPT experiment. 13C NMR DEPT experiment has suggested the presence of three methyl carbons, two methylenes, four methine carbons, nine quaternary carbons and two carbonyls. The position of methoxy group is fixed based on their biogenetic considerations and from HMBC experiment. Based on the above data, along with its mass spectrum the structure of the compound-J was confirmed as 4-methoxy derivative of kiglinol, a new compound named as sterequinone-H.
All the napthoquinones isolated have been screened for their antibacterial activity against different strains of bacteria and are found to exhibit moderate to good level of activity. 
The second chapter describes the isolation and charecterisation of six compounds from the acetone extract of the Hyndnocarpus wightiana. Compounds A, B, C, E and F are identified as apigenin, naringenin, luteolin, Hydnocarpin and isohydnocarpin.
The compound-D was obtained as pale yellow solid, mp  260-262C, molecular formula deduced as C26H22O9 (M+ 464). The IR spectrum has shown a broad band at 3350cm-1 and 1650 cm-1 indicating the presence of hydroxyl groups as well as carbonyl group. The 200MHz 1H NMR spectrum in DMSO-D6 has shown a doublet at δ 6.40 (J = 1.6 Hz) and a doublet at δ 6.82 (J = 1.6 Hz) each integrating for one proton respectively are attributed for H-6, H-8 protons of flavonoid B ring. A sharp singlet at δ 6.90 integrating for one proton was attributed to the olifinic proton 3C-H. A doublet at δ 7.70 (J = 1.7Hz) integrating for one proton was attributed for 2’ proton. The peaks observed at δ 7.62, δ 7.05, δ 6.88 and δ 6.80 are in similar pattern as that of hydnocarpin. The sharp singlets at δ3.82, δ 3.77 each integrating for three protons suggested that two aromatic methyl groups were present, the peak at δ 3.77 is attributed for D ring of a flavanolignan. The 13C NMR spectrum has shown 26 signals and the signal assignments were made on the basis of 13CNMR and DEPT experiments. The 13C NMR spectrum is similar to that of hydnocarpin except for the presence of one extra methoxy peak. This suggested that compound-D should be a methoxy derivative of hydnocarpin. The position of methoxy group was established based on the correlations of proton and carbons from HMBC spectrum of compound-D and shift of proton values of H-6 and H-8 in proton spectrum. Thus compound D is identified as 7-O-metyl ether of hydnocarpin. In HMBC spectrum the 6C-H has shown the correlation with C-8 and C-10, methoxy group of δ 3.80 has shown correlations with C-7 suggesting that the methoxy group is present on C-7. Based on the above data and along with its mass spectrum the structure of compound-D was confirmed as 7-O-methyl hydnocarpin. This is the first report of occurrence of 7-O-methyl hydnocarpin in nature.  
The compounds obtained from extract are in substantial yield namely luteolin, isohydnocarpin, hydnocarpin were tested for their antioxidant activity and α-glucosidase inhibitory activity. Hydnocarpin, isohydnocarpin did not exhibit much antioxidant activity on DPPH test model, the strong radical scavenging activity of the acetone extract might be due to the presence of luteolin in substantial amount.
The extract and compounds were also tested for their radical scavenging potential on ABTS, a radical model representing free radicals originating in hydrophilic media. The concentrations of ABTS and test materials were kept identical as in DPPH method. It was observed that all the compounds including extract were strong in scavenging this radical. The order of their ABTS scavenging potency observed as luteolin > extract > isohydrocarpin > hydnocarpin. It is also important note that ABTS radical scavenging capacity of the extracts as well as compounds were 6 to 15 times more these that of their DPPH scavenging potential.
Using yeast -glucosidase technique we observed that acetone extract, luteolin and isohydnocarpin were potent inhibitors of -glucosidase. However hydnocarpin showed mild inhibitory activity. In the order of -glucosidase inhibitory potential, luteolin was stronger then extract followed by isohydnocarpin. However interms of molecular strength luteolin and isohydnocarpin were equipotent. The kinetics of inhibition of these compounds for -Glucosidase at 10 g /ml concentration reveals that acetone extract inhibits the enzyme in a competitive manner while the inhibitory patterns of luteolin and isohydnocarpin were of mixed type.
Chapter III section A describes the chemical and antibacterial activity studies on Piper nigrum (pepper black). Isolation of eight compounds designated as A, B, C, D, E, F, G and H from the petroleum ether extract of the berries of Piper nigrum is described. Compounds A, B, C, E, G and H are identified as pellitorine, (2E, 4E, 8Z)-N-isobutyleicosatrienamide, guineensine, tricostachine and piperine.  Compounds D and F are identified as trachyone and pergumidiene which are isolated for the first time from piper nigrum.
All the alkamides isolated have been screened for their antibacterial activity against different strains of bacteria and are found to exhibit good level of activity.
Chapter III section B describes a synthetic route in order to synthesis a series of piper amides and their mimics derived out of ferullic, caffeic and cinnamic acids. Several syntheses are reported in literature for making the amide bond formation. All the known procedures involve the usage of harsh reagents and reaction conditions. Hence a simple and effective method is developed for the amide formation involving the usage of silica chloride and potassium fluoride. The amides synthesized were subjected to antibacterial assay and their structure activity relationship is studied. The amides are particularly active against bacterial strain Staphylococous aureus and Chromobacterium  violaceum among gram +ve strains. One intresting observation is that amides with no substitution on benzene ring are more potent than amides with substitution on the ring. Among amides prepared with substitutions on the ring, amides with free hydroxyl group at fourth position seems to enhance the activity















