Open Access Repository of Indian Theses

D, Prasanna

APPENDIX   2



Title	STUDIES ON BIODEGRADATION OF NITROPHENOL ISOMERS BY MIXED BACTERIAL CULTURES
Author	Shabana Basheer
Guide	Dr. S. Divakar
Degree	Ph.D. in Biotechnology
University	Mysore University
Institute/lab	Central Food Technological Research Institute, Department of Fermentation Technology and bioengineering
Date of Submission	30-10-2003
No. of pages	222
Subject/Area	Biodegradation/Bioremediation
Abstract/Synopsis	SYNOPSISIn general, the presence of nitroaromatic compounds in nature is a major consequence of anthropogenic activities. Nitroaromatic compounds are used a herbicides, fungicides, bactericides, explosives and dyes (Higson, 1992; Marvin-Sikkema and deBont, 1994; Pieper et al., 1996). The combustion of petroleum products and other organic substances also generate nitroaromatics (Rosenkranz and Mermelstein, 1983). Considerable amount of  these compounds have been found to accumulate in soil by the hydrolysis of organophosphorus insecticides such as parathion or of herbicide nitrophenols (Kearney and Kaufman, 1975,). Nitroaromatic compounds are detected as contaminants in river water (Golab etal.,1979) and in large amounts in industrial waste waters (Hallas and Alexander, 1983). Mononitrophenols  such  as o-Nitrophenol and p-Nitrophenol have been listed as priority pollutants by the US Environmental Protection Agency (Keith and Telliard, 1979). The presence of the nitro group causes such compounds to be more resistant to biodegradation than the unsubstituted analogs and they tend to accumulate in the environment and cause deleterious effects to the biological systems due to their toxicity. Fresh and marine waters, sewage and soils posses highly diverse microbial communities that exhibit degradative capacities and species within these communities transform many organic compounds aerobically or anaerobically.Nitroaromatic compounds have been reported to be used by microorganisms as carbon, energy and nitrogen sources (Spain 1995; Meulenberg et al., 1996). A mixed bacterial system isolated from a contaminated soil sample was employed during the present study on individual and simultaneous degradation of mononitrophenol isomers - o-Nitrophenol(ONP), m-Nitrophenol(MNP) and p-Nitrophenol (PNP).A brief introduction to nitroaromatic compounds and detail review of literature regarding the biodegradation of these compounds both aerobically and anaerobically by various microorganisms and specially bacteria has been discussed. Emphasis is also laid on the different mechanisms involved in degradation. Factors that affect biodegradation such as carbon and nitrogen sources, inorganic nutrients, acclimation period, inoculum size, substrate concentration, adaptation, varying pH and temperature conditions have been dealt with. It also includes a brief introduction on the characteristics of the mononitrophenol isomers and the objective of studying their degradation.Degradation of the three mononitrophenol isomers and their mixture using a consortium have been studied. The mixed bacterial culture successfully degraded individual isomers of mononitrophenol and their mixture. The consortium degraded subtoxic concentrations ( 0.5mM) of ONP, MNP and PNP by 54h, 12h and 36h on short periods of induction. Nitrite was released during ONP and PNP degradation indicating an oxidative mechanism whereas production of ammonia during MNP breakdown suggested a reductive mechanism. Simultaneous degradation of a mixture of the isomers occurred by 18h during which more than 90% substrate disappearance was observed. Differential rates of degradation of the three isomers from a mixture indicated that MNP was catabolized faster than PNP and ONP as evident by HPLC. Only nitrite release could be recorded during simultaneous degradation of subtoxic concentrations probably due to the  presence of two sources of nitrite production in the form of ONP and PNP in the mixture. Pre-growth of the consortium on related aromatic compounds such as isomers of cresol and phenol drastically affected the rate of degradation of all the mononitrophenol isomers. Pre-growth on non-aromatic compound such as sodium acetate resulted in retardation of degradation of ONP, MNP and PNP. Pre-exposed cells of the consortium degraded ONP, MNP and PNP by 24h whereas non-exposed cells could catabolize only 50% in the same period. Presence of an additional nitrogen source (NH4NO3) did not affect the degradation rates. The consortium could tolerate toxic concentrations (0.5mM-3mM) and bring about complete degradation of 1.5mM concentrations.The degradative abilities of the individual bacterial cultures of the consortium wee tested. Eight bacterial cultures were isolated from the consortium and they were identified as Bacillus licheniformis (SNP-1), Xanthomonas maltophila (SNP-2), Serratia liquefaciens (SNP-3), Pseudomonas putida (SNP-4), Pseudomonas sp. (SNP-5), Psuedomonas alcaligenes (SNP-6), Psuedomonas sp. (SNP-7) and Sarcina maxima (SNP-8) of which Sarcina  maxima, an unreported culture was employed for degradation studies. 	              All the cultures except SNP-4 and SNP-7 showed around 50% reduction in substrate concentration by 96h. Cultures SNP-2, SNP-5 and SNP-6 brought around 90% reduction in substrate concentration within 96h followed by SNP-3, SNP-8 and SNP-1 which degraded 50% of the mixture. SNP-4 and SNP-7 could degrade only about 20% of the isomers.  Well  induced cells of Sarcina maxima (SNP-8) exhibited both oxidative and reductive mechanisms in degrading mononitrophenol isomers as evident by the release of nitrite and ammonia respectively. Interestingly cells induced for a long periods could degrade around 50% of 0.5mM of a mixture of the isomers while cells induced for comparitively short periods showed only 34% reduction in substrate concentration.A brief study on the enzymatic activities of cell extracts of the mixed bacterial culture was conducted. Dioxygenase activities were observed in cell extracts of the consortium induced with ONP, MNP and PNP separately. Pronounced catechol 1,2-dioxygenase activity was observed in ONP and PNP induced cell extracts indicating an ortho cleavage pathway in their degradation during which catechol was oxidized to cis, cis-muconic acid. Catechol 2,3-dioxygenase activity could be recorded only in MNP induced cell extracts and an absorbance maxima at 375nm consistent with the formation of -hydroxy muconicsemialdehyde was observed.An extensive study on the biochemistry of the pathways employed by a mixed bacterial culture and one of its constituent culture Sarcina maxima, hitherto unreported, using NMR spectroscopy was carried out. Intensive 1HNMR and 2D HMQCT studies revealed the pathways followed by the degrading consortium and the single culture Sarcina maxima. 1HNMR spectra of the ONP sample degraded by the consortium revealed the presence of metabolites such as catechol, cis, cis-muconic acid,  -hydroxy muconic semialdehyde, maleyl acetate and -ketoadipate. The spectra of ONP reaction mixture degraded by Sarcina maxima showed that formation of maleylacetate from -hydroxy muconic semialdehyde should go through a new metabolite -hydroxy maleylacetate, hitherto unreported. A deviation in MNP degradation of the consortium and the single culture was observed. The consortium seemed to breakdown MNP to 4-aminocatechol indicating it came from 3-hydroxyl aminophenol. 1,2,4-benzenetriol and -ketoadipate being the other metabolites. Sarcina maxima seemed to convert to 2-nitrohydroquinone as indicated by its presence along with  -hydroxy muconic semialdehyde, muconolactone and maleyl acetate. The pathway followed by the consortium during PNP degradation was by the formation of 4-nitrocatechol, maleyl acetate and -ketoadipate which was confirmed by the 1H NMR spectra. Both 1H NMR and Carbon-13 signals from 2D HMQCT confirmed the presence of maleyl acetate, -hydroxy muconic semialdehyde and -ketoadipate in the PNP reaction mixture of Sarcina maxima. The pathway is expected to go through the formation of p-hydroquinone as the initial metabolite as no 4-nitrocatechol was detected. Conclusions from the degradation studies of mononitrophenol isomers carried out by the mixed bacterial culture and the individual culture Sarcina maxima (SNP-8) and the scope for future research in the field of biodegradation studies have also been discussed.ReferencesGolab T., Althaus WA and Wooten HD (1979). Fate of  (13C) trifluralin in soil. J Agric Food Chem. 27: 163-179Hallas LE and Alexander M (1983). Microbial transformation of nitroaromatic coumpounds in sewage effluent. Appl. Environ. Microbiol. 45: 1234-1241Higson FD (9192). Microbial degradtion of nitroaromatic compounds. Adv. Appl.  Microbiol. 37: 1-19Kearney  PC and Kaufman DD (1975). Herbicides: Chemistry, Degradation and Mode of Action,  Marcel Dekker Inc: New YorkKeith LH and Telliard WA (1979). Priority pollutants- a perspective view. Environ. Sci.Technol. 13: 416-423Marvin-Sikkema FD and deBont JAM (1994). Degradation of nitroaromatic compounds by microorganisms. Appl. Microbiol. Biotechnol. 42: 499-507Meulenberg R, Pepi M and deBont JAM (1996). Degradtion of 3-Nitrophenol by Psuedomonas putida occurs via 1,2,4-benzenetriol. Biodegradation. 7: 303-311Pieper DH, Timmis KN and Ramos JL (1996). Designing bacteria for the degradtion of nitro and chloroaromatic pollutants. Natur Wisenschaften Aufsatze. 83:201-203Rosenkranz HS and Mermelstein R (1983). Mutagenicity and genotoxicity of nitroarenes: all nitro- containing chemicals were not created equal. Mutant Res. 114:217-267Spain JC (1995). Biodegradation of nitroaromatic compounds. Annu Rev Microbiol. 49: 523-555
Papers	
Patents	




