Open Access Repository of Indian Theses

Acharyya, Tamoghna


	I 
Synopsis 


SYNOPSIS
	The thesis entitled ‘Total Synthesis of Fostriecin’ has been divided into three chapters.
Chapter-I	: Introduction to cancer and the approaches cited in the literature towards the synthesis of fostriecin.
Chapter-II	: Formal synthesis of fostriecin by a carbohydrate-based approach.
Chapter-III	: Synthesis of tyrphostin analogues anti-tumour active molecules.

CHAPTER-I
An introduction to cancer, some of anti-cancer molecules synthesized in the group and the previous approaches towards the synthesis of fostriecin described. The stereocontrol strategies utilized in the reported total syntheses are summarized in Figure 1.
 





















Figure 1: Summary of the stereocontrol strategies for the total syntheses of fostriecin.
CHAPTER-II: FORMAL SYNTHESIS OF FOSTRIECIN BY A CARBO-HYDRATE-BASED APPROACH
Introduction: Fostriecin (CI–920) 1, a cytotoxic phosphate ester natural product isolated from Streptomyces pulveraceus possesses potent anticancer activity against leukemia and many other cell lines. The cytotoxic properties of 1 are attributed to its selective inhibition of protein phosphatase 2A (PP2A). The relative and absolute stereochemistry of 1 was established by Boger, who also disclosed the first synthesis of the natural product as well as preliminary SAR studies of this molecule. A number of total syntheses as well as synthetic approaches to fostriecin have been reported in the literature. However, it continues to be a challenging endeavor to synthesize this molecule using inexpensive and readily available raw materials via shorter routes.
Present work: A chiral pool approach to the C1–C13 fragment 2, a key intermediate in the Takeshi synthesis of fostriecin is described (Figure 2). 


















Figure 2: 

Stereoselective construction of the four chiral carbons of fostriecin is the key for the success of the total synthesis. A strategy different from earlier approaches to construct the four chiral centers is disclosed (Scheme 1).





































Scheme 1: Retrosynthetic Analysis of Fostriecin.
The retrosynthetic approach to 2 is illustrated in Scheme 1, in which the key fragment 10, plays a central role, serving not only as the source of the C-9 and C-11 stereocentres, but also sets the stage for introducing C-8 and C-5 stereocentres via two routes.
1.	Chelation-controlled addition of anion 11 (M=MgBr) to ketone 10 followed by Wittig rearrangement of 7a should afford 6 with the full set of chiral centers (route 1).
2.	Wittig olefination of ketone 10 using a stable ylide followed by ester reduction would set the stage for Sharpless asymmetric epoxidation. Asymmetric allylation of 8 and generation of the Z-olefin by iodomethylenation would complete the synthesis 2 (route 2).
Synthesis of C-8 to C-12 fragment of fostriecin (10) 
The compound 10 was obtained from D-glucose via two routes, among which route b is shorter and gives a higher overall yield (59%) than route a (44%).
Route a:


















Scheme 2: Reagents and conditions: (a) CuSO4, H2SO4 (catalytic), acetone, r.t., 16 h, 56%; (b) NaH, BnBr, DMF,0 оC to r.t., 4 h, 93%; (c) 0.8% H2SO4, MeOH, r.t., 6 h, 91%; (d) NaIO4 on silica gel, CH2Cl2 : H2O (8:2), r.t., 2 h, 96%; (e) MeMgI (generated from Mg and MeI), Ether, 0 оC, 2 h, 90%; (f) PCC, NaOAc, CH2Cl2, r.t., 16 h, 83%; (g) Pd on charcoal (10%), H2, 40 psi, Ethanol, 5 h, 98%; (h) MsCl, Et3N, DMAP (catalytic), CH2Cl2, 0 оC to r.t., 2 h, (i) DBU, CH2Cl2, 12 h, 83% (two steps); (j) Raney Ni, H2, 1atm, n-hexane, 90%.
1,2-5,6-di-O-isopropylidene--glucofuranose 13 was prepared from D-glucose 12 by using CuSO4, acetone and catalytic H2SO4 at room temperature for 16 h.  The free hydroxyl group of 13 was protected as its benzyl ether 14 and selective deprotection of 5,6-O-isopropylidene moiety in 14 using 0.8% H2SO4 in methanol at room temperature for 6 h afforded the diol 15 in 91% yield.
Sodium meta periodate mediated oxidative cleavage of the diol 15 followed by Grignard reaction of the resulting aldehyde with methylmagnesium iodide in ether at 0 oC gave a diastereomeric mixture of alcohol 16 as a white solid in 90% yield, which was oxidized using PCC and NaOAc in CH2Cl2 to give the ketone 17 as a white crystalline solid in 83% yield.
Deprotection of the benzyl group 17, using palladium on charcoal (10%) in absolute ethanol under hydrogen atmosphere at 40 psi pressure for 5 h afforded the alcohol 18 as a crystalline white solid in 98% yield. Mesylation of the alcohol 18, followed by elimination of the mesylate by treatment with DBU in the same pot afforded olefin 19 in 83% yield. Subsequent hydrogenation of 19 in presence of Raney-Ni, n-hexane at atmospheric pressure, room temperature for 2 h afforded the single diastereomer 10 with C-4 inversion in 90% yield (Scheme 2).  
Route b:
The free hydroxyl group of glucose diacetonide 13 was protected as its mesylate 20 by using standard procedure, elimination of the mesylate 20 by treatment with DBU in DMF at reflux temperature for 24 h afforded an olefin 21 in 69% yield. The elimination of the triflate derived from glucose diacetonide 13, by treatment with DBU also afforded olefin 21 in 94% yield. Subsequent hydrogenation of 21 in the presence of Raney-Ni in ethanol at 40 psi gave 22 in 98% yield as a white crystalline solid. 
The structure assigned to compound 22 was further confirmed by comparing its spectral data with its possible diastereomer 26 synthesized as follows. Glucose diacetonide 13 was converted to xanthate 25 and deoxygenated using Barton-McCombie protocol to afford the 3-deoxyglucose  26 in 75% yield as a colorless oil. The compound 26 in its 1H NMR spectrum revealed the presence of a multiplet (apparent quartet) at 3.87-3.75 ppm for H-4 proton (0.55 ppm upfield as compared to the chemical shift for the same proton of 22) and the other protons resonating at their expected chemical shift values.


























Scheme 3: Reagents and conditions: (a) MsCl, TEA, DMAP (catalytic) CH2Cl2, 0 оC to r.t., 3 h, 89%; (b) DBU, DMF, reflux, 12 h, 69%; (c) Tf2O, Pyridine, DMAP (catalytic), CH2Cl2, 0 оC, 30 min; then DBU, CH2Cl2, 12 h, 94%; (d) Pd on charcoal (10%), H2, 1atm, ethanol, 12 h, r.t.,  90%; (e) 40% AcOH, r.t., 6 h, 83%; (f) NaIO4 on silica gel, CH2Cl2 : H2O (8 : 2), 96%; (g) MeMgI, ether, 0 оC, 2 h, 83%; (h) PCC, NaOAc, CH2Cl2, r.t., 12 h, 89%; (i) NaH, CS2, MeI, THF, 0 оC-r.t., 1 h, 91%; (j) Bu3SnH, AIBN (cat), toluene, reflux, 8 h, 75%.