 EMBED ISISServer  






IONOSPHERIC RESPONSE TO SOLAR PHENOMENA AND THUNDERSTORMS

V., NAGA RAMACHANDER T.

Abstract


ABSTRACT

The Thesis entitled “Advanced Synthetic Applications of Baylis-Hillman Chemistry: Stereoselective Synthesis of Functionalized Trisubstituted Olefins Including Some Insects Pheromones and Potent Pharmaceutical Agents” consists of four chapters.

CHAPTER I

The Baylis-Hillman Reaction: An Overview 
This Chapter is divided into two sections.
SECTION A: Introduction
Construction of a C–C bond is most fundamental requirement in the synthetic organic chemistry from its origin. The well-known C–C bond forming reactions are 
Aldol condensation reaction (1872), Friedel-Craft reaction (1877), Diels-Alder reaction (1928), Wittig reaction (1954) and Heck reaction (1968).
Very recently Baylis-Hillman reaction got serious attention as a novel and versatile C–C bond forming protocol after it’s discovery by two German scientists A. B. Baylis and M. E. D. Hillman in 1972.
Baylis-Hillman Reaction:
This is essentially the coupling of the -position of activated alkenes with carbon electrophiles under the catalytic influence of a tertiary amine providing a simple process for synthesis of densely functionalized molecules (Eq.1).

Equation. 1 








R = alkyl, aryl, heteroaryl; R1 = H, COOR, Alkyl; X = O, NCOOR, NTS, NSO2Ph
EWG = Electron withdrawing group; COR, CHO, CN, COOR, PO(OEt)2 SO2Ph, SO3Ph, SOPh


Essential Components
1) Activated alkenes: A variety of activated alkenes such as alkyl vinyl ketones, alkyl (aryl) acrylates, acylonitrile, vinyl sulfones, acrylamides, allenic esters, vinyl sulfonates, vinyl phosphonates and acrolien couple with a number of carbon electrophiles to provide a wide range of multifunctional molecules.
2) Electrophiles: Aldehydes have been primary source of electrophiles. Tthus, various aliphatic, aromatic and hetero-aromatic aldehydes have been extensively employed in obtaining various interesting Baylis-Hillman adducts. Also -keto esters, non enolizable 1,2-diketones, aldimine derivatives and activated alkenes have been employed as electrophilies in this reaction.
3) Catalyst: Although DABCO (1) has been the catalyst of choice, various other tertiary amine catalysts such as quinuclidine (2), 3-HQD (3), 3-quinuclidone (4) and indolizine (5) were also employed to perform the Baylis-Hillman reaction (Eq. 1). 

