Open Access Repository of Indian Theses

MULCHANDANI, MEENA

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 Summary                                                                                                                         



Summary
The thesis describes the phytochemical investigation, structural determination of bioactive compounds from well known medicinal plants used in Indian traditional system of medicine and synthesis of two new furanoflavanoids. Synthesis of various furanochalcones, biological activity evaluation along with a brief study of SAR is discussed. Series of Pongamol analogues were prepared and their biological activity evaluation was studied.
The first chapter deals with the isolation and characterization of 5 compounds designated as A, B, C, D and E from the hexane extract of the heartwood of Ailanthus excelsa. Compounds A, B and E are identified as Ocotillone, Epoxymalabaricol and Malabaricanediol are first time isolated from the heartwood of Ailanthus excelsa. Compounds C and D are new dammarane triterpenoids and named as Ailexcelone and Ailexcelol respectively. 
               Compound C was isolated as a colourless solid, with the negative optical rotation [α]D28 –39.59o (c = 2.0, CHCl3), mp 120 °C. The HRESI-MS revealed a molecular ion peak corresponding to [M++Na] at m/z 481.3654 indicating the molecular formula C30H50O3. The IR spectrum displayed characteristic bands for hydroxyl group (3415 cm-1) carbonyl function (1698 cm-1) and olefin double bond (1618 cm-1) functionalities. The 1H NMR spectrum showed eight quaternary methyls as singlets at        0.78 (H-19), 0.82 (H-18), 0.86 (H-30), 0.98 (H-21), 1.02 (H-28), 1.08 (H-29), 1.18              (H-26), 1.22 (H-27). It further displayed a two proton triplet at 2.10 indicating the presence of a methylene adjacent to carbonyl carbon. A broad singlet for one proton at 5.23 is due to an olefinic proton. The signal at  3.25 (dd, J = 8.3, 1.5 Hz) is due to an oxymethine proton. The 13C NMR spectrum of compound D showed the presence of 30 carbons. Further the 13C NMR spectrum showed peaks characteristic to a dammarane type triterpene.  The DEPT experiment showed the presence of 8 methyls, 9 methylenes, 6 methines and 7 quaternary carbons. Furthermore, the sodiated molecular ion peak appeared at m/z 481.3654 [M++Na] in HRESI-MS. The peak at  217.1 is due to a carbonyl carbon. The 13C NMR, DEPT and HSQC experiments revealed the side chain C20-C27 to be having three methyls (24.5, 26.5 and 28.8), one methylene (23.1), one oxymethine carbon (79.5) and two quaternary carbons (73.2 and 148.9). All the carbon values are assigned based on DEPT and HSQC experiments. In the HMBC spectrum the olefinic methine at  5.25 showed strong correlation with C-21 carbon (24.5), C-20 carbon at  148.9 and the C-24 carbon at  79.5. The oxymethine proton at  3.25 showed strong correlations with C-25 at  73.2, C-26 at  26.5 and C-27 at  28.8. Based on these data, compound C is identified as a new dammarane triterpenoid named as Ailexcelone (24,25-dihydroxydammar-20-en-3-one).
Compound D was isolated as a yellow viscous liquid, with the negative optical rotation [α]D25 –34.2o (c =2.0, CHCl3). Its positive ion HRESI-MS spectrum displayed a molecularion peak [M+Na]+ at m/z 483.3827 suggesting a molecular formula of C30H52O3. The IR spectrum revealed the presence of hydroxyl group at 3450 cm-1 and olefin double bond at 1617 cm-1. The 1H NMR spectrum displayed eight quaternary methyls as singlets at  1.28 (s, H-21), 1.22 (s, H-29), 1.18 (s, H-28), 0.98 (s, H-27), 0.92 (s, H-26), 0.88 (s, H-30), 0.82 (s, H-18), 0.78 (s, H-19). It further displayed two doble doublets at  3.25 (dd, J =10.5, 4.5 Hz, H-3), 3.32 (dd, J = 7.5, 4.5 Hz, H-24) each integrating for one proton is due to an oxymethine protons. A broad doublet for one proton at  5.26 is due to an olefinic proton. The 13C NMR spectrum of compound D showed the presence of 30 carbons. The DEPT experiment showed the presence of 8 methyls, 9 methylenes, 7 methines and 6 quaternary carbons. Furthermore, the sodiated molecular ion peak appeared at m/z 483.3827 [M++Na] in HRESI-MS. The 13C NMR, DEPT and HSQC experiments revealed the side chain C20-C27 to be having three methyls at  22.4 (C-21), 31.4 (C-26) and 26.6 (C-27 ). One methylene at  27.7 (C-23), two oxymethine carbons at  78.4 (C-3) and 78.1 (C-24) and two quaternary carbons at 72.5 (C-25) and 145.3 (C-20). All the carbon values are assigned based on DEPT and HSQC experiments. In the HMBC spectrum the olefinic methine at  5.26 showed strong correlation with C-21 carbon  (22.4), C-20 carbon at  145.3 and the C-24 carbon at  78.1. The oxymethine proton at  3.32 (dd, J = 7.5, 4.5 Hz, H-24) showed strong correlations with C-25 at  72.5, C-26 at  31.4 and C-27 at  26.6. Based on these correlations, the side chain at C-17 is deduced as shown in below. Based on these data, compound D is identified as a new dammarane triterpenoid named as Ailexcelol (dammar-20-ene-3b,24,25-triol).
All the isolated triterpenoids were tested for their antifungal activity and exhibited moderate levels of activity. New compound C was moderately active against Aspergillus niger and Rhizopus oryzae with an inhibition zone diameter of 12 mm at 100 µg/ml. New compound D also displayed a moderate degree of activity against Aspergillus niger and Candida albicans with an inhibition zone diameter of 10 and 11 mm, respectively. 
    