Selective deprotection of 5,6-O-isopropylidene of 22 using 40% acetic acid at room temperature for 12 h afforded the diol 23 in 83% yield (8% starting material recovered). The diol 23 was further subjected to oxidative cleavage to afford the aldehyde as a white syrup in 96% yield using NaIO4 in CH2Cl2 : H2O (8:2) at room temperature for 1 h. The aldehyde was treated with MeMgI in dry ether at 0 oC for 1 h to afford the diastereomeric mixture of alcohol 24 as a colorless liquid in 83% yield. The mixture of alcohols 24 has oxidized using pyridinium chloro chromate, sodium acetate in CH2Cl2 at room temperature for 16 h to afford a colorless liquid in 89% yield and its spectral and analytical data were in agreement with the assigned structure 10 (Scheme 3).
Installation of C-8 and C-5 stereocenters of fostriecin using chelation-controlled addition and Wittig rearrangement: (route 1)





















Scheme 4: Reagents and conditions: (a) 11, -78 to  -50 оC, 2 h, 91% (5a alone 73%); (b) n-BuLi (5 eq), THF, -100 оC, 3 h; (b) LiAlH4, THF, 0 оC to r.t., 2 h; 75% ( two steps, 5a alone 50%).

The stereocenter at C-8 of fostriecin was introduced by the chelation-controlled addition11 of 11 (M =MgBr), generated from allyl propargyl ether and EtMgBr in THF at 0 oC to ketone 10 in THF at –78  –50 oC for 3 h to afford a chromatographically separable mixture of diastereomers 7a and 7b in 91% yield in a 8:2 ratio. Additionally, the reaction of the lithium anion of 11 (M=Li) with ketone 10 was also examined under the same reaction conditions, which gave a mixture of 7a and 7b in a 3:7 ratio (Scheme 4).
 Compound 7a was subjected to Wittig rearrangement by treatment with excess n-BuLi at – 100 oC for 3 h to afford 6. Reduction of triple bond using LiAlH4 in THF afforded a separable mixture of diastereomers 5a and 5b in a 2:1 ratio (Scheme 4). 


Confirmation of C-8 and C-5 stereocenters of fostriecin using Sharpless asymmetric epoxidation and chiral allylation reactions: (route 2)
Compound 5a has the full set of chiral centers and it was synthesized following another route from ketone 10, in which the C-8 and C-5 stereocenters were installed by Sharpless asymmetric epoxidation and Chiral allylation respectively (Scheme 9).




















	




Scheme 5: Reagents and conditions: (a) Ph3PCHCOOEt (3 eq), C6H5COOH (cat), toluene, reflux, 16 h, 91% (E-isomer alone 83%); (b) DIBAL-H, THF, -78 to 0 oC, 2 h, 92%; (c) (+)-DIPT, Ti(O-i-Pr)4, TBHP, 4Ao molecular sieves, CH2Cl2, -23 oC, 24 h, 90%; (d) TPP, CCl4, reflux, 6 h; (e) n-BuLi, THF, -23 oC, 84% (two steps, 76%, 9a); (f) EtMgBr, THF, 9a, 0 oC, 1 h, then (CH2O)n, reflux, 3 h, 79%; (g) LiAlH4, THF, 0 oC to r.t., 2 h, 91%; (h) Dess-Martin periodinane, NaHCO3, CH2Cl2, r.t., 94%;  (i) (+)-Ipc2BOMe, allylmagnesium bromide, Et2O, -100 to 23 oC, 1.5 h; 30% H2O2, pH 7 buffer, 23 oC, 12 h, 79%. 

	The ketone 10 was subjected to Wittig olefination with (carboethoxymethylidene) triphenylphosphorane in the presence of catalytic benzoic acid in refluxing toluene for 16 h to afford E-ester 27 in 83% yield after chromatography on silica gel without contamination of the Z-ester (8%). The ester 27 was reduced to allylic alcohol 28 using DIBAL-H in THF at 0 оC to room temperature for 2 h in 92% yield (Scheme 5).
Sharpless asymmetric epoxidation of 28 proceeded efficiently to produce epoxide 29 in 90% yield with a 10:1 ratio of diastereomers. Although the mixture was not separated at this stage, transformation by Yadav’s methodology afforded acetylene alcohols 9a and 9b in 74% and 7% yields, respectively after chromatography on silica gel.
	Compound 9a was subjected to formylation by treatment with 2.0 equivalents of EtMgBr (freshly prepared from EtBr and Mg), 1.0 equivalent of acetylene 9a in THF at 0 оC for 1 h followed by the addition of 1.5 equivalents of paraformaldehyde to afford the propargyl alcohol 30 in 79% yield. Further protection of 30 by treatment with base and allylbromide gave a compound identical to 7a.
	Triple bond reduction of 30 with LiAlH4 in THF at 0 оC to room temperature over 2 h provided the allylic alcohol 31 in 91% yield. Oxidation of 31 with Dess-Martin periodinane gave aldehyde 8 in 94% yield, which underwent asymmetric allylation using (+)-β-methoxydiisopinocamphenyl borane and allylmagnesium bromide in ether at –100 оC to 23 оC for 2 h to afford  5a in 79% yield (Scheme 5). 















Scheme 6: Reagents and conditions: (a) acryloyl chloride, DIPEA, CH2Cl2, 0 oC, 2 h; (b) (PCy3)2RuCl2(=CHPh) (0.1 eq), Ti(O-i-Pr)4 (0.3 eq), CH2Cl2, reflux, 12 h, 92% (two steps); (c) 30% AcOH, 50 oC, 2 h, 92%; (d) Ph3P+CHI I-, NaHMDS, HMPT, -78 oC to r.t., 2 h, 63% (Z:E ratio = 3:1).

Chemoselective esterifiction of the diol 5a using acryloyl chloride and diisopropyl ethylamine in dichloromethane at 0 оC for 2 h gave an ester alcohol 32 in 96% yield, which was subjected to ring closing metathesis (RCM) with Grubbs 1st generation catalyst (0.1 equiv) in the presence of Ti(O-i-Pr)4 (0.3 equiv) in refluxing CH2Cl2 for 12 h to afford  lactone 33 in 92 % yield. Cleavage of the 1,2-O-isopropylidene group in 33 with 30% aq AcOH at 50 oC for 2 h afforded the lactol 4 in 92% yield. Iodomethylenation of lactol 4 gave Z-isomer 2 as a major product (Z:E = 3:1; Scheme 6). 

CHAPTER-III: SYNTHESIS OF TYRPHOSTIN ANALOGUES
Introduction: Tyrphostins are  low molecular weight compounds having the benzylidenemalononitrile moiety as the active pharmacophore and are known to inhibit EGF dependant proliferation and EGFR autophosphorylation. Due to their easy synthetic access through Knoevenagel condensation of a substituted benzaldehyde with a substituted nitrile, hundreds of tyrphostins derivatives have been synthesized by several research groups ( e.g., Levitzki et al., Rhone-Poulenc Rorer, Bruke et al., etc). 







Figure 3

Present work: Several Tyrphostin analogues have been synthesized using the Knoevenagel condensation reaction.









Scheme 7: General Scheme for Knoevenagel condensation reaction.