English

Bioremediation of polycyclic aromatic hydrocarbons in slurry phase bioreactor

Jebaraj, Cathrine Sumathi

Ecology of fungi in the denitrification zones of the Arabian Sea

Sajid, Andaleeb

Abstract


This thesis entitled “Design and Synthesis of /-Hybrid Peptides: Identification of Novel Secondary, Super Secondary and Tertiary Structures” mainly describes the design and synthesis of peptides with heterogeneous backbone from alternating C-linked carbo-β-amino acids (-Caas) and L-Ala. The peptides containing C-terminal ‘L-Ala--Caa-L-Ala’ (--) sequence have shown unprecedented novel 9/11-helical structures. Similarly, the studies on the peptides with C-terminal ‘-Caa-L-Ala--Caa’ (--) sequence resulted in two new three residue turns, besides helix-turn motifs,  that were successfully utilized for the creation of tertiary structural motif, such as Helix-Turn-Helix. Further, the peptides made from (R)-β-Caa and D-Ala, have also shown the presence of secondary structures. 
An overview on peptides with homogeneous and heterogeneous backbones and their conformational properties
A brief review is presented on -peptides, -peptides and peptides with heterogeneous backbones. The discussion encompasses the aspects of conformational analysis and protein architecture. -Peptides and most of the foldamers1 reported so far have homogeneous backbones. Peptides with heterogeneous backbones were also recently found to display well-defined folding behavior, resulting in helical and skeletal diversity. 
Design and synthesis of /-hybrid peptides with novel mixed helices from alternating L-Ala and (S)-C-linked carbo--amino acids [(S)--Caa]
β-Amino acids are part structures of several natural products and very important components. Their importance and nonavailability from ‘chiral pool’, prompted the development of new and efficient strategies2 for their synthesis, enroute` to peptidomimetics. Seebach et al.3 and Gellman et al.4 were the first to report β-peptides with novel helical structures, which followed subsequent reports5 on a variety of novel secondary structures in β-peptides with different designs. In a further work on foldamers, peptides with heterogeneous backbone6 were reported to show novel helical patterns.
The research in the recent past indicated that the attachment of carbohydrate7 to peptides improves bioavailability and water solubility, increases resistance to proteases, or enables them to enter the blood-brain barrier. Based on the structures of nikkomycins8, our group has reported the synthesis of C-linked carbo-β-amino acids9 (β-Caa), with carbohydrate moieties as side chains. Making use of these monomers, earlier our group has reported β-peptides with novel helical patterns.10
The β-Caa residues were made from D-glucose. Accordingly, the Michael acceptor 2, prepared (Scheme 1) from the corresponding aldehyde 1,11 on reaction with benzyl amine12 at room temperature gave esters 3 (46%) and 4 (22%), which are epimeric at the amine stereocentre (Scheme 1).
Scheme 1
	
Hydrogenolysis (10% Pd-C) of 3 and 4 in methanol under hydrogen atmosphere gave 5 and 6 respectively, which on reaction with (Boc)2O and Et3N in THF gave 7 (72%) and 8 (72%) respectively. Further, 7 and 8 on hydrolysis with NaOH in methanol afforded respective acids 9 (85%) and 11 (86%), while, exposure to trifluroacetic acid (TFA) in CH2Cl2 furnished the respective amine salts 10 and 12.
As a part of our ongoing program on foldamers, to create skeletal and novel secondary structural diversity, peptides with heterogeneous backbone using L-Ala and -Caa were envisaged to result in /β-hybrid peptides. Based on the earlier findings from β-Caas resulting in 10/12-mixed helices, the new design with heterogeneous backbone was anticipated to result in 9/11-mixed helices.
This new design to synthesize /β-hybrid peptides is based on the 1:1 alternating use of (S)-β-Caa and L-Ala (Figure 1). The peptides were prepared using standard peptide coupling13 conditions in the presence of water soluble EDCI and HOBt, with DIPEA as a base in dry CH2Cl2.
 
Figure 1: (S)-β-Caa ((S)-C-linked carbo-β-amino acid), HCl.NH2-L-Ala-OMe (Alanine methyl ester hydrochloride) and Boc-L-Ala-acid 

Accordingly, the dipeptide 13, tetrapeptide 14 and hexapeptide 15 were prepared from (S)-β-Caa 7 and L-Ala by adopting segment condensation method in the presence of EDCI, HOBt and DIPEA in CH2Cl2. NMR, CD and MD studies clearly indicated the pre-





	





Figure 2: A) Circular dichroism spectrum of 15 in CH3OH at concentration of 0.2 mM. B) Superimposed 25 minimum energy structures of 15 (sugars are replaced with methyl groups and protons have been removed for clarity)

sence of novel right-handed 9/11-mixed helical pattern in the /β-hybrid peptides 14 and 15 (Figure 2). Due to the conformational preferences, (S)-β-Caa 7 and L-Ala participated in 9- and 11-membered H-bonding respectively. Thus, the 9/11-helical pattern that was indicated in these /β-peptides is newly found and unprecedented in the literature.
	Having met with success on the above new ‘design’ with (S)-β-Caa at the N-terminus, it was proposed to synthesize new /β-peptides with L-Ala at the N-terminus. Based on the above observations, the new design with L-Ala at N-terminus is anticipated to give a different helical structure such as an 11/9-helix.
Accordingly, tripeptide 16, pentapeptide 17 and heptapeptide 18 were prepared from L-Ala and (S)-β-Caa 7 under standard peptide coupling conditions (EDCI, HOBt 






	
	



and DIPEA) in CH2Cl2. NMR, CD and MD studies evidently proved the presence a right-handed 11/9-mixed helical pattern in /β-hybrid peptides 16, 17 and 18. Further, this study also proved the presence of a 11/9-helix in as short as a tripeptide 16.   
	Thus, the new designs on /β-hybrid peptides, resulted in a new class of peptides for the first time with robust helical structures with heterogeneous backbones, besides the report of a novel and unprecedented 9/11-mixed helical pattern. 
Synthesis of /β-hybrid peptides with new three residue turns and their application for Helix-Turn (HT) and Helix-Turn-Helix (HTH) motifs
The preceding discussion described the design and synthesis of /-hybrid peptides with ‘L-Ala--Caa-L-Ala’ (--) sequence at the C-terminus, from L-Ala and (S)--Caa, forming very robust 11/9-mixed helices. Further studies were undertaken on the synthesis of /-peptides having C-terminus ‘-Caa-L-Ala--Caa’ (--) three residue component. In comparison to the earlier /-peptides, in the newly designed peptides, the C-terminal (S)--Caa residue does not participate in the helix formation. It thus would be interesting to understand the implications of such a residue on the helix forming capability. 
	Accordingly, tetraptide 19, hexapeptide 20 and octapeptide 21 were prepared with L-Ala (at N-terminus) and (S)-β-Caa 7, under standard peptide coupling conditions (EDCI, HOBt and DIPEA) in CH2Cl2. NMR, MD and CD studies clearly indicated that /-hybrid peptides 20 and 21 fold into helix-turn super secondary structural motifs, with an 11/9-mixed helical pattern at N-terminus and 7/13-turn at C-terminus (Figure 3). This study amply indicated that the C-terminal three residue sequence has resulted on nucleation with a 11/9-helix into a novel ‘turn’ serendipitously. 









Figure 3: Characteristic nOes and H-bonds defining the turn in 20 (Dotted lines indicate the nOes, dark lines indicate the H-bonds in the turn region and italic represents residue number)

Having successfully synthesized the /β-hybrid peptides with C-terminus ‘-Caa-L-Ala--Caa’ (--), which resulted de novo in ‘helix-turn’ motifs (super secondary structure), the attention was then focused on the synthesis of the /-hybrid peptides with (S)--Caa at the N-terminus to result in peptides with new helix-turn patterns.
Accordingly, tripeptide 22, pentaptide 23 and heptapeptide 24 were prepared with  in the presence of EDCI, HOBt and DIPEA in CH2Cl2. NMR and MD studies clearly indicated that the peptides 23 and 24 with a -Caa at the N-terminus also resulted in the helix-turn motif, nucleated by a 9/11-helix (Figure 4).








Figure 4: (A) Circular dichroism spectrum of 24 in CH3OH at concentration of 0.2 mM. MD structure of 24; (B) Stereoview (bundle of the twenty five conformers lowest in energy determined by restrained molecular dynamics, sugars are replaced with methyl groups and protons have been removed for clarity)
		
		This study thus resulted in a new and novel three residue turn in ‘-Caa-L-Ala--Caa’. A literature survey revealed that the three-residue turns are the second most frequently observed class of hairpin forming loops after two-residue loops. Unlike the D- 






Figure 5: Ball and stick models of turns in: A) -helix; B) D-Pro-L-Pro-D-Ala; C) -Caa-L-Ala--Caa
Pro-L-Pro-D-Ala turn14 (Figure 5B), the new ‘-Caa-L-Ala--Caa’ turn (Figure 5C), is devoid of any constrained amino acids and differs in the directionality of the 13-memebered H-bond. Further, both these turns differ from the -turns15 (Figure 5A) observed in proteins.
Thus, the above study resulted in the discovery of a new three residue turn motif in C-terminus ‘--’ sequence, with 7- and 13-membered H-bonded pseudo rings. Further, it is a serendipitous discovery to find the ‘--’ sequence as a turn motif upon nucleation by helix, which is the first such example of a three residue turn in linear peptides, devoid of any constrained amino acid. This study has thus resulted in new ‘helix-turn’ motif. 
Having successfully completed the synthesis of /β-hybrid peptides with novel ‘Helix-Turn’ (HT) motifs, the HT motifs were envisaged as an attractive option to design Helix-Turn-Helix (HTH) tertiary structures. 
Accordingly, peptide 25, derived by appending (S)-β-Caa at the C-terminus in 23, was investigated first. NMR and MD studies suggested that for 25, the three residue turn in the C-terminal appears to be disturbed (Figure 6). Although the signatures of the helix at the N-terminus are retained, additional H-bonding was observed between NH(5)-O(6) of furanoside ring.
       