The second chapter also deals with the isolation and characterization of 13 compounds designated as A, B, C, D, E, F, G, H, I, J, K, L and M  from the DCM+MeOH (1:1) extract of the roots of D. indica. Compounds A, B, C, D, E, F, H, I and K are identified as Karanjin, Pongamol, Ovalitenone, Desmethoxykanugin, Pongachromene, Fisetin tetramethyl ether, Pinnatin, 7-O-methyl-5-hydroxy flavone and Pongapinone B.  
Compounds L and M are new furanoflavanoids named as 3′,4′-dihydroxy-4H-furo[2,3-h]chromen-4-one and 3,3′,4′-trihydroxy-4H-furo[2,3-h]chromen-4-one respectively.
                 Compound L was obtained as a pale yellow amorphous solid, mp 284○C. Its molecular formula deduced as C17H11O5 from its molecular ion peak at [M+-H] at m/z 295.0604 in the HRESI-MS. Its IR spectrum (Fig-6) showed absorption bands at 3393 cm-1 suggested the presence of hydroxyl group, a sharp band at 1694 cm-1 indicated the presence of α,β-unsaturated carbonyl group. The 300 MHz 1H NMR spectrum of compound L taken in DMSO-d6 (Fig-7) revealed the presence of a singlet at δ 6.72 integrating for one proton was attributed to an olefinic proton, which is adjacent to the carbonyl group. Two doublets at δ 6.92 (J = 8.4 Hz, H-5′), 7.45 (J = 2.4 Hz, H-2′) and a multiplet at δ 7.46 (H-6′) each integrating for one proton suggested the presence of 1,3,4-tri substituted aromatic ring. A doublet at δ 8.10 (J = 1.8 Hz) and a double doublet at δ 7.34 (J = 1.8 Hz) integrating for one proton each indicated the H-2″, H-3″ of the furan ring. Two doublets at δ 7.88 (J = 8.6 Hz, H-5), 7.59 (d, J = 8.6 Hz, H-6), each integrating for one proton indicated the presence of a 1,2,5,6-tetra substituted aromatic ring. Two signals at δ 9.62 and 9.98 showed no connectivity with any carbon nucleus in the HSQC plot, thus suggesting the presence of two aromatic hydroxyl groups. The explanation is further supported by the disappearance of two peaks at δ 9.62 and 9.98 in a heavy water exchange condition. 
The appearance of H-5 and H-6 protons as doublets strongly indicated that the annellation of furan ring to be at C-7/C-8 positions. The 13C NMR spectrum in DMSO-d6 of compound L exhibits the presence of 17 carbon atoms whose multiplicity is explained using DEPT 135 and HMBC correlations. DEPT 135 spectrum clearly shows there are eight methines and nine quaternary carbon atoms (including carbonyl). A signal at δ 177.2 indicated the presence of an α,β-unsaturated carbonyl group. A signal at 104.6 and 147.7 indicated the presence of a furan ring. The DEPT experiment revealed the presence of eight methines and nine quaternary carbons. The HMBC correlations of H-2″ with C-7 and C-8 indicated that the furan ring should be fused at C-7 (oxygenated) and C-8 of the aromatic ring. The structure of compound L was conformed as 3′,4′-dihydroxy-4H-furo[2,3-h]chromen-4-one.
Compound M was obtained as a pale yellow amorphous solid, mp 320 oC. Its molecular formula deduced as C17H9O6 from its molecular ion peak at [M+-H] at m/z 309.0395 in the HRESI-MS. It’s IR spectrum showed absorption band at 3283 cm-1 suggested the presence of hydroxyl group, a sharp band at 1692 cm-1 indicated the presence of carbonyl group. The 500 MHz 1H NMR spectrum of compound M taken in CDCl3 + DMSO-d6 revealed the presence of two doublets at δ 8.07 (J = 8.6 Hz, H-5) and 7.58 (J = 8.6 Hz, H-6) each integrating for one proton suggested the 1,2,3,4-tetrasubstituted aromatic ring. Two doublets at 7.96 (J = 2.0 Hz) and 7.32 (J = 2.0 Hz) each integrating for one proton indicated the H-2″, H-3″ of the furan ring. Two doublets at 6.95 (J = 8.4 Hz, H-5′), 7.87 (J = 2.1 Hz, H-2′) and double doublet at 7.70 (J = 2.1, 8.4 Hz, H-6′) each integrating for one proton indicated the presence of a 1,3,4-trisubstituted aromatic ring. Three signals at 8.85, 9.15 and 9.36 showed no connectivity with any carbon nucleus in the HSQC plot, thus suggesting the presence of three hydroxyl groups. This explanation is further supported by the disappearance of three peaks at 8.85, 9.15 and 9.36 in a heavy water exchange condition. The appearance of H-5 and H-6 protons as doublets strongly indicated that the annellation of furan ring to be at C-7/C-8 positions. 
               The 13C NMR spectrum in DMSO-d6 of compound M exhibits the presence of 17 carbon atoms whose multiplicity is explained using DEPT 135 and HMBC correlations. DEPT 135 spectrum clearly shows there are seven methines and ten quaternary carbon atoms (including carbonyl). A signal at δ 171.3 indicated the presence of a carbonyl group. A signal at δ 102.8 and δ 144.7 indicated the presence of a furan ring. The DEPT experiment revealed the presence of seven methines and ten quaternary carbons. The HMBC correlations of H-2″ with C-7 and C-8 indicated that the furan ring should be fused at C-7 (oxygenated) and C-8 of the aromatic ring. Based on these data the structure of compound M was identified as 3,3′,4′-trihydroxy-4H-furo[2,3-h]chromen-4-one. Further, these compounds (L & M) were also confirmed by the synthetic approach.
	       Resacetophenone was treated with bromoacetaldehyde diethyl acetal in the presence of potassium carbonate in anhydrous DMF to give compound 2 (O-alkylated resacetophenone), which on treatment with acidic Amberlyst-15 in toluene at 90 ○C yielded 4-hydroxy-5-Acetyl coumarone (3) and 6-hydroxy-5-Acetyl coumarone (4). Compound  4-hydroxy-5-Acetyl coumarone (3) was condensed with benzyl protected aldehyde 5 in the presence of KOH (60%) in anhydrous ethanol to give furanochalcone 6 which was cyclized  followed by debenzylated using I2 in trigol at 120 ○C to afford 3′,4′-dihydroxy-4H-furo[2,3-h]chromen-4-one (L).
Chalcone (6) was converted to the flavanol using Algar-Flynn-Oyamada reaction by treatment of hydrogen peroxide in the presence of KOH in Ethanol for 24 hours to afford benzylated flavanoid 7 with the yield of 65%. Debenzylation is carried out using TiCl4 in dry DCM at -10 ○C for 15 mints to afford Compound 3′,4′-trihydroxy-4H-furo[2,3-h]chromen-4-one (M).
All the isolated compounds displayed moderate intestinal α-glucosidase inhibition and DPPH scavenging activity. Pongamol and Karanjin identified as potent α-glucosidase inhibitors. Compounds L and M displayed moderate α-glucosidase inhibition and potent DPPH scavenging activity. This is the first report identifying presence of intestinal α-glucosidase inhibitory and DPPH scavenging activity from D. indica.
The third chapter deals with the synthesis and biological evaluation of furanochalcone derivatives. In the course of our efforts in identifying antihyperglycemics from traditional Indian medicinal plants, we isolated two new intestinal α-glucosidase inhibitor furanoflavonoids (L & M) (described in the Chapter-II) from antihyperglycemic extract of Derris indica Lam (Leguminosae). In the process of reconfirmation of the structures of two new furanoflavonoids (L & M) through synthetic approach, we observed that furanochalcone, the intermediate in synthetic route displayed better intestinal α-glucosidase inhibitory activity than the two new furanoflavanoids (L & M). This observation led us synthesize several new furanochalcones.

Claisen-Schmidt condensation of acetophenone derivatives (1 & 5) (described in the chapter-II and also in the experimental section) with various substituted benzaldehydes (2a–2g) & (6a–6g) in the presence of aqueous KOH in anhydrous ethanol to give angular and linear furanochalcones (3a–3g) & (7a–7g) respectively. Compounds 3a, 3b and 3c are previously reported from natural origin. Amongst angular and linear furanochalcones some benzyl protected chalcones (3e–3g) & (7e–7g) were further debenzylated using TiCl4 in dry DCM for 15 minutes under N2 atmosphere to afford angular and linear hydroxy furanochalcones (4a–4c) & (8a–8c) respectively.
Furanochalcone derivatives were synthesized and screened for intestinal α-glucosidase inhibitory and free-radical (DPPH) scavenging activity. All the compounds showed very good α-glucosidase enzyme inhibitory and DPPH free-radical scavenging activities. Among the all compounds 3c showed potent α-glucosidase inhibitory activity. This is the first report assigining α-glucosidase inhibitory, DPPH free radical scavenging and antihyperglycemic activity to furanochalcones.          
The fourth chapter deals with the synthesis and cytotoxic activity evaluation of pongamol derivatives. Pongamol is the lead compound from the hexane extract of roots of Derris indica. It has shown significant α-glucosidase inhibitory and cytotoxic activity. To enhance the activity (Cytotoxic activity) of pongamol, we have modified keto-enol pharmacophore in pongamol and prepared different oxazole and pyrazole analogues (2-4j). 
All the compounds were tested for the cytotoxic activity against human cancer cell lines Neuroblastoma (IMR-32), T-lymphocytes (Jurkat) and cervical cancer cells (HeLa). Among the all compounds 2, 4b & 4c were showed good cytotoxic activity. 












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English

THE ROLE OF MEMBRANE ESSENTIAL POLYUNSATURATED FATTY ACIDS IN SCHIZOPHRENIA OUTCOME

N., SATYAVANI

Seismic Reflection data modeling to generate useful information to study the structure and tectonics of Aravalli fold belt.

DAS, TAPASI

Studies on sperm antigen and antibody and their role in sperm function.

Fernandes, A.A.