Tyrphostin analogues:











































Figure 4

All the tyrphostin analogues were prepared by using Knoevenagel condensation, in which the substituted aromatic aldehyde was treated with  the active methylene compound (malanonitrile or ethyl cyanoacetate or cyano acetamide) and catalytic piperidine in ethanol at reflux temperature for 1 h to afford condensed product (tyrphostins). The structures of all the products were confirmed by 1H NMR, IR, MASS spectroscopy.
The substituted aromatic aldehydes were prepared by a sequence of reactions and some of directly purchased from manufacturer.  

Preparation of substituted aromatic aldehyde:






















Scheme 8: Reagents and conditions: (a) DMS, K2CO3, acetone, reflux, 7 h, 53%; (b) CHCl3, hot NaOH (aq), 70 – 80 оC, 1 h, 43%; (c) allyl bromide, K2CO3, THF, reflux, 7 h, 70% 59; (d) 2,3-dibromo propene, K2CO3, THF, reflux, 7 h, 52%, 60; (e) BnBr, K2CO3, THF, reflux, 7 h, 52%.

	p-Methoxy phenol 56 was obtained from hydroquinone 55  by mono methylation, which was subjected to Reimer-Tiemann reaction to afford 57. The free phenolic functionality of 55 was protected as its methyl ether 58 and as its allyl ether (59 and 60). Hydroquinone was also protected as its mono-benzyl ether, and subjected to Reimer-Tiemann reaction providing 62 (Scheme 8).






Scheme 9: Bromination and Vilsmaeyer reaction.
	
Bromination on salicylaldehyde using bromine in acetic acid at 0 оC afforded the dibromo derivative 64 in 80% yield after recrystalyzation from acetic acid. 2-Hydroxy acetophenone 65 in DMF was treated with POCl3 at –20 оC to room temperature for 13 h to afford aldehyde 67 (Scheme 9). 


































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English

Variability of phytoplankton pigment composition in a tropical monsoon driven estuary (Godavari, India) : natural and anthropogenic influences

ANDRADE, VANESSA MARY RACHEL

SYNOPSIS

The thesis entitled “Design and Synthesis of New Pyrrolo[2,1-c][1,4]benzodiazepine Hybrids as DNA Interactive Antitumour Agents“ has been divided into four chapters. Chapter I gives the general introduction about cancer chemotherapy, covalent, non-covalent interactions of drug-DNA, particularly of pyrrolo[2,1-c][1,4]benzodiazepine antitumour antibiotics and objectives of the present work. Chapter II comprises of two sections; section A consists of the design, synthesis and DNA binding ability of novel anthraquinone-pyrrolo[2,1-c][1,4]benzodiazepine hybrids and their activity. While section B describes the design and synthesis of a first example of bifunctional PBDs with an intercalater and their biological activity. Chapter III is also divided into two sections; section A describes the synthesis, DNA binding affinity and in vitro anticancer activity of a series of pyrrolobenzodiazepine hybrids in which DC-81 has been linked to naphthalimide through piperazine moiety comprising of different alkyl chain lengths. Section B of chapter III describes the design, synthesis and biological activity of novel fluorenone-, and flavone-PBD hybrids. Chapter IV describes the development of a new methodology for the protection of carbonyls as thioacetals, its application in the synthesis of novel podophyllotoxin-PBD hybrids and their in vitro cytotoxicity.

Chapter I: General introduction

Cancer is a disease where cells grow and divide in an uncontrolled manner. The four types of treatment for cancer are surgery, radiation, chemotherapy and biological therapy. A major advantage of chemotherapy is its ability to treat cancer cells that have metastasized (spread) to other parts of the body, whereas surgery and radiation therapy are used to treat localized cancers. The major categories of chemotherapeutic agents are antitumour antibiotics, DNA topoisomerase I and II inhibitors, DNA interactive agents and other miscellaneous agents. Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a well known class of antibiotics. PBDs have potential as regulators of gene expression with possible therapeutic application in the treatment of genetic disorders including cancer and as probes and tools for use in molecular biology.
The pyrrolo[2,1-c][1,4]benzodiazepine (PBD) class of antitumour antibiotics are derived from various Streptomyces species. Lemgruber et al described the first PBD antitumour antibiotic anthramycin in 1968 and since then a number of compounds have been developed based on PBD ring system.  These compounds exert their biological activity by covalent binding to the N2 of guanine in the minor groove of DNA through the imine or imine equivalent functionality at N10-C11 of PBD. These molecules have a right-handed twist, which provides the appropriate three dimensional shape for the isohelicity with the minor groove of B-form DNA leading to a snug fit at the binding site. 





Chapter II-Section A: Design, synthesis and biological evaluation of novel anthraquinone-pyrrolo[2,1-c][1,4]benzodiazepine hybrids

Recently, there has been growing interest in modifying and extending the recognition patterns of DNA binding ligands. Pyrrolo[2,1-c][1,4]benzodiazepine antitumour antibiotics bind covalently to the N2 of guanine in the minor groove of DNA. In the past few years, several PBD analogues have been designed and synthesized with the aim of finding related compounds showing better antitumour activity. The PBDs have been used as a scaffold to attach EDTA, epoxide, (+)-Cyclopropapyrroloindole and Cyclopropapylbenzindole moieties leading to novel unsymmetrical hybrids of PBD, which have exhibited novel sequence selective DNA cleaving and cross-linking properties.
Anthracenediones represent an important class of intercalating antitumour compounds. Mitoxantrone, which is the lead compound in this series is routinely used in clinic for the treatment of certain hematological malignancies, as well as ovarian and breast cancers.
The objective of the present work is to combine the features of both intercalating and DNA binding properties in the same molecule. Therefore, it has been considered of interest to couple anthraquinone to the C8-position of the PBD through its amino functionality. In the present chapter the synthesis, DNA binding affinity and in vitro cytotoxicity of the novel anthraquinone-PBD hybrids have been described. 
	Synthesis of these novel anthraquinone-PBD hybrids has been carried out by employing N-(9,10-dihydro-9,10-dioxo-1-anthracenyl)-1-bromo-alkanamide (1a-b) as one of the precursors, which has been obtained by the reaction of an appropriate acid chloride with 1-aminoanthraquinone (Scheme-1). 
	The other precursor (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrro-lidine-2-carboxaldehyde diethyl thioacetal 9 has been prepared by employing commercially available vanillin. Oxidation of vanillin followed by esterification employing literature method provides vanillin methyl ester 2. This on benzylation gives 3, which upon nitration followed by ester hydrolysis affords 5. L-Proline methyl ester has been coupled with 5 to give the nitro ester 6. This nitro ester upon treatment with DIBAL-H followed by protection of aldehyde with ethanethiol gives diethyl thioacetal 8. This upon debenzylation provides the compound 9. This nitrothioacetal has been coupled with N-(9,10-dihydro-9,10-dioxo-1-anthracenyl)-1-bromo-alkanamide (1a-b) to give 10a-b. This nitrothioacetal has been reduced to give 11a-b. The deprotection of the thioacetal group afforded the desired compounds 12a-b (Scheme 2).