Figure 6: (A) Stereoview of 25 (bundle of the twenty five conformers lowest in energy determined by restrained molecular dynamics, sugars are replaced with methyl groups and protons have been removed for clarity except that of residue-6) 

	To understand the above results conclusively, higher oligomers 26, 27 and 28 obtained by extending 23, 20 and 24 at the C-termini with Boc-NH-β-Caa-L-Ala-β-Caa-L-Ala-OMe, respectively were studied. The NMR studies on 26, 27 and 28 though indicated the presence of stable helices at the two termini, with the characteristic H-bonding and the nOe patterns, the geometry of the ‘turn’ region differed from that in helix-turn peptides. In 26, the turn region involving the residues 4-7, displayed new nOes; NH(5)/C4H(6), NH(5)/C1H(6), NH(5)/NH(6), CH(4)/NH(6) and CH(4)/NH(7). The MD calculations provided compelling evidence for the involvement of CO(3) in a three



																			
















centered H-bonding with NH(6) and NH(7), having 11- and 15-membered pseudo rings respectively. H-bonding was also observed between NH(5)-O(6) of the furanoside ring, further stabilizing the structure. This three residue turn, imparts an angle of about 90º between the two helices. The features described above for the peptide 26 are reiterated in 27 and 28, thus resulting in Helix-Turn-Helix tertiary structures. 	

Figure 7: Characteristic nOes and H-bonds defining the turn in 26 (Dotted lines indicate the nOes, dark lines indicate the H-bonds in the turn region and italic represents residue number)










Figure 8: (A) Circular dichroism spectrum of 26 in CH3OH at concentration of 0.2 mM. MD structure of 26; (B) Stereoview; (bundle of the twenty five conformers lowest in energy determined by restrained molecular dynamics, sugars are replaced with methyl groups and protons have been removed for clarity except that of residue-6) 

The H-bonding in the new ‘L-Ala--Caa--Caa’ turn (Figure 9B) differs from the ‘-Caa-L-Ala--Caa’ turn (Figure 9A), wherein ‘L-Ala--Caa--Caa’ is having a new three-centered or bifurcated H-bonding.16 Thus, the Helix-Turn-Helix (HTH) peptides 26, 27 and 28 have been identified with three centered (bifurcated) hydrogen bonding between two donar N-H groups and one acceptor C=O.






Figure 9: Ball and stick models of turns in: A) -Caa-L-Ala--Caa and B) L-Ala--Caa--Caa	
Synthesis of /-hybrid peptides from alternating D-Ala and (R)-C-linked Carbo--amino acids [(R)--Caa]
	In the preceding discussions, the /β-hybrid peptides with heterogeneous backbone derived from L-Ala and (S)-β-Caa, having C-terminus ‘-β-’ and ‘β--β’ sequence resulted in novel right-handed 9/11-helical structures and new turns/helix-turn and helix-turn-helix motifs respectively. In a further study to explore the heterogeneous backbone peptides with helical diversity, a new design was envisaged based on the alternating use of (R)-β-Caa and D-Ala (Figure 10). 
	
Figure 10: (R)-β-Caa (R -C-linked carbo-β-amino acid), (D)-Alanine methyl ester hydrochloride and Boc-D-Ala-acid 
	Accordingly, dipeptide 29, tripeptide 30, tetrapeptide 31, pentapeptide 32 and hexapeptides 33 were prepared having D-Ala at the N-terminus and (R)-β-Caa at the C-terminus, with the concept of alternating chirality using standard peptide coupling conditions (EDCI, HOBt and DIPEA) in CH2Cl2. 


Similarly, new /β-hybrid peptides having N-terminus (R)-β-Caa were also prepared. Accordingly, tripeptide (35), tetrapeptide (36), pentapeptide (37) and hexapeptides (38) were prepared under the standard peptide coupling conditions. 











The NMR studies on these new /β-hybrid peptides from (R)-β-Caa and D-Ala indicated the presence of H-bonding by downfield shift for the amide protons. Besides, the solvent titration studies confirmed their participation in H-bonding. Further, the CD spectral data obtained for these peptides in MeOH gave proof for the presence of secondary structural elements in these /β-hybrid peptides. However, though the signatures are directing towards the presence of 9/11-mixed helical pattern, sufficient nOe data could not be obtained from NMR studies to do the MD simulation studies to conclusively prove the conformations that are present in these peptides.
Conclusion
Thus, the work reported in the thesis resulted in the design and synthesis of a new class of /β-hybrid peptides from alternating L-Ala/(S)-β-Caa and D-Ala/(R)-β-Caa. The first design (L-Ala/(S)-β-Caa) with C-terminus -β- sequence resulted in robust 9/11-helical structures, while the peptides with C-terminal β--β sequence resulted initially in a novel three residue ‘turn’ having 7- and 13-membered H-bonding and gave Helix-Turn (HT) peptides. These HT peptides were converted into Helix-Turn-Helix peptides wherein, yet another new three residue turn (-β-β) with bifurcated (11- and 15-membered pseudo rings) H-bonding was observed. A further study on the peptides made from (R)-β-Caa and D-Ala, eventhough has shown the signatures for the presence of secondary structure (mixed helical pattern), sufficient nOe data could not be obtained to conclusively prove the secondary structure.
References
1.	Reviews: a) Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 2015. b) Gellman, S, H. Acc. Chem. Res. 1998, 31, 173. c) DeGrado, W, F.; Schneider, J. P.; Hamuro, Y. J. Pept. Res. 1999, 54, 206. d) Gademann, K.; Hintermann, T.; Schreiber, J. V. Curr. Med. Chem. 1999, 6, 905. e) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. Rev. 2001, 101, 3219. f) Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.; Aguilar, M-I. Curr. Med. Chem. 2002, 9, 811.
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3.	Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta. 1996, 79, 913.
4.	Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071.
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Role of Transmembrane Serine/Threonine Protein Kinases in Signal Transduction Pathways of Mycobacterium tuberculosis

Krishnan, Anita

Chemical investigation and structural determination of Bioactive compounds from Marine organisms and Development of Synthetic Methodologies.
_______________________________________________





ABSTRACT


SUBMITTED FOR THE DEGREE OF


Doctor of Philosophy

IN CHEMISTRY


TO

OSMANIA UNIVERSITY







BY

MASUNA SRINIVASULU




ORGANIC CHEMISTRY DIVISION-I
INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY
HYDERABAD-500 007, INDIA
MAY-2007


ABSTRACT
Nature has been as a source for therapeutics. Despite the fact that we live in an oceanic planet, a number of technical factors have historically hampered the evolution of a marine-based chamanic medicine. With the implementation of scuba diving tools and the development of sophisticated instruments for the isolation and elucidation of structures of natural products from marine organisms, major advances have been made in the discovery of marine derived therapeutics. In this context, it is important to consider that the major anti-infective, anti-cancer, analgesics and immunosuppressive compounds are of natural origin.
By contrast, the historical relationship between humankind and the sea is usually appreciated as the basis for traveling, trading and as a nutritional source. We live in a planet of oceans. The marine ecosystem covers more than 70% of the earth’s surface but represents 95% of the biosphere. The first living organisms appeared in the sea more than 3500 million years ago and evolutionary development has equipped many marine organisms with the appropriate mechanisms to survive in a hostile milieu in terms of extreme temperatures, changes in salinity and pressure, as well as overcoming the effects of mutation, bacteria and viral pathogens. Marine organisms have developed exquisitely complex biological mechanisms showing cross phylum activity with terrestrial organisms. Another important differential factor deals with biodiversity. All but 2 of the 28 major animal phyla are represented in aquatic environments and some of them are exclusive of the marine ecosystem, such as Ctenophore, Echinidermata, Porifera, Phoronidea, Brachiopoda and Chaetognata. In terms of evolution and biodiversity, the sea appears to be superior to the terrestrial ecosystem-one has to consider that the most important biological explosion took place in the marine ecosystem during the Cambrian period 600 million years ago and marine species comprise approximately a half of the total biodiversity, thus offering a vast source from which to discover useful therapeutics. In this context, and our ongoing research programme, “drugs from the sea”, we have collected the red colonial ascidian Didemnum obscurum From Tiruchandur coast, Tamilanadu, India, during March 2003. 

Chapter-I: Chemical investgation of ascidians 
Didemnum obscurum : The ascidian Didemnum obscurum was preserved in methanol at the site of collection until work-up. The initial methanol extract was decanted and the ascidian material was extracted with freshly 1:1 DCM: MeOH (2x1L) and concentrated under reduced pressure to predominantly an aqueous suspension which was extracted in to ethyl acetate (3x250 ml), the organic layer was concentrated to give crude extract (1.5g.). The combined crude extract of the ascidian was subjected to Sephadex LH-20 gel filtration chromatography and grouped in to two fractions, fraction-I and fraction-II. The fraction-I was further subjected to silicagel column chromatography followed by reversed phase (C-18) HPLC, using MeOH : H2O (80:20) as eluent to afford eight lamellarin alkaloids,  as DO-6, DO-7 and DO-8. Fraction-II was acetylated (Ac2O/NaOAc) and purified sequentially on silicagel chromatograohy, followed by reversed phase (C-18) HPLC using MeOH : H2O (60:40) as eluent, to afford two new lamellarin alkaloids DO-1(lamellarin-φ) and DO-2 (lamellarin-χ)  and three known lamellarin alkaloids DO-3, DO-4, and DO-5 as their acetates. 









Didemnum granulatum (IIC-618): This is an ascidian Didemnum granulatum   collected from the Tiruchandur coast (Tamil Nadu) the Gulf of Mannar during October 2003. The organism was preserved in excess of methanol until workup. The resulting aq. methanol was removed from the ascidian and freshly extracted with 1:1 DCM: MeOH (1x3L) at room temperature. Concentration of the combined extracts including initial methanol extract concentrated under reduced pressure afforded crude aqueous extract. The crude aqueous extract was further extracted into ethyl acetate (3x250 ml). The combined ethyl acetate extract was concentrated to obtain brownish gummy extract (3 gm).  It was subjected to gel filtration (Saphadex LH-20, 1:1 DCM: MeOH) followed by silica gel column chromatography eluting with hexane through hexane-ethyl acetate mixtures to ethyl acetate. The individual compounds were purified on reversed phase(C-18) HPLC in water methanol (80:20) to afford compounds 1-2which were characterized by the study 1H NMR, NOESY and 13C NMR spectral data.




