Open Access Repository of Indian Theses

R., HARISH

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Abstract


HETEROGENEOUS TRANSITION METAL CATALYSTS FOR C-C AND C-N BOND FORMING REACTIONS
The work in the thesis has been presented in five chapters. 
Chapter-I describes a detailed introduction on the state of the art of heterogeneous catalysts preparation, uses and advantages as well as the disadvantages.
Chapter-II describes the preparation, characterization and catalytic application of nanocrystalline magnesium oxide supported palladium catalyst (NAP-Mg-Pd0). The catalyst was synthesized by counter ion stabilization of PdCl42- on to the high surface area aerogel prepared nanocrystalline magnesium oxide followed by reduction with hydrazine hydrate (Scheme 1). The NAP-Mg-Pd0 was characterized by using FTIR, XRD, XPS, TEM, ICP-AES, UV-VIS DRS, SEM-EDX etc. NAP-Mg-Pd0 was the employed in different carbon-carbon bond forming reactions namely Heck, Suzuki, Stille, and Sonogashira coupling reactions (Scheme 2). The catalyst showed excellent reusability over several cycles. 

Scheme 1. Preparation of NAP-Mg-Pd0.
 
Scheme 2. Catalytic applications of NAP-Mg-Pd0 in Heck, Suzuki, Stille, and Sonogashira reactions.
Chapter-III describes the preparation, characterization and catalytic application of layered double hydroxides (LDHs) supported rhodium catalyst (LDH-Rh0). The catalyst was synthesized by grafting of Rh3+ on to the LDHs followed by reduction with molecular hydrogen at 250 °C. The thus synthesized catalyst was characterized by using different techniques such as UV-VIS DRS, XPS, XRD, TEM, SEM-EDX, TPR, ICP-AES etc. LDH-Rh0 was employed in Heck, Suzuki, and Stille coupling of aryl halides (iodo- and bromoarenes). The catalyst is easily separated by filtration and recycled with almost consistent activity. 
Chapter-IV describes the preparation, characterization and catalytic application of modified silica supported nanorhodium catalyst (SiO2-Rh0). The catalyst was synthesized by complexation of RhCl3 with 3-aminopropyl functionalized silica gel followed by reduction with aqueous NaBH4. SiO2-Rh0 was characterized by using ICP-AES, XPS, FTIR, XRD etc. SiO2-Rh0 was employed in the Heck-type coupling of alkenes and arylboronic acids to afford the corresponding Heck product in moderate to excellent yields (Scheme 3). SiO2-Rh0 was separated by simple filtration and reused for 5 cycles with slightly diminished activity.


Scheme 3. Heck-type coupling of alkenes with arylboronic acids catalyzed by SiO2-Rh0.

Chapter-V is divided into two sections. Section-A describes the preparation, characterization and catalytic activity of silica immobilized copper complexes in N-arylation of N(H)-heterocycles and benzyl amines with aryl halides and arylboronic acids (Scheme 4).

Scheme 4. N-arylation of N(H)-heterocycles and benzyl amine.
In Section-B, the three component coupling of aldehyde, amine and terminal alkyne catalyzed by silica immobilized copper(I) iodide was described (Scheme 5). The catalyst is easily separated by simple filtration and reused with consistent activity for several cycles.

Scheme 5. Three component coupling of aldehyde, amine, and alkyne catalyzed by silica immobilized CuI.





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English

ISOLATION OF BIOACTIVE MOLECULES FROM THE ROOTS OF DECALEPIS HAMILTONI AND THEIR MODE OF ACTION

JAIN, VIKAS

Stringent Response in Mycobacteria: Molecular Dissection of Rel

K., MANIKANDAN

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                                                   ABSTRACT 
 
The thesis entitled “Studies towards the synthesis of fumagillin & ovalicin and development of new synthetic methodologies” is divided into three chapters.

CHAPTER I: This chapter is further divided into two sections.
Section A : This section describes introduction and previous synthetic approaches for fumagillin.
Section B : This section describes introduction and previous synthetic approaches for ovalicin.

CHAPTER II: This chapter includes our contribution for the synthesis of fumagillin and  ovalicin and is further divided into two sections.
Section A : This section describes the synthesis of a key intermediate for the total synthesis of fumagillin .
Section  B : This section describes the formal total synthesis of ovalicin.

CHAPTER III: This chapter deals with the development of new methodologies and is further divided into two sections
Section  A : This section describes introduction of α- amino phosphonates, α- hydroxyl amino phosphonates and previous approaches for the synthesis α- amino phosphonates
Section B : This section describes the present work for the synthesis  α- amino phosphonates and α- hydroxyl amino phosphonates.


Abstract

Chapter II. 
Scection–A: This section describes the stereo selective sythesis of a  key intermediate for the total synthesis of fumagillin .
         In 1990 Judah Folkman and co-workers discovered that fumagillin 1 isolated from aspergilles fumigatus and  its semi-synthetic drug TNP-470, 2 (also known as AGM 1470) have the remarkable capacity to inhibit the proliferation of endothelial cell in vitro and tumor induced angiogensis in vivo (Fig 1). 
          Fumagillin and its less toxic semisynthetic derivative TNP-470 are potential anti-malarial and anti-leishmaniasis drugs and are the only treatments for intestinal microsporidiosis in HIV-infected patients







Fig 1
Retro synthetic strategy : 

         The  retro synthetic strategy was devised and outlined in Scheme 1. In this the main core cylohexane moiety 3 could be obtained from 4 by extending the aldehyde group. The compound 4 could be obtained from olefinic alcohol 5 which in turn could be synthesized from our well known ring opening reaction of alcohol 6. It was also envisioned that the alcohol compound 6 could be easily obtained from 7, which was accessed from the Diels-Alder reaction of protected furfuryl alcohol 8 and bromo methyl propiolate 9.


Abstract

Scheme 1
        Our first approach is outlined in Scheme 2. The Diels-Alder reaction of TBS protected furfuryl alcohol 8a and bromo meyhyl propiolate 9 in refluxing benzene  gave the adducts 10 and 7a in 3:7 ratio. The regiochemistry of the two adducts was assigned from their ​1H -NMR spectroscopy. The adduct 7a when treated with NaOMe in MeOH gave the dimethoxy compound 11 in good yields. Compound 11 on hydrogenation followed by TBS deprotection with TBAF in THF gave the alcohol 6. Where as the Diels-Alder reaction of PMB protected furfuryl alcohol 8b and bromo meyhyl propiolate 9 under similar conditions  gave the adduct 7b as a single regioisomer. To check the regio chemistry of the adduct, we applied the same set of reactions as above and obtained the  same alcohol 6.
Scheme 2
Abstract
The free 1o-alcohol was transformed to iodide using iodine, TPP and imidazole in refluxing toluene. This iodo compound 14 when treated with TMSOTf in DCM gave the keto compound 15. NaBH4 reduction followed by methylation resulted the iodo methoxy compound 17. Zn mediated ring opening reaction of iodo methoxy compound 17 in refluxing ethanol gave the olefinic alcohol 5. Protection of the sec-alcohol as TBS ether by using TBS-Cl followed by dihydroxylation of the double bond with OsO4-NMO resulted in diol 19. Acetonide protection of the diol followed by DIBAL-H reduction gave the aldehyde compound 21 which is in contrast to desired aldehyde 4 from the  noe studies, which revealed that the two sec-hydroxyl groups were not in the same plane which is mandatory for the target molecule, instead the methoxy group was in the same plane with aldehyde (Scheme 3).

Scheme 3



Abstract

            As the above synthetic route gave the unexpected and unwanted epi compound (at the methoxy center) a small modification was applied in the retro synthetic strategy as given in Scheme 4. In this the epoxide compound 3 could be obtained from the key inter mediate 22 by extending the 10-alcohol. The key intermediate 22 could be obtained from olefin 23 which in turn could be obtained from compound 6 in a similar manner as mentioned above.
Modified retro synthetic strategy.
. 
Scheme 4                                                               
The alternate strategy devised was delineated in Scheme 5. Zn mediated ring opening reaction of iodo compound 14 gave the dimethoxy olefinic alcohol 23. Dihydroxylation of the double bond followed by acetonide protection gave compound 25. This compound 25 when subjected to either protection of the hydroxyl group or deprotection of the dimethoxy ketal  resulted in decomposition
Scheme 5
  
Abstract

As the above conversion was unsuccessful, we tried to synthesize the epoxide intermediate 26 which may be converted to the target molecule 1 and also can be converted to 27 which is biologically active (Fig-2). The details were outlined in Scheme 6.