SECTION B: Synthesis of Functionalized Trisubstituted Olefins and Baylis-Hillman Chemistry
The Baylis-Hillman adduct is densely functionalized with several groups in close proximity. A hydroxyl or amino group, a highly activated double bond, an electron withdrawing group ranging from –CHO, -COR, -COOR, -CN, -PO(OR)2 to -SO2Ph are the core functionalities. Normally the adduct is having an allyl alcohol moiety, a chiral center and a Michael acceptor. Applications of this adduct needs proper tuning of the functional groups.








		





Baylis-Hillman adducts are mostly exploited for nucleophilic substitution (SN2 and SN2’) and addition reactions. Nucleophilic substitution reactions of Baylis-Hillman adducts are commonly associated with the concomitant allylic rearrangement of the double bond which led to the formation of a variety of trisubstituted olefins stereoselectively. Various adducts have been employed for the stereoselective synthesis of different naturally occurring bioactive compounds including several alkaloids, terpenoids, macrolides and pheromones. All of these molecules contain a stereodefined trisubstituted olefin moiety as the central structural unit which have been well documented in the literature.

CHAPTER II

Stereoselective Synthesis of Functionalized Trisubstituted Olefins Introducing Hydride Nucleophile to the Baylis-Hillman Adducts
This chapter is divided into three sections. 

SECTION A: Stereoselective Synthesis of [2E]-2-Methylalk-2-enoates and Its Applications
[2E]-2-Methylalk-2-enoates and [2E]-2-methylalk-2-enoic acids (or its other derivatives) are the important skeletons present in a wide range of biologically active molecules. The presence of [2E]-2-methylalk-2-enoates and [2E]-2-methylalk-2-enoic acids moieties in various biologically active molecules has attracted our attention. We have visualized that the Baylis-Hillman adduct derived from methyl acrylate could be easily transformed into the desired [2E]-2-methylalk-2-enoates. Accordingly, we have treated methyl 3-hydroxy-2-methylene-alkanoate 1 with NaBH4 in the presence of a series of metallic chlorides. Using NaBH4/CuCl2.2H2O in MeOH at room temperature, the resulting trisubstituted alkene 2 was obtained in high yield and in 100% [E]-selectivity (Scheme 1).
Scheme 1


The sole [E]-stereoselectivity of the reaction can possibly be explained by considering the transition state models A & B (Figure 1). The transition state A is more 

Figure 1

favoured than B due to steric demand and the R group (alkyl, aryl) prefers to stay trans to the –COOMe group. Thus, [E]-products were formed exclusively.
With a view to prove the efficacy of this methodology, we have undertaken the practical synthesis of five insect pheromones. (4S, 2E)-2,4-dimethyl-2-hexenoic acid 3, a caste-specific substance present in the mandibular glands of the male carpenter ants in the genus Camponotus, (+)-(S)-manicone 4 and (+)-(S)-normanicone 5, the mandibular gland alarm pheromone components of the ants in the genus Manica, and (+)-(S)-1-methylbutyl (E)-2-methyl-2-pentenoate (dominicalure-I) 6 and (+)-(S)-1-methylbutyl (E)-2,4-dimethyl-2-pentenoate (dominicalure-II) 7, the aggregation pheromones of lesser grain borer Rhyzopertha dominica (F) have been synthesized here.

We have synthesized the pheromone 3 starting from (S)-2-methyl butanol via the Baylis-Hillman adduct 9 according to the Scheme 2.
Scheme 2

The pheromones 4 and 5 have been synthesized through a common sequential route of six steps via the compound 3 according to Scheme 3. 
Scheme 3

We have undertaken the synthesis of pheromones 6 and 7 starting from n-propanal and 2-methylpropanal respectively according to the procedure presented in Scheme 4.
Scheme 4

Thus, we have successfully utilized the potential of Baylis-Hillman chemistry for the practical synthesis of five important insects pheromones.