Chapter-II: Chemical investigation of sponges and soft corals
Sinularia kverittinesis : This is a soft coral collected from Mandapam coast during February2004.The organism was preserved in excess of methanol until workup. The resulting aq. methanol was removed from the coral and extracted freshly with 1:1 DCM : MeOH at room temperature. The combined extract including initial methanol was concentrated to give crude gummy extract (53gm.). It was subjected to gel filtration (Sephadex LH-20, 1:1 DCM : MeOH) followed by silica gel column chromatography eluting with hexane through hexane-ethyl acetate mixtures to ethyl acetate to afford six known compounds 1-6.



























Psammaplysilla purpurea : This is a sponge Psammaplysilla Purpurea  collected from the Mandapam coast (Tamilnadu) the Gulf of Mannar during February 2002.The organism was preserved in excess of methanol until workup. The resulting aq. methanol was removed from the sponge and extracted with 1:1 DCM : MeOH (2x3L) at room temperature. The combined extracts including initial methanol extract  concentrated under reduced pressure afforded crude aqueous extract. The crude aqueous extract was further extracted into ethyl acetate. The ethyl acetate extract was concentrated to obtain brownish gummy extract  (14 gm).  It was subjected to gel filtration (Sephadex LH-20, 1:1 DCM : MeOH) followed by silica gel column chromatography eluting with hexane through hexane-ethyl acetate mixtures to ethyl acetate to afford known compounds 1-4,  Which were characterized by the study 1H NMR and 13C NMR spectral data.











Chapter-III: Chemical modification of ingol diterpenes and Development of novel synthetic methodologies:

Several natural bioactive molecules have been converted into their analogues in recent chemical and biochemical means. Actually, the modifications of lead molecules to novel compounds for new biological leads. Some of the semisynthetic analogues have been found to have better activity than their natural parent molecules. 
Development of new synthetic methodologies is an important subject in organic chemistry for synthesis and conversion of bioactive natural compounds under mild reaction conditions with suitable reagents. Several classical synthetic methodologies involve expensive reagents and catalysts, which are not easily available. To replace such reagents and catalysts, different improved processes have been discovered with inexpensive materials. Research in this direction is also of immense importance especially while dealing with sensitive functionalities within the molecule. As all the reagents cannot satisfy all the criteria, the newer reagents have high scope for getting recognition in the list of the existing ones in satisfying specific needs. Modern methodologies are also concerned with the yield and selectivity of the products.

1) Lewis acid-catalyzed selective cyclopropane ring opening in ingol   diterpene Derivatives

The unique reactivity of cyclopropanes is due to its high level of ring strain, which offers considerable utility in organic synthesis. Many imaginative applications of cyclopropanes as useful building blocks have been predicted on regio and stereo controlled ring opening reactions of substituted cyclopropanes. Typically, electrophilic ring opening has been achieved by transition metals (Pd, Pt, Rh, and Ir), halide ions, and Lewis acids. 



The electron donor (particularly alkoxy, siloxy and arylthio) substituted cyclopropanes have found increasing use, primarily due to facile and regio controlled cyclopropane ring opening. During our studies on chemical modification of various secondary metabolites from plant sources and marine organisms, we isolated ingol diterpenes from a plant euphorbia nivulia some of these ingol diterpenes contain cyclopropane ring for example when ingol diterpenoid, compound (1) 3,12-diacetyl-8-methoxyl-7-angeloylingol (1) reacted with boron trifluoride diethyl etherate to yield rearranged compounds nivulianol-A (3) and nivulianol-B (4), and compound (2) 3, 12-diacetyl-8-methoxylingol (2) gave nivulianol-C (5) and nivulianol-D (6) simililarly when reacted with scandium triflate gave only nivulianol-B (4) and nivulianol-D (6) the reaction mechanism has been discussed. 

2) A mild and efficient chemoselective synthesis of acetals and geminal diacetates (acylals) from aldehydes using lanthanum (III) nitrate hexahydrate
Selective protection and deprotection of carbonyl compounds plays an important role in the multi step organic synthesis of complex natural products. Acetals are among the most popular protecting groups for carbonyl compounds. Acetals as a functional group which is stable under neutral and basic conditions are not only the most widely used protecting groups, but are also efficient chiral auxiliary groups for enantio selective synthesis. Numerous procedures have described the formation of acetals under acidic
conditions. However, several reported methods are associated with drawbacks involve low chemo selectivity, incompatible with other protecting groups, strong acidic conditions, long reaction times, tedious procedure for the preparation of catalyst and most of the methods demanding dry reaction media. All the Lewis acids requires dry media and large amounts of reagent if the substrate contains nitrogen, as the Lewis acids are trapped by the nitrogen. Thus there is a need for suitable, mild and selective alternative for the synthesis of acetals.




We have developed a mild reagent lanthanum nitrate hexahydrate for protection of aldehydes with ethylene glycol, the results obtained for the protection of aldehydes as their corresponding acetals, we turned our attention towards another important protection of aldehydes as their geminal acetates (acylals)(Scheme-II). Acylals are also one of the useful protecting groups for carbonyls compounds due to their stability under neutral and basic media as well as aqueous acids.





3) Lanthanum(III) nitrate hexahydrate catalyzed chemo selective Thioacetalization   of  aldehydes under solvent free conditions

1,3 dithiane plays an important role in the multi step organic synthesis of complex natural products due to group inherent stability under both acidic and basic conditions.






 

we describe an efficient chemoselective method for thioacetalisation of aldehydes (Scheme I) using catalytic amounts of commercially available, inexpensive lanthanum (III) nitrate hexahydrate. This method does not require expensive reagents or special care to exclude moisture from the reaction medium. Initially, we reacted benzaldehyde with 1,3dithiane and ethanethiol in presence of catalytic amounts of lanthanum(III) nitrate hexahydrate to yield corresponding thioacetal in 90% yield.



























