Open Access Repository of Indian Theses

Mukherjee, Shashwata

 
SUMMARY 
 
III 
 
Wilson disease (WD, OMIM# 277900) is a rare inborn error of copper accumulation inherited 
in an autosomal recessive manner. It is caused by mutations in the copper transporting P-
type ATPase gene (ATP7B) (Thomas GR et al., 1995a). The disease is manifested at a 
median age of 12 to 23 years as hepatolenticular degeneration resulting from accumulation of 
copper primarily in the liver and brain. Patients mostly present with hepatic, neuropsychiatric 
or mixed symptoms (Das SK and Ray K, 2006). Varied clinical and biochemical 
manifestations often complicate diagnostic workup and diagnosis can be challenging 
especially for patients who are asymptomatic or present with liver disease. Failure to 
diagnose WD before its substantial progression is the leading cause of morbidity and 
mortality. But if patients can be diagnosed early enough, lifelong de-coppering with 
appropriate chelating agents can lead to gradual improvement and make them largely 
symptom free for the rest of their lives (Walshe JM, 2007). There is no single clinical or 
biochemical test to exclude WD. Genetic analysis still remains to be the confirmatory 
approach to detect the disease especially in sporadic cases with no family history and 
ambiguous clinical indications. 
When the work for this dissertation was initiated, several studies have revealed the 
ATP7B mutation spectrum from North Indian (Kumar S et al., 2005), South Indian (Santhosh 
S et al., 2006) and East Indian (Gupta A et al., 2005, 2007a) (our group) WD population. 
However, the WD mutation pattern from West India was yet to be elucidated. Hence, the 
genetic, clinical and functional studies leading to this dissertation have sequentially focussed 
on the following areas (i) further characterization of molecular defects present in ATP7B gene 
in East Indian WD patients and exploration of the ATP7B mutation spectrum from West India 
(ii) screening of the ATP7B regulatory region in patients with single detectable coding 
mutations or lack of any, and followed it up with functional characterization of variants in the 
promoter and 5'UTR, (iii) Investigating the genotype-phenotype relationships in WD by 
generating a matrix for exploration of the clinico-genetic interface, followed by evaluation of 
candidate modifier loci, and (iv) functional evaluation of variants detected in the ATP7B 
coding region by localization and trafficking studies. 
A total of 199 WD patients (174 East Indian and 25 West Indian) mostly with 
neurological problems from 178 unrelated families comprising of 687 individuals have been 
included in this study prospectively. This group is an extension of a previous study from our 
lab where 25 East Indian patients had been characterized with both ATP7B mutations and 
single mutations found in a few (Gupta et al., 2007a). ATP7B mutation screening has been 
undertaken in 174 patients (East Indian: 149, West Indian: 25). Finally, a comprehensive 
mutation profile of all 199 patients is described. In this study, we detected 29 reported and 22 
novel mutations after screening most of the ATP7B coding region (exons 2 to 21) and flanking 
intron-exon boundaries. Of the reported ones, 19 defects had been reported from other 
populations but detected for the first time in our patient cohort and 10 variants were previously 
described by our group (Gupta A et al., 2007a) in addition to finding those during the study 
related to my dissertation. Adding up all mutations, our patient cohort harbors a total of 10 
 