Compounds 12a-b have been evaluated for their in vitro cytotoxicity in selected human cancer cell lines of colon (HT-29, HCT-15), lung (A-549, HOP-62) and cervix (SiHa) origin. Compounds 12a and 12b exhibit significant cytotoxicity against some of the cancer cell lines. Compound 12a suppresses HT-29 and HCT-15 cell growth by 68% and 59% respectively. It is inhibiting HOP-62 cell growth by 74%. Compound 12b suppressing HT-29 and HCT-15 cell growth by 73% and 61%. It is also inhibiting A-549 and HOP-62 cell growth by 56% and 73%. 
The DNA binding affinity of these novel PBD hybrids has been evaluated through thermal denaturation studies with duplex-form of calf thymus DNA (CT-DNA). Melting studies show that these compounds stabilize the thermal helix to coil transition of the CT-DNA efficiently. In this assay, compound 12a has melting temperature of 9.1 C after incubation for 18 h at 37 C. Compound 12b elevates the melting temperature of CT-DNA by 5.2 C after incubation for 18 h. On the other hand, the naturally occurring DC-81 exhibits a ∆Tm of 0.7 C. Thus demonstrating that these PBD hybrids have significant DNA binding ability.

Chapter II-Section B: Design, synthesis and biological activity of novel bifunctional PBDs with intercalating anthraquinone

The cytotoxicity and antitumour activity of PBDs are attributed to their ability to form covalent adducts. PBD monomers span three base pairs with a preference for Pu-G-Pu motifs (where Pu = purine, G = guanine).  In an attempt to extend the number of base pairs spanned by these molecules, PBD dimers have been synthesized, with the hope that enhanced sequence selectivity might increase selectivity for tumour cells.
Since, Suggs and coworkers have reported the first PBD dimer comprising of two PBD units joined through their A-C7/A-C7’ positions, a number of PBD dimers have been designed and synthesized that exhibited varying degree of cytotoxicity and DNA cross-linking activity. These PBD dimers have been joined through different positions A-C8/A-C8’, C-C2/C-C2’, A-C8/C-C2’. Among these, A-C8/A-C8’ linked PBD dimers have shown promising cytotoxicity and efficient cross-linking properties. Thurston and coworkers synthesized A-C8/A-C8’ alkane diyldioxy bridged PBD dimers (DSB-120) and C2/C2’ exo-unsaturated PBD dimer (SJG-136) which span six base pairs of DNA and exhibit high DNA binding affinity and antitumour activity. 
The main objective of this work is to synthesize the bifunctional PBDs with an intercalater to enhance sequence selectivity and anticancer activity. Therefore, the synthesis of new PBD dimers in which two PBD units have been joined to the 1 and 4 positions of anthraquinone through their A-C8 positions via alkane diyldioxy bridge with linkers of varying lengths has been carried out. 
Synthesis of these novel PBD hybrids has been carried out by employing 1,4-bis-(n-bromoalkyloxy)anthracene-9,10-diones (13a-c) as one of the precursors, which have been obtained by etherification of 1,4-dihydroxy anthraquinone with dibromoalkanes. The other precursor (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal (9) has been prepared as described in section-A. Compounds 13a-c have been coupled with 9 to give 1,4-bis-{n-[(2S)-N-(4-oxy-5-methoxy-2-nitrobenzoyl)-pyrrolidine-2-carboxaldehyde diethyl thioacetal]alkyloxy}anthracene-9,10-diones (14a-c). These anthraquinone coupled nitro thioacetal intermediates 14a-c have been efficiently reduced to afford the corresponding amino thioacetals 15a-c. Deprotection of the thioacetal group afforded the desired bifunctional PBDs with an intercalating anthraquinone moiety in compounds 16a-c (Scheme 3).  



	Compounds have been evaluated for their in vitro cytotoxicity in selected human cancer cell lines of colon (Colo205), lung (HOP-62), prostate (PC3) and cervix (SiHa) origin. Compound 16a has promising cytotoxicity in Colo205 and SiHa cell lines. Compound 16b has not shown significant cytotoxicity. Compound 16c has potent cytotoxicity in Colo205, HOP-62, PC3 and SiHa cell lines.

Chapter III-Section A: Synthesis, DNA binding affinity and in vitro anticancer activity of naphthalimide-pyrrolo[2,1-c][1,4]benzodiazepine hybrids

Cancer chemotherapy continues to be an important research avenue. Complex structures built out of groove binders linked to other moieties which interact with the DNA via intercalation or groove interactions or molecules which cleave DNA, are of interest in terms of inter alia. These compounds are capable of recognizing heterogeneous DNA sequences.
Many drugs that possess chemotherapeutic activity intercalate with DNA.  The orientation in the geometry of the limited drug-DNA complexes has been studied using X-ray diffraction, NMR spectroscopy and traditional solution methods. It has also been shown that a wide variety of planar ring systems can intercalate with DNA to exert their antitumour activity. 
In the present work, the design and synthesis of new PBD hybrids in which naphthalimides have been linked to PBD through alkyl piperazine moiety side armed by identical or mixed alkane chain spacers has been carried out. This study is with an objective to improve the lipophilicity, antitumour activity and DNA sequence specificity.
The synthesis of these new PBD hybrids has been carried out by employing piperazine linked naphthalimides 20a-c as one of the precursors. These have been obtained by alkylation of naphthalimide with dibromo compounds followed by coupling with N-boc piperazine and deprotection of boc with trifluoroacetic acid (Scheme 4).



Synthesis of the other precursor (2S)-N-[4-(n-bromoalkyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal 25a-c has been carried out by employing the commercially available vanillin. Oxidation of vanillin followed by esterification employing literature methods provides the vanillin methyl ester (21). The mono alkylation of 21 has been achieved by using three molar equivalents of the dibromo alkanes. Nitration of 22a-c followed by ester hydrolysis and coupling of (2S)-pyrrolidine-2-carboxaldehyde diethyl thioaetal affords 25a-c. This nitro thioacetal has been coupled with 2-[n’-(piperazine-1-yl)alkyl]benz[de]isoquinoline-1,3-dione (20a-c) to give C8-linked naphthalimide nitro thioacetal 26a-e. This compound has been efficiently reduced by employing SnCl2.2H2O to afford the corresponding amino thioacetal. Deprotection of the thioacetal group with HgCl2 and CaCO3 afforded the desired naphthalimide-PBD hybrids 28a-e (Scheme 5).



Compounds have been evaluated for their in vitro cytotoxicity in selected human cancer cell lines of colon (HT-29, HCT-15), lung (A-549, HOP-62) and cervix (SiHa) origin. These compounds exhibit potent cytotoxicity. These compounds exhibit more than 75% inhibition even at 10-6 mol/L concentration in some of the cell lines. Compound 28b suppresses HT-29 and HOP-62 cell growth by 91% and 72% while compound 28c suppressing HT-29 and HOP-62 cell growth by 80% and 86% at 10-6 mol/L concentration. Compound 28d is inhibiting HT-29 cell growth by 63% and compound 28e inhibiting HT-29 and HOP-62 cell growth by 67% and 80% at the same concentration. 
The DNA binding affinity of these novel PBD hybrids has been evaluated through thermal denaturation studies with duplex-form of calf thymus DNA (CT-DNA). The results show that these compounds stabilize the thermal helix to coil transition of CT-DNA duplex more efficiently. There is an increase in the melting temperature when incubation time is increased from 0 to 18 h at 37 C. Compounds 28a and 28c elevate the helix melting temperature of CT-DNA by 22.7 C and 23.9 C respectively after incubation for 18 h at 37 C. Compounds 28d and 28e have shown a Tm of 12.9 C and 20.8 C. In this series, compound 28b has shown highest Tm value and it is increasing the helix-melting temperature by 26.5 C. In the same experiment, the naturally occurring DC-81 exhibits a ΔTm of 0.7 C and its dimer (DSB-120) gives a ΔTm of 15.4 C. This demonstrates that these new PBD hybrids have remarkable DNA binding affinity.