ROLE OF RNAi IN HETEROCHROMATIN FORMATION AND CHROMATIN REMODELLING

Mallik, Moushami

Abstract/Synopsis:
All eukaryotic genomes contain, besides the coding information for amino acids in different proteins, a significant amount of noncoding sequences, which may or may not be transcribed. In general, the more evolved or biologically complex the organisms are, greater is the proportion of the noncoding component in their genomes. A resurgence of studies on the noncoding sequences in various genomes during the past several years makes it clear that the complex biological organization demands much more than a rich proteome. Although the more popularly known ncRNAs are the small microRNAs and other similar species, other types of larger ncRNAs with critical functions in regulating gene activity at various levels are being increasingly identified and characterized. A comprehensive understanding of the significance of noncoding DNA/RNA sequences in eukaryotic genomes is essential for understanding the origin and sustenance of complex biological organization of multicellular organisms.
For the past few decades our laboratory has been working on the noncoding hsrω gene in Drosophila. This developmentally active, stress-inducible gene situated at the 93D4 band on the right arm of chromosome 3, produces multiple transcripts with no protein coding potentiality (Mohler and Pardue 1982, Lakhotia and Mukherjee 1982, Garbe and Pardue 1986, Hovemann et al. 1986, Fini et al. 1989). It plays a crucial role in survival and normal development in the fly, since nullisomy for this gene leads to embryonic lethality. Among the major heat shock loci in Drosophila, 93D or hsrω forms one of the transcriptionally most active puffs after a temperature shock (Mukherjee and Lakhotia 1979) and is unique in its inducibility by a number of amides (Lakhotia and Mukherjee 1970, 1980, 1984, Tapadia and Lakhotia 1997). Of the several ncRNAs produced by this locus, the nucleus limited >10 Kb long  hsrω -n transcripts form dynamic structures called omega speckles which harness a variety of heterogeneous nuclear RNA binding (hnRNPs) and other RNA processesing proteins and regulate their availability during normal as well as stress conditions (Lakhotia et al. 1999, Prasanth et al. 2000). The omega speckles are thus believed to function as storage sites for unengaged hnRNPs and depending upon the cellular needs, hnRNPs are dynamically released from and sequestered in the omega speckles (Lakhotia et al. 1999, Prasanth et al. 2000, Lakhotia 2003). Evolution of such RNA-based communication networks which perform hsrω -like functions and act as platforms for the association of specific proteomes with each of the varied nuclear sub-structures appear to be a fundamental prerequisite of all cells and organisms subjected to stress. Based on structural and functional similarities, it has been proposed that the large noncoding Sat III transcripts in human cells are functional analogues of the hsrω transcripts (Jolly and Lakhotia 2006).
To better understand physiological roles of the hsrω gene, both gain-of-function and loss-of-function genetic studies were employed to over-express and ablate this gene’s transcripts by using tissue-specific GAL4 drivers. In accordance with previous reports using hsrω deficient lines, the present study (Chapter 3) also showed that the hsrω -n transcripts are critical for survival since ubiquitous over-expression or RNAi-mediated ablation of hsrω transcript levels resulted in organismal lethality. Levels of these transcripts apparently regulate sex-specific responses in the fly with global reduction in levels of hsrω transcripts resulting in death of more female than male progeny and over-expression causing greater mortality of males. Absence of the large nuclear hsrω -n RNA leads to loss of omega speckles resulting in a diffuse nuclear distribution of the hnRNPs associated with these transcripts. On the other hand, EP-mediated augmentation of endogenous hsrω  transcript levels enhanced sequestration of the hnRNPs in a manner reminiscent of hnRNP distribution during stress conditions. Furthermore, RNAi-mediated depletion of  hsrω transcripts following a 40 min heat treatment at 370C was found to impede trafficking of hnRNPs from the 93D locus to transcriptionally active sites on the chromatin. Over-expression of these transcripts results in functional depletion of cellular pools of hnRNPs and other RNA processesing proteins known to associate with the hsrω RNA. Conversely, in the absence of this RNA and thus omega speckles, these proteins cannot be efficiently directed to their proper cellular targets (transcriptionally active chromatin sites). Either of these conditions result in dysregulation of downstream events. I have further discovered that the 93D locus produces a fourth noncoding transcript which is essentially identical to the nuclear hsrω-n RNA, except that it does not contain the 700 bp intron. Immunoprecipitation experiments revealed that this novel transcript is also a component of the omega speckles. However, functional characterization of this splice variant of hsrω n needs further studies.  
During the course of my work I have established a new mechanism of how levels of the Drosophila Inhibitor of Apoptosis Protein 1 (DIAP1), and thus apoptosis, are regulated. Data presented in Chapter 4 demonstrate that depletion of this ncRNA dominantly suppressed apoptosis-induced by ectopic expression of the key regulators/effectors of caspase-mediated cell death in Drosophila by augmenting the levels of DIAP1. Further, suppression of induced cell death following depletion of hsrω transcripts was abrogated when the DIAP1-RNAi transgene was co-expressed. These results indicate that the hsrω transcripts regulate cellular levels of DIAP1 via the hnRNP Hrb57A which physically interacts with DIAP1 and any alteration in levels of the hsrω transcripts in eye disc cells enhances association between these two proteins. In addition, I also show, for the first time, a genetic interaction between the hsrω transcripts and the JNK signaling pathway since down regulation of hsrω transcripts suppressed JNK activation. Data presented here revealed a novel regulatory role of the hsrω ncRNA at multiple levels in the apoptotic cell death pathways. 
In earlier studies, over-expression of the hsrω transcripts was shown to dominantly enhance polyQ-induced cytotoxicity in Drosophila (Sengupta and Lakhotia 2006). In the present study (Chapter 5), I show that down regulation of hsrω-n transcripts ameliorates pathogenesis in several Drosophila models of human polyQ neurodegenerative diseases. Loss of hsrω RNA not only suppressed the eye-specific degeneration mediated by GMR-GAL4 driven expression of the 127Q or MJDtr-Q78 or ataxin1 82Q or httex1p Q93 transgene, but also rescued premature death of flies expressing the expanded polyQ proteins pan-neuronally. I further demonstrate that the morphological and functional rescue of polyQ toxicity observed upon depletion of hsrω-n transcripts is associated with substantial reduction of polyQ protein aggregation without affecting transcription of the 127Q transgene. Reduction in hsrω transcript levels also abolished the induction of Hsp70 in the polyQ expressing cells. These results suggest that the hsrω transcripts have a role in early stages of polyQ aggregate formation. I have also investigated possible pathway/s through which the hsrω transcripts modulate polyQ-induced neurodegeneration in Drosophila. I show that down regulation of  hsrω -n transcripts in a polyQ background suppressed the enhancing effects brought about by reduced availability of the hnRNP Hrb87F and of the transcriptional regulator, CREB Binding Protein (CBP). This study revealed, for the first time, that CBP and hnRNPs like Hrb87F and Hrb57A physically interact with each other and depletion of hsrω -n RNA enhances cellular levels of CBP possibly through its association with hnRNPs. Over-expression of hsrω and/or polyQ proteins disrupted the proteasomal activity. On the other hand, while hsrω-RNAi partially rescued eye damage following compromised proteasome activity, its ability to rescue polyQ toxicity was affected by sub-normal proteasomal activity. Thus, depletion of  hsrω transcripts seems to rescue polyQ toxicity by simultaneously targeting several pathways which in turn suppress formation of polyQ aggregates and transcriptional dysregulation in polyQ expressing cells to prevent neurodegeneration and preserve neuronal function. 
Identification and elucidation of diverse novel roles played by ncRNAs such as the hsrω transcripts thus provide a new paradigm for understanding the complex regulation of nuclear RNA processing activities. The present study clearly demonstrates that such large ncRNA species contain additional information or functions which are as important as the protein coding genes. It is intriguing that these transcripts influence intricate networks of gene expression that result in precise organization of living organisms, not only in the nucleus but also in the cytoplasm. These transcripts apparently function as endogenous communication systems or hubs, which are central to the genetic control architecture not only within but also between cells, for coordination of many genes at different levels and thus ensure homeostasis. Such multiple networking interactions provide a basis for the context-dependent actions of the same molecule in different cells or in same cell under different conditions



Molecular Genetic Analysis of Roles of the Non-coding hsrω Gene in Development, Induced Apoptosis and PolyQ toxicity in Drosophila melanogaster

Thakur, Abhay Kumar

PAGE  




i
Synopsis		


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

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

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

Chapter 2

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

Chapter 3

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

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

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

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

Chapter 4

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

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

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


Chapter 5

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

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



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








PRION PROTEIN: PHOTO-OXIDATION, AMYLOIDOGENESIS AND INTERACTION WITH SMALL HEAT SHOCK PROTEINS

Kumar, Suprit

 1 
SYNOPSIS 
 
STUDIES ON THE PREPARATION AND CHARACTERISATION OF 
PROTEIN HYDROLYSATES FROM GROUNDNUT AND SOYBEAN 
ISOLATES 
 
Proteins are important in food processing and food product development, as they are 
responsible for various functional properties that influence consumer acceptability. Both 
animal and plant proteins are used commercially as functional ingredients. Plant proteins are 
the most abundant in the world. A number of vegetable proteins have been tried for 
incorporation in various food products as functional and nutritional ingredients.  
Oilseed protein products are rapidly gaining importance in protein supplementation 
because of their unique functional properties. The intrinsic properties of proteins like the amino 
acid composition and conformation of the proteins, methods and conditions for their isolation, 
degree of purification and processing alterations are some of the important factors that 
influence the functional properties of food proteins. Various approaches like chemical 
modification, physical treatments and enzymatic modification have been tried to improve 
functional characteristics of proteins. Chemical modifications such as succinylation, 
acetylation and mild alkali hydrolysis have been reported to improve the functional properties.   
One of the major drawbacks of these approaches is the deterioration of the nutritional quality 
owing to the blocking or destruction of essential amino acids. One of the important ways to 
enhance the functional properties of oilseed proteins is enzymatic modification. Enzymatic 
modification occur under mild conditions retaining nutritional value and offer a convenient 
means for improving functional properties of proteins. By controlling the extent of hydrolysis 
it is possible to enhance various functional properties to develop new functional ingredients to 
fabricate new food analogs simulating traditional foods.    
 2 
Protein hydrolysates find application in special foods such as those designed for 
children, old people, athletes and also in pharmaceutical preparations developed for 
convalescents and those who suffer from digestive disorders. Foods based on highly 
hydrolysed proteins are useful in controlling food allergies. The major ways of supplying 
tailored amount of amino acids are i) enzymatic protein hydrolysates and ii) a mixture of 
synthetic amino acids. They are preferred over synthetic amino acids at moderate cost because 
of availability on commercial scale and high quality of enzyme hydrolysed products. 
Enzymatic protein hydrolysates containing short chain peptides with defined amino acid 
composition and molecular size are preferred for specific formulations. These protein 
hydrolysates score over elemental diets in which the protein component consists exclusively of 
a mixture of free amino acids. The short chain peptides are absorbed preferentially over free 
amino acids in the gut. Protein hydrolysates offer as an alternative to intact proteins and 
elemental formula in the development of special formulations designed to provide nutritional 
support .    
Soybean and groundnut are the most widely cultivated oilseeds all over the world. 
Dehulled soybean contains 17-18% oil and 25-35% protein depending on the variety. 
Similarly, dehulled groundnut depending on the variety contains 50-60% oil and 30-35% 
protein. Defatted soybean flour contains 50-55% protein of good nutritional quality. Soybean 
proteins are rich in lysine and deficient in methionine. The major intrinsic anti nutritional 
factor is trypsin inhibitor, which affects its utility. Groundnut proteins are deficient in lysine 
and methionine. The protein digestibility corrected amino acid score for soy protein and 
groundnut protein is 0.92 and 0.52 respectively. The protein ingredients such as defatted flour, 
protein concentrate and protein isolates from these sources have a good potential for preparing 
speciality foods under native and modified conditions. The protein hydrolysates obtained from 
these sources can effectively replace commercially available milk protein hydrolysates because  
 3 
of their good nutritional quality. 
In general, oilseed proteins offer resistance to enzymatic hydrolysis. Studies on 
hydrolysis of pure proteins would help in understanding of the various structural features of the 
protein both at surface and subunit levels. Though the resistance to hydrolysis of seed proteins 
by enzymes is well documented, work on the relative effectiveness of the major proteins with 
different proteolytic enzymes is less. Glycinin and conglycinin are the major fractions of 
soybeans and make up to 70% of the total proteins. Arachin and conarachin are the major 
fractions, which make nearly 80% of the total groundnut proteins. The individual roles of 
major protein fractions are important in order to understand the overall functional profile of the 
total proteins and protein ingredients. Glycinin is poorer in functional properties compared to 
conglycinin. This has been attributed to the compact structure of the high molecular protein 
fraction in which hydrophobic groups are buried inside. The functional properties of protein 
isolates and isolated fractions can be modified either by chemical means or enzymatic 
hydrolysis.  Although sufficient information is available on the enzymatic modification of 
soybean or groundnut flour and its effect on functional properties, the information on the 
controlled hydrolysis of major globulins of oilseeds are limited. 
In this work, protein hydrolysates from soybean and groundnut were prepared using 
different proteolytic enzymes starting with different materials like defatted meal, protein 
isolates and purified fractions. With the use of different enzymes and by varying the 
experimental parameters like E/S ratio, pH, temperature and combinations of proteolytic 
enzymes, it was possible to tailor the functional characteristics of various protein hydrolysates. 
Attempt has also been made to correlate various functional characteristics with some of the 
biophysical and biochemical parameters. 
 