                        
Fig-2
Reduction of the ester in 23 gave homoallylic alcohol 28. The m-CPBA epoxidation of the homo allylic alcohol 28 gave the epoxide 29. This epoxide moiety decomposed when subjected to any reaction i.e either protection of the 1o-alcohol or deprotection of the ketal. Therefore the p-alcohol in 28 was first protected as its TBDPS ether using TBDPS-Cl followed by epoxidation of the double bond  with m-CPBA afforded the epoxide compound 31. Deprotection of the ketal to ketone 32 was carried out using CSA, acetone and water in 9:1 ratio.  Chelation controlled reduction of the ketone 32 with Et3B and NaBH4 gave the syn diol 26. unfortunately the noe studies revealed that this epoxide compound is epi at the epoxide center 33.   

                                                             Scheme 6
Abstract

As the above synthetic route again gave the unexpected and unwanted epi compound (at the epoxide center)  the strategy was slightly modified as given in  Scheme 7.
Dihydroxylation of the double bond in 28 with OsO4-NMO gave the tetrol 34. Protection of the 1,2- di hydroxyl groups and deprotection of the ketal to ketone was achieved in one pot by treating with CSA in acetone. The 1o-hydroxy group in 35 was masked as its TBS ether and chelation controlled reduction of the ketone with Et3B and NaBH4 gave the syn diol 37. Protection of the two sec-hydroxyl groups with 2,2 DMP followed by deprotection of the TBS group with TBAF in THF resulted in required cyclohexane moiety 22. The stereo chemistry of the key intermediate 22 was further confirmed by the NOESY and COSY studies. Further Studies are under progress in our lab to complete the total sythesis of fumagillin 1. 


Scheme 7






Abstract
                                                           
CHAPTER II, Section-B: This section describes the  formal synthesis of ovalicin by carbohydrate approach.
 	During recent years abnormal angiogenesis has been recognized as a common feature for many proliferate diseases viz. diabetic retinopathy, psoriosis, rheumatoid arthritis including cancer. Thus it has become increasingly importance to inhibit angiogenesis as one of the alternative promising method for cancer therapy.
 	Towards these studies, ovalcin 3 isolated from cultures of Pseudorotium ovalis stock was found to be non-toxic, non-inflammatory and stable at room temperature for years bearing equal potency as AGM 1470 2 and enhanced potency compared to fumagillin 1.  






Retro synthetic strategy:
Our synthetic approach towards the ovalcin  is based on chiron approach and utilizes two key steps, an olefin metathesis reaction and the zinc mediated ring-opening reaction to afford the chiral olefinic alcohol. 
 Our retrosynthetic approach revealed a key intermediate 4 which is formed by the disconnection of the side chain and our main target was to synthesise this  fragment 4  as the conversion of 4 to final target  was well documented in the literature. It was envisioned that the key fragment 4  could be easily prepared by Grubbs RCM of 5, which 
in turn is obtained from fragment 6. The fragment 6 is easily prepared from D-ribose via a sequence of reactions (Scheme 1).


Abstract
Retro synthetic stategy:









                                                              Scheme 1
       Commercially available D(-)ribose was protected as its 2,3-O-isopropylidene derivative and the primary hydroxyl group was protected as tert-butyldimethylsilyl ether with TBDMSCl and imidazole in DCM. The resulting compound 8 was homologated to compound 9 by using Corey-Chekoviskies reagent. The compound 9 was taken and protected as its MPM ether 10 with para-methoxybenzyl bromide. Desilylation of compound 10 with TBAF afforded free alcohol 11,which was further converted to iodide 6 with iodine, triphenyl phosphine and imidazole (scheme 2). 

                                                             Scheme 2
Abstract

             By applying the commonly used protocol for zinc mediated ring opening reaction compound 12 was obtained in good yields.  The resulting free alcohol in 12 was masked with NaH and benzyl bromide as its benzyl ether 13. Deprotection of the acetonide group from compound 13 was achieved using pTSA to give diol 14.  The diol 14 was selectively protected as the corresponding TES ether 15 at the allylic position under lower temperatures and simultaneously as methyl ether 16 with methyl iodide. The compound 16 on PMB deprotection with DDQ provided primary alcohol 17 which was oxidized to aldehyde 18 using TPAP and NMO conditions. Vinylation of the aldehyde 18  afforded allyl alcohol 5, which could be used for the metathesis reaction to get the required basic skeleton (Scheme 3).

                                                             Scheme 3

     

Abstract

Thus compound 5 was subjected to metathesis using Grubb’s 1st generation catalyst to afford the compound 19.The allylic alcohol 19 was converted to enone 20 with iodoxybenzoic acid.  One pot reduction of the unsaturated olefin and deprotection of benzyl ether was achieved with Pd(OH)2 under hydrogen atmosphere to afford the compound 21. One carbon Wittig reaction on ketone with methyltriphenylphosphonium iodide yielded olefin 22.  Epoxidation of the olefin with m-CPBA  afforded the known key intermediate 4 (Scheme 4).  
              
                                                               Scheme 4



This intermediate 4 was already converted to ovalicin in further few steps as reported earlier by Barton et al.   Thus we have accomplished formal total synthesis of ovalicin.
In conclusion, we have described an efficient formal total synthesis of ovalicin by chiron approach. Since all the reactions are high yielding, this strategy could be applied for multigram scale synthesis of the key intermediate, which can be derivatized further for the synthesis of several other analogues for various biological activities.



Abstract

Chapter-III. Section-A: This section describes introduction -amino phosphonates, -hydroxyl amino phosphonates and previous approaches to the synthesis of -amino phosphonates.
Section-B: This section describes present work on -amino phosphonates and -hydroxyl amino phosphonates.
The synthesis of -amino phosphonates has attracted much interest because of their biological activity and structural analogy to -amino acids. As a result, a variety of synthetic approaches have been developed for the synthesis of -amino phosphonates during the past two decades. 
         Recently, ionic liquids have particularly gained recognition as possible environmentally safe and alternative solvent to conventional molecular organic solvents for the development of waste-free chemical processes. Ionic liquids are widely employed as novel and recyclable reaction media for a variety of transformation including asymmetric hydrogenation reaction, oxidation, Friedel-Crafts alkylation and acylation, Diels-Alder reaction, esterfication reaction, Heck-reaction, Baylis-Hillman reaction, Wittig reaction, aromatic nitration, Baker’s yeast reduction, lipase induced transetserfication, acetylation reaction, alkene metathesis, asymmetric epoxidation, O-alkylation, hydroformylation, Fries rearrangement, allylation reaction, Pechmann reaction, Knoevenagel condensation, single-electron transfer reaction, and polymerization reaction, etc.










bmimBF4- : 1-Butyl -3-methyl imidazolium tetraflouroborate.
bmimPF6-  : 1-Butyl -3-methyl imidazolium  hexaflourophosphate.
        Abstract

	Herein, the ionic liquids (bmimBF4 & bmimPF6) have been proved as an efficient catalyst and reaction medium for the three component coupling of a carbonyl compound, amine and diethyl phosphite to yield various -amino phosphonates (Scheme 1). This method describes a general procedure for producing biologically important -amino phosphonates.



Scheme 1

	We first investigated the reaction between a simple, commercially available benzaldehyde 1a, amine 2a and diethyl phosphite 3 by stirring equimolar quantities of the three components in ionic liquids (ILs) for an appropriate time (Table-1) and isolated the desired  -amino phosphonate 4a in  excellent yields (Table 1) (Scheme 2).  


Scheme 2

         Similarly several aldimines (generated in situ from aldehydes and amines) reacted with diethyl phosphite under similar reaction conditions to afford the corresponding -amino phosphonates in high yields. In all cases, the reaction proceeded smoothly at ambient temperatures with high selectivity (Table 1). 


Abstract
                                                             Table-1
a Products were characterized by 1H NMR, IR and mass spectroscopy
b Isolated yields of products
           
                 Abstract
          
              Both aromatic and aliphatic aldimines produced excellent yields of products. However, the reactions of aliphatic aldimines took longer reaction time compared to aromatic imines because of the low reactivity of aliphatic aldehydes than aromatic. The reactions are clean and complete within 5-12 hours. The reaction conditions are very mild  and amino phosphonates are exclusively formed without the formation of any undesired side products.
              Encouraged by the above results, we tried to apply the same ILs system for the synthesis of α-hydroxyl amino phosponates. Although a large number of methods is available for the synthesis of -amino phosphonates, only few methods are reported for -hydroxylamino phosphonates. Further more there are no examples on the use of ILs as promoters for the three component coupling of aldehydes, hydroxylamines and diethyl phosphite to produce -hydroxylamino phosphonates .
              The treatment of benzaldehyde and phenyl hydroxylamine with diethyl phosphite in ILs afforded the corresponding -hydroxylamino phosphonate in excellent yield (Scheme 3).