Summary 
 
IV 
 
common and 48 rare lesions in the coding region of ATP7B. Three mutations (p.G1061E, 
p.N1270S and p.A1049A-fs) are shared between East and West. The common mutations 
have been found to account for 74% of characterized mutant alleles with p.C271X (63/260) 
and p.G1101R (7/31) being the most prevalent in East and West Indian patient cohorts, 
respectively. Comparison of mutation pools across India suggested that the exons 2, 8, 9, 13, 
14, 15, 18 and Intron 4 harbor the prevalent mutations and should be screened with priority in 
the Indian WD population. In addition to the mutations, 28 innocuous variants could be 
detected during this study. Mutations in both the alleles could be characterized in 114 
patients, while in 63 patients only one mutation could be detected in the coding region. No 
coding mutations could be detected in 22 patients. Further studies on these patients with 
missing mutation(s) are described in relevant sections below. 
WD is one of the few genetic diseases, the progression of which can be controlled if 
diagnosed early enough. Knowledge of common mutations in a population would pave the 
way for rapid diagnosis, even in some suspect case without family information, before the 
manifestation of overt clinical symptoms. But mutation screening is a time consuming 
process. So, a parallel strategy was employed to identify the carriers and presymptomatic 
individuals in 41 East Indian and 19 West Indian WD affected families with multiple sibs to 
provide relevant information faster without waiting for determination of the underlying causal 
mutation. By studying the inheritance of linked polymorphic microsatellite markers D13S314 
and D13S316 in the affected pedigrees, we could detect 5 presymptomatic, 40 carrier and 36 
normal individuals. Mutation information, when available was utilized to resolve the 
ambiguous cases. 
Most genetic studies reported earlier focussed on studying the ATP7B coding region 
but the gene promoter, though characterized, remained largely unexplored. As already 
described, 85 patients (63 + 22) in our pool could not be characterized for either one or both 
ATP7B mutations even after screening much of the coding region. In order to identify the 
missing mutations, we screened the ATP7B promoter and both the 5'- and 3'-UTRs in these 
patients. We detected 5 novel and 6 reported variants in the promoter and 5'UTR. Promoter 
cloning, site-directed mutagenesis and dual luciferase assay was carried out for functional 
evaluation of these variants. Five variants and one haplotype in the 5' regulatory region could 
be implicated in WD pathogenesis based on significant down regulation of the promoter 
activity. We carried out segregation studies in pedigrees to dissect the phase of alleles with 
respect to promoter variants and downstream coding mutations. We further hypothesized that 
ATP7B promoter variations altering its activity might also be present in patients where both 
coding mutations have been detected and influence the expression of the mutated protein. 
The differences in promoter strengths could have the potential to add on to the observed 
clinical heterogeneity in addition to the influence of other confounding genetic and 
environmental factors. Thus, the role of promoter variants in the regulation of ATP7B merits 
further investigation. 
 
Summary 
 
V 
 
Till date, no nucleotide substitutions in the ATP7B 3'UTR have been associated with 
WD phenotype. This study reports 3 novel variations in this region in WD patients, however, 
further experiments are required to ascertain their status as mutations or rare innocuous 
variants. We reached a mutation detection frequency of 77% in our study. 
We devised a Neurological Involvement Score (NIS) to explore the spectrum of 
neurological association across cognition, behaviour and motor domains in WD patients with 
preferential neuropsychiatric manifestation. The scale was found to have high internal 
consistency (Cronbach’s α 0.802) and inter-rater reliability (ICC 0.888). Thus NIS was highly 
reproducible. Subsequently we undertook a novel unified approach of genotype-phenotype 
(G2P) correlation by utilizing NIS as a simple tool to explore the neuropsychiatric arena of WD 
patients in light of the mutations identified across different ATP7B protein domains. Using age 
at onset and neurological involvement score, we generated a G2P matrix. The scheme of 
G2P matrix provided opportunities to examine and compare the patient phenotypes with 
respect to same genotype and also across different protein domains. We observed that the 
truncation mutations could be equally deleterious irrespective of the site of termination being 
early or late in the polypeptide chain. Compound heterozygotes bearing combination of 
missense and truncation mutations indicated missense mutations to retain more functionality 
in the mutated protein as reported previously (Nicastro E et al., 2009). Specific mutations in 
the nucleotide binding domain were found to attribute hepatic predominance. 
Patients with same/similar mutation background would be expected to have similar 
presentation. The most interesting observation was made when we encountered a few 
patients who depicted stark difference in their clinical manifestation as compared to other 
patients in the same mutation cluster. We counted the patients with divergent phenotypes as 
“outliers” in the matrix. Possible roles of COMMD1 and ATOX1, two genes in copper 
homeostasis pathway, were also investigated for their potential to act as modifier loci in the 
outliers, though no variant could be implicated for the cause. Since the ‘basal level 
heterogeneity’ of presenting symptoms in WD is not exclusively explained by the ATP7B 
mutations, and cases of misdiagnosis and phenotypic overlap have been reported implicating 
additional genes such as NPC1, SLC33A1, and SLC30A10 (Connemann BJ et al., 2012; 
Huppke P et al., 2012a; Quadri M et al., 2012), we propose that these loci should be studied 
for their putative role in imparting WD features by some novel pathophysiological 
mechanisms. 
The diagnostic delay emerged to be a confounding factor for G2P analysis. The mean 
diagnostic delay in our study was 19.5 months (0.2 to 180 months) and there is opportunity 
for further improvement. 
Missense mutations might have a spectrum of consequences on the mutant protein. 
In this context, we made an attempt to explore the effect of missense mutations on cellular 
localization and anterograde trafficking of ATP7B. GFP fusion constructs for 9 different 
ATP7B mutants selected from different domains were investigated for their localization and 
 