 4 
CHAPTER 1:  INTRODUCTION 
 
The introduction reviews the information regarding the physico-chemical 
characteristics, nutritional, and functional properties of soybean and groundnut proteins. This 
also includes the review of literature on enzymatic hydrolysis of food proteins, methods of 
isolation and physico-chemical characteristics of the purified high molecular weight protein 
fractions of soybean and groundnut (glycinin and arachin). 
CHAPTER 2:  MATERIALS AND METHODS 
 This presents details regarding the materials and experimental methods used in the 
investigation. 
CHAPTER 3:  RESULTS AND DISCUSSION 
This has the results followed by the relevant discussion, which are given in the 
following Sections. 
PART – A: STUDIES ON SOYBEAN PROTEINS 
SECTION 1: ENZYMATIC HYDROLYSIS OF SOYBEAN FLOUR 
The maximum degree of hydrolysis (DH) obtained with papain, alcalase and fungal 
protease enzymes were 18.6%, 29.6% and 32.4%, respectively. A comparison of the DH values 
obtained with different proteases showed that fungal protease was more effective among the 
proteases used for the hydrolysis of defatted soy flour (DSF). The overall effectiveness of 
different proteases in getting higher DH was in the order fungal protease > alcalase > papain. 
Enzymatic modification of DSF to low DH (4-6%) resulted in remarkable increase in 
emulsification capacity (EC) and marginal increase in foaming capacity (FC). The extent of 
improvement in EC followed by limited proteolysis was almost same for different proteolytic 
enzyme modified flours. The fat absorption capacity (FAC) and water absorption capacity 
(WAC) of enzyme hydrolysed DSF was higher than that of intact DSF. Extensive hydrolysis 
impaired the overall functionality of DSF. The trypsin inhibitor activities of low DH and high 
 5 
DH enzyme modified freeze dried DSF were similar to control, suggesting that trypsin 
inhibitors were resistant to enzymatic attack. The nitrogen content of spray-dried protein 
hydrolysate obtained with papain, alcalase and fungal protease was almost same (9-9.2 %). The 
nitrogen content of DSF increased to 11.5% after acid wash. The protein hydrolysate prepared 
by hydrolysis of wet protein isolate obtained by alkali extraction followed by iso-electric 
precipitation had higher nitrogen content (14.5%).Amino acid composition of protein 
hydrolysates showed that the nutritional quality of protein was retained after enzymatic 
hydrolysis. The bitterness of protein hydrolysates was in the order papain< alcalase=fungal 
protease. The spray dried protein hydrolysate of DSF obtained by different proteolytic enzymes 
showed inactivation of lipoxygenase and urease activity. The trypsin inhibitor activity of DSF 
hydrolysate was in the range 20-22 TIU/mg sample. The lower trypsin inhibitor activity of 
spray dried hydrolysates compared to DSF may be due to application of heat in spray drying 
process. The protein hydrolysate was soluble over a wide range of pH (2.0-11.0); at iso-electric 
pH, the solubility was >98%. 
SECTION 2: ENZYMATIC MODIFICATION OF  SOY PROTEIN ISOLATE  
The DH of soy protein isolate obtained with papain, alcalase and fungal protease was 
7.5%, 9.5% and 18.9% respectively. The effectiveness of proteolytic enzymes for hydrolysis of 
soy protein isolate (SPI) was lower compared to DSF. The overall effectiveness of proteases 
towards hydrolysis of SPI was in the order fungal protease>alcalase>papain. Comparison of 
the hydrolysis curves of freeze dried and spray dried SPI showed their susceptibility towards 
proteolytic enzymes was almost same. The solubility of SPI followed the typical U-shape 
pattern. The minimum solubility was found at pH 4.5 (iso-electric pH). A low DH of 3-5% 
obtained with papain, alcalase and fungal protease increased the solubility of modified SPI up 
to 29-35% at pH 4.5 and 97-98% at pH 7.0. Extensive hydrolysis of SPI increased the 
solubility at pH 4.5 up to 49-54% with different proteolytic enzymes. Limited hydrolysis of 
 6 
SPI with different proteolytic enzymes increased the EC. Among the proteases papain modified 
SPI showed more EC compared to alcalase and fungal protease modified SPI. The FC of low 
DH modified SPI was higher compared to unmodified SPI. Fungal protease modified SPI 
showed higher FC compared to papain and alcalase modified SPI. Extensive hydrolysis of SPI 
with proteolytic enzymes brought drastic reduction in EC. Although the FC of SPI extensively 
hydrolysed using papain, alcalase and fungal protease was higher than intact SPI, the FC 
values were lower than the corresponding low DH modified SPI. The maximum DH obtained 
with SPI with proteolytic enzymes was lower than that of DSF. SPI with limited proteolysis 
showed remarkable increase in FC but the increase was marginal with DSF.  
SECTION 3: CONTROLLED ENZYMATIC HYDROLYSIS OF GLYCININ 
Glycinin, the major protein fraction of soybean when hydrolysed with different 
proteolytic enzymes showed that papain had the least effect followed by alcalase and fungal 
protease. The enzymatic hydrolysis followed typical Michaelis-Menten pattern. The affinity of 
glycinin to proteolytic enzymes was in the order fungal protease > alcalase  > papain as shown 
by the Km values. The SDS-gel electrophoretic pattern of glycinin showed bands corresponding 
to acidic (30-33kD) and basic subunits (29-22kD). The pattern observed for enzymatically-
modified glycinin suggested the preferential cleavage of acidic subunits compared to basic 
subunits. The hydrolysis of isolated acidic and basic subunits of glycinin with fungal protease 
showed that basic subunits were less susceptible. A maximum DH of 26% was obtained with 
acidic subunits at the end of 4h hydrolysis compared to 9-10% DH with basic subunits. The Km 
values for acidic and basic subunits for fungal protease correlated well with cleavage 
susceptibility. Glycinin possess poor functional properties; enzymatic modification with 
proteolytic enzymes improved a few of the functional characteristics. Papain with limited 
proteolysis increased the FC almost three fold with good foam stability. There was no 
difference in the EC of low DH modified glycinin samples. The FAC of papain, alcalase, and 
 7 
fungal protease modified glycinin decreased compared to unmodified glycinin. Limited 
proteolysis of glycinin did not bring significant differences in the WAC. The molecular sieve 
chromatography on Sepharose-6B gel showed single peak for glycinin. In the case of modified 
glycinin it could be resolved in to two peaks. The first peak had same Ve/Vo as that of glycinin 
but the second peak had higher Ve/Vo. This indicated that even low DH degraded glycinin into 
low molecular weight peptides. The electrophoresis pattern of gel filtration chromatographic 
peaks suggested that peak1 and peak 2 did not correspond to native glycinin; acidic subunits 
were readily hydrolysed compared to basic subunits. Peak 2 did not give bands on the 10% gel 
suggesting that peak 2 was extensively hydrolysed and the molecular weight of the resulting 
peptides were low. A low DH of 4-5% resulted in drastic reduction in mean residue ellipticity 
of modified glycinin for all the enzymes tested. However, both glycinin and modified glycinin 
exhibited characteristic near UV CD peaks at 263, 275, 283 and 291nm. This suggests that 
even a low DH disrupts the tertiary structure of glycinin. The EC and FC of glycinin were 
lesser than that of SPI. The poor functionality of glycinin may be due to the closely packed 
conformation of glycinin in which the hydrophobic groups are buried inside. The enhancement 
in functionality by limited proteolysis could be due to the exposure of hydrophobic groups.  
PART- B: STUDIES ON GROUNDNUT PROTEINS 
SECTION 4: ENZYMATIC MODIFICATION OF GROUNDNUT PROTEIN ISOLATE 
  
Groundnut protein isolate (GPI) was more susceptible to hydrolysis with papain, 
alcalase and fungal protease compared to SPI. The maximum DH of GPI obtained with papain, 
alcalase and fungal protease was 18.6%, 17.4% and 26.6% respectively. The comparatively 
high affinity of GPI for proteolytic enzymes may be because groundnut proteins are less 
hydrophobic compared to soybean proteins. The effectiveness of different enzymes was in the 
order fungal protease > papain > alcalase. The solubility curve of GPI followed U-shaped 
pattern with the minimum at pH 4.5. The low DH modified GPI with different proteolytic 
 8 
enzymes at isoelectric pH showed solubility of 27-28%.  However, high DH increased the 
solubility up to 47-55% at pH 4.5.The EC and FC of GPI increased after limited proteolysis. 
The effectiveness of different proteolytic enzymes in enhancing the EC was almost same. 
However, alcalase and fungal protease enzymes were more effective compared to papain in 
enhancing the FC by limited proteolysis. Extensive hydrolysis of GPI resulted in a remarkable 
reduction of EC. Although the FC of high DH modified GPI with different enzymes were 
higher than unmodified GPI, the values were lower than that of low DH modified GPI.  
SECTION 5: ENZYMATIC HYDROLYSIS OF ARACHIN AND ITS EFFECT ON                         
           FUNCTIONAL PROPERTIES  
Arachin, the major protein fraction of groundnut purified by ammonium sulfate 
precipitation eluted as a single peak with Ve/Vo =1.5. Arachin with a low DH of 3-5% showed 
two peaks, the first peak at Ve/Vo similar to arachin and second peak with a Ve/Vo =1.8 This 
suggested that even at low DH arachin degraded into low molecular weight peptides. The Km 
values for arachin with different proteases were 0.83-0.931%. The Km values were lower when 
compared to the values obtained with glycinin as substrate. Arachin was the preferred substrate 
over glycinin for the proteolytic enzymes used. The maximum DH of arachin obtained with 
papain, alcalase and fungal protease was 23.6%, 18.6% and 25.5% respectively. The 
effectiveness of these enzymes was in the order fungal protease > papain > alcalase. These 
results obtained with arachin were comparable to that of GPI. This suggests that the proteolytic 
enzymes have got equal effectiveness for GPI and arachin. The solubility of modified arachin 
to low DH (3-5%) was 14-16%. Extensive hydrolysis increased the solubility up to 55-60%. 
Hydrolysis increased the solubility of arachin considerably. Limited proteolysis increased the 
EC of arachin. Among the different enzymes papain was more effective in enhancing the EC. 
The FC of alcalase-modified arachin was remarkably high (two fold). Papain and alcalase 
modified arachin showed marginal increase in FC. Excess hydrolysis impaired the 
functionality of arachin except solubility, The SDS-PAGE pattern of arachin was similar to 
 9 
those already reported in the literature. Papain, alcalase and fungal protease degraded high 
molecular weight subunits. The pattern for alcalase and fungal protease was similar. The action 
of papain on arachin subunits was different. The low molecular weight subunit disappeared 
only after hydrolysis for 1h with different enzymes. The EC of arachin was higher than that of 
glycinin. There was considerable difference in the FC between the two fractions. The overall 
effect of proteases in enhancing the functionality differed considerably. Comparison of the DH 
obtained with flour, isolate and purified fractions of groundnut and soybean showed that the 
different proteases acted differently in getting high DH and in changing functional 
characteristics. DSF gave high DH with proteolytic enzymes compared to SPI. The effect of 
proteases on GPI and arachin was similar. In general, the study has indicated that groundnut 
and soybean proteins are resistant to hydrolysis by proteolytic enzymes. The bitterness of 
hydrolysate was more with soy proteins than groundnut proteins. The affinity of proteases 
towards groundnut proteins was more than that of soybean proteins. Thus by using appropriate 
proteolytic enzymes under specified conditions the functional characteristics of seed proteins 
can be tailored to meet our requirements. 
CHAPTER 4: SUMMARY AND CONCLUSIONS 
 