SECTION B: Stereoselective Synthesis of [E]-α-Methylcinnamic Acids and Its Applications
[E]--Methyl cinnamic acids moiety is an important and central structural unit present in various biologically active molecules, for example, 1-[p-(myristyloxy)--methylcinnamoyl]glycerol (LK-903) 1 is a very active hypolipidemic agent. 
N-Allyl-N-[4-{(4-amidinophenoxy)carbonyl}--methylcinnamoyl] glycine methane sulfonate 2  and its analogues are potent orally active serine protease inhibitors. Also [E]-2-methyl-3-(4-(myristyloxy)-phenyl)prop-2-enoic acid 3 itself shows good hypolipidemic activity. [E]-2-Methyl-3-(4-carbomethoxyphenyl)-prop-2-enoic acid 4  is a valuable synthon for the synthesis of serine protease inhibitor 2.


The presence of [E]--methyl cinnamic acid moiety (or derivatives) in various biologically active molecules has attracted our attention. We observed that, the treatment of Baylis-Hillman adduct, methyl-3-hydroxy-3-aryl-2-methylene propanoates 5 with NaBH4/CuCl2.2H2O in methanol or NaBH4 in combination with molecular I2 in THF at room temperature, followed by hydrolysis with KOH/MeOH and crystallization afforded the corresponding [E]--methylcinnamic acids 7 in high yields via the formation of the intermediates 6 (Scheme 1).
Scheme 1



The stereochemistry of products was solely [E]- as evident by considering the same transition state models described in Section A (Fig. 1). Encouraged by this observations, we have then synthesized a series of (E)--methyl cinnamic acids directly from various adducts without isolating the intermediates 6. The used reagents i.e. NaBH4/CuCl2.2H2O or I2/NaBH4 are comparably useful for the synthetic purposes as per as the operational simplicities, overall yields and stereoselectivies are concerned. Here, we presented NaBH4/CuCl2.2H2O reagent system in synthetic applications. Infact, the efficiency of the protocol have been proved by the practical synthesis of compound 1, 3, & 4 which have been discussed below. 
We have undertaken the synthesis of synthon 4 starting from 4-carboxybenzaldehyde in four steps (Scheme 2).
Scheme 2


Potent anti-cholesterol drug LK-903 (1) was synthesized starting from 
4-hydroxybenzaldehyde in six steps via the p-myristyloxy)-[E]--methyl cinnamic acid 3 which also showed good hypolipidemic activity (Scheme 3). 
Scheme 3


SECTION C: Stereoselective Synthesis of Trisubstituted [E]-Alkenones From Unmodified Baylis-Hillman Adducts: Improved Synthesis of (+)-(S)-Manicone and (+)-(S)-Normanicone
Trisubstituted alkenone is an important and key structural unit present in various biologically active molecules and is a versatile intermediate for the synthesis of various natural products. α, β-Unsaturated ketones are enormously useful synthons in organic synthesis. 3-Alkenyl–2-ketones as well as 4-alkenyl-3-ketones are common metabolites in insects and other arthropods where they serve a range of communicatory and ecological functions. As For examples, (+)-(S)-manicone (1) and (+)-(S)-normanicone (2) are the two mandibular gland alarm pheromones of Manica ants. (+)-(S)-homomanicone (3) is another alarm pheromone found in the mandibular gland secretions of two sympatric harvester ants, Pogonomyrmex salinus and Messor lobognathus.






In the section-A of this chapter, we have already discussed the six steps synthetic procedures for both the pheromones 1 and 2. Though, those were simple and practically useful, we were still in search for the shorter routes applying Baylis-Hillman chemistry for their synthesis. We envisaged that a chemoselective reduction of B-H adduct (4) derived from an aldehyde and vinyl ketone would lead to the formation of a trisubstituted alkenone (5) what we required for the synthesis of the pheromones. After several attempts, it was observed that sodium borohydride in presence of catalytic amount of indium chloride in acetonitrile acted as the best catalyst to afford the desired product in high yield within 3 hours (Scheme 1).