Summary 
 
VI 
 
copper responsive redistribution. While 6 of 9 mutants showed normal behavior, 2 mutants 
(p.G1061E and p.G1101R) were found to be grossly mislocalized to endoplasmic reticulum 
while 1 mutant (p.S1362A) was found to be unresponsive to copper induction. Mutations 
might affect several properties of ATP7B which could alter its precise conformation, catalytic 
and kinase mediated phosphorylation, ATPase activity, copper binding and transport rate 
across membranes etc. Each of these factors might have deleterious consequences on 
normal copper distribution and may lead to WD. Thus, a detailed functional exploration of the 
effect of missense variants on other properties of the protein would provide deeper insight of 
their effect on the clinical phenotype of the patients. 
The studies leading to this dissertation have resulted in the following publication as 
noted below and additional manuscript describing unpublished work is under preparation: 
Mukherjee S, Dutta S, Majumdar S, Biswas T, Jaiswal P, Sengupta M, Bhattacharya A, 
Gangopadhyay PK, Bavdekar A, Das SK, Ray K. Genetic defects in Indian Wilson disease 
patients and genotype-phenotype correlation. Parkinsonism and Related Disorders 2013; (In 
press); DOI, 10.1016/j.parkreldis.2013.09.021 


English

Studies on Molecular Basis of Wilson Disease by Genetic and Functional Approaches

Sengupta, Debashree

Root System Plasticity to Water Stress Tolerance in a Food Legume Mungbean,Vigna radiata (L.) Wilczek

Joshi-Navare, Kasturi

xiii 
 
 
Abstract 
Sophorolipids (SLs) belong to the category of glycolipid biosurfactants. They 
consist of a dimeric sugar- sophorose as a carbohydrate head and a long hydroxy 
fatty acid chain as tail. This amphiphilic nature allows them to form unique 
structures such as micelles and bilayers in heterogenous systems. SLs find 
applications in wide range of fields including Petroleum, food, pharmaceuticals, 
cosmetics, laundry and bioremediation. SLs are synthesized by employing non 
pathogenic yeast strains and they confer several physiological advantages to the 
producer. Biosurfactants are coming up as emerging class of biomedical 
compounds. They are a suitable alternative to synthetic medicines and 
antimicrobial agents, and could be used as safe and effective therapeutic agents 
or probiotics, especially at a time when drug resistance among causal organisms 
for many life-threatening diseases is on the rise. SLs offer the advantages of 
biodegradability, low ecotoxicity and the production based on renewable-resource 
substrates. The US FDA has also approved biosurfactants/sugar esters for the 
use in food and pharmaceuticals. Keeping this in mind; several basic and applied 
aspects of sophorolipids have been explored and the work has been reported in 
the thesis. 
The thesis has been divided into 5 chapters 
Chapter I. Introduction 
The first chapter is an introduction to the thesis. It includes brief account on 
surfactants, current market share of Biosurfactants, types, their advantages over 
chemical ones and producing microorganisms. SL stands as a promising 
biosurfactant. The features which make it so are described along with its 
physiological role and the biosynthetic pathway. Owing to the unique nature; 
sophorolipids find applications in wide range of areas. The chapter gives brief 
literature review of the applications. With this background, the scope and 
objectives of the thesis have been defined.  
 
Chapter II. Production of sophorolipids using non edible oils and their use as an 
alternative/ additive to laundry detergents 
The second chapter is about the attempt to reduce the SL production cost by 
using non-edible oils namely Jatropha and Pongamia. Through optimization of 
xiv 
 
 
parameters and resting cell method, the yields 15.25g/l and 19.3g/l could be 
achieved for Jatropha oil derived SL (SLJO) and Pongamia oil derived sophorolipid 
(SLPO) respectively. Both SLs showed good surfactant property with the CMC 
values 9.5mg/l for SLJO and 62.5mg/l for SLPO. Keeping the prospective use of 
these SLs in mind, the physicochemical characterization, emulsion stability, 
antibacterial and stain removal studies in comparison with commercial detergent 
were done. Based on the results, it can be said that SLs have utility as fabric 
cleaner with advantageous properties such as skin friendly nature, antibacterial 
action and biodegradability. SLs enhance the detergent performance, so less 
quantity of detergent can be sufficient for desired cleaning effect. Thus the harm 
caused to environment through detergent usage can be reduced by replacing 
synthetic surfactants with green surfactant molecules. 
 