This has the general summary of the investigation focusing on the important findings of 
the investigation. The references are arranged in alphabetical order. 
 
 
H. SRINIVAS         GOVINDARAJU.K 
(Guide)        (Candidate) 
Retd. Scientist 
Department of Protein Chemistry and Technology 
Central Food Technological Research Institute 
Mysore- 570013, INDIA. 


Hydrological Modelling of the West Coast of India

G, VENU GOPAL

DEVELOPMENT OF IN VITRO CULTURE SYSTEM FOR MARINE SPONGES (DEMOSPONGIAE):NEW PERSPECTIVE FOR PRODUCING BIOACTIVE SUBSTANCE OR BIOMOLECULES

Rao, Venugopal

Abstract


ABSTRACT
Synopsis of the thesis entitled “Chemoenzymatic synthesis of biologically active entities, synthesis of both the stereoisomers of dihydrokawain-5-ol and development of novel synthetic methodologies” has been divided into four chapters.
Chapter-I: Biotransformations: Introduction and Background Information 
Chapter-II 
Section-A: A new chemoenzymatic Baylis-Hillman approach for the synthesis      	    of enantiomerically enriched umbelactones.
Section-B: Chemoenzymatic synthesis of (R)-GABOB and (R)-carnitine.
Chapter-III 
Section A: Chemoenzymatic synthesis of (-)-centrolobine.
Section B: Chemoenzymatic synthesis of (R) and (S) enantiomers of timolol.
Chapter-IV
Section-A: Chemoenzymatic synthesis of enantiomerically enriched           kawalactones.
Section-B: Enantioselective total synthesis of both the stereoisomers of
 dihydrokawain-5-ol.
Section-C: Cu(BF4)2·xH2O catalyzed one-pot synthesis of Biginelli and Strecker reactions.








Chapter-I: Biotransformations: Introduction and Background Information 
This chapter describes a concise and general account of enzymes. It also brings about a brief account of importance of chirality, classification of lipases and their applications in organic synthesis for various stereoselective transformations.
Chapter-II 
This chapter has been devided into two sections. Section-A describes a new chemoenzymatic Baylis-Hillman approach for the synthesis of enantiomerically enriched umbelactones and section-B describes chemoenzymatic synthesis of (R)-GABOB and (R)-carnitine.
Section-A: A new chemoenzymatic Baylis-Hillman approach for the synthesis of enantiomerically enriched umbelactones

(R)-Umbelactone-1 is one example of a naturally occurring -(hydroxymethyl)--butenolide which was isolated from Memycelon umbelatum Brum. The crude extracts of this plant have shown antiviral (activity against Ranikhet disease virus), antiamphetory and spasmolytic activity as a result, it has been considered that the synthesis of umbelactone is of particular interest.
The synthesis was commenced from compound 3 as a precursor, which was prepared from aldehyde 2 upon treatment with with methyl acrylate and quinuclidine applying Baylis-Hillman strategy in quantitative yields. The hydroxyl of 3 was protected as TBS ether using TBSCl and imidazole to give 4 in high yield. Unsaturated ester 4 was reduced to the alcohol 5 by DIBAL-H in CH2Cl2 in 95% yield, which subsequent tosylation with using tosyl chloride and triethly amine in CH2Cl2 (in the presence of catalytic amount of DMAP) as tosyl ether 6 in 75% yield. Further, 6 was reduced to methyl group with LiAlH4 in THF to give 7, this upon deprotection of TBS group using TBAF in THF gave the required intermediate 8.
Having successfully completed the synthesis of 8, our focus was aimed at the development of lipase-catalyzed transesterification of racemic hydroxy group to obtain enantiomerically pure (R)-9 and (S)-8, which would be useful for the synthesis of enantimerically enriched umbelactones (R)-1 and (S)-1 (Scheme 1).
Scheme 1
 Reagents and conditions: i. Methyl acrylate, quinuclidine, MeOH, 90%, 8 h; ii. TBDMSCl, imidazole, CH2Cl2, DMAP 0 °C to rt; 96%; iii. DIBAL-H, CH2Cl2, -78 °C, 95%; iv. TsCl, Et3N, CH2Cl2, DMAP, rt, 4 h, 75%; v. LiAlH4, THF, 88%; vi. TBAF, THF, 0 °C to rt, 85%; vii. Lipase, vinyl acetate, hexane; viii. K2CO3, MeOH, 90%; ix. Acryloyl chloride, Et3N, CH2Cl2, DMAP, 0 °C to rt, 30 min, 90%; x. II, CH2Cl2, 35 °C; xi. TiCl4, CH2Cl2, 0 °C to rt, 10 min, 90%.
Section-B: Chemoenzymatic synthesis of (R)-GABOB and (R)-carnitine
Amino-hydroxybutyric acid (GABOB) is a compound of great pharmacological importance because of its biological function as a neuromodulator in the mammalian central nervous system. The (R)-form of GABOB is shown to have greater biological activity than its S-enantiomer. Moreover, the (R)-isomer of GABOB serves as a precursor for (R)-carnitine, a compound having high level of medical significance. Carnitine is a vitamin like substance and plays an important role in converting stored body fat into energy. Its primary physiological function is to transport long chain fatty acids through the mitochondrial membrane into the cellular compartments for oxidation where these fats can be converted into energy. Moreover, there are several medical indications for which carnitine has been prescribed these include its usefulness as a pharmaceutical for hemodialysis (hypolipidemic agent), heart diseases etc. 
An efficient method for the preparation of (±)-N-(3-cyano-2-hydroxy propan-1-yl)phthalimide 17 and its successful enzymatic resolution have been described. This lipase-mediated transesterification process has been optimized with respect to different lipases and solvents. Furthermore, enantiomerically pure N-(3-cyano-2-hydroxy propan-1-yl)phthalimide-(R)-17b has been employed in the preparation of (R)- GABOB-(R)-12 and (R)-carnitine (R)-13.




Chapter III 
This chapter has been divided into two sections. Section A describes the chemoenzymatic synthesis of (-)-centrolobine 19 and Section B describes the Lipase-catalysed resolution of 1-chloro-3-[(4-morpholin-4-yl-1,2,5-thiadiazole-3-yl)oxy) propan-2-ol. Synthesis of (R) and (S)-timolol.
Section A: Chemoenzymatic synthesis of (-)-centrolobine


(-)-Centrolobine-19

Reagents and conditions: i. BnBr, K2CO3, acetone, reflux, 10 h, 90%; ii. (COCl)2, DMSO, -78 °C, 2 h, then NEt3,  -78 °C to –10 °C, 88%; iii. Allyl bromide, Zn, THF, 3 h, 90%; iv. Lipase, vinyl acetate, diisopropyl ether; v. (a) 4-Nitro-benzoic acid, PPh3, DEAD, 95%; (b) K2CO3, MeOH, rt, 1 h 90%; (c) Acryloyl chloride, NEt3, CH2Cl2, 1 h, 90%; vi. Grubbs’ 2nd generation catalyst, CH2Cl2, reflux, 30 h, 80%; vii. Pd/C, H2, EtOAc, 4 h, 90%; viii. (a) p-MeO-C6H4-MgBr, THF, -78 °C, 1 h, 76%; (b) BF3.Et2O, Et3SiH, CH2Cl2, -78°C then rt, 1 h, 65 %. 
(-)-Centrolobine 19 was isolated from the heartwood of Centrolobium robustum and from the stem of Brosinum potabile. Recently, (-)-centrolobine 19 and related natural products have been shown to be active against Leishmania amazonensis promastigotes; a parasite associated with leishmaniasis, a major health problem in Brazil. Leishmaniasis is a parasitic disease transmitted by the sand fly, and is related to Indian dum dum fever. Once in its human host, the parasite attacks the spungiform organs of the body, especially the liver and spleen, where the initial symptoms include a high fever, meaning that the disease is often mistaken for malaria. If the parasite attacks the skin, similar symptoms to leprosy arise, again, often leading to the wrong treatment being given to the patient. For the past 80 years, the only available drug for this distressing disease has been the pentavalent antimonials, which have been recently, linked to cardiac and renal toxicity. It is for this reason that Leon and co-workers, conducted a screen of traditional remedies from the Amazon rainforest to find new antileishmanial compounds. Interestingly, (-)-centrolobine 19 had already been shown to be one of the active ingredients in a herbal tea made from the wood of Centrolobum robustum that is used by the native peoples of the Amazon as a tonic cure for a variety of ailments.
(-)-Centrolobine 19 was synthesized from 20 as a starting material. Aromatic alcohol of its 20 was protected as benzyl ether 21 by benzyl bromide (K2CO3, acetone, reflux, 10 h) in quantitative yield, and this was oxidized to aldehyde 22 under Swern oxidation conditions. This aldehyde 22 on allylation using allyl bromide and zinc in THF afforded the homoallylic alcohol 23 (Barbier allylation conditions).
This homoallylic alcohol 23 upon lipase resolution gave the (R)-acetate (R)-25a and (S)-alcohol (S)-23a, and these compounds were separated by column chromatography. Enantiomeric excess has been calculated by HPLC on AS-H chiral daicel column. The effect of different solvents was also studied.
After separating (R)-25a and (S)-23a by column chromatography the alcohol (S)-23a was subject to the Mitsunobu reaction4 followed by hydrolysis gave alcohol (R)-23b and the acetate (R)-25a was hydrolysed using potassium carbonate in methanol gave the alcohol (R)-23b. This upon esterificaton using acryloyl chloride and after the ring closing metathesis using Grubbs’ 2nd generation catalyst gave unsaturated lactone (R)-27. This, upon treatment with hydrogen in the presence of Pd/C in ethyl acetate gave (R)-28 via debenzylation and hydrogenation of the double bond in one pot. This lactone (R)-28 upon treatment with p-MeO-C6H4-MgBr gave lactol, which was further treated with BF3.Et2O and Et3SiH to afford the required product 19 (Scheme 3). Its 1H, 13C NMR spectra as well as its optical rotation closely match with the previously reported data for the natural product 19.
Section B: Chemoenzymatic synthesis of (R) and (S) enantiomers of timolol