Scheme 1





The stereochemistry of the products was found to be solely [E]. The formation of stereodefined [E]-alkenones in the present reaction can possibly be explained by considering the transition state models I and II. The transition state I is more favored than II since –COR3 is bulkier than –CH2R2 group and causing more steric crowding to R1 group.


	


Exclusive chemoselectivity and [E]-stereochemistry of the products enabled us to synthesize (+)-(S)-manicone (1) and (+)-(S)-normanicone (2) applying our methodology according to Scheme 2. 
Scheme 2









The synthetic routes demonstrated here are simple, convenient, shorter and more improved compared to the previously described methods in section-A of this chapter.

CHAPTER III

Stereoselective Synthesis of Functionalized Trisubstituted Olefins Introducing Carbon Nucleophiles to the Baylis-Hillman Adducts
This chapter is divided into two sections. Section A is further divided into two parts.

SECTION A/PART I: Zn-Mediated Alkylation of Baylis-Hillman Adducts in Aqueous Media: Introduction of Alkyl Nucleophiles 
In continuation of work for the synthetic applications of B-H chemistry, we wanted to synthesize different stereodefined trisubstituted oefins of synthetic need. Herein we have reported an efficient alkylation on activated Baylis-Hillman adducts to produce trisubstituted alkenes in water. In the presence of zinc and saturated aqueous NH4Cl, simple alkyl iodides reacted with activated Baylis-Hillman adducts at room temperature to yield [E] and [Z] trisubstituted alkenes with moderate to high yield and excellent stereoselectivity (Scheme 1).
Scheme 1

	

	





During the studies, several 3-acetoxy-2-methylene-alkanoates (1) and 3-acetoxy-2-methylene-alkannitriles (2) were treated with various alkyl iodides in the presence of zinc in saturated aqueous NH4Cl solution at room temperature to generate different trisubstituted alkenes. The electron withdrawing groups present in the adducts directed the stereochemistry of the products which is a well known fact in in the B-H chemistry. When EWG = -COOMe (1) the conversion afforded the olefins (3) with [E]– stereoselectivity exclusively while when EWG = -CN (2) the olefins (4) were formed with high [Z]– stereoselectivity. The regioselective alkylation could be explained by a 1,4 addition type mechanism involving a -acetoxy elimination. This mechanism explained the [E]– selectivity with ester (forming a chelated reaction intermediate, A, Figure 1) and [Z]– selectivity with nitriles (forming a non-chelated intermediate, B, Fig. 1).

Figure 1. 

​[1]​

PART II: Friedel-Crafts Reaction of Baylis-Hillman Adducts: Introduction of Aryl Nucleophiles
Stereoselective synthesis of [2E] and [2Z]-2-benzyl substituted trisubstituted alkenes (7, 8) has been achieved by employing Friedel-Craft reaction of benzene with unactivated Baylis-Hillman adducts (6) in the presence of HClO4.SiO2 as a heterogeneous catalyst. When EWG = -COOMe, [E]- alkene was the major product whereas [Z]- alkene was obtained as the major product when EWG = -CN (Scheme 2).

Scheme 2 




.

                                                                                      



SECTION B: Applications of Alkylation of Baylis-Hillman Adducts in Aqueous Media: Stereoselective Synthesis of Biologically Active Molecules
We have already demonstrated a novel Zn-mediated alkylation of the B-H adducts in aqueous medium for the synthesis of methyl [2E]-2-alkylalkenoates. With a view to prove the efficacy of our above mentioned methodology, we have undertaken the synthesis of some biologically active molecules. Synthesis of [2E]-2-butyloct-2-enal, an alarm pheromone component of the African weaver ant, Oecophylla longinoda (1), [2E]-2-tridecylheptadec-2-enal (2), an unusual metabolite from the red alga Laurencia species and a potent anti alzheimer’s drug (R)-2-propyloctanoic acid (3) have been demonstrated here.

  

  


Accordingly, we have synthesized the alarm pheromone [2E]-2-butyloct-2-enal (1) by preparing the B-H adduct 3-hydroxy-2-methyleneoctanoate (4) starting from commercially available n-hexanal and methyl acrylate as per the procedure described in Scheme 1.