Chapter III(a). Sophorolipid biosurfactants act in synergy with antibiotics to 
enhance their efficiency  
In the third chapter (part a) the effect of co-administration of SL and antibiotics 
has been evaluated. SLs are amphiphilic in nature and therefore can span 
through the bacterial cell membrane. Thus the co-administration is expected to 
facilitate the entry of antibiotic molecules. During the experiments, effect of SL-
antibiotic co-administration was evaluated against 3 different index bacteria 
namely, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa. Also 
the antibiotics differed in their mode as well as site of action. In accordance with 
the anticipation, SL-antibiotic combination achieved the bacterial inhibition within 
shorter period as compared to the antibiotic alone. The Scanning electron 
micrographs of the bacterial cells treated with combinations confirmed the 
damage to cell membrane with all 3 test bacteria at sublethal concentrations of 
both inhibitory agents.   
 
Chapter III(b). Exploration of antiviral activity of SLs 
The third chapter (part b) describes antiviral action of Oleic, Linoleic acid derived 
SL against viruses differing in their genetic makeup such as plus stranded RNA, 
segmented single negative stranded RNA and double stranded DNA viruses. The 
antiviral activity was tested in 3 different modes- direct treatment of virus, rapid 
xv 
 
 
culture assay for Influenza virus and treatment of host cell line with SL prior to 
viral challenge in order to check if SL gives any antiviral immunity. 
Coxsackieviruses CV (B1-CVB6, CA7, CA9), murid herpesvirus, strain MHV-68, 
Influenza virus A/Mississippi/1/85 (H3N2) have been used for the studies. 
It was observed that direct treatment of virus indicated 1 log10- 4.5 log10 
reduction in the virus titers. Pretreatment of cell cultures GMK and Hep-2 prior to 
Coxsackie virus infection showed a reduction in virus titer 1log10 - 2 logs10. Similar 
results were obtained on the VERO, BHK and 3T3 cells with gamma-herpesvirus 
MHV-68. Visible reduction of Influenza A virus replication on MDCK cells was 
obtained at the concentration 100 μg/ml of SLLA (SL derived from Linoleic acid). 
Thus it can be concluded that direct treatment of viruses was more effective than 
indirect treatment and the inhibitory action can be attributed to the amphiphilic 
structure of SL which might be killing the viruses by disturbing the membrane lipid 
order. 
 
Chapter IV. Differentiation inducing ability of SLs against glioma cells 
The forth chapter deals with the differentiation inducing ability of SLs. The effects 
have been investigated on LN229 - a glioma cell line which has been reported for 
the first time. In response to different SL forms, marked difference in cell density 
was observed as compared to the control cells along with various morphological 
changes such as formation of long thread like extensions arising from ends of the 
cells, cell alignment, cell elongation and bundle formation in dose dependent 
manner.  In this chapter the morphological evidence of the potential of SLs as 
differentiation inducers has been presented. The finding suggests the utility of 
SLs as a pharmaceutical agent for the treatment of glioblastomas. With the use of 
SL, the cancerous cells undergoing uncontrolled proliferation can be forced to 
differentiate thus tumor progression may be arrested. 
 
 
Chapter V(a). Glycolipid production by a novel yeast- Pichia caribbica (HQ222812) 
with xylose as a head group and its advantageous properties 
The fifth chapter (part a) deals with the use of a new xylose fermenting yeast- 
Pichia caribbica for biosurfactant production so as to achieve the less explored 
head group diversity in SL structure. The media and fermentation parameters 
xvi 
 
 
have been optimized to achieve maximum yield of 7.5g/l. The physicochemical 
properties of the xylolipid biosurfactant have been assessed. It reduced the 
surface tension of distilled water from 70mN/m to 35.9mN/m with the 
remarkably low CMC value 1.0 mg/l as compared to typical SLs (reported CMC 
range-40-100mg/l). Structural characterization was done using FTIR and HR-MS 
to identify the structure putatively. 17-L-[(β-D-xylopyranosyl)-oxy]-Δ9-
heptadecanoic acid correlated to m/z 415 majorly constituted the product. 
Control experiment was performed in which glucose was provided as the 
hydrophilic carbon. This product was also subjected to HR-MS analysis to 
determine its chemical nature and found to be different from xylolipid. Presence 
of xylose as head group was anticipated to give altered physicochemical and 
biological activities. In accordance to the same, low CMC value and better 
inhibitory action was demonstrated against Staphylococcus aureus, a gram 
positive bacterium. 
 