                                                           Scheme 3
              In a similar fashion, various aldehydes and aryl hydroxylamines reacted smoothly with diethyl phosphite to give the corresponding -hydroxylamino phosphonates in excellent yields (Table 2). The reactions proceeded efficiently at ambient temperature with high selectivity. No trace amounts of -hydroxy phosphonates are obtained as a result of the reaction between the aldehyde and diethyl phosphite. However in the absence of ILs the reaction did not yield any product even after a long reaction time.
Abstract
Table-2
a Products were characterized by 1H NMR, IR and mass spectroscopy
b Isolated yields of products
Abstract

     In addition to its simplicity and milder reaction conditions, this method is even effective with aliphatic and unsaturated aldehydes which normally produce low yields. The present method does not require any additives or promoters to proceed the reaction.
        Another important feature of this reaction is the survival of a variety of  functional groups such as olefins, ethers, and halides under the reaction conditions. The advantage of the use of ILs as novel reaction media for these two transformations is that these ILs were easily recovered and reused.
        In summary, we have demonstrated a novel and efficient protocol for the synthesis of -amino phosphonates and -hydroxylamino phosphonates which can serve as peptide mimetics by using ILs as efficient catalysts and reaction medium. The method is effective for aromatic, aliphatic, unsaturated aldehydes and provides excellent yields of the products, which makes it useful and attractive process for the synthesis of α-amino phosphonates and -hydroxylamino phosphonates.                                                                    
       It is believed that this method presents a better and more practical alternative to the existing methodologies for the synthesis of -amino phosphonates and -hydroxylamino phosphonates.




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CRYSTAL STRUCTURES OF NATIVE AND XYLOSACCHARIDES-BOUND ALKALI THERMOSTABLE XYLANASE FROM AN ALKALOPHILIC BACILLUS SP. NG- 27: STRUCTURAL INSIGHTS INTO ALKALOPHILICITY. ANALYSIS OF C-H...O HYDROGEN BONDS IN HELICES OF GLOBULAR PROTEINS

SRIVASTAVA, ANUP


Abstract



    Multifunctional molecules are often employed as synthons in organic synthesis. Development of new synthetic methodologies in which synthons are prepared is an important subject of recent research in organic chemistry. Several methods have been developed to obtain these precursors, which involve more tedious jobs or contain circuitous routes. Generally the classical synthetic methodologies involve expensive reagents and catalysts, which are not easily available and require harsh reaction conditions. Thus there is a need to replace such reagents and catalysts. Recently there is also a much greater demand on organic chemists for innovation of new mild synthetic methodologies in view of the stipulations laid down by the environmental systems. The threat to ecological and environmental synthesis due to the damages caused by the chemicals has put forward a new area of the so called “Green Chemistry”. 

    Different improved processes are now being discovered to carry out the reactions efficiently and conveniently with readily available inexpensive materials. During the present study some important synthetic methodologies involving C-N or C-O bond formation reactions and halogenation reactions have been developed by utilizing heterogeneous as well as homogeneous catalysts. Some chemical reagents have also been applied in novel ways for useful synthetic transformations. To discuss systematically, all these investigations have been divided to two chapters (Chapter-I and Chapter-II).

Chapter-I: Development of new synthetic methodologies involving C-N or C-O bond formation reactions

1. Synthesis of coumarins via Pechmann condensation using heterogeneous catalysts

    Coumarins are ubiquitous in nature and have an important place in both natural and synthetic organic chemistry. They are usually synthesized by several methods such as Pechmann, Perkin, Knoevenagel, Reformatsky and Wittig reactions. We recently observed that the synthesis of coumarins via Pechmann condensation can easily be carried out with the heterogeneous catalysts, silica supported sodium hydrogen sulfate (NaHSO4∙SiO2) and silica chloride. A phenol and ethyl acetoacetate underwent condensation in the presence of either of a catalyst under solvent free conditions to produce the coumarin conveniently (Scheme IA).


                         9 examples
Scheme IA

    Several coumarins were successfully synthesized in high yields by following the above method. The experimental simplicity, high yields, application of less costly heterogeneous catalysts and absence of solvent are the advantages of the present procedure. The method is environmentally benign.

2. Highly efficient, mild and chemo- and stereoselective synthesis of enaminones and enamino esters using silica supported perchloric acid under solvent-free conditions

    -Enaminones and -enamino esters are useful synthones for the synthesis of various pharmaceuticals and bioactive heterocycles. They are the intermediates for the synthesis of several aminoacids, aminols, peptides and alkaloids. Classically, -enaminones are prepared by direct condensation of -dicarbonyl compounds with amines under reflux in an aromatic solvent with azeotropic removal of water. 

    We have recently observed that silica supported perchloric acid (HClO4·SiO2) is a highly efficient catalyst for the preparation of -enaminones and -enamino esters from -dicarbonyl compounds by treatment with amines (Scheme IB).


                              25 examples
Scheme IB

    The method was found to be highly chemoselective. Amine attacks only at the ketone carbonyl for both diketones and -ketoesters. The (Z)-selectivity was observed in the products derived from acyclic diketones and -ketoesters but (E)-selectivity in the case of 5,5’-dimethyl-1,3-cyclohexadione.

    The solvent-free conditions, mildness of the conversion, simple experimental procedure, clear reaction profiles, high yields and chemo- and stereoselectivities, short reaction times and reusability of the catalyst are the noteworthy advantages of the protocol.

3. A highly chemoselective Boc protection of amines using sulfonic-acid-functionalized silica as an efficient heterogeneous recyclable catalyst

    The tert-butoxycarbonyl (Boc) group is one of the most useful functionalities for protection of amines and amine derivatives. Different base-mediated reactions for Boc protection of amines have been developed. Recently, some Lewis acids have also been applied as catalysts for this purpose.

    Here we have described a simple method for Boc protection of amines using sulfonic-acid-functionalized silica as heterogeneous catalyst at room temperature (Scheme IC). Selectivity of the method was extended by using a mixture of different amines (Scheme ID).


                                                                                                Sulfonic-acid-functionalized silica
                  24 examples
Scheme IC


                         3 examples
Scheme ID
 
   The protocol is highly chemoselective offering potential in different applications. The method also has several other advantages such as simple experimental procedures, mild reaction conditions, excellent yields of mono Boc protected amines and reusability of the catalyst.

4. An efficient and convenient protocol for the synthesis of quinoxalines and dihydropyrazines via cyclization-oxidation processes using HClO4·SiO2 as a heterogeneous recyclable catalyst

    Quinoxalines and pyrazines are important class of heterocycles in medicinal chemistry. A number of methods have been developed for the synthesis of substituted quinoxalines and dihydropyrazines (DHPs) involving condensation of 1,2-diamines with α-diketones, 1,4-addition of 1,2-diamines to diazenylbutenes, oxidation-traping of α-hydroxy ketones with 1,2-diamines, cyclization-oxidation of phenacyl bromides and o-phenylenediamines through solid-phase and oxidative coupling of epoxides with ene-1,2-diamines.

    Recently, we investigated an efficient method for the synthesis of quinoxalines and DHPs from α-bromo ketones and 1,2-diamines in the presence of silica supported perchloric acid (HClO4·SiO2) at room temperature (Scheme IE).

                         14 examples
Scheme IE

    The mild reaction conditions, simple experimental procedure, clear reaction profiles and reusability of the catalyst are notable advantages of the present method. To our knowledge, this is the first report of the synthesis of quinoxalines and DHPs from 1,2-diamines and α-bromo ketones using heterogeneous catalyst at room temperature.

5. A simple and efficient chemoselective benzyloxycarbonylation of amines in water

    The protection of amines with benzyloxycarbonyl (Cbz) group is frequently used in organic synthesis. The earlier methods reported for Cbz protection of amines are associated with different drawbacks such as strongly basic conditions, low or high temperatures, long reaction times, unsatisfactory yields and requirement of an organic solvent.

    Here we report a simple method for Cbz protection of amines by a treatment with Cbz-Cl in water in the absence of any acid / base catalyst at room temperature (Scheme IF).

                                          28 examples
Scheme IF
    The mild and eco-friendly reaction conditions, organic solvent free conversion, interesting chemoselectivity in the case of two different amines as well as symmetrical amines and amine in the presence of various functionalities and impressive yields are the notable advantages of the method.

Chapter-II: Development of synthetic methodologies involving halogenation reactions

1. α-Bromination of carbonyl compounds using N-bromosuccinimide and heterogeneous catalysts

     -Bromination of carbonyl compounds is an important transformation in organic synthesis as the -brominated products are useful synthetic intermediates. Previously NBS has been utilized for α-bromination of carbonyl compounds using a radical initiator [such as, azobisisobutyronitrile (AIBN) or dibenzyl peroxide (BPO)], or in strongly basic media or in the presence of Mg(ClO4)2 or NH4OAc.