Scheme 1















[2E]-2-tridecylheptadec-2-enal (2), the unusual metabolite of red algae was synthesized in six steps starting from n-pentadecanol according to the route depicted in Scheme 2.

Scheme 2













	
Synthesis of potent Anti-Alzheimer Drug, (R)-2-Propylocta-noicacid (3):
Alzheimer’s disease (AD) is the most common form of dementia in the elderly. It is a fatal disorder that robs its victims of their most precious organ, the brain by slowly destroying the complex web of neuronal connections that support cognitive processes such as thought and memory. Thus, research into fighting this ever-growing disease is directed towards new therapeutic agents, such as neuroprotective agents or antioxidents. One novel emerging compound is (R)-arundic acid (3), a neuroprotective agent, which modulates astrocytic activation by inhibiting the enhanced astrocytic synthesis of S-100β, responsible for inducing neuronal death.
We envisaged a synthetic route for compound 3 applying our ecofriendly alkylation protocol of preparing [2E]-2-alkylalkenoates as the key step. We thought to generate the desired configuration of the chiral center (‘R’ at C-2 position) by asymmetric hydrogenetion of double bond of the trisubstituted alkene. Noyori reported that [2E]-2-alkylalk-2-enoic acids upon hydrogenation by (R)-BINAP-Ru (II) dicarboxylate complex (16) as catalyst, affords (R)- configurated 2-alkylalkanoic acid. This prompted us to think for the asymmetric hydrogenation as our target molecule is having the chiral center with ‘R’-configuration. In this case, we have started with the B-H adduct (4) prepared from the commercially available n-hexanal (Scheme 3). 

Scheme 3














The key intermediate, [2E]-2-propyloct-2-enoate (13) was synthesized by the alkylation of B-H acetate (5) with ethyl iodide in presence of zinc in saturated aqueous NH4Cl solution. Finally asymmetric hydrogenation by Ru (II) [(R)-BINAP] dicarboxylate catatlyst (15) furnished the target molecule (R)-2-propyloctanoic acid (3) in 88% yield and 84%optical purity.

CHAPTER IV

Stereoselective Synthesis of Functionalized Trisubstituted Olefins Introducing Heteroatoms (Cl, Br, I and O) as Nucleophiles to the Baylis-Hillman Adducts.
This chapter is divided into two sections. 

SECTION A: Stereoselective Synthesis of [2Z]-2-(halomethyl)alk-2-enoates and [2E]-2-(halomethyl)alk-2-enenitriles
[2Z]-2-(halomethylalk-2-enoates have been used as valuable synthons in the synthesis of a variety of important molecules such as micanecic acid, kijanolide, rennin inhibitor 
A-72517, and β-lactams. Similarly others bioactive molecules like, α-metylene-γ-butyrolactones and flavonoids have also been synthesized using [Z]-allyl halides derived from Baylis-Hillman adducts.	
The importance of [Z]- and [E]-allyl halides in the synthesis of several natural products deserved our attention. We thought to synthesize these important class of synthons directly from unmodified B-H adducts using metal-halides in the presence of some heterogeneous Lewis acid catalysts.
Introduction of Br¯ and I¯ Nucleophiles:

Stereoselective synthesis of [2Z]- and [2E]- allyl bromides and iodides have been achieved by the treatment of LiBr and LiI with Baylis-Hillman adducts in presence of NaHSO4. SiO2 as the heterogeneous catalyst according to Scheme 1 and 2 respectively.

Scheme 1





Scheme 2







The stereochemistry of products was exclusively [Z]- when EWG = -COOMe, whereas [E]- was the major isomer (94-97%) when EWG = -CN. 
Introduction of Cl¯ ion Nucleophile:



We wanted to synthesize allyl chloride moieties under heterogeneous conditions. It was observed that the Baylis-Hillman adduct 1 could efficiently be transformed into [2Z]-2-(chloromethyl)alk-2-enoates (4) using InCl3 in CH2Cl2 at room temperature (Scheme 3).