Study of the directional spectrum of ocean waves using array, buoy and radar measurements

PAWAR, SNEHALATA

Molecular evidence for anticancer activity of aloe emodin in colon cancer cells

Aloe emodin (1, 8-dihydroxy-3-(hydroxymethyl)-anthraquinone), a natural anthraquinone, is
reported to have antiproliferative activity in various cancer cell lines. In this study we made
an effort to analyze the molecular mechanisms involved in the growth inhibitory activity of
this hydroxy anthraquinone in colon cancer cell line, WiDr. In our observation aloe emodin
inhibited cell proliferation by arresting cell cycle at G2/M phase through induction of p21 and
downregulation of cyclin B1. It also modulated the expression of MAPKs by inhibiting
phosphorylation of ERK1/2 and activating phospho JNK1/2 and phosphor p38. Aloe emodin
appreciably induced cell death specifically through apoptotic pathway, activating caspases
9/6 and by inactivating PARP. Aloe emodin induced apoptotic execution in WiDr cell was
found to be solely dependent on the caspase- 6 than caspase- 3 or 7. Apart from these
apoptosis specific molecular changes, morphological observations also revealed the
apoptotic potential of aloe emodin by loss of plasma membrane and mitochondrial
membrane integrity, DNA fragmentation etc. Our results also show that at a relatively non
toxic level of aloe emodin suppressed the phorbol-12-myristyl-13-acetate (PMA) induced
migration and invasion. On analysis of the molecules involved in the migration and invasion,
we found mRNA expression and promoter function of MMP-2, 9, Cyclin B1 and RhoB were
downregulated. It is also found that PMA induced gelatinolytic activity of MMP-2/9 and
protein expression of RhoB was downregulated on treatement with aloe emodin. We have
also analyzed the involvement of specific transcription factors in aloe emodin mediated down
regulation of MMP-2/9. Among the two transcription factors analysed, NF-κB and AP1, NF-
κB was having significant role in aloe emodin induced pharmacological changes in WiDr
cells. Binding of NF-κB to specific DNA sequence was inhibited by the treatment of aloe
emodin and it also found to reduce the translocation of p50, one of the subunits of NF-κB, in
to the nucleus. Aloe emodin also inhibited VEGF expression and Human Umbilical Vein
Endothelial cells (HUVECs) migration/invasion and in vitro tube formation.



MOLECULAR AND BIOCHEMICAL ASPECTS OF GLUCOSE / XYLOSE ISOMERASE FROM STREPTOMYCES sp.

Telang, Sangeeta

Abstract/Synopsis              Xylanases are xylan-degrading enzymes, belong to glycosyl hydrolases (GH). Xylanases from the two major families 10 (GH10) and 11 (GH11) catalyze the hydrolysis of internal -1, 4 bonds of xylan backbone. Xylan is the second most abundant polysaccharide in nature. Nearly one third of the dry weight of the higher plants is xylan and therefore, xylanases have an important role in biomass conversions. Currently, the most effective application of xylanases is in prebleaching of kraft pulp to minimize the use of environmentally hazardous chemicals in the subsequent treatment stages. In recent years, therefore, attention is focused on to isolate and/or engineer the xylanases for the industrial requirements. The desirable properties of xylanases in paper industry are stability and activity at high temperatures and alkaline pH. While the factors responsible for the thermal stability of GH10 xylanases have been analyzed, factors governing the alkaline stability of GH10 xylanases remain poorly understood.	The present thesis reports the crystal structures of an alkali thermostable GH10 extracellular endo-xylanase (BSX) from an alkalophilic organism, Bacillus sp. NG-27 in free and xylosaccharides-bound form. The enzyme was purified from the native organism and crystallized. The structure was solved by molecular replacement method. The 2.2 Å crystal structure of the native BSX enzyme is the first structure of an alkali thermostable GH10 family xylanase from an alkalophilic organism. It has unveiled unique protein properties that can form the basis for improving the thermal, alkaline stability and activity by site directed mutagenesis. The comparative study, especially in relation to GH10 xylanases, deciphered important structural features which are likely to be responsible for the alkaline stability of the enzyme. The work exemplifies the mechanism of adaptation of enzymes to function under polyextreme conditions through changes in the nature and composition of solvent-exposed residues. As apparent from the comparative study, the enhanced stability of the protein can be attributed to the surface rich in acidic residues and less number of solvent-exposed Asn as seen in BSX.  This situation which may be roughly described as “acidic residues outside and Asn inside”, is a notable feature of alkali-stable GH10 xylanases from alkalophilic organisms.	In addition, the thesis describes the results of the comprehensive database analysis of the occurrence of C-H…O hydrogen bonds in helices and helix termini of globular proteins. The study provides a compelling evidence that the main-chain C and the side-chains CH which participate in C-H…O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N-H…O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins.



Crystal structures are known for several glycosyl hydrolase family 10 (GH10) xylanases.  However, none of them is from an alkalophilic organism that can grow in alkaline conditions. We have determined the crystal structures at 2.2 Å, of a GH10 extracellular endo-xylanase (BSX) from an alkalophilic Bacillus sp. NG-27, for the native and complex with xylosaccharides. The industrially important enzyme is optimally active and stable at 343 K and at a pH of 8.4. The comparison of structure of BSX with those of other thermostable GH10 xylanases optimally active at acidic or close to neutral pH, showed that the solvent exposed acidic residues, Asp and Glu, were markedly enhanced in BSX, while solvent exposed Asn were noticeably depleted. The BSX structure when compared with putative three-dimensional homology models of other extracellular alkalophilic GH10 xylanases from alkalophilic organisms suggests that protein surface rich in acidic residues may be an important feature common to these alkali thermostable enzymes. A comparison of the surface features of the BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in poly-extremophilicity of proteins, where understanding structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. The work brings to the fore the role of nature and composition of solvent exposed residues in the adaptation of enzymes to poly-extreme conditions as in BSX.

Cellulases catalyze the hydrolysis of beta-1,4-glycosidic linkages within cellulose, the most abundant organic polymer on earth. The cellulase (TSC; EC 3.2.1.4) from an alkalothermophilic Thermomonospora sp. has a low molecular weight of 14.2 kDa. It is optimally active at 323 K and stable over the wide pH range of 5-9. Moreover, it has bifunctional activity against cellulose and xylan polymers. In this study, TSC was purified from the native source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.9, b = 79.5, c = 99.7 angstroms, and diffract to better than 2.3 angstroms resolution

Xylanases (EC 3.2.1.8) catalyze the hydrolysis of beta-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 A, beta = 131.2 degrees, and diffract to better than 2.2 A resolution.

A comprehensive database analysis of C--H...O hydrogen bonds in 3124 alpha-helices and their corresponding helix termini has been carried out from a nonredundant data set of high-resolution globular protein structures resolved at better than 2.0 A in order to investigate their role in the helix, the important protein secondary structural element. The possible occurrence of 5 --> 1 C--H...O hydrogen bond between the ith residue CH group and (i - 4)th residue C==O with C...O < or = 3.8 A is studied, considering as potential donors the main-chain Calpha and the side-chain carbon atoms Cbeta, Cgamma, Cdelta and Cepsilon. Similar analysis has been carried out for 4 --> 1 C--H...O hydrogen bonds, since the C--H...O hydrogen bonds found in helices are predominantly of type 5 --> 1 or 4 --> 1. A total of 17,367 (9310 of type 5 --> 1 and 8057 of type 4 --> 1) C--H...O hydrogen bonds are found to satisfy the selected criteria. The average stereochemical parameters for the data set suggest that the observed C--H...O hydrogen bonds are attractive interactions. Our analysis reveals that the Cgamma and Cbeta hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic beta-branched residue Ile to participate in 5 --> 1 C--H...O hydrogen bonds involving methylene Cgamma 1 atom as donor in alpha-helices. This may be an enthalpic compensation for the greater loss of side-chain conformational entropy for beta-branched amino acids due to the constraint on side-chain torsion angle, namely, chi1, when they occur in helices. The preference of amino acids for 4 --> 1 C--H...O hydrogen bonds is found to be more for Asp, Cys, and for aromatic residues Trp, Phe, and His. Interestingly, overall propensity for C--H...O hydrogen bonds shows that a majority of the helix favoring residues such as Met, Glu, Arg, Lys, Leu, and Gln, which also have large side-chains, prefer to be involved in such types of weak attractive interactions in helices. The amino acid side-chains that participate in C--H...O interactions are found to shield the acceptor carbonyl oxygen atom from the solvent. In addition, C--H...O hydrogen bonds are present along with helix stabilizing salt bridges. A novel helix terminating interaction motif, X-Gly with Gly at C(cap) position having 5 --> 1 Calpha--H...O, and a chain reversal structural motif having 1 --> 5 Calpha-H...O have been identified and discussed. Our analysis highlights that a multitude of local C--H...O hydrogen bonds formed by a variety of amino acid side-chains and Calpha hydrogen atoms occur in helices and more so at the helix termini. It may be surmised that the main-chain Calpha and the side-chain CH that participate in C--H...O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N--H...O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins.

Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component of the biosphere. The crystal structure of this F/10 family xylanase, which has a triosephosphate isomerase (TIM) barrel (beta/alpha)(8) fold, has been solved to small-molecule accuracy at atomic resolution (1.11 A) at 293 K (RTUX) and at ultrahigh resolution (0.89 A) at 100 K (CTUX) using X-ray diffraction data sets collected on a synchrotron light source, resulting in R/R(free) values of 9.94/12.36 and 9.00/10.61% (for all data), respectively. Both structures were refined with anisotropic atomic displacement parameters. The 0.89 A structure, with 177 476 observed unique reflections, was refined without any stereochemical restraints during the final stages. The salt bridge between Arg124 and Glu232, which is bidentate in RTUX, is water-mediated in CTUX, suggesting the possibility of plasticity of ion pairs in proteins, with water molecules mediating some of the alternate arrangements. Two buried waters present inside the barrel form hydrogen-bond interactions with residues in strands beta2, beta3, beta4 and beta7 and presumably contribute to structural stability. The availability of accurate structural information at two different temperatures enabled the study of the temperature-dependent deformations of the TIM-barrel fold of the xylanase. Analysis of the deviation of corresponding C(alpha) atoms between RTUX and CTUX suggests that the interior beta-strands are less susceptible to changes as a function of temperature than are the alpha-helices, which are on the outside of the barrel. betaalpha-loops, which are longer and contribute residues to the active-site region, are more flexible than alphabeta-loops. The 0.89 A structure represents one of the highest resolution structures of a protein of such size with one monomer molecule in the asymmetric unit and also represents the highest resolution TIM-barrel fold structure to date. It may provide a useful template for theoretical modelling studies of the structure and dynamics of the ubiquitous TIM-barrel fold.




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

D, Shankar

Abstract 
 
I 
 
The thesis entitled “Synthetic studies on R207910, aza-Diospongin A and 
hydroxylamine derivatives” is divided into three chapters. 
Chapter I: Chapter I is divided into two sections. 
Section A: Introduction to Tuberculosis and the drugs available for treatment. 
Section B: Describes the practical synthesis of anti-TB clinical molecule (2S)-R207910 
and its (2R) isomer. 
Chapter II: Deals with the introduction to analogues and asymmetric synthesis of aza (-)-
Diospongin A as an iNOS inducer. 
Chapter III: Describes the Palladium-catalyzed addition of hydroxylamine derivatives to 
Baylis–Hillman acetate adducts. 
------------------------------------------------------------------------------------------------------------- 
Chapter I 
Section A: Introduction to Tuberculosis and the drugs available for treatment. 
Tuberculosis (TB) is a deadly infectious disease caused by Mycobacterium 
tuberculosis (MTb). It is primarily an infirmity of the respiratory system and is spread 
through air via coughing and sneezing of the infected person. In conjunction with the 
spread of HIV infection, tuberculosis is today amongst the worldwide health intimidations. 
Nearly 1/3
rd
 of world’s population has been infected with Mtb. Globally 9.2 million new 
cases and 1.7 million deaths occur every year albeit widespread vaccination and 
chemotherapy.
 
As a result, TB has become a major health and socioeconomic problem in 
most of the countries. Although the treatment with current first line drugs: isoniazid (1), 
rifampicin (2) and pyrazinamide (3) and ethambutol (4) (Fig 1) was able to cure most of 
the disease, a major problem in TB treatment is the development of multidrug-resistant 
tuberculosis (MDR-TB), which can be defined as strains that are resistant to at least 
isoniazid (1) and rifampicin (2), two
 
important first line drugs used in TB treatment. 
Another serious problem, in the context of MDR-TB, is the emergence of extensively 
drug-resistant tuberculosis (XDR-TB), which can be defined as strains resistant to first and 
second line anti-TB drugs. As a consequence many second line drugs were discovered and  
used in the treatment depending upon the diagnosis and patient response. But present 
Abstract 
 
II 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1 
situation demands a continuous need of new drugs, which can cure all the active, latent and 
resistant forms with novel mechanism of action.  
Section B: Practical synthesis of anti-TB clinical molecule (2S)-R207910 and its (2R) 
isomer. 
The search for new entities to treat M. tuberculosis and related strains in all their 
forms led to quinoline nucleus, which constitutes an important class of heterocyclic 
compounds found in many synthetic and natural products with a wide variety of important 
pharmacological activities. R207910 (5a) (TMC 207) (Fig 2) is one such compound 
structurally dissimilar to isoniazid and rifampicin and consisting of diarylquinoline 
scaffold. This compound was developed at Johnson & Johnson Pharmaceutical Research 
and Development, and possesses a new mechanism of action based on the interaction with 
the enzyme adenosine triphosphate (ATP) synthase. It is active against both drug-resistant 
and drug-susceptible M. tuberculosis, as well as other mycobacterial species. It has a 
relatively longer half life in plasma and tissues, of nearly 24 h. The compound stops the 
energy production by acting on the proton pump of adenosine triphosphate (ATP) synthase 
of M. tuberculosis but not on other bacterial ATP systems, which explains its high 
specificity for mycobacteria. In the mouse model, 2-month treatment regimes containing 
R207910 and PZA led to sterilization, which shows the potential for reduction of treatment 
Abstract 
 
III 
 
time to two months. Its fascinating biological activity interested us to develop an 
asymmetric and practical synthesis of R207910.  
     
 
 
 
Figure 2 
According to our retrosynthetic analysis, 5a could be obtained from 6a in two steps 
(Scheme 1). The stereocenter at the tetrasubstituted carbon was introduced by chelation 
controlled Barbier type reaction. The enantiomerically enriched ketone 8 could be obtained 
by Sharpless asymmetric epoxidation followed by ring opening with PhMgBr. 
Retrosynthetic analysis of (2S) R207910. 
 
 
 
 
 
 
 
 
 
 
 
 
Scheme 1 
Abstract 
 
IV 
 
Results and Discussion 
The synthesis commenced with a known intermediate 6-bromo-2-chloroquinoline-3-
carbaldehyde 10, prepared from 4-bromo-acetanilide using a modified Vilsmeier-Haack 
reagent. It was subjected to Horner-Wadsworth-Emmons olefination using lithium enolate 
of the phosphonate [(OEt)2 P(O)CH2CO2Et] to afford α,β-unsaturated ester 11 in 89 % 
yield with complete E-selectivity. Reduction of compound 11 with DIBAL-H at 0 
o
C 
furnished allyl alcohol 12. Reaction of 12 with sodium methoxide gave 2-methoxy 
derivative 13.
 