    We have recently found that silica-supported sodium hydrogen sulfate (NaHSO4∙SiO2) (Scheme IIA, Condition A) or sulfonic-acid-functionalized silica (Scheme IIA, Condition B) is an efficient catalyst for α-bromination of carbonyl compounds using NBS.


                                                                                       0.25 – 3h            10 – 99%
                                                    16                                                  17
Condition A: NBS, NaHSO4∙SiO2, ether or CCl4
Condition B: NBS, sulfonic-acid-functionalized silica, ether or CCl4
                 40 examples
Scheme IIA

    The present protocol is associated with several advantages such as mild reaction conditions, short reaction times, excellent yields of monobrominated products, simple experimental procedure and reusability of the catalyst. The used catalysts make the process more eco-friendly compared to the homogeneous acid catalysts. A novel important application of sulfonic-acid-functionalized silica is also discovered.

2. Regioselective nuclear bromination of aromatics and heteroaromatics with NBS using sulfonic-acid-functionalized silica or ammonium acetate as a catalyst

    Brominated arenes and heteroarenes are useful as pharmaceuticals, agrochemicals, flame retardants and specialty chemicals. Several aryl and heteroaryl bromides are potential antitumour, antibacterial and antioxidant agents. Earlier several methods for the bromination of aromatics and heteroaromatics involving the utilization of NBS are associated with certain drawbacks including long reaction time (NBS-SiO2), high temperature (NBS-TBAB and NBS-HZSM-5) and complex experimental procedure (NBS-HBF4·Et2O).

    We have recently found that several activated arenes can easily be brominated with NBS in the presence of sulfonic-acid-functionalized silica or ammonium acetate at room temperature (Scheme IIB).



Condition A: NBS, sulfonic-acid-functionalized silica, CH3CN-Et2O (1:3), r.t.
Condition B: NBS, ammonium acetate, CH3CN, r.t.
             67 examples
Scheme IIB

    The method is highly regioselective offering potential in various synthetic applications. The mild reaction conditions, simple experimental procedure, rapid conversion and excellent yields are notable advantages of the method.

3. A rapid and efficient stereoselective synthesis of (Z)- and (E)- allyl bromides form Baylis–Hillman adducts using bromodimethylsulfonium bromide

    The allyl halides prepared form Baylis–Hillman adducts have been used in the synthesis of various natural and biologically active molecules and their analogues such as α-methylidene-γ-butyrolactones, α-alkylidene-β-lactams and flavonoids. The direct conversion of the Baylis–Hillman adducts into their corresponding halides has been previously accomplished with different halogen-containing reagents including strong acids (HBr/H2SO4, HI/H3PO4), organic acid halides (oxalyl chloride MsCl), HCA/PPh3, Lewis acids (FeCl3, InCl3) and metallic (Na and Li) halides.

    We have recently discovered that the Baylis–Hillman adducts can easily be converted into the corresponding allyl bromides by treatment with bromodimethylsulfonium bromide (Me2S+Br Br–) in acetonitrile at room temperature (Scheme IIC).


                       15 examples
Scheme IIC

    The method is characterized by mild reaction conditions, short conversion times, high yields and excellent stereoselectivities [(Z)-configuration, when EWG = ester and (E)-configuration in the case of EWG = CN].

4. Ammonium acetate catalyzed improved method for the regioselective conversion of olefins into bromohydrins and bromoethers at room temperature

    Selective vicinal functionalization of alkenes with the functional groups such as hydroxy or alkoxy and halogen finds applications in various useful organic transformations. The most common method for the preparation of halohydrins involves ring opening of epoxides or cyclic sulfate by hydrogen halides or metalhalides. 

    We observed that olefins are rapidly converted into bromohydrins and bromoethers using N-bromosuccinimede and catalytic amount of ammonium acetate (NH4OAc) (Scheme IID).


              18 examples
Scheme IID

    The mild reaction conditions, simple experimental procedures, rapid conversion, clear reaction profiles, excellent yields and high regioselectivity are the noteworthy advantages of the present protocol.

5. An improved method for the regioselective conversion of olefins into iodohydrins and iodoethers using NIS at room temperature catalyzed by ammonium acetate

    Synthesis of bromohydrins has been reported directly from dilute aqueous solutions of bromine. The preparation of iodohydrins using the same procedure is hampered by the ready reversibility of the addition of hypoiodous acid. The preparation of iodohydrins from alkenes also is reported by the use of N-iodosaccharin, H5IO6/NaHSO3, triodide ion, N-iodoimide, dimethyldioxirane/CH3I, bis(pyridine)iodine(I)salts and iodine mediated by clay.

    Here we report the synthesis of iodohydrins and iodoethers form alkenes by treatment with NIS in the presence of NH4OAc catalyst in water-acetone (1:4) at room temperature (Scheme IIE).


             12 examples
Scheme IIE

    The mild reaction conditions, interesting chemoselectivity and simple experimental procedures are the noteworthy advantages of the present protocol.

6. Synthesis of β-chlorohydrins in water

    The transformation of epoxides into halohydrins can be carried out with halogens, hydrogen halides, and metal halides in organic solvents such as MeCN or CH2Cl2. However, many of these methods are associated with several disadvantages, including harsh reaction conditions, unsatisfactory yields and poor selectivity. Thus, an improved protocol for the conversion of epoxides into the corresponding vicinal halohydrins is essential.

    We observed that the ring opening of epoxides can efficiently be carried out with TCT (2,4,6-trichloro-1,3,5-triazine) in the presence of morpholine in H2O at room temperature to form the corresponding β-chlorohydrins (Scheme IIF).



                                 12 examples
Scheme IIF

    The efficiency and the economic and environmental benefits of this reaction are the notable features of our synthetic protocol.

Chapter-III: Studies on natural polycyclic compounds – Chemical investigation on Pulicaria wightiana

    Natural Products are increasingly gaining importance and attention from chemists and pharmacologist alike. Many of the medicinal and biological agents used on a worldwide basis are either natural products or derivatives of natural products. Natural product chemistry is experiencing a dramatic evolution presently due to recent technological development. Natural products have been the major source of medicines and much of the world’s population still rely upon the remedies of natural product origin. The searching for pharmacologically active agents obtained by screening natural sources such as plant extracts has lid to the discovery of many clinically useful drugs those play a major role in the treatment of human diseases. Several complex biologically promising molecules have been isolated and characterized from different plant sources. However, nature continues to offer unrivaled and virtually random structural diversity to produce various types of bioactive compounds. Therefore the chemical investigation on medicinal plants have always been given considerable importance. The plants not only continue to retain their historical significance as important sources for development of new drugs, but also are extremely useful as sources of lead compounds for structural modification and optimization which can also be employed as specific probes in biochemical studies. Plant derived compounds which have recently undergone development include the anticancer agents such as taxol and camptothecin and the antimalarial drug artemisinin.

    Inspired by the tends in the modern phytochemical research, the author has made a modest effort to investigate on a medicinal plant, Pulicaria wightiana. In this thesis the results of chemical investigation on this plant has been discussed. Chemical investigation of the plant led to the isolation of some new polycyclic compounds along with several reported known compounds. The isolation and structure elucidation of the constituents have been discussed. The biological activities of some of the compounds have been presented.

Chemical investigation on Pulicaria wightiana

    Various species of Pulicaria are known for their medicinal properties. The metabolities obtained from different species of Pulicaria are also chemically interesting. These metabolities are of varied types, viz., sesquiterpenoids, diterpenoids, triterpenoids, flavonoids, phenolic compounds and steroids. 

    The present investigation afforded three new clerodane diterpenoids, one new benzofuranoid along with several known compounds. The structures of all the new molecules have been confirmed by their 1D and 2D NMR (COSY, NOESY, HMBC and HSQC) and MS spectroscopic studies. Antibacterial and antifungal activity of all the new and some reported molecules have been studied. The structures of all the known compounds were established from their detailed spectroscopic (IR, 1H NMR, some cases 13C NMR and MS) data and by comparing the spectral values with those reported in literature.





    The following compounds have been isolated from the plant Pulicaria wightiana.