Chapter III-Section B: Synthesis and biological activity of novel fluorenone- and flavone-pyrrolo[2,1-c][1,4]benzodiazepine hybrids 

The biological activity of certain low molecular weight compounds appears to be related with their mode and specificity of interaction with particular DNA sequences. In the search of antitumour compounds with improved biological activity, an approach of hybrid ligands combining two modes of binding to DNA has been developed. Coupling of a polyaromatic heterocyclic to an addressing molecule does not only provide extra strength of binding but can also influence the sequence selectivity of the conjugate. With such hybrid molecules, it has been demonstrated that DNA is able to accept two types of binding in close proximity, despite their unique distortions. Furthermore, the length and flexibility of the spacer arms in conjugates seems to be critical for optimal positioning of both parts of the molecule. In view of the importance of the conjugates, synthesis of fluorenone- and flavone-PBD conjugates has been carried out to improve DNA binding ability and anticancer activity.
Synthesis of these novel hybrids has been carried out by employing (2S)-N-[4-(n-bromoalkyloxy)-5-methoxy-2-nitrobenzoyl]-pyrrolidine-2-carboxaldehy-de diethyl thioacetal (25b-d) as a precursor. This nitro thioacetal 25b-d has been coupled with 2-hydroxy fluorenone or 6-hydroxy flavone to give 29a-f. This intermediate has been reduced with SnCl2.2H2O to give amino thioacetal 30a-f. The deprotection of the thioacetal group afforded the desired compounds 31a-f (Scheme 6).


The DNA binding affinity of these novel PBD hybrids has been evaluated through thermal denaturation studies with duplex-form of calf thymus DNA (CT-DNA). In fluorenone series, compound 31a has shown a ΔTm of 6.1 C while compounds 31b and 31c elevate the helix melting temperature by 3.8 C and 3.9 C after incubation for 18 h. In flavone series, compound 31d has shown melting temperature of 3.8 C at 0 h and the melting temperature increases to 6.2 C after incubation for 18 h at 37 C. Compounds 31e and 31f elevate the helix melting temperature of the CT-DNA by 1.8 C and 2.6 C after incubation for 18 h. 
Compounds have been evaluated for their in vitro cytotoxicity in selected human cancer cell lines of colon (HT-29, HCT-15), lung (A-549, HOP-62) and cervix (SiHa) origin. Compound 31d has a strong effect to HT-29, HCT-15 and HOP-62 cell lines. Compound 31e has not shown any significant cytotoxicity. Compound 31f is suppressing HCT-15 and A-549 cell growth by 69% and 63%.

Chapter IV: Synthesis and in vitro cytotoxicity of novel podophyllotoxin-pyrrolo[2,1-c][1,4]benzodiazepine hybrids

The protection of carbonyl groups as acetals and thioacetals is most commonly used as an important synthetic technique in the course of preparation of many organic compounds including multifunctional complex molecules. Thioacetals are comparably more stable than the corresponding acetals under acidic conditions and are useful in organic synthesis as acyl carbanion equivalents in carbon-carbon bond forming reactions. Many of the reported methods suffer from some disadvantages.  The present chapter describes a simple and convenient method for the protection of aldehydes as thioacetals using Cu(BF4)2.xH2O under solvent-free conditions. The present chapter also describes the application of this methodology in the synthesis of novel podophyllotoxin-PBD hybrids and their in vitro anticancer activity.
Various aldehydes have been subjected to thioacetal formation by the treatment of carbonyl compound with ethanethiol under the catalytic influence of 5 mol% Cu(BF4)2.xH2O. 



	Synthesis of these novel podophyllotoxin-PBD hybrids has been carried out by employing (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbox-aldehyde as one of the precursors, which has been obtained as described in chapter II. Protection of this aldehyde with ethanethiol in presence of Cu(BF4)2.xH2O afforded diethyl thioacetal 32. This upon debenzylation provides the compound (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal (33). Etherification of compound 33 by methyl bromoalkanoate provides 34a-b. Basic hydrolysis of these esters gives the desired precursor acids 35a-b. The other precursor 4’-O-demethyl-4-amino-4-deoxy podophyllotoxin has been prepared by literature method. Amidation of this with acid 35a-b in presence of EDCI/HOBt affords the corresponding podophyllotoxin coupled nitro thioacetal intermediates 36a-b. This upon reduction affords 37a-b. The deprotection of the thioacetal group using HgCl2/CaCO3 resulted in the formation of the desired podophyllotoxin linked PBD hybrids 38a-b (Scheme 8).
Compounds 38a-b have been evaluated for their in vitro cytotoxicity in a panel of six human cancer cell lines. Compound 38a exhibit GI50 values in the range of 9.5-38.7 mM and GI50 values of compound 38b is 13.7-36.6 mM.
	In conclusion, the thesis describes the design, synthesis and biological activity of novel pyrrolo[2,1-c][1,4]benzodiazepine analogues with the potential to develop as anticancer agents.





PAGE  



I





Seismotectonics of the Andaman-Nicobar plate boundary and evaluation of the 2004 deformational and depositional features towards assessing past tsunamigenic earthquakes.

Mogili, Nirupa

8. Conclusions 
The salient features of the results obtained and their summary are illustrated along with 
the important conclusions drawn in the present thesis program. 
 The CDM activities are carried out in a fixed bed quartz micro-reactor operated in 
a down flow mode at temperatures of 500-900 C and ambient pressures. 
 The CDM process is influenced by parameters like amount of active metal, nature 
of support material, reaction temperature, catalyst calcination temperature, mode 
of reduction, precipitation pH and Ni precursor. 
 The catalytic activity is also influenced by the amount of second metal component 
i.e. Cu, which could enhance methane decomposition activity of the active Ni 
component at higher temperatures. 
 20 wt% Ni loading is found to be optimum loading over HY zeolite, that posses 
higher activity than other zeolite systems. 
  Ni-Al2O3-SiO2 (4:0.5:1.5) catalyst is reduced with N2H4 showed superior activity 
over all the Ni-based systems at 550 C reaction temperature. 
 It is observed that the addition of Cu to Ni supported systems enhances methane 
decomposition activity when it is added in an appropriate amount. It is also 
observed that the Ni-Cu-Al2O3 (60:25:15) catalyst pose higher hydrogen yields 
over other Ni-Cu-Al2O3 compositions.  
 Decomposition results revealed that the quantity of Cu presence in Ni-Cu-SiO2 
catalysts have significantly influenced methane decomposition activity of Ni. The 
catalyst Ni-Cu-SiO2 (60:25:15) calcined at 450 C for 5 h showed high carbon 
accumulation of 801 gC/gNi at 650 C. 
 