Scheme 3






Here InCl3 worked under heterogeneous condotion as it was poorly soluble in CH2Cl2. 
The EWG present in the adduct directed the stereochemistry of the allyl halides which can be explained by transition state models A, B and C. Model A is favored compared to B when EWG = -COOR’ and thus [Z]-alkenes are formed exclusively whereas, Model C is favored when the EWG = -CN as it is not facing any steric boundary due to its linear disposition. 










SECTION B: Stereoselective Synthesis of [E]-Cinnamyl Alcohol Derivatives Introducing Oxygen Nucleophile to The Baylis-Hillman Adducts
The Baylis-Hillman adducts, 3-aryl-3-hydroxy-2-methylene-alkanoates and 3-aryl-3-hydroxy-2-methylene-alkanenitriles have been efficiently isomerized to the corresponding [E]-cinnamyl alcohols by treatment of the adducts with Ac2O in the presence of HClO4.SiO2 followed by hydrolysis of the intermediate acetates with K2CO3/MeOH. The first step occurs under solvent-free conditions and the catalyst has been found to be reusable (Scheme 1).
Scheme 1








The stereochemistry of the conversion can possibly be explained by considering the favourable transition state models A and B, which suggest the formation of [E]-cinnamyl alcohols predominantly in the present reaction.








In conclusion, we have developed a simple and efficient protocol for stereoselective synthesis of [E]- cinnamyl alcohols starting from Baylis-Hillman adducts containing both ester and nitrile moieties. The simple experimental procedure, utilization of a heterogeneous recyclable catalyst and high yields and stereoselectivity of the products are the advantages associated with the protocol.










































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Characterization of a 5.0kb genomic DNA fragment from Streptomyces aureofaciens NRRL 2209 involved in poly-3-hydroxybutyrate (PHB) synthesis

BHAMIDI, SURESH

Abstract
	The study throws light on the production of betalaines from hairy roots of        B. vulgaris and biotransformations using C. frutescens normal roots for the production of vanillin related metabolites. Various aspects viz. polyamines, MJ and down stream process methodologies have been studied to enhance the metabolite production.

	It may be concluded from the present study that hairy roots can be used as a potential system for the production of betalaines. The organized nature and stable metabolite production makes them more amenable for bioreactor cultivation and scale-up studies. Among all bioreactors studied, acoustic mist bioreactor performed well with high growth and metabolite production. There was no mass transfer limitation observed in the acoustic mist bioreactor unlike others such as nutrient sprinkle and bubble column reactors. Osmolarity measurements proved reliable than conductivity for indirect estimation of biomass when the hairy roots were cultivated in bioreactor systems. 

	The studies on product enhancement (polyamines and MJ) and on-line extraction of pigment (using CTAB) provided an insight for integrating unit operations and developing a process for continuous operation and higher production of phytochemicals in bioreactor systems. In the present study it was found that the roots were still viable and showed normal growth even after CTAB treatment (0.002%) which would be an important observation to make this process a continuous one. 

	This study has shown the production of DOPA and dopamine, which are very important pharmaceutical compounds from hairy roots of B. vulgaris. Further studies with tyrosine feeding led to higher production of DOPA and dopamine. This study is of importance in understanding the biosynthetic pathway of betalaines derived from DOPA and dopamine. Use of oxygenase inhibitors such as caffeic acid and catechin formed the basis to envisage the enzymatic pathways in betalaine production. The higher production of DOPA and dopamine under the influence of caffeic acid and catechin might be because of the inhibitory action of these chemicals on tyrosinase preventing further conversions.

Future work would be oriented to the studies on metabolic block of enzymes such as DOPA dioxygenase to produce more DOPA, which is an important pharmaceutical compound.  Antisense RNA methodology for the enzyme DOPA dioxygenase would also be helpful probably leading the higher production of DOPA and dopamine.

The studies on biotransformation of phenylpropanoid compounds to various vanillin related metabolites using C. frutescens normal roots led to understand the biosynthetic pathways involving various chemical reactions. 