Compound code	Compound name	Compound nature	Remarks
PWV-1	Stigmasterol	White crystals	First report from the species
PWV-2	β-Sitosterol	White solid	First report from the species
PWV-3	6-Benzofuran carboxylic acid-5-formyl methyl ester	Dark yellow needles	New compound
PWV-4	Methyl 15,16-epoxy-6α-hydroxy-3,13 (16),14-clerodatrien-18-oate	Viscous mass	Known compound
PWV-5	Phyllemblin	White powder	First report from the species
PWV-6	Methyl 6-oxo-3,13-clerodadien-15,16-olid-18-oate	Colorless crystals	New compound
PWV-7	Pectolinarigenin	Pale green crystals	First report from the species
PWV-8	Isopimara-8(14),15-diene-7-keto-2α-ol	White solid	Known compound
PWV-9	2β-(2-Methylbutanoyl)-3,13-clerodadien-15,16-olid-18-oic acid	White solid	New compound
PWV-10	Methyl 6α-hydroxy-3,13-clerodadien-16,15-olid-18-oate	Viscous mass	Known compound
PWV-11	Chrysin	Pale yellow powder	First report from the species
PWV-12	Methyl 6,15-dihydroxy-3,13-clerodadien-15,16-olid-18-oate	Viscous mass	Known compound
PWV-13	6α-Hydroxy-3,13-clerodadien-16,15-olid-18-oic acid	Colorless needles	First report from the species
PWV-14	Methyl 6α-hydroxy-3,13-clerodadien-15,16-olid-18-oate	Pale green crystals	Known compound
PWV-15	Methyl 6α,7α-dihydroxy-3,13-clerodadien-15,16-olid-18-oate	Viscous mass	Known compound
PWV-16	6α-Hydroxy-3,13-clerodadien-15(16),4(7)-diolide	White crystals	Known compound
PWV-17	Oxyanin B	Pale green crystals	Known compound
PWV-18	Methyl 6α-hydroxy-3,13-clerodadien-18-oate	Viscous mass	New compound
PWV-19	Fraxetin	Pale green crystals	First report from the species
PWV-20	Gallic acid	White powder	First report from the species
PWV-21	Daucosterol	White powder	First report from the species
PWV-22	Bergenin	Colorless crystals	First report from the species

    All the isolated compounds have been examined (TLC experiments) to be present in the original extract of the plant.
         


                                              





         

 
                         


    Thus the chemical investigation on Pulicaria wightiana offered four new compounds along with several known compounds, some of which are reported first time from the species.















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i





ANTIOXIDANT PROPERTIES AND CHEMOPREVENTIVE POTENTIAL OF THE BIOACTIVE CONSTITUENTS OF THE ROOTS OF DECALEPIS HAMILTONII

ALAMI, MEHRAN

PHYSICO-CHEMICAL PROPERTIES AND SPAGHETTI MAKING QUALITY OF INDIAN DURUM WHEAT

R., MAHALAKSHMI

In the present study, the medicinal plant, Soymida febrifuga, was selected based on the rural people practice to cure various diseases and Ayurvedic (Indian System Medicine) literature available. Since, bark, root and leaf of this were used as a folklore medicine for inflammation, diabetes, tumor and hepatic diseases, these plant parts have been selected for the study. Though S. febrifuga received ample of attention in Ayurveda, only some of the therapeutic uses have been evaluated scientifically, such as anti-inflammatory, antiplasmodial and sexual vitality in women. Hence, remaining therapeutic uses, diabetes, hepatoprotetive and tumor suppression, was evaluated scientifically in this study.
Plant fingerprinting has done using the HPTLC system. All the extracts of S. febrifuga have shown the presence of various phytochemicals such as alkaloids, flavonoids etc. In the acute oral toxicity studies, Maximum Tolerated Dose was determined based on the visual, hematological and biochemical parameters. All the extracts did not show any toxicity even at higher doses (2000mg/kg), except root bark methanolic extract that has shown severe toxicity even at low doses (LD-50: 102 ± 25 mg/kg).
In vitro antioxidant activity of S. febrifuga extracts has been evaluated taking total phenolic content, reducing power, metal chelating activity, superoxide, hydroxyl, hydrogen peroxide, DPPH and nitric oxide scavenging activity as parameters. Results have shown that, methanolic and aqueous extracts of bark and leaf have significant antioxidant activity than the root and root bark extracts.
The results obtained in the in vitro antioxidant study, prompted us to evaluate the protective effects of S. febrifuga extracts on oxidative damage. Liver cell line, HepG2 was selected for the study and oxidative damage was induced with tert-butyl hydroperoxide (t-BHP). Initially, HepG2 cells were incubated with extracts/t-BHP to evaluate the cytotoxicity followed by co-incubation with extracts and t-BHP using MTT, ALT, LDH as parameters. Results have shown that, all the extracts have little toxicity even at higher concentrations (250µg/ml) except root bark methanolic extract. T-BHP has shown singinificant toxicity on HepG2 cell and IC-50 value found to be 3.90 mM. In co-incubations with S. febrifuga extracts, the viability of the cells was significantly increased indicating the protective roles of extracts on oxidative damage. To elucidate the molecular mechanism behind the protective roles of S. febrifuga extracts, all the glutathione related enzymes, catalase and superoxide dismutase were evaluated. Results have shown that all the antioxidant enzymes were increased in a dose dependant manner. Especially, glutathione was significantly up regulated. To understand the prime target of the extract that specifically up regulated the levels of glutathione, glucose-6-phosphate dehydrogenase, which pumps NADPH to convert GSSG to GSH, protein levels were monitored using western blot. Glucose-6-phosphate dehydrogenase (G6PD) was highly up regulated in the extracts treated cells. Especially, bark and leaf extracts were more active in increasing the G6PD levels. With all results the following conclusion was made for the protective role of extracts.
In co-incubations with extracts and t-BHP, ALT, LDH levels were restored in the medium due to stabilization of the plasma membrane by the extract and all the antioxidant enzymes were also elevated. In co-incubations G6PD levels were significantly elevated which led to the elevation of NADPH. Elevated levels of NADPH were utilized by glutathione reductase to convert GSSG to GSH. The elevated levels of GSH protected the cells. When DHEA, inhibitor of G6PD, was co-incubated protective roles were diminished, indicating extracts prime target is G6PD.
To evaluate the hepatoprotective activity of the extracts, animals were pretreated with extracts for 14 days. Since bark and leaf extracts were active in protecting HepG2 cells from oxidative damage, only these two plant parts have selected for the in vivo hepatoprotective activity. On day 15 animals received single dose of CCl4, orally. After 48 hr, plasma ALT, AST and glutahione related enzymes were estimated.  Results have shown that, serum ALT, AST and all the glutathine related enzymes as well as catalase and superoxide dismutase were also restored to normal levels, indicating the hepatoprotective roles of S. febrifuga extracts. Histopathological studies confirmed the same.
In vitro hypoglycemic activity of the S. febrifuga extracts was evaluated in 3T3-L1 preadipocytes. 3T3-L1 cells were induced to adipocyte by various external insults like dexamethasone, insulin and IBMX. Adipocytes were incubated with extracts for 4 hr (pretreatment) and media was replaced with fresh media containing rdiolebeled glucose. After incubation for additional 6 hr, radiolebeled glucose concentration was estimated in the cell lysate. Results have shown that, al the extracts significantly increased the glucose uptake in basal and insulin stimulated conditions, in a dose dependant manner.. Extracts could also stimulate the glucose uptake in liver cell line HepG2, in a dose dependant manner. Insulin secretogague activity of the extracts was evaluated in RIN cells, but no extract has shown insulin secretogague activity.
In vivo hypoglycemic activity of the extracts was evaluated in three animal models, normohypoglycemic, glucose tolerance and streptozotocin induced model. Though, extracts have shown significant hypoglycemic activity in 3T3 cell, but they are mild active in in-vivo.
Cytotoxicity studies of the extracts have shown that root bark methanolic extract is highly toxic. To know the molecular mechanism behind extracts toxicity, extracts was screened for apoptosis inducing activity. Morphological characters, cell cycle analysis, western blot analysis was used to assess the apoptosis inducing activity of the extract. Results have shown that, extracts at lower concentrations (50 µg/ml) has shown all the typical apoptosis characters such as DNA fragmentation, nuclear condensation, elevated levels of cytochrome C and cleaved caspase 3 and decreased levels of Bcl2. At higher concentrations (>100 µg/ml) extracts induced necrosis.
Further prospects of the study are, though root bark methanolic extracts has shown significant apoptosis inducing activity in cell culture models it has to validate in vivo models such as in nude mice to assign anticancer property.
Bark and leaf extracts of the Soymida febrifuga have shown significant hypoglycemic activity in cell culture model but they were found to be mild active in in-vivo model. The reasons might be inability of the active principle to reach target site or active principle might be breaking down to non-active by its metabolism, further investigation is needed to confirm.
The methanolic extracts of Holoptelea integrifolia (Roxb.) (Urticaceae) leaves (MLE) and stem bark (MSBE) were studied for the woundhealing potential. Since wound healing is severely hampered by microbial infection and reactive oxygen species (ROS), this study was undertaken to evaluate antimicrobial and antioxidant activity apart from wound-healing activity. The antimicrobial property of the Holoptelea was studied against the six bacterial and five fungal strains using the agar well diffusion method and minimum microbicidal concentration and minimum inhibitory concentration were determined for each strain, in which methanolic extract of stem bark (MSBE) has shown bigger zone of inhibition (11.3–20.4 mm) than methanolic extract of leaves (MLE) (9.6–14.9 mm). The anti-oxidant activity was evaluated by DPPH free radical scavenging activity using HPLC method. The IC50 values obtained for MSBE (TPC: 78.53±1.26 mg/g) and MLE (TPC: 57.71±1.45 mg/g) were 37.66±0.48 and 50.36±0.59µg/well, respectively. In excision wound model, more than 90% wound healing was recorded in treated groups by 14 days of post surgery, where as only 62.99% was observed in the control group. In incision model, higher breaking strengths and higher hydroxyproline content in treated groups suggested higher collagen re-deposition than the control group. Finally, histopathology studies conformed wound-healing activity of Holoptelea integrifolia.