 
 
 
 
 
 
 
 
 
 
Scheme 2 
Reagents and conditions: (a) (EtO)2P(O)-CH2CO2Et, LiHMDS, THF, 0 
o
C-rt, 2 h, 89 %; (b) DIBAL-H, 
CH2Cl2, 0 
o
C-rt, 2 h, 84 %; (c) NaOMe, MeOH, reflux, 8 h, 92 %; (d) (+)-DIPT, Ti(O
i
Pr)4, TBHP, CH2Cl2, 
−20 oC, 4 h, 86 %; (e) PhMgBr, CuCN, THF, −40 oC, 4 h, 86 %. 
The next operation was the Sharpless asymmetric epoxidation
 
which was carried out 
with (+)-DIPT, Ti(O
i
Pr)4, TBHP at −20 
o
C to afford epoxy alcohol 9 in 86 % yield (95 % 
ee). Subsequent exposure of the epoxide to PhMgBr in the presence of CuCN furnished 
diol 14 in 86 % yield (Scheme 2).
 
Diol 14 was oxidatively cleaved with NaIO4 
impregnated over silica gel to furnish aldehyde 15. The crude aldehyde was treated with 1-
naphthylmagnesium bromide to obtain secondary alcohol 16 as a mixture of isomers which 
Abstract 
 
V 
 
on oxidation with Dess-Martin periodinane afforded the keto derivative 8 in 81 % yield 
with high enantiomeric purity (95% ee). Our next concern was to construct the 
tetrasubstituted carbon center through allylation of carbonyl group present in 8. Our 
attempts to introduce the tetrasubstituted carbon through conventional nucleophiles like 
allylmagnesium, allylaluminium, allylindium, allylboronate, and allylstannane derivatives 
were not successful. A recent development of solvent free addition reaction of allylzinc 
bromide to carbonyl compound attracted our attention in this regard. Thus, with a slight 
modification compound 8 was treated with allylzinc bromide in the presence of 
CuBr.Me2S in THF to afford an inseparable mixture of required homoallyl alcohol 7a and 
7b in almost equal amounts of diastereomers (by HPLC) (Scheme 3). 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scheme 3 
Reagents and conditions: (a) NaIO4, CH2Cl2, 0 
o
C-rt, 1 h, 98 %; (b) 1-naphthylmagnesium bromide, Et2O, 0 
o
C, 1 h, 92 %; (c) DMP, CH2Cl2, 3 h, 87%; (d) Allylzinc bromide, CuBr.DMS, THF, rt, 1 h, 90 %. 
Abstract 
 
VI 
 
The diastereomers were separated by preparative HPLC (Pheonomenex Luna-C18 
(250x10mm, 10µ), ACN/water 9/1, flow 1.5 mL, detection- DAD): tR 10.0 min (syn) and 
9.25 min (anti). Both the diastereomers were subjected to a sequence of routine steps 
individually to end up in final compounds. Thus Oxidative cleavage
 
of olefin using OsO4-
NaIO4 in the presence of 2,6-lutidine afforded aldehyde 17a, which was treated with 
NaBH4 in methanol at 0 
o
C to obtain the diol 6a in 82% yield over two steps. Finally, O-
mesylation followed by displacement of the mesylate group in 18a with dimethylamine 
furnished (2S)-R207910 (5a) in 79% yield over two steps (Scheme 4). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scheme 4 
Reagents and conditions: (a) NaIO4, 2,6-lutidine, OsO4, dioxane:H2O (3:1), rt, 2 h; (b) NaBH4, MeOH, 0 
o
C-rt, 82 % (over two steps); (c) MsCl, Et3N, CH2Cl2, 0 
o
C-rt, 3 h, 88 %; (d) Me2NH, THF, 45 
o
C, 24  h, 90 
%. 
Abstract 
 
VII 
 
The spectral and analytical data of 5a ([α]D
25
 −165.2 (c = 0.8, DMF); ref.12: ([α]D
25
 
−166.98 (c = 0.5, DMF) was in good agreement with the reported data. The same set of 
reactions was performed on the other diastereomer (7b) to furnish (2R)-R207910 (5b) 
which was also in good agreement with the literature data. 
 
          
 
 
In a more practical approach the diastereomeric mixture (7a & 7b) obtained in the 
allylation step was carried to the final step with the same set of reaction protocol and the 
viability of chromatographic separation at the final stage has been tested. To our 
satisfaction the products were separable easily by silica gel column chromatography (ethyl 
acetate:hexane = 1:6). In fact this approach is operationally simple and amenable for scale 
up at affordable cost. 
 
 
 
 
In conclusion the synthesis of (2S)-R207910 (5a) and (2R)-R207910 (5b) was 
achieved in an overall yield of 12 % each in 10 steps. The key steps involved are Sharpless 
asymmetric epoxidation, regioselective epoxide opening and modified allylzinc bromide 
addition.  
 
 
 
Abstract 
 
VIII 
 
Chapter II: Asymmetric synthesis of aza (-)-Diospongin A as an iNOS inducer. 
Introduction: 
Nature has been the rich source of bioactive natural products and has laid basis for 
drug leads and new medicines since pre-historical era. Nearly half of the currently used 
drugs are of natural product origin derived either directly or designed based on 
pharmacophore.
 
However the structural complexity, paucity, side effects of, these 
pharmacologically active natural compounds has set limitations to use them as drug 
candidates. Thus modification of bioactive natural products to structurally similar synthetic 
analogues with minimum molecular complexity while retaining pharmacological activity 
and reduced side effects has gained importance over time. In this context we chose to 
prepare aza-analogue of diospongin-A (1a). 
 Diospongins 1a and 1b are a new family of diaryl heptanoids, which were isolated 
from the rhizomes of Dioscorea spongiosa in 2004 by Kadota et al. They are known to 
exhibit potent anti-osteoporotic activity in a bone organ culture. The structure of the 
diospongins containing a tetrahydropyran ring with two phenyl groups and three 
asymmetric centres is a tunable scaffold for the synthesis of various analogues. Our 
interest in the synthesis of bio-active molecules and their analogues has directed us 
towards the synthesis of diospongin analogues. Replacing the oxygen atom of the pyran 
ring in diospongin-A (1a) with nitrogen atom has resulted in the substituted piperidine ring 
of aza diospongin-A (2) whose biological activity was evaluated otherwise in the induction 
of inducible nitric oxide synthase (iNOS) enzyme and was found to be better when 
compared to its natural counterpart (Fig 3).  
 
 
 
 
 
 
Figure 3 
Abstract 
 
IX 
 
Our retrosynthetic analysis led us to consider the key synthon to be 3 which could be 
synthesized easily from cross metathesis between phenyl vinyl ketone (9) and 4 which in 
turn could be obtained from a known intermediate (R)-1-Phenyl-3-buten-1-ol (5) (Scheme 
5).  
 
 
  
 
 
 
 
Scheme 5 
Results and discussion 
The synthesis began with (R)-1-Phenyl-3-buten-1-ol (5), which can be readily 
obtained from benzaldehyde via a Keck allylation. Alcohol 5 underwent Mitsunobu 
inversion with hydrazoic acid to provide azide 6 in 54% yield. The reduction of the azide 
functionality to an amine using LiAlH4 followed by protection of the primary amine with 
(Boc)2O afforded the protected homoallyl amine 7. To prepare the precursor for olefin 
cross-metathesis, the terminal olefin of protected homoallyl amine 7 was subjected to 
dihydroxylation followed by periodate cleavage to give aldehyde 8, which was 
immediately treated with allyl bromide in the presence of zinc. The allylation of aldehyde 
8 provided a mixture of diastereomers 4a and 4b in a 1:1 ratio, which were separated by 
column chromatography (EtOAc/Hexane: 3:17). The required isomer 1, 3-amino alcohol 
4a was submitted to olefin cross-metathesis with phenyl vinyl ketone (9) using Grubb’s 
2nd generation catalyst 10 (20 mol %) in dichloromethane. The reaction proceeded in 
excellent yield of the enone product 3, which was ready for aza-Michael addition reaction. 
As expected, amino enone 3 underwent Boc-deprotection followed by aza-Michael 
Abstract 
 