The higher biotransformation rates in cultures fed with ferulic acid complexed with BCD would be because of the increased solubility and availability of precursor. The formation of vanillin from ferulic acid might be via feruloyl-CoA through a reaction similar to -oxidation of fatty acids. Vanillic acid was formed by further oxidation of vanillin, whereas reduction yielded vanillyl alcohol. In the present study, biotransformation resulted in higher concentrations of vanilla flavour compounds in the medium than in roots. Thus, it would be appropriate to use a bioreactor system for down stream processing of the biotransformed products. The formation of vanillin from protocatechuic aldehyde involved O-methylation reaction and oxidation to produce vanillin and protocatechuic acid respectively.

It was observed that ferulic acid formation was more in the early stages after caffeic acid feeding as reflected by O-methylation reaction. When MJ was fed along with caffeic acid, it probably enhanced the CAOMT activity leading to increased biotransformed product. Further, ferulic acid upon oxidation probably led to vanillin production along with other metabolites. Veratraldehyde fed cultures showed maximum vanillin accumulation than other precursors. Veratraldehyde was demethylated to vanillin and parahydroxy benzoic acid. 
There was no capsaicin accumulation recorded in the studies indicating the non-existence of capsaicinoid synthase that is considered as a key enzyme in the synthesis of capsaicin.  The efficiencies of biotransformation in all the biotransformation studies ranged from 2-9% suggesting the possible channeling of precursors resulting in the accumulation of other compounds such as lignin.

Further work would involve the use of inhibitors of lignin biosynthetic pathway to divert the phenylpropanoid precursors to vanillin related metabolite pathways.
























INVESTIGATION ON THE PRODUCTION OF FOOD INGREDIENTS AND BIOTRANSFORMATION USING HAIRY ROOT CULTURES

RAO, K. C. SEKHAR

Abstract

Lipoxygenase (LOX) inhibitors are used as preservatives or antioxidants in food. In medicine, they are used as anti-asthmatic agents, anti-inflammatory, anti-angiogenic, anti-cancer and as anti-atherosclerotic agents. Literature search revealed that inhibitors against LOX from microbial sources were scanty. 

The present investigation has lead to the isolation of methylbisphenol and asperenone as LOX inhibitors from Aspergillus niger through submerged fermentation. 

Another LOX inhibitor from the fermented wheat bran of Aspergillus niger-CFR-W-105 has lead to the identification of the novel metabolite as 2-amido-3-hydroxy-6-methoxy-5-methyl-4-(prop-1’-enyl)benzoic acid. Due to the novel inhibitory nature and new structure, the compound was designated as nigerloxin. Since, nigerloxin was produced at reasonably good concentrations in SSF, it was taken for optimization of; physical, nutritional and downstream process parameters for the maximum production of nigerloxin. An analytical method by HPLC was developed to determine the nigerloxin concentration in crude samples. It was found that, at the optimum levels of physico-chemical and down stream process parameters nigerloxin yield was found to be 5.1 mg/g dry wheat bran, which was 2.4 fold higher compared to unoptimized medium. 

Biochemical studies on nigerloxin revealed that, it inhibits LOX and rat lens aldose reductase with an IC50 values of 79 M and 69 M, respectively. Nigerloxin also shows, a free radial scavenging property using 1,1-diphenyl-2-picryl hydrazyl (DPPH) with an ED50 value of 66 M and invitro antitumor property on EAT cell lines. Studies on the mode of inhibition of nigerloxin against LOX-1 had revealed that it is a reversible and mixed type of inhibitor with a Ki value of 13 M, finally it was proved that nigerloxin inhibits LOX by the similar mechanism as that of the standard antioxidant NDGA. As an academic interest, the binding of nigerloxin to bovine serum albumin (BSA) was investigated using fluorescence spectroscopy. The fluorescence of BSA was quenched following interaction with nigerloxin and this property was used to determine binding constant (1.006 x 106 M-1) and nigerloxin formed a 1:1 complex with BSA.




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