Synopsis:
The present study was designed to evaluate the antioxidant and antimicrobial properties of hexane (LH), methanolic (LM) and aqueous (LA) extracts of Soymida febrifuga (Maliaceae) leaves, which is a traditional folk medicine in India. No pharmacological evaluation has been reported on this plant (except antiplasmodial activity) till date. Antioxidant activity of different extracts was evaluated by DPPH free radical scavenging activity, taking Total Phenolic Content (TPC) as an index. Antimicrobial activity was tested against 6 bacterial and 5 fungal strains using Agar Hole Diffusion method and Minimum Inhibitory Concentrations (MIC) and Minimum Microbicidal Concentration (MMC) were determined for all the test organisms against the extracts. Results have shown that, methanolic and aqueous extracts of leaf had higher antioxidant activity and total phenolic content than the hexane extract. The antioxidant activity and TPC of the extracts were highly correlated. Extracts had also shown several degrees of antimicrobial activity against different microbes. Methanolic extract was more potent against Aspergillus fumigatus and Candida tropicana. Lowest MIC values obtained for LM, LA and LH were 78, 156, 625µg/ml against A. fumigatus C. tropicana and C. albicans, respectively. Hence, this study conforms Soymida febrifuga leaves possess potent antioxidant and antimicrobial activity.




AROMATIC INTERACTIONS IN PEPTIDES : DESIGNED HELICES AND β-HAIRPINS

Saxena, A

Synopsis 
Adaptation to any undesirable change in the environment dictates the 
survivability of many microorganisms. Such changes generate a quick and suitable 
response, which guides the physiology of bacteria. Stringent response is one of the 
mechanisms that can be called a survival strategy under nutritional starvation in 
bacteria and was first observed in E. coli upon amino acid starvation, when bacteria 
demonstrated an immediate downshift in the rRNA and tRNA levels (Stent and 
Brenner 1961). Mutations that rendered bacteria insensitive to amino acid levels were 
mapped to an ‘RC gene locus’, later termed relA because of the relAxed behavior of 
the bacteria (Alfoldi et al. 1962).  Later on, Cashel and Gallant, showed that two 
“magic spots” (MSI and MSII) were specifically observed in starved cells when a 
labeled nucleotide extract of these cells was separated by thin layer chromatography 
(Cashel and Gallant 1969). These molecules were found to be polyphosphate 
derivatives of guanosine, ppGpp and pppGpp (Cashel and Kalbacher 1970; Sy and 
Lipmann 1973), and were shown to be involved in regulating the gene expression in 
the bacterial cell, demonstrating a global response, thus fine-tuning the physiology of 
the bacterium. Two proteins in E. coli, RelA and SpoT, carry out the synthesis and 
hydrolysis of these molecules, respectively, and maintain their levels in the cell 
(Cashel et al. 1996; Chatterji and Ojha 2001). On the other hand, Gram-positive 
organisms have only one protein Rel carrying out the functions of both RelA and 
SpoT (Mechold et al. 1996; Martinez-Costa et al. 1998; Avarbock et al. 1999).  
Although Rel or RelA/SpoT has been studied from several systems in detail 
pertaining to the physiological adaptation, less information is available on the 
regulation of the protein activity under different conditions. Our studies show that the 
RelMsm is composed of several domains (HD, RSD, TGS and ACT) with distinct 
function. HD and RSD domains, present in the N-terminal half of the protein, harbor 
catalytic sites for the hydrolysis and the synthesis of (p)ppGpp, respectively. TGS and 
ACT domains, on the other hand, are present at the C-terminal half of the protein and 
have regulatory function. It, therefore, appears that a communication exists between 
these domains, to regulate protein activity. It was shown earlier, while studying Rel 
from S. equisimilis, that there exists an interaction between the C-terminal and the N-
terminal of the protein which determines the kind of activity (synthesis/hydrolysis), 
 xii
the protein should demonstrate (Mechold et al. 2002). Later, the N-terminal half 
crystal structure of the same protein suggested an inter-domain “cross-talk” between 
the HD and the RSD domain that controls the synthesis/hydrolysis switch depending 
on cellular conditions (Hogg et al. 2004). 
In the present work, studies have been carried out to understand a Gram-
positive Rel in greater detail and to find out how the opposing activities of Rel are 
regulated so that a futile cycle of synthesis and hydrolysis of (p)ppGpp, at the expense 
of ATP, can be avoided. The work has been divided into several chapters describing 
studies on various aspects of the protein. 
Chapter 1 outlines the history of the stringent response and summarizes the 
information available about the stringent response in various systems including plants. 
Several roles that (p)ppGpp plays in different bacteria have been examined. A special 
mention on the crystal structure of RelSeq has been made with respect to the regulation 
of activity. Also, the information available regarding the effects of (p)ppGpp on RNA 
polymerase has been documented. Role of ppGpp in plants has been discussed in 
great detail with special emphasis on abiotic stresses. 
Since different functional domains have been identified in RelMsm, the protein 
has been divided into two halves and they have been discussed separately in the form 
of two chapters. 
Chapter 2 describes the N-terminal half of the Rel protein of M. smegmatis in 
greater detail. Out of the several domains identified, the role of the two domains 
present in the N-terminal half of the protein has been studied. The N-terminal half 
shows both synthesis and hydrolysis activities. Importantly, we find that the protein is 
active even in the absence of accessory factors such as ribosome and uncharged 
tRNA, unlike RelA of E. coli. Moreover, deletion of the C-terminal half of the protein 
leads to a much higher synthetic activity, clearly indicating that the C-terminus is 
involved in regulating the activity of the protein. Both TGS and ACT domains (the 
two domains found in the C-terminal half of the protein) have been found to play a 
regulatory role. The results also indicate that all the deleted constructs are active both 
in vitro and in vivo. 
Chapter 3 discusses the C-terminal half of the protein and its role in the 
multimerization observed in RelMsm. We show that multimerization of Rel protein is 
due to the inter-molecular disulfide cross-linking. Furthermore, we find that the 
 xiii
monomer is the active species in vivo. One of the fascinating points about the C-
terminal half is that it is largely unstructured. Additionally, the C-terminal half cannot 
complement the N-terminal part of the protein when provided in trans, demonstrating 
further, the requirement of an intact protein for bringing about regulation of Rel 
activity. This requirement in cis suggests the presence of an intra-molecular 
communication between the N- and the C-termini, as a mediator of protein regulation. 
Further, presence of uncharged tRNA increases pppGpp synthesis and down-regulates 
its hydrolysis in the wildtype protein. However, the uncharged tRNA-mediated 
regulation is absent in the deleted construct with only the N-terminus half, indicating 
that uncharged tRNA binds to the C-terminal half of the protein. Several cysteine 
mutants have been constructed to understand their role in the regulation of Rel 
activity. The results suggest that one cysteine, present at the C-terminus, is required 
for intra-molecular cross-talk and the uncharged tRNA-mediated regulation. 
A detailed characterization of the communication between the two halves of 
the protein has been attempted in Chapter 4. Surface plasmon resonance experiments 
carried out on the different cysteine mutants discussed in Chapter 3, for uncharged 
tRNA binding indicate that all the mutants bind to uncharged tRNA with near-equal 
affinities as the wildtype protein. This study suggests that the non-responsiveness for 
tRNA seen in one of the cysteine mutants is due to the loss of inter-domain 
interaction, while the binding of protein to accessory factors is unaffected. 
Fluorescence resonance energy transfer has been carried out to observe domain 
movement in the presence of accessory factors. Distances between the different 
domains scattered in this ~90 kDa protein, measured by FRET technique, are 
suggestive of an inter-domain cross-talk, specifically between C338 and C692, 
thereby regulating the activity of this enzyme. We show, for the first time, that the 
product of this protein, (p)ppGpp can bind to the C-terminal half making it 
unstructured, and can, therefore, regulate the protein activity. 
Chapter 5 is an effort to characterize the promoter of rel from M. tuberculosis. 
This study was undertaken in order to develop an expression system in mycobacteria. 
The +1 transcription and the translation start sites have been identified. The –10 
hexamer for the RNA polymerase binding has also been mapped using site-directed 
mutagenesis and is found to be TATCCT. This promoter is also unusually close to the 
+1 transcription start site. The promoter is specific for mycobacteria and does not 
 xiv
function in E. coli. Additionally, the promoter is found to be constitutive in M. 
smegmatis; however, the possibility of it being regulated in M. tuberculosis cannot be 
ruled out. 
Appendix section discusses, in short, the phylogenetic analysis of the 
mycobacterial Rel sequences. Diagrams of the plasmids used in this study have been 
provided. Mass spectra recorded for the in vitro synthesized and purified pppGpp and 
the trypsin digest of the full-length Rel protein have also been given. 