Chapter VIII. Conclusions 
 168 
 Over all, the hydrogen (or) carbon yields are increasing with the systems in the 
order of 20 wt% Ni/HY < Ni-Al2O3-SiO2 (4:0.5:1.5) < Ni-Cu-Al2O3 (60:25:15) < 
Ni-Cu-SiO2 (60:25:15) combinate systems.   
 The Ni metal surface areas are calculated from O2 pulse chemisorption that 
behaves linear relationship with H2 yields. 
 The XRD patterns of oven dried Ni-Cu-Al2O3 and few Ni-Al2O3-SiO2 samples 
reveal the formation of hydrotalcite like catalyst precursors. However, the HT-like 
structure is collapsed with the thermal treatment at 450 C for 5 h in static air.  
 XRD spectra of all the fresh samples revealed Ni containing phase is only NiO 
irrespective of the method of preparation, however, the crystallinity is varied with 
the preparation method and the amount of Ni. In addition to NiO phase, CuO 
phase is also observed in few fresh Ni-Cu based samples, where Cu, is present at 
high loadings.  
 All the XRD patterns of deactivated samples have revealed the appearance of 
metallic Ni phase and is dominated by graphitic carbon phase.  
 TPR profiles of impregnated Ni based catalysts showed single reduction center 
i.e. Ni2+  Ni0 (~ 450 C), in contrast to two reduction centers observed for co-
precipitated Ni based catalysts. Also a less intense low temperature peak is 
observed in between 250-300 C and is attributed to the reduction of Ni2+ species 
that are not interacted with the support. The second reduction peak is more intense 
at high temperature peak in the region of 450-700 C with a Tmax of 580 C and is 
ascribed to the reduction of NiO interacted with the support material presumably 
due to the formation of amorphous nickel aluminates/silicates.  
 
Chapter VIII. Conclusions 
 169 
 The TPR profiles of Ni-Cu based catalysts showed three reduction centers, the 
low temperature reduction peak (240-260 C) is due to phase transfer of Cu2+ to 
Cu0, whereas the moderate temperature reduction peak (425-520 C) belongs to 
reduction of Ni2+ and is interacted with Cu. The high temperature reduction peak 
(600-620 C) is due to the presence of Ni2+ interacted with Al2O3, SiO2 supports. 
Furthermore, the Tmax of moderate temperature reduction peak is shifted towards 
low temperature with increase in Cu content. The shift in Tmax is due to the added 
Cu that produces spillover hydrogen, which considerably accelerates the 
nucleation of the Ni metal in these reduction conditions, and enhances the 
reducibility of Ni. This suggests the role of Cu, which enhances the reducibility of 
dispersed Ni2+ species resulting in the shift of Tmax towards low temperatures. 
 XPS results suggests the presence of Ni2+ in all the catalysts, however a shift in 
the binding energy towards higher side with increase in the amount of Al3+ or Si4+ 
amounts are observed. 
 The relative surface compositions obtained by XPS analysis has indicated that Ni-
Cu-Al2O3 (60:25:15) sample is enriched with Ni when compared to other Ni-Cu-
Al2O3 catalysts, that could be one of the reason to pose higher activity. 
 The XPS analysis of Ni-Cu-SiO2 has indicated the main line of Ni 2p at BE of 
855 eV that implies a change in the chemical state of NiO  NiSiO3. 
 SEM and TEM images of deactivated catalysts are revealed that the deposited 
carbon is filamentous in nature and the Ni has occupied at the tip of the filaments. 
However, the size and length of the filaments are varied with the catalyst 
compositions. 
 
Chapter VIII. Conclusions 
 170 
 Raman spectra reveals the deposited carbon is filamentous in nature and can be 
distinguished the presence of ordered (G-band) and defective (D-band) structures 
of carbon. The high CDM activity over Ni-Cu-Al2O3 (60:25:15) catalyst could be 
possibly due to deposition of high amount of ordered carbon when compared to 
other Ni-Cu-Al2O3 catalysts. 
 Methane decomposition results in the decrease of surface area, pore volume, 
micro-pore volume and increase of average pore diameter revealing that 
adsorption and decomposition of methane occurs mainly in the micropore of 
activated carbons.  
 The activation energies over activated carbons for methane decomposition are in 
the range of 170-175 kJ/mol. 
 The carbon catalyst COC showed highest initial methane conversion rate of    
1.27 mmols/min.g, and a sustainability factor of 0.40 with a carbon yield of      
890 mg/g-cat at 900 ºC. 
The salient features of all the results obtained in the present thesis work are summarized 
in Table 8.1. 
 
 
Chapter VIII. Conclusions 
 171 
Table 8.1: Results at a glance on “Study and development of methane decomposition catalysts for COX-free hydrogen.” 
 
 
 
S.No Catalyst Weight  
(mg) 
GHSV 
(L/h. g) 
Temp.  
(C) 
Run time 
(min) 
Carbon yields 
(gC/g-cat) 
                   Publication 
 
1 
 
20 wt% Ni/HY 
 
 
50 
 
72 
 
550 
 
850 
 
50 
 
J Power Sources, 164 (2007) 809-814 
Catalysis Letters, 118 (2007) 139-145 
 
2 
 
Ni-Al2O3-SiO2 
(4:0.5:1.5) 
 
50 
 
72 
 
550 
 
2700 
 
146 
 
J Natural Gas Chemistry, 17 (2008) 113-119 
Int J Hydrogen Energy, 33 (2008) 4809-4818   
 
 
3 
 
Ni-Cu-Al2O3 
(60:25:15) 
 
20 
 
180 
 
650 
 
900 
 
310 
 
Int J Hydrogen Energy, 33 (2008) 2704-2713 
 
 
4 
 
Ni-Cu-SiO2 
(60:25:15) 
 
 
20 
 
180 
 
650 
 
1800 
 
480 
 
Energy Fuels, In press, 2008 
 
5 
 
AC-COC 
 
200 
 
18 
 
900 
 
180 
 
0.89 
 
Catalysis Communications, 9 (2008) 164-169 
 
N. B.:- The total work carried out under this thesis program is published as “Development of methane decomposition 
catalysts for COX free hydrogen” in Catalysis Surveys from Asia 12 (2008) 229-237 (Review) 


Molecular cloning and characterization of cDNAs encoding Glyceraldehyde - 3 - phosphate dehydrogenase and NC1 domain of Collagen XVIII from Indian shrub frog, Polypedates maculatus