X 
 
reaction in one-pot to afford the target product, aza-diospongin A (2) in 96% yield (with 
99% enantiomeric purity) with the exclusive formation of cis-isomer (Scheme 6). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scheme 6 
Reagents and conditions: (a) HN3, DIAD, TPP, benzene, rt, 3 h, 54%; (b) (i) LiAlH4, THF, 0 
o
C to rt, 1 h, 
(ii) (Boc)2O, 3 h, 90% (for two steps); (c) (i) NMO, OsO4 (cat), acetone–water (4:1), rt, overnight, (ii) NaIO4, 
THF, rt, 15 min, 95% (for two steps); (d) Allyl bromide, Zn, DMF, rt, 30 min, 70%.(e) 9, 10 (20 mol %), 
CH2Cl2, rt, 1 h, 70%; (f) TFA, CH2Cl2, 0 
o
C, 5 h, 96%. 
Abstract 
 
XI 
 
The structure of the product was confirmed by IR, Mass, 
1
H NMR and 
13
C NMR 
spectroscopic data. The cis-configuration of 2,6-substituents was confirmed by nOe 
studies. 
A biological profile of this analogue was also evaluated towards the induction of 
inducible nitric oxide synthase. Table 1 clearly illustrates that 2 showed the best induction 
of inducible nitric oxide synthase/nitric oxide synthase 2 (iNOS) at 1 µM. The decreased 
biological effect observed at 10 µM might be related to the cytotoxic properties of 2. At the 
same time, the compound failed to elicit any detectable increase in the immunostimulatory 
potential at 0.1 µM demonstrating the requirement of minimum threshold concentration 
required for the transcriptional activation of iNOS gene. It might be possible to argue that 
the immunomodulatory potential of compound 2, which maintains improved bioactivity 
while showing reduced levels of cellular toxicity when compared to 1a, is superior and can 
be enhanced by synthesizing the corresponding analogues. The table next illustrates the 
quantitative responses of the two compounds tested towards the induction of inducible 
nitric oxide synthase (iNOS). 
Compound name  
Dose tested 
(µM) 
Nitrite (mM) 
produced 
Fold induction with respect to 
control 
2 10 8.88 1.605786618 
  1 24.65 4.457504521 
  0.1 16.4 2.965641953 
1a 10 8.81 1.593128391 
  1 13.44 2.430379747 
  0.1 11.79 2.132007233 
IFN-γ   12.8 2.314647378 
LPS+IFN-γ   60.93 11.01808318 
Controls   11.01 1.990958409 
 Controls DMSO   5.53 1 
Table 1: Nitrite production and corresponding fold activation of compounds 2 and 1a. Cells were 
treated with 1ng/mL IFNγ in order to prime the cells before treatment with compounds. 
Abbreviations: DMSO: Dimethyl Sulfoxide; iNOS/NOS2: inducible Nitric Oxide Synthase/Nitric 
Oxide Synthase 2; LPS: Lipopolysaccharide; IFNγ: Interferon gamma; ng: Nano gram.  
Abstract 
 
XII 
 
In conclusion, the aza-derivative of (-)-Diospongin A has been synthesized with the 
key steps being Mitsunobu inversion, cross olefin metathesis and intramolecular aza-
Michael addition reactions. The, aza-Diospongin A was tested towards the induction of the 
inducible nitric oxide synthase enzyme by comparison with the natural Diospongin-A and 
it was found that the aza-analogue showed a better activity when compared to the natural 
one. 
 
Chapter III: Palladium-catalyzed addition of hydroxylamine derivatives to Baylis–
Hillman acetate adducts. 
Introduction:  
The carbon-carbon, carbon-halogen, carbon-heteroatom bond forming process are 
invariably the fundamental and most important synthetic tools for synthetic community in 
the construction of complex natural products, functional advanced materials,  
pharmaceutical lead compounds, and other high-value commercial products. Among many 
known and available methods for these bonds forming processes allylic substitution was 
well explored in terms of transition metal catalysis. Palladium-catalysed allylic substitution 
has been used in a wide variety of synthetically useful reactions involving various 
nucleophiles. However the use of hydroxylamines as oxygen atom (aminoxy equivalent) 
nucleophiles for allylic substitution reactions has been studied limitedly. The products of 
Baylis–Hillman reactions (BH adducts) are multifunctional allylic alcohols and have been 
used as precursors in synthesizing several useful compounds. In particular, the acetates of 
Baylis–Hillman adducts have proved to be very useful synthons for addition reactions with 
different nucleophilic reagents. In continuation of our interest in the reactions involving 
Baylis–Hillman adducts, we studied the nucleophilic addition of hydroxylamine 
derivatives to Baylis–Hillman acetate adducts in the presence of 10 mol % Pd(PPh3)4 
(Scheme 7). The products obtained also serve as intermediates for making useful allyloxy 
amines/aminoxy esters and their derivatives. 
We first examined the reaction of N-hydroxyphthalimide 1b with Baylis–Hillman 
adduct 1a in the presence of 10 mol % Pd(PPh3)4 in acetonitrile at room temperature to 
afford the corresponding allyloxy phthalimide 1c. The reaction was complete in 1 h and  
Abstract 
 
XIII 
 
 
 
 
 
 
 
Scheme 7 
product 1c was obtained in 80% yield with exclusive (E)-selectivity (entry 1). The 
stereochemistry of the product was established based on extensive 2D NMR studies. In the 
absence of a catalyst, no reaction occurred even at reflux. Next, the reaction of Baylis–
Hillman acetate adduct 2a, derived from acrylonitrile, with N-hydroxyphthalimide under 
similar reaction conditions gave the corresponding product, 2c in 71% yield with exclusive 
(E)-selectivity (entry 2). To prove the generality of the method, other Baylis–Hillman 
acetate adducts with N-hydroxyphthalimide 1b and succinimide 2b were engaged in this 
method (entries 3–11). The results are summarized in Table 1. In all cases, the reaction 
proceeded smoothly and afforded the products in reasonably good yields with good 
selectivity. As shown in Table 1, Baylis–Hillman acetate adducts derived from ethyl 
acrylate gave the corresponding products having the aryl group trans to the ester, whereas 
the adducts derived from acrylonitrile gave products having the aryl group cis to the nitrile. 
The olefin geometry was established based on 
1
H and 
13
C NMR chemical shifts and by 
comparison with literature values. However, reaction with N-Boc-hydroxylamine and N-
benzyl hydroxylamine gave low yields of the desired products with (E)-selectivity (entries 
12 and 13).  
We also investigated the transformations of 1c into useful substituted allyloxy 
amine/aminoxy ester 1d and its derivatives 1e and 1f (Scheme 8). Further applications of 
these products to prepare pseudo-γ-amino acids are underway. 
 
 
Abstract 
 
XIV 
 
 
 
 
Abstract 
 
XV 
 
 
 
 
 
 
 
 
 
Scheme 8 
 
In conclusion, we have developed a palladium-catalysed addition of hydroxylamine 
derivatives to Baylis–Hillman acetate adducts. The selective formation of substituted 
allyloxy amine products was observed and these products may find use in organic 
synthesis. 
 