Environmental studies of the Arabian Sea using remote sensing, GIS and GPS techniques

K.V., SATYANARAYANA

CLONING AND CHARACTERIZATION OF N-METHYLTRANSFERASE GENE ASSOCIATED WITH CAFFEINE BIOSYNTHESIS IN COFFEE

-, SHADANAIKA








Abstract


C-C COUPLING REACTIONS CATALYZED BY NANOCRYSTALLINE MAGNESIUM OXIDE 

The pictorial flow of the thesis and a brief description of the chapters are outlined below:
	

The thesis mainly deals with the applications of nanocrystalline magnesium oxide for selective organic transformations. The first chapter mainly deals with the importance and applications of heterogeneous catalysis, importance of nanocrystalline materials in catalysis for selective organic transformations. The importance and the need for the design and development of environmentally cleaner catalytic methodologies is also highlighted. Chapter 2 which is divided into two sections, deals with the application of nanocrystalline magnesium oxide for the one-pot Wittig reaction and Wadsworth-Emmons reaction for the synthesis of α,β-unsaturated esters and nitriles, for the stereoselective formation of C=C double bonds. Chapter 3 which is divided into two sections, describes the application of nanocrystalline magnesium oxide for C-C bond formation reactions in Mannich and aldol reactions for the synthesis of α-thioalkyl-β-amino acid derivatives and α-thioalkyl-β-hydroxy esters respectively. Chapter 4 which is divided into two sections, describes the application of nanocrystalline magnesium oxide for the Strecker and cyanosilylation reactions to produce α-amino nitriles and cyanohydrins respectively. Chapter 5 deals with the one-pot synthesis of pyridines and 1,4-dihydropyridines by the three component coupling reaction of aldehydes, malononitrile and thiols using nanocrystalline magnesium oxide.
CHAPTER-I: Introduction
This chapter describes the various catalysts/process options available for an industrial chemist to effect different organic transformations. It includes a brief introduction of homogeneous catalysis, heterogeneous catalysis, nanomaterials and nanocatalysis. The need and development of cleaner and greener alternative technologies using truly heterogeneous catalytic system in the synthesis of pharmaceuticals and fine chemicals is described. This chapter also describes the basic concepts of nanocrystalline magnesium oxide and evaluation of this material for organic synthesis. 
CHAPTER-II: Stereoselective C=C double bond formation reactions catalyzed by nanocrystalline MgO
The development of methods for the stereoselective formation of C=C double bonds represents one of the most important challenges in organic synthesis. 
This chapter is divided into two sections.
Section A: One-pot Wittig reaction: A facile synthesis of α, β-unsaturated esters and nitriles by using nanocrystalline MgO
The most important intermediates for several biologically active molecules have been synthesized through Wittig reaction. α,β-Unsaturated esters and nitriles have attracted increased interest since they can serve as useful intermediates in the synthesis of variety of biologically active compounds.
The most impressive variant in the Wittig reaction is the replacement of the three step-process, involving the preparation of phosphonium salt, followed by base treatment to give ylide, subsequent reaction with carbonyl compounds to give olefinic products with the one pot synthesis.  The α,β-unsaturated esters are obtained directly in one-pot in good yields by the reaction of aldehyde with methyl bromoacetae using n-Bu3P/Zn at 100 0C, or catalytic amounts of tributylarsine or dibutyl telluride, in the presence of triphenyl phosphite. The use of ethyl diazoacetate (EDA) instead of -bromo esters in the above protocol is also reported by using catalytic amounts of iron, ruthenium, cobalt porphyrin complexes in the presence of PPh3, P(OMe)3, P(OEt)3. 
In this chapter, one-pot Wittig reaction to afford α,β-unsaturated esters in excellent yields with high E-stereoselectivity and Z-α,β-unsaturated nitriles with excellent yields by the reaction of aldehydes with α-bromoacetaes and α-bromoacetonitrile using nanocrystalline magnesium oxide is described.


A fluorine atom substitution, adjacent to the ester moiety increases significantly the biological activity of these compounds, as exemplified in vitamin A and pheromone chemistry. -Fluoro-α,β-unsaturated esters, a useful class of intermediates in the synthesis of a variety of biologically active fluoro compounds were also synthesized through one-pot Wittig reaction under these optimized conditions by the reaction of aldehydes and α-fluoro,α-bromoacetae in moderate yields with Z selectivity.
             

In one-pot Wittig reaction ethyl bromoacetate reacts with triphenylphosphine forms phosphonium salt, and it was converted to phosphorous ylide by using NAP-MgO, was proved with 31P-NMR, and ylide reacted with aldehyde to afford olefin. 
In Wittig reaction olefins are obtained with high selectivity towards E isomer, due to the surface of the catalyst and its high reactive sites. It was assumed that the formed intermediate “betaine” forms a complex with the unsaturated  Mg2+/Mg+ site (Lewis acid type) of NAP-MgO. In betaine, aldehyde R group and ylide COOEt group should be in different plane for minimizing their steric interactions. Therefore, trans-oxaphosphetane

will be formed by the planar four centered transition state to relieve a dominating 1,2 interaction between R group on the aldehyde and COOEt group on the ylide, which predominates E-,-unsaturated esters. When the hydrogen is  substituted by fluorine atom in the same E-form of ,-unsaturated esters, the product -fluoro-,-unsaturated ester is considered as Z-form according to atomic number priority and the same Z-isomer of fluoro compound was predominantly obtained in the optimized conditions. Whereas in ,-unsaturated nitriles, the increase in Z isomer is due to the lesser steric requirement of the linear cyano group.
Section B: Wadsworth-Emmons reactions catalyzed by nanocrystalline MgO
The versatile Wadsworth-Emmons (WE) reaction belongs to a class of C=C double bond formation reactions, which find wide applications in the elegant synthesis of intermediates for fine chemicals such as perfumes, fragrances, analgesics, insecticides, carotenoids, pheromones, pharmaceuticals and prostaglandins etc.
In WE reaction, the phosphonate anions are strongly nucleophilic and react readily with aldehydes to form olefins. Generally, the WE is performed in the presence of relatively strong bases such as n-butyllithium, potassium tert-butoxide, sodium hydride, LDA, DBU, Triton B, N-ethylpiperidine etc., in homogenous conditions.
The recyclable nanocrystalline magnesium oxide was used for WE reaction of aldehydes with various phosphonates to afford E-α,β-unsaturated esters and Z-α,β-unsaturated nitriles with excellent yields under mild conditions without the formation of by-products. Due to the formation of trans-oxaphosphetane anion as intermediate, the products, olefins are obtained as stereoselective E-isomers.	




CHAPTER-III: Application of nanocrystalline MgO for C-C bond formation reactions 
A carbon-carbon bond forming methodology for the formation of β-amino acid derivatives or β-hydroxy acid derivatives is of prime importance in the synthetic organic chemistry. Since the reaction between nucleophile and imine or aldehyde is the most powerful and convenient process, for a carbon-carbon bond formation.
This chapter is divided into two sections.
Section A: Synthesis of -thioalkyl--amino acid derivatives by Mannich-type reaction between N-sulfonyl aldimines and sulfonium salts 
Mannich reaction is one of the direct approaches for the preparation of β-amino acid derivatives and produces two adjacent stereogenic centers, constructed simultaneously with a concomitant C-C bond formation. This provides a unique opportunity for directly and selectively introducing subtituents at both α-and β-positions through the choice of eletrophiles and nucleophiles employed. Specially, α-mercapto-β-amino acid derivatives are prepared through Mannich reaction and are shown to be biologically active. 
α-Thioalkyl acetates reacted with N-sulfonyl aldimines in the presence of strong base LDA to afford α-thioalkyl-β-amino esters in moderate yields. Recently, ring opening of aziridinium ion intermediates with thiolate nucleophiles to give the α-thiosubstituted, β-amino esters in high yields in the presence of K2CO3 in acetonitrile at 60 0C was reported.
The novel Mannich-type reaction between N-sulfonyl aldimines and various sulfonium salts to afford α-thioalkyl-β-amino esters/nitriles/ketones/amides in moderate to high yields with moderate to good diastereoselectivities by using nanocrystalline MgO, instead of predicted aziridination products under basic conditions is described.
    