K.V.N.S., Lakshmi

CULTURED DIVERSITY OF PURPLE ANOXYGENIC PHOTOTROPHIC RHIZOBACTERIA OF PADDY AND THEIR PLANT GROWTH PROMOTING ATTRIBUTES

Almal, Suhani Hariram


Genetic Variations in Indian Population: Implication for Health 

In past, our knowledge about human genetic variation was limited to mainly the rare changes in the number and structure of chromosomes which included aneuploidies, heteromorphisms, rearrangements and other polymorphism, large enough to be visualized by microscopic techniques. With the advances in molecular biology came the single nucleotide polymorphism (SNPs) in DNA, which is the most prevalent type of genetic variation and identified mainly using PCR-based methods. Recently, copy number variants (CNVs) are highlighted to be a significant source of human genetic variation accounting for disease susceptibility and disease development, thus revealing the gap between long-recognized microscopic chromosomal alterations and invisible changes at nucleotide level. Copy number variation (CNV) can be defined as segments of DNA ranging in size from kilobases (larger than 1 kb) to several megabases (Mb) varying in copy number in comparison to the reference genome. Deletions, duplications, segmental duplication, insertions, and translocations all result in CNVs. Rapid advances in the technologies have enabled the detection of CNVs on larger scale. Thus, depending on the method of detection, it has been estimated that CNVs may cover about 12% of the human genes. 
The discovery of submicroscopic copy-number variations (CNVs) present in human genome has dramatically changed our perspective on DNA structural variation and development of disease. Advancement in technology has allowed for the analysis of CNVs in thousands of individuals with or without a specific disease. Till date many researchers have addressed the presence of CNVs in the human genome and their association with common human diseases including neuropsychiatric, autoimmune, infectious, and many more, whereas others found evidence that CNVs are not associated with common diseases. CNVs and their involvement in disease susceptibility and drug response have been widely studied in other populations but not to any extent in Indian population. Therefore, the main aim of our study was to evaluate the distribution of copy number variable genes in Indian population and their link, if any, to health, disease and drug response. Based on our selection criteria, few copy number variable genes were selected and studied for their role in human diseases like cancer (breast and head and neck cancer), psoriasis, asthma, HIV-1/AIDS and others. In this study, the candidate copy number variable genes studied, MTUS1 for breast cancer and head and neck cancer; LCE3C and LCE3B for psoriasis susceptibility; GSTT1 and GSTM1 for asthma susceptibility; and CCL3L1 for HIV-1/AIDS susceptibility. The copy number frequency distribution of FCGR3B and SULT1A1 involved in SLE susceptibility and drug metabolism respectively, was evaluated in the Indian population. In this study CNV for these selected genes were analyzed in 100 – 280 normal healthy individuals from Western Indian population and in variable number of patients suffering from respective diseases. In many cases the frequency of CNV variants was significantly different in Indian population as compared to studies reported in other population. Also, significant inter-ethnic differences were observed in the distribution of variants in comparison to other population. The association of the MTUS1 deletion variant in susceptibility or decreased risk to head and neck and breast cancer was evaluated where indicative correlation with deletion of MTUS1 gene was observed only in breast cancer patients. Further, the significant association between CCL3L1 copy number and risk to HIV-1 is observed in our study. The sample size in the above mentioned study was very small, therefore the results needs to be further validated in large sample size before making firm conclusion about the link between the CNV of MTUS1 and CCL3L1 and risk to breast cancer and HIV-1 respectively. The discovery of submicroscopic copy-number variations (CNVs) present in human genome has dramatically changed our perspective on DNA structural variation and development of disease. Advancement in technology has allowed for the analysis of CNVs in thousands of individuals with or without a specific disease. Till date many researchers have addressed the presence of CNVs in the human genome and their association with common human diseases including neuropsychiatric, autoimmune, infectious, and many more, whereas others found evidence that CNVs are not associated with common diseases. CNVs and their involvement in disease susceptibility and drug response have been widely studied in other populations but not to any extent in Indian population. Therefore, the main aim of our study was to evaluate the distribution of copy number variable genes in Indian population and their link, if any, to health, disease and drug response. Based on our selection criteria, few copy number variable genes were selected and studied for their role in human diseases like cancer (breast and head and neck cancer), psoriasis, asthma, HIV-1/AIDS and others. In this study, the candidate copy number variable genes studied, MTUS1 for breast cancer and head and neck cancer; LCE3C and LCE3B for psoriasis susceptibility; GSTT1 and GSTM1 for asthma susceptibility; and CCL3L1 for HIV-1/AIDS susceptibility. The copy number frequency distribution of FCGR3B and SULT1A1 involved in SLE susceptibility and drug metabolism respectively, was evaluated in the Indian population. In this study CNV for these selected genes were analyzed in 100 – 280 normal healthy individuals from Western Indian population and in variable number of patients suffering from respective diseases. In many cases the frequency of CNV variants was significantly different in Indian population as compared to studies reported in other population. Also, significant inter-ethnic differences were observed in the distribution of variants in comparison to other population. The association of the MTUS1 deletion variant in susceptibility or decreased risk to head and neck and breast cancer was evaluated where indicative correlation with deletion of MTUS1 gene was observed only in breast cancer patients. Further, the significant association between CCL3L1 copy number and risk to HIV-1 is observed in our study. The sample size in the above mentioned study was very small, therefore the results needs to be further validated in large sample size before making firm conclusion about the link between the CNV of MTUS1 and CCL3L1 and risk to breast cancer and HIV-1 respectively.  




Genetic Variations in Indian Population: Implication for Health

Chari, N. V. Harikrishna

Fluorescent Dissolved Organic Matter (FDOM) studies in the western Bay of Bengal.

Sudharsanam, Suchithra

Abstract
Anthrax toxin, secreted by Bacillus anthracis, consists of three monomeric proteins that assemble after secretion from the bacterium into toxic hetero-oligomeric complexes. These proteins are: Protective Antigen (PA; 83 kDa), Lethal Factor (LF; 90 kDa) and Edema Factor (EF; 89 kDa).  LF and EF are the catalytic moieties, which have intracellular targets within mammalian cells, while PA delivers these catalytic molecules (LF and EF) to the cytosol.  EF is an adenylate cyclase and together with PA forms a toxin referred to as edema toxin that causes cellular edema due to increase in intracellular cAMP levels.  LF and PA together form a toxin termed as lethal toxin which is lethal to mammalian cells and causes death of cultured mammalian cells by a yet unknown mechanism.  These three proteins are individually non-toxic. 
PA, a monomeric 83 kDa protein, binds to the cell-surface receptor on mammalian cells and is activated by cellular endoproteases such as furin.  The resulting N-terminal 20 kDa fragment is released into the extracellular milieu and plays no further role in the intoxication process.  The receptor-bound 63 kDa fragment (PA63) manifests certain properties which are not found in the native protein.  PA63 binds LF and oligomerizes to a ring-shaped heptamer.  In the endosome, acidic pH triggers membrane insertion by PA63 and subsequent translocation of LF into the cell cytosol.  X-ray diffraction analysis of PA revealed the presence of a pair of adjacent calcium ions coordinated to a variant of EF hand motif in domain I.  The exchange of intrinsic calcium ions with extrinsic labeled calcium ions is very slow indicating that these ions are tightly bound to PA.  However, the biochemical properties and structural transitions of protein in Ca2+ ion bound and unbound state have not been studied in detail. 
The present study was undertaken to delineate the role of calcium with an assumption that calcium might be providing PA a conformation, required for its specific activation and biological activity.  The role of calcium ions in the biological activity and structural stability of PA was investigated by proteolysis, spectroscopic analysis, denaturation studies and mutational analysis.  On comparing PA and apo-PA it was found that binding of calcium provides stability to PA, as evident by the enhanced susceptibility of apo-PA towards proteases like trypsin and chymotrypsin.  The binding and removal of calcium was associated with detectable conformational changes in the molecule as seen by intrinsic tryptophan fluorescence and CD studies.  In order to study the role of calcium binding residues, PA mutants with single, double and triple point mutations were constructed.  The mutated PA proteins were unstable with drastically reduced activity suggesting the critical role of calcium binding region of domain I in protein assembly and initial folding.  We hypothesize that the binding of calcium ions and protein folding may occur as a concerted mechanism or may involve unstable intermediates.  The urea denaturation studies of PA showed a typical biphasic curve suggesting the presence of an intermediate in the unfolding of PA at 2-M urea.  Furthermore, proteolysis and spectral studies of this intermediate suggested that apo-PA may be an intermediate in the unfolding pathway of the protein.
We next examined the effect of acidic pH on the unfolding of PA.  The pH plays an important role in the PA mediated delivery of LF and EF into the cytosol of mammalian cells.  PA is able to insert into phospholipid bilayers and cell membranes to form ion-permeable channels in a pH-dependent fashion.  Macrophages pre-treated with agents, which elevate the endosomal pH are protected from the effect of lethal toxin.  Internalization of LF is experimentally blocked by incubation at 4˚C, whereas acidifying