 


Low-Frequency Variability of Sea Level Along the Coast of India

PATWARDHAN, ROHINI


					
The work carried in the research tenure has been compiled in the form of a thesis entitled “Synthesis and Apoptosis Inducing Ability of Pyrrolobenzodiazepine Conjugates and Heterocyclic Chalcones”. The main aim of this work has been to design and synthesize biologically active 2,5-diaryloxadiazole-pyrrolobenzodiazepine conjugates and imidazo/ benzimidazothiazole-pyrrolobenzodiazepine conjugates, to explore their DNA binding ability and anticancer activity, including mitochondrial mediated apoptosis. Furthermore, the biologically active imidazo[2,1-b]thiazole chalcone derivatives have been prepared and evaluated for their anticancer activity apart from the cell cycle effects on MCF-7 cell lines. The thesis has been divided into four chapters as shown below.
	Chapter I: This chapter gives the general introduction about cancer chemotherapy, covalent interactions of drug-DNA, in particular for pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumour antibiotics, and the objectives of the present work.
	Chapter II: This chapter describes the synthesis of a series of 2,5-diaryloxadiazole-PBD conjugates connected through simple alkane spacers and these conjugates have been evaluated for their biological activity. It is mainly focused on the mitochondrial mediated apoptotic pathway responsible for the significant anticancer activity.
	Chapter III: This chapter deals with the synthesis of new PBD hybrids by linking imidazothiazole/benzimidazothiazoles through stable alkane spacers to the C8-position of the A ring of the PBD scaffold. The present chapter is more focused on the DNA-binding affinity and anticancer activity of the newly synthesized PBD hybrids. 
	Chapter IV: A new class of imidazo[2,1-b]thiazole chalcone derivatives have been synthesized and evaluated for their anticancer activity. Further these derivatives are evaluated for the cell cycle effects on MCF7 cell lines. Moreover, these compounds have shown the characteristic features of apoptosis like enhancement in the levels of p53, p21 and p27, inhibition of NF-kB.


SYNOPSIS





REACTIVE HYDROGELS EXHIBITING CATALYTIC ACTIVITY

MARATHE, SUJATA

INVESTIGATIONS ON THE EFFECT OF HEAT STRESS ON TWO PHOTOSYSTEMS IN PHOTOSYNTHESIS

Chapter 1 of the thesis deals with the introduction in which review of literature, national and international scenario and objectives of the present work have been presented. Chapter 2 deals with the description of various techniques and protocols employed in the proposed work. Measurements were performed in isolated thylakoid membranes as well as on intact wheat [Triticum aestivum] leaves.  Chapter 3 deals with the effect of high temperature stress on rates of electron transport mediated by PSII and PSI. Results from polarographic measurements suggest that high temperature stress [upto 45oC] lead to decline in PSII rates and no change in PSI rates were observed. The results obtained from electron transport measurements and O2 evolution showed that the donor side of PSII has been affected and that the OEC has been damaged. Low temperature [77 K] Chl a fluorescence studies indicated that under high temperature conditions, no energy transfer took place between the two photosystems. Chapter 4 deals with the individual effect of pH on PSII and PSI activity as well as the comparative study of action of pH and high temperature on thylakoid membranes. The results lead to the conclusion that the regulation of PSII photochemistry and the inhibition of PSII caused by high temperature and low pH follow different and independent mechanisms. Chapter 5 deals with the effect of high temperature on intact wheat plants and detached leaves. The OJIP induction kinetic curve was converted into OKJIP after high temperature treatment. The appearance of an additional K step indicated that the OEC has been damaged. JIP test revealed that the ratio Fv/Fm which is a stress indicator decreased as the temperature was increased. This decrease showed that the photochemical yield of PSII has been affected. The value of Fo increased which attributed to the physical separation of PSII RC from associated pigment antennae, resulting in blocked energy transfer of the PSII traps. The decreased ratio of Fv/Fo depicts that structural changes occurred at the donor side of PSII due to high temperature stress. Recovery studies were also performed which revealed that at 40oC slight recovery was observed but at 45oC no recovery was observed and a permanent damage to PSII was observed.
Chapter 6 deals with the effect of high temperature stress on PSII heterogeneity. Two types of PSII heterogeneity i.e. Reducing side heterogeneity and Antenna size heterogeneity was studied. The reducing side heterogeneity [QB reducing and QB-non-reducing centers] was calculated using the Double hit method. In the leaves treated with high temperature the amount of QB-non-reducing centers increased while amount of QB-reducing centers decreased. It led to the conclusion that the active centers of PSII were converted into the inactive centers. In the next part of this chapter antenna size heterogeneity was studied. After high temperature treatment it was observed that the number of β centers increased on expense of α center which indicated that inactive centers have been increased. Thus, high temperature stress led to changes in PS II heterogeneity which may be one of the adaptive mechanisms to cope with high temperature stress. Chapter 7 deals with the characteristic of fluorescence transients in wheat plants grown in natural conditions on a hot summer day. In this study we measured fluorescence transients after every 2 hrs from 9 AM till 7 PM. High temperature for a short period of time (2-3 hrs) during a day is tolerated by decreasing the number of active reaction centre and by increasing the dissipation of energy. However these changes are restored largely when the stress conditions are over. Mid-day depression which results in the declined rate of photosynthesis seems to be a strategy to cope with environmental stresses and is advantageous for the survival of the plant.
	 
 Thus we have concluded that high temperature [upto 45oC] caused a decline in PSII activity but no change was observed in PSI activity. In case of detached wheat leaves high temperature caused detrimental effects which were irreversible at high temperature. High temperature also caused changes in PSII heterogeneity which is probably an adaptive mechanism through which plants survive under stress conditions. Mid-day depression which results in the declined rate of photosynthesis seems to be a strategy to cope with environmental stresses and is advantageous for the survival of the plant in case of high temperature stress in natural conditions of the plant. In dual stress, the salt treatment could not completely protect the wheat leaves from high temperature damage.



HOMO- AND COPOLYMERIZATION OF BICYCLIC OLEFINS USING ZIEGLER-NATTA CATALYSTS

SONPATKI, MILIND

Abstract
The thesis entitled “Variability of phytoplankton pigment composition in a tropical monsoon driven estuary (Godavari, India): impact of natural and anthropogenic influences” includes seven Chapters which are again compiled in five parts. 
Chapter 1 is the introduction which is all about the comprehensive literature survey on various investigations carried out so far in Godavari estuary after discussing briefly about riverine estuaries, their classification and importance. It also deals with the various implications of estuarine phytoplankton and unique role played by them at community level.
	Godavari estuary is the largest estuary of peninsular India largely fed by Indian Summer Monsoon (ISM) rainfall (July-September). It has been dammed at several locations in the upstream reaches for crop irrigation and other domestic and industrial consumptive usages. Availability of fresh water in the downstream estuaries is thus regulated by dam authorities at Rajahmundry (situated at about 70 km upstream of the estuarine mouth opening into  the adjacent Bay of Bengal) who allow excess water to flow down by lifting the sluice gate when water holding capacity of dam reservoirs surpass. During the monsoon period, huge volume of fresh water is released from the dam reservoir (at times greater than the entire volume of estuary) that flushes entire estuary less than a day. In contrast, enhanced stratification sets in during the lean discharge period with longer flushing time since river runoff virtually ceases as all the upstream tributary rivers dry up and water is retained in the reservoir. Such strong seasonality in river discharge and peakiness in the discharge pattern puts Godavari under unique suit of estuaries called monsoonal estuaries. Apart from this unique discharge pattern, Godavari estuary experiences variable stratification pattern such as well mixed, partially mixed and salt wedge type during different periods. 
                 Phytoplankton constitutes the majority of particulate organic carbon fraction in the estuary and act as the base of food web. Even though they are adapted through ages to withstand the dynamic environment of estuaries, they are also extremely sensitive to subtle variations. Such variations may occur through long term climate change or any short term anthropogenic impacts such as damming; dredging of estuarine mouth etc. Indian monsoonal estuaries (Godavari for example) are the best suited to study such impacts on phytoplankton since they experience highly variable discharge pattern both in short term (daily) and long term (inter annual) time scales and are highly regulated by the irrigation authorities though multiple dams.
Present investigation was carried out to answer some of these outstanding questions such as how short term (such as tidal variations, diurnal variations) and long term variabilities (inter annual) affect inhabiting phytoplankton biomass and community composition in Godavari river estuary, what is the atmosphere-estuary coupling in regulating phytoplankton in Godavari, how transient salinity variations shape phytoplankton community, how and why trace metals play dual role ( nutrient vs. toxicant) in the group level and community level of phytoplankton. This investigation, to the best knowledge of the author, is the first ever attempt to understand natural and anthropogenic variabilities of phytoplankton community composition through fast and robust HPLC pigment analysis approach in Indian estuaries. Above all this study was designed and conducted in an integrated approach so that phytoplankton variability could be explained in terms of physics, chemistry, meteorology, geology etc. 
Chapter 2 comprise with Materials and methods adopted in the study. Physiography, station locations, sampling strategies and brief methodologies of all the analytical and sensor-based measurements have been outlined in this Chapter. Since this thesis majorly concentrates on HPLC pigment analysis, it has been described in detail. An inter-calibration exercise of Chlorophyll a measurements with different instruments has also been incorporated in this Chapter.