The stereochemistry assignment for these compounds was unambiguously determined by literature and spectral analysis that the major diastereomer was confirmed to be anti isomer, and minor diastereomer was to be syn. Further corroboration for the correct configuration assignment for the major isomer was unambiguously obtained by single-crystal X-ray crystallographic analysis, to be anti isomer. 
The abnormal bond cleavage of sulfonium salt was observed by using NAP-MgO in the reaction. To evaluate this, the evolved gas fragments m/z = 47, 60, 61, 62 and 76 due to SCH3, S(CH)CH3, S(CH2)CH3, S(CH3)2, SCH2(CH3)2 respectively, were observed in TGA-DTA-MS of sufonium salt 2A treated NAP-MgO. All these m/z fragments clearly indicate that the ylide and salt was coordinated to Mg2+/Mg+ and/or O2-/O- of NAP-MgO. 

However, sulfur of the ylide was coordinated to O2-/O- (Lewis base) of NAP-MgO, resulting is one of the alkyl subtituents on sulfur being polarized. Simultaneously, O2-/O- (Lewis base) of NAP-MgO dominants on that substituent, and coordinates with that alkyl group to encourage that substituent to leave from the sulfur atom. This type of C-S bond cleavage with demethylation was earlier observed in the hetaryl(dimethyl)sulfonium salts formed in the course of heterocycles with (CH3)2S+-SO2CF3CF3SO3- in the synthesis of methylthio-substituted heterocycles, in the presence of triethylamine base.
Section B: Synthesis of -hydroxy--thioalkyl esters by aldol-type reaction between aldehydes and sulfonium salts 
The aldol reaction is the most frequently used tool for constructing C-C bond formation in the synthetic organic chemistry. Accordingly, the aldol products, β-hydroxy sulfides and β-hydroxy-α-thioalkyl carboxylic acid derivatives have found widespread use in the synthesis of natural products and pharmaceuticals.
Aggarwal et al. reported an aldol condensation of cyclohexanecarboxaldehyde with α-thiomethoxy-tert-butyl-acetate by using strong base LDA, furnished mixture of β-hydroxy-α-thiomethyl esters in favour of anti product. The readily enolizable α-thio substituted esters can be easily transformed by the TiCl4/triethyl amine protocol into the corresponding titanium enolates, and these compounds undergo the aldol condensations with aldehydes to afford β-hydroxy-α-thioalkyl esters in good yields with syn selectivity.
In this section, the aldol-type reaction between aldehydes and various sulfonium salts to afford β-hydroxy-α-thioalkyl esters in moderate to good yields by using nanocrystalline MgO, instead of predicted epoxides under basic conditions is described. 
   
The stereochemistry assignment for these compounds was unambiguously determined by H1 NMR NOE studies. Relative stereochemistry of these aldol products was determined as follows. The diastereomer (syn or anti) obtained from 4-cyano benzaldehyde and sulfonium salt 2A, was reduced by lithium aluminium hydride (THF, 0 oC, 1 h) to give the corresponding 1,3-diol, which was converted to acetonide-A or B respectively (2,2-dimethoxypropane, cat. p-toluenesulfonic acid, dry acetone, rt, N2 atm) as depicted in the scheme given below. 


	
The NOE experiments of acetonide-A and B showed that the diastereomer (acetonide-A) with high Rf value was confirmed to be syn isomer and the diastereomer (acetonide-B) with low Rf value to be anti.  
CHAPTER-IV: Strecker and Cyanosilylation reactions catalyzed by nanocrystalline MgO
 This chapter is divided into two sections. 
Section A: Synthesis of -amino nitriles through Strecker reaction of aldimines and ketoimines by using nanocrystalline MgO
The nucleophilic addition of cyanide ion to the imines, instead of the three component Strecker synthesis, represents a popular and widely used alternative route, is of great importance to modern organic chemistry as it offers one of the most direct and viable methods for the synthesis of α-amino nitriles. These bifunctional compounds have subsequently shown to be versatile intermediates in synthetic organic chemistry.
Strecker reaction between N-tosylimines and TMSCN proceeded smoothly in the presence of Lewis base, AcOLi in dry DMF or water-containing DMF and the corresponding α-amino compounds were obtained in high yields. Feng et al. reported that the bifunctional N,N’-dioxide catalyst derived from L-prolinamide catalyzed the enantioselective Strecker reaction of N-sulfonyl ketoimines. 
In this chapter, an effective Strecker reaction of aldimines and ketoimines with TMSCN to afford α-amino nitriles and α,α-disubstituted α-amino nitriles respectively in high yields by using nanocrystalline magnesium oxide is described.


As to the mechanism, it was assumed that the cyanation of imines with TMSCN proceeds through the formation of hypervalent silicate intermediate, rather than nucleophilic cyanide ion as active species. 29Si NMR spectral evidence proved that the Strecker reaction proceeds through the hypervalent silicate species by coordination to O2-/O- (Lewis basic site) of nanocrystalline magnesium oxide. 
Section B: Cyanosilylation of aldehydes and ketones catalyzed by nanocrystalline MgO
 The cyanation reaction of carbonyl compounds is one of the most important methods to obtain poly-functional molecules in organic synthesis. Cyanohydrins or cyanohydrin trimethylsilyl ethers are useful buiding blocks and highly versatile synthetic intermediates in organic synthesis. 
The cyanosilylation reaction has been studied extensively by using various promising Lewis acids and Lewis bases. Verkade et al. reported the cyanosilylation of aldehydes and ketones in the presence of catalytic amounts of the non-ionic strong bases P(RNCH2CH2)N (R =Me, iPr) in THF at 0 oC or room temperature.
In this section, cyanosilylation of aldehydes and ketones with TMSCN to afford cyanohydrin trimethylsilyl ethers in good to high yields by using nanocrystalline magnesium oxide is described. The cyanohydrin trimethylsilyl ethers obtained from aldehydes, afforded cyanohydrins in good to high yields on treatment with 2N HCl.




As to the mechanism, it was assumed that the cyanosilylation reaction with TMSCN proceeds through the formation of hypervalent silicate intermediate, rather than nucleophilic cyanide ion as active species. 29Si NMR spectral evidence proved that the cyanosilylation reaction proceeds through the hypervalent silicate species by coordination to O2-/O- (Lewis basic site) of nanocrystalline magnesium oxide. 


CHAPTER-V: One-step three-component synthesis of highly substituted pyridines and 1,4-dihydropyridines using nanocrystalline MgO
In one-pot multicomponent coupling reactions (MCRs) are an attractive strategy in organic synthesis. Three-component coupling of aldehyde, malononitrile and thiol affording pyridine derivatives is one of the best examples of such type of reactions and received much attention in recently. 
The pyridine ring is found in a number of bioactive compounds, both naturally occurring and synthetic compounds and are often of considerable complexity. The highly substituted pyridine derivatives, like 2-amino-4-aryl-3,5-dicyano-6-sulfanyl pyridines, the “privileged medicinal scaffolds” having significant and diverse medicinal utility with suitable pharmacological profiles. 
The one-step, three-component synthesis of 2-amino-3,5-dicyano-6-sulfanyl pyridines and the corresponding 1,4-dihydropyridines from aldehydes with various thiols and malononitrile using triethyamine or DABCO under ethanol reflux conditions, and using a basic ionic liquid [bmIm]OH at room temperature was reported.
In continuation of the application of nanomaterials in organic transformations, an effective three-component condensation of aldehyde, malononitrile and thiol to afford 2-amino-4-aryl-3,5-dicyano-6-sulfanylpyridines P and the corresponding 1,4-dihydro- pyridines DP in moderate to high yields by using nanocrystalline magnesium oxide is described in this chapter. 


Significant Achievements
	Evaluation of single site catalyst for development of simplified and eco-friendly processes for the production of several organic intermediates such as α, β-unsaturated esters, nitriles, α-amino nitriles and cyanohydrins.
	An efficient synthesis of α-thioalkyl-β-amino acid derivatives, and β-hydroxy-α-thioalkyl esters using nanocrystalline magnesium oxide.
	Development of truly heterogeneous nanocrystalline magnesium oxide for synthesis of highly substituted pyridine derivatives, like 2-amino-4-aryl-3,5-dicyano-6-sulfanyl pyridines, the “privileged medicinal scaffolds” having significant and diverse medicinal utility.
	The use of simple and bifunctional nanocrystalline magnesium oxide catalyst permits the employment of cheaper raw materials and reduces the cost of unit operation thus influencing the economics of the process.
	   Easy separation of the catalyst by simple filtration and reusable for several cycles without loss or minimal loss of activity.













































































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