Protective antigen (PA) is the central receptor binding
component of anthrax toxin, which translocates catalytic
components of the toxin into the cytosol of mammalian cells.
Ever since the crystal structure of PA was solved, there have
been speculations regarding the possible role of calcium ions
present in domain I of the protein. We have carried out a systematic
study to elucidate the e¡ect of calcium removal on the
structural stability of PA using various optical spectroscopic
techniques, limited proteolysis and mutational analysis. Urea
denaturation studies clearly suggest that the unfolding pathway
of the protein follows a non-two state transition with apo-PA
being an intermediate species, whereas the folding pathway
shows that calcium ions may be critical for the initial protein
assembly

Acidic pH plays an important role in the membrane insertion of protective antigen (PA) of anthrax toxin leading to the
translocation of the catalytic moieties. The structural transitions occurring in PA as a consequence of change in pH were investigated
by .uorescence and circular dichroism measurements. Our studies revealed the presence of two intermediates on-pathway of
acid induced unfolding; one at pH 2.0 and other at pH 4–5. Intrinsic .uorescence measurements of these intermediates showed a red
shift in the wavelength of emission maximum with a concomitant decrease in .uorescence intensity, indicative of the exposure of
tryptophan residues to the bulk solvent. Furthermore, no signi.cant change was seen in the secondary structure of PA at a pH of
2.0, as indicated by far UV–CD spectra. The low pH intermediate of PA was characterized using the hydrophobic dye, 8-anilino-1-
naphthalenesulfonate, and was found to have properties similar to those of a molten globule state.



Aeromicrobiological study of indoor air quality in hospitals ? characterisation of bioaerosols and their association with nosocomial infections

SANDIPAM, SRIKANTH

Abstract

The advent of new century has witnessed an unchecked and over exploited use of fossil fuels as well as their depletion which consequently resulted in the alarming environmental pollution with resultant rise in global warming, causing an unusual increase in surface temperature. This has necessitated, more than ever rapid development of bioenergy to solve the looming energy crisis as well as to save the planet from the brink of an environmental catastrophe. Biofuels present an exciting and sustainable alternative to the fossil fuels which can defend the worldwide energy crisis and environmental pollution problems. Recently, biohydrogenesis through acidogenic fermentation and bioelectrogenesis through microbial fuel cell (MFC) technology are representing new and promising biological processes for bioenergy generation. Utilization of wastewater as substrate for both the processes and mixed culture as biocatalyst makes these technologies as sustainable for energy generation as well as waste management. Various studies have already been conducted in the direction of utilizing wastewater as substrate. However, there still remain certain limitations to overcome for these technologies to become alternatives for the traditional energy production processes. Lower substrate conversion efficiency, accumulation of acid intermediates and rapid pH drop are the major concerns which should be addressed prior to the upscaling of biohydrogenesis. On the other hand, increasing the anodic electron transfer along with the effective utilization of reducing equivalents [protons (H+) and electrons (e-)] at cathode and recovery of other value added products or treatment of complex pollutants during bioelectrogenesis can be considered as crucial in the MFC sector. Irrespective of the nature of microbial metabolism (aerobic or anaerobic), all the microbes will involve in the utilization of available substrate (fermentation) generating the reducing equivalents in the form of redox carriers (NAD+, FAD+, FMN+, etc.) which will help in generating the energy during respiration. However, the energy can be transformed into utilizable form in the absence of oxygen (as terminal electron acceptor (TEA)) by providing the required conditions. Henceforth, the anaerobic microbial metabolism is considered as highly energy conservative, where a diverse range of useful products can be obtained for human usage. When hydrogenase acts as TEA during acidogenic fermentation, biohydrogenesis is feasible by the re-oxidation of reduced redox carriers. Similarly, by introducing the external solid electron acceptor s and separating the fermentation from respiration by ion selective membrane, power can be harvested (bioelectrogenesis) from microbial metabolism. It is presumed that by regulating these fermentation and respiration processes, the product formation can be enhanced. 

Present thesis deals with some of the strategies to regulate the microbial fermentation and respiration processes towards enhancement of hydrogen (H2) and power outputs. The change in dehydrogenase activities with the function of microenvironment associated with operating pH was studied for the first time to evaluate the importance of dehydrogenase during H2 production. Further, feed replacement strategy (25% and 50% feed replacements) was studied to overcome the feedback inhibition of H2 production by the accumulated metabolites (organic acids, H2, CO2, etc.) of acidogenic fermentation. Reuse of CO2 generated during fermentation was studied in different time intervals were also studied to maintain the system buffering capacity. The influence of different chemical additions (Na-salts of hydroxide, carbonate and phosphates) to regulate the feeding pH during acidogenic fermentation was also evaluated. Secondary integration of photobiological H2 production was studied to utilize the acid rich effluents from the acidogenic fermentation, where the effluents with higher organic acids showed higher additional H2 during integration. Enhancing the electron transfer at anode was evaluated in detail by varying electrode materials and their synergistic interaction with the anodic biocatalyst, where stainless steel and nickel were proved to be alternative after graphite as effective anode materials. The influence of anode placement from the TEA was also studied where the 3cm distance depicted lower electron losses and enhanced electrogenesis. Influence of initial anodic poised potential was studied to enrich electrochemically active consortia on the anode surface, where 0.6 V of applied potential showed its positive impact on the microbial metabolism. Change in exocllular electron transfer was evaluated in detail by the augmentation of pure strains (Pseudomonas and Bacillus) at anode. The cathodic reduction mechanism was evaluated under chemical respiration (abiotic cathode) as well as microbial respiration (biocathode) under different microenvironments. Biocathode operation under aerobic reduction microenvironment showed higher electrogenesis and treatment efficiency over anoxic and anaerobic microenvironments. The potential of photosynthetically generated oxygen as TEA over mechanical aeration was evaluated using mixed microalgae at cathode. Feasibility of recovering bioplastics and biodiesel from the anoxic and algal biocathodes respectively at the end of operation was also studied in detail. In conclusion, Application of different strategies to fermentation and respiration processes to overcome the inherent limitations of the mixed microbial metabolisms will increase the practical feasibility of the bioenergy generation from wastewater.














STRATEGIES TO ENHANCE BIOHYDROGENESIS AND BIOELECTROGENESIS BY REGULATING MICROBIAL FERMENTATION AND RESPIRATION PROCESSES

D, Raju

Studies on metal accumulation and synthesis of inorganic nanomaterials in plants and their applications

ASTHANA, ABHISHEK

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.




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