Timolol is a non-selective -blocking drug used for the topical treatment of open-angle glaucoma, an eye disease characterized by increased intraocular pressure, which results in defects in the field of vision. Like that of most of the family of aryloxypropanolamines, the -blocker activity of 29 is almost entirely due to the S enantiomer, eudismic ratio ER = 50 to 90. Nevertheless, the weakly active isomer (R)-29 maintaines a beneficial effect of bronchial constriction caused by non-selective action of (S)-29 on 1-2 receptors, and has therefore been proposed as an alternative for the treatment of glaucoma.
Optically pure timolol was prepared from 31 as a material, which was prepared from compound 30 in two steps. Compound 31 was then treated with dichloroacetone in dry DMF gave 32, which was reduced to 33 using NaBH4 in methanol almost quantitative yields. The reacemic halo hydrin 33 was then subjected to lipase-catalysed resolution. The resolution was carried out using in different lipases.
After enzymatic resolution, alcohol and acetate were isolated by column chromatography. Compounds (R)-33 and (S)-33 were converted into epoxide, which was then treated with tbutyl amine gave (S)-timolol-29 (R)-timolol-29. 


Chapter IV
 This chapter has been divided into three sections. Section-A describes the chemoenzymatic synthesis of enantiomerically enriched kavalactones, section-B describes the enantioselective total synthesis of both the stereoisomers of dihydrokawain-5-ol and section-C describes the Cu(BF4)2.xH2O catalyzed one-pot synthesis of multi component reactions (Biginelli and Strecker) under solvent-free conditions.
Section-A: Chemoenzymatic synthesis of enantiomerically enriched kawalactones
Kawalactones are a class of -pyrones and 5,6-dihydropyrones isolated from the kava plant Piper methysticum (Piperaceae), which grows widely in the South Pacific islands including Fiji and Hawaii. The extracts of its root and stem are used as a folk medicine or as a ceremonial drink in this region. Various structurally related analogues possess significant biological activities such as anticonvulsive, muscle-relaxing, sedative, analgesic, and antithrombotic, and have attracted attention in both the pharmaceutical and chemical research sectors. 
Figure 1


Enantiopure dihydrokawain 36a and 36b have been synthesized by employing β-keto ester 39 as the starting material. β-Keto ester 39 has previously been prepared by the alkylation of the dianion of methylacetoacetate 38 with benzyl bromide 37. Reduction to β-hydroxy ester 40 was achieved using sodium borohydride in methanol. The racemic β-hydroxy ester 40 was resolved enzymatically using different lipases. After separation, alcohol (R)-40 was treated with lithium diisopropyl amide (LDA) and following addition of t-butyl acetate gave hydroxy -keto ester (R)-42. The compound (R)-42 was lactonised with TFA to give (R)-43, which upon treatment with dimethyl sulphate in acetone in the presence of potassium carbonate to afford (R)-dihydrokawain (R)-36a. (S)-Dihydrokawain (R)-36b was obtained using the same procedure after hydrolysis of the acetate (S)-41 using potassium carbonate in methanol (Scheme 5).

Reagents and conditions: i. NaH, n-BuLi, THF, -50 to 0 °C, 85%; ii. NaBH4, MeOH, 0 °C to rt, 80%; iii. Lipase PS-C, vinyl acetate, diisopropyl ether; iv. K2CO3, MeOH, 90%; v. MeCO2tBu, LDA, THF, -78 °C, 85%; vi. TFA, CH2Cl2, 10 h; vii. K2CO3, acetone, (MeO)2SO2, 76% for two steps.
Racemic 46 was obtained as a pale yellow liquid by reaction of cinnamaldehyde 44 with the dianion of ethylacetoacetate 45. Racemic hydroxyl of 46 was resolved using lipases PS-C, PS-D and different acylating agents. Optically pure (R)-46 was applied for the synthesis of (R)-1 (Scheme 6).
Scheme 6

Reagents and conditions: i. NaH, n-BuLi, THF, 0 °C, 80%; ii. lipase, vinyl chloroacetate, diisopropyl ether, 20 h; iii. aq. NH3 (25%), MeOH, 0.5-1 h; iv. (a) K2CO3, MeOH, rt, 3 h; (b) acetone, (MeO)2SO2, rt, 10 h, 80% for two steps.
Section-B: Enantioselective total synthesis of both the stereoisomers of dihydrokawain-5-ol

We commenced our target molecule dihydrokawain-5-ol 36e from commercially available hydrocinnamaldehyde 47 as a starting material, which upon Wittig homologation gave unsaturated ester 49. The ester 49 was reduced to allylic alcohol 50 using DIBAL-H in CH2Cl2 in quantitative yield. Compound 50 upon Sharpless asymmetric dihydroxylation using AD-mix  at 0 °C furnished triol 51a in 90% yield. Selective 1,3 protection of 51a into six membered as p-methoxy benzylidene acetal 52 using PMB acetal, CSA in CH2Cl2 (85%), along with 1,2 protection of the five membered PMB acetal 54 in 10%. These two compounds 52 and 54 were separated by column chromatography.
Scheme 7

Reagents and conditions: i. Ph3PCHCO2Et, benzene, 6 h, reflux, 95%; ii. DIBAL-H, -78 °C, 2 h, 93%; iii. AD-mix-, t-BuOH-H2O, MeSONH2, 20 h, 0 °C, 90%; iv. PMB(OMe)2, CH2Cl2, CSA, 0 °C to rt, 1 h, 85%; v. BnBr, NaH, THF, 0 °C to rt, 4 h, 88%; vi. DIBAL-H, CH2Cl2 0 °C, 1 h, 88%; vii. Dess-Martin periodinate, CH2Cl2, 0 °C to rt, 1 h; viii. N2CHCO2Et, anhydrous SnCl2, CH2Cl2, 0 °C to rt, 85%; ix. ZrCl4, CH3CN, 0 °C to rt, 2 h, 85%; x. K2CO3, MeOH, 0 °C to rt, 2 h, then acetone, Me2SO4, 10 h, 75%; xi. TiCl4, CH2Cl2, 0 °C to rt, 15 min, 90%.
Scheme 8

i. AD-mix-, t-BuOH-H2O, MeSONH2, 20 h, 0 °C, 90%.

The hydroxyl group of 52 was protected as benzyl ether 53 (BnBr, NaH, THF) in quantitative yield. Compound 53 upon regioselective reduction with DIBAL-H gave alcohol 55. Compound 55 upon oxidation using Dess-Martin periodinane in CH2Cl2 gave aldehyde 56 in good yields. The aldehyde 56 without further purification was treated with N2CHCO2Et in the presence of anhydrous SnCl4 in CH2Cl2 gave -keto ester 57. The compound 57 upon deprotection of PMB group using ZrCl4 in acetonitrile at 0 °C gave -hydroxy--ketoester 58. This upon lactonization and methylation gave benzyl ether of dihydrokawain-5-ol 59, which upon debenzylation using TiCl4 in CH2Cl2 gave dihydrokawain-5-ol 36e. Another isomer of dihydrokawain-5-ol 36d was obtained from 50 using AD-mix .
Section-C: Cu(BF4)2·xH2O catalyzed one-pot synthesis of Biginelli and strecker reaction
Copper(II)tetrafluoroborate hydrate Cu(BF4)2·xH2O is a corrosive, moisture-sensitive, blue crystalline solid readily soluble in water and sparingly soluble in alcohols. Cu(BF4)2·xH2O is readily available and it does not require special storage conditions and is safe and stable under normal environmental conditions, Cu(BF4)2·xH2O has emerged as one of most useful reagent in various transformations such as acylation reactions and 1,1-diacetate formations. In all the cases, this reagent has been found to be superior to other available Lewis acids such as triflates, tetrafluoroborates and perchlorates with regard to short reaction times, requirement of stoichiometric amount of reagent, mild reactions conditions, solvent-free conditions and low cost. Using this catalyst we have developed novel synthetic methodologies (Biginelli and Strecekr) in one-pot, under solvent-free conditions.







(i)	Copper (II) tetrafluoroborate as a mild and efficient catalyst for the one-pot synthesis of 3,4-dihydropyrimidin-2(1H)-ones under solvent–free conditions
Scheme 9

(ii)	Copper(II)tetrafluoroborate as an efficient catalyst for the one-pot synthesis of -amino nitriles





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