Chapter 3 compiles the discussion on Study area and hydrography. It starts with the importance of Godavari River and its estuary from biogeochemical and socio-economic point of view. In depth discussion has also been made on hydrography (temperature, salinity, total suspended matter etc.) and meteorology (atmospheric temperature, rainfall, relative humidity, wind speed and direction etc.) at the daily sampling location, Yanam.

Chapter 4 Tidal Variation: Chapter 4 is all about the variability of different parameters during two tidal cycles; one in February (non-monsoon month) and the other in June (monsoon month) in the year 2010. This study shows how tidal variability of phytoplankton biomass and communities are controlled by tidal currents and their axial distribution along the estuarine channel.Godavari estuary is a micro tidal estuary (tide range from 0.5 to 1.8 m) with semi-diurnal cyclicity; i.e. it experiences two high tides and two low tides in a day. Temperature followed diurnal pattern with higher temperature during the day time and lower temperature during the night and showed no variability with tides. Estuary was more stratified with respect to salinity during the moderate discharge period (June) compared to the dry period (February). Salinity followed tidal rhythm closely. All the nutrients significantly increased during the ebb period which was consistent feature during both wet and dry periods. Phytoplankton biomass, in terms of   chlorophyll a (Chl a), increased during the ebb period showing that upstream pool of phytoplankton advects down the estuary along with the tidal current. Diatom and cyanobacteria were more prevalent during the dry and wet period respectively and followed similar tidal pattern as that of the Chl a.

Chapter 5 is the compilation of a long term daily observation (2007 - 2009) of phytoplankton biomass and community composition at daily sampling location in Godavari. Major emphasis has been laid on how different parameters like river discharge, salinity, nutrients, flushing time which varies in annual and inter-annual time scale regulate the quantity of biomass and composition of phytoplankton in Godavari estuary. It was found that significant amount of nutrients (>80 µM of dissolved inorganic nitrogen (DIN) and >12 µM of dissolved inorganic phosphate (DIP)) were associated with high river discharge in the Godavari estuary. However, during that time phytoplankton biomass remained low (<2 mg m-3) due to severe light limitation. Daily observation over three years revealed that decrease in precipitation over the Indian subcontinent from 2007 to 2009 resulted in the lowering of mean annual discharge in Godavari estuary from 748.63 m3s-1 in 2007 to 218.40 m3s-1 in 2009. The reduced water discharge slowed the flushing of the estuary from 1.2 days to 6.3 days, respectively. The consequent increase in stability of water column and reduced suspended material load gave rise to intense phytoplankton blooms (Chla 18 μg l-1 in 2007 to 28 μg l-1 in 2009).  This study suggested that reduced river discharge creates conducive conditions for phytoplankton to bloom through enhanced stratification and reduced flushing rates. 
Chapter 6 was based on the laboratory based bottle experiment to understand the response of phytoplankton communities due to the transient salinity changes by natural (such as fresh water runoff) or various anthropogenic perturbations. Subtle changes in salinity of the freshwater by one practical salinity unit (PSU) completely removed green algae from the system and allowed the cyanobacteria to come into dominance. The cyanobacteria were found to tolerate higher osmotic stress until the salinity reaches to 16. The higher salinity tolerance range of the cyanobacteria was attributed to the enhanced synthesis of Zeaxanthin as protective xanthophylls against the osmotic stress. The energy cost associated with the Zeaxanthin synthesis is probably balanced by the adaptability of cyanobacteria to tolerate a wider range of salinity fluctuation which helps them to occupy the niche left by the green algae (green algae disappeared from the system at the salinity of 1). However the increase in zeaxanthin (cyanobacteria) concentration, to combat the progressively higher salinity stress, is not indefinite and after a certain salinity maxima (16 PSU, in this study) the population cannot tolerate the stress and they crash. Therefore modification of salinity in the estuary through dredging and channelling the freshwater flow would bring significant impact on ecosystem structure in the monsoonal estuaries. 
Chapter 7 describes another bottle experiment where stress and toxicity of different trace metals Trace metals (Copper-Cu, Zinc-Zn, Nickel-Ni and Cobalt-Co) were investigated on phytoplankton biomass and community composition at the head estuary. Trace metals are micronutrients which are typically occur at concentrations less than1ppb (parts per billion).  They are indispensable as integral cofactors for a range of important enzymes essential for various cellular machineries. However, these metals may turn highly toxic above their threshold levels. Bioavailability of metals play a prominent role as this property determines what fraction of the metal would be available to be utilised as nutrient or to act as a toxicant. 
Trace metals are delivered into the estuary mostly via land run-off. We conducted a laboratory based incubation experiment to understand how four biologically important trace metals (Co, Ni, Cu and Zn) impart stress and toxicity on natural phytoplankton assemblage inhabiting in the estuarine head.   It is evident from the data that all metals acted as a nutrient when 1 x 10-7 M of metals was exposed into the system. The variation in biomass in the freshwater system under exposure to different metals at concentrations of 1 x 10-7 M indicates that they follow the following order as a nutrient.
                                                   Cu > Co > Ni > Zn. 

This order clearly shows that Cu acted as a nutrient and helped to increase the biomass followed by Co, Ni and Zn. The variation in biomass in the freshwater system under exposure to different metals at concentrations of 1 x 10-6 M indicates that they follow the following order as toxicant.

                                                   Cu > Co > Zn > Ni

 In general it is found that phytoplankton communities in Godavari River station have different tolerance for different metals. Cu and Zn were found to be lethal at high concentration. Cyanobacteria were found to be very sensitive to slight variation in Ni concentration and Co was found to be less toxic than Cu but more toxic than Zn at high exposed concentration. Estuary receives variable metal pollution at different locations and different phytoplankton groups have variable threshold levels to withstand. Once the level of the metal concentration crosses its limits, it would have lethal effect on phytoplankton and shift in community structure is inevitable that would have significant impact not only in the primary but propagate to secondary and tertiary producers. 

Estuaries are extremely important in processing the in-situ and land driven materials to the adjacent coastal ocean and phytoplankton play pivotal role in the entire process. Present investigation showed that monsoonal estuaries such as Godavari are extremely sensitive towards fresh water availability since fresh water brings nutrients; both macro and micronutrients such as trace metals, churn out sediments, modify salinity, alter flushing and residence time of the water parcel and all these factors directly or indirectly control phytoplankton biomass and community composition. Since phytoplankton constitute the base of the food chain and zooplankton exhibit strong feeding preference, any change in the phytoplankton biomass and community composition could detrimentally affect other higher trophic levels ( fish for example) in the estuary and thus humans on land. Evidences are also piling up that fresh water delivered by all the east coast estuaries control the unique biogeochemical setting of the Bay of Bengal which in turn regulates formation of depression/cyclones that drives rainfall in the entire east coast of India. On the river Godavari, construction of several dams is underway and more have been proposed by the Indian Government. In addition to this climate change is also going to bring significant modification in the precipitation pattern over Indian subcontinent. The east coast estuaries (including Godavari) are linked with the Bay as well as with the atmosphere via fresh water supply in a complex feedback loop. Any jeopardy in this fine loop, either natural or manmade, would bring irreversible changes in these sensitive ecosystems.  




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