Chapter V(b). Crystalline xylitol production by a novel yeast- Pichia caribbica 
(HQ222812) and its application for quorum sensing inhibition in gram negative 
marker strain Chromobacterium violecium CV026 
The fifth chapter (part b) is about quantitative production of xylitol from D-xylose 
with the yield of 0.852 gm/gm and volumetric productivity of 1.83 gm/l/h. A safe 
procedure for product extraction has been described. The ability of xylitol to act as 
a quorum sensing antagonist in gram negative marker strain Chromobacterium 
violaceum CV026 has been demonstrated for the first time.  
 
Conclusions 
The last chapter summarizes the work presented in the thesis and emphasizes on 
possible further research in this area. 


Biosynthesis of novel Glycolipids: Basic and applied aspects

Malik, Garima

ABSTRACT 
 
Rice (Oryza sativa), a member of Gramineae family, is a major crop plant that feeds almost 
half of the global human population. In recent years, this plant has emerged as a model 
system for functional genomics studies among the monocot plants. The availability of its 
complete genome sequence, easy transformation protocols and various mutant lines have 
been instrumental in unravelling the genetic and epigenetic molecular mechanisms that 
regulate gene expressions and various developmental pathways including reproductive 
development in rice. However, despite these advances, one of the important epigenetic 
regulators, the process of DNA methylation, has largely remained unexplored partly 
because of increased complexity in higher plant organization. With the view to get a clearer 
picture of the role of this regulatory process and to be able to understand the roles of its 
molecular components in modulating gene expression patterns, we selected an early land 
plant Physcomitrella patens as the second model organism for the present study. P. patens 
is a simple moss plant with fully sequenced genome. This study in the two model 
organisms has provided significant insight into the conservation and diversity among the 
methylation machinery components and their evolutionary relationships in land plants.  
The objective of this Ph.D. work was to first establish conclusively the role of DNA 
methylation in regulating growth, development and differentiation in both, Physcomitrella 
and O. sativa and then to identify and characterize the core component of the DNA 
methylation machinery, cytosine DNA methyltransferases, in these plants. As a first step in 
this direction, the effect of global hypomethylation induced by cytidine analog, zebularine, 
on growth and development of O. sativa and Physcomitrella was examined. Thereafter, a 
genome-wide in silico study was undertaken to identify the genes encoding cytosine 
methyltransferases in these plants. A comprehensive structural and phylogenetic analysis of 
rice and moss methyltransferase gene families was then carried out. This was followed by 
expression analysis of genes during different developmental stages and in response to 
abiotic stress conditions. Thereafter, putative functions of the genes were speculated by 
studying in vivo subcellular localization patterns of methyltransferase-GFP fusion proteins 
in onion epidermal cells and moss protonema cells. Finally, functional characterization of 
selected rice methyltransferase genes (OsCMT2, OsMET1-2, OsDRM2, OsDRM3 and 
OsDNMT2) was undertaken using gene silencing and overexpression strategies.  
The concise review of literature presented at the beginning of the thesis is aimed to discuss 
and highlight the evolutionary relatedness among the methyltransferase genes in eukaryotes 
and the pivotal roles played by cytosine DNA methylation in plant development. The 
experimental procedures used in this study and the results thus obtained have been 
summarized in separate sections. The new and recent findings and their relevance to the 
current research scenario has been discussed and significant conclusions have been drawn. 
References cited throughout this study and the research publications earned through this 
work have been compiled at the end. 
This research not only enhances our understanding of the evolution and functional 
conservation/diversification of DNA methylation and components of the methylation 
machinery, but it also provides evidence for the role of methyltransferases in modulating 
various developmental pathways regulating growth and development in both, O. sativa and 
Physcomitrella patens.  
  
Abstract 


Understanding the Role of DNA Methylation in Regulating Gene Expression during Development in Oryza sativa and Physcomitrella patens.

Joish, Madhusudhana Murthy

128 
CHAPTER 6 
Summary and Conclusions 
 
The brief summary and following conclusions can be drawn from this work that, the 
tested plant materials (Eichhornia crassipes, Plumbago capensis and Piper betle) still 
have new antifeedant/ toxic compounds. Moreover the bioactive compounds isolated 
from these plants were easily conjugated with silica nano particles to prepare effective 
formulations. The prepared nanoformulations are successful in exhibiting improved shelf 
life and higher controlled release profile isolated compounds. In addition nano-
formulations also enhance the antifeedant activity of the individual isolated compounds 
against Spdoptera litura F., Achaea janata L. and Helicoverpa armigera H.  
 
 There have been many methods employed to isolate the principle bioactive 
compounds form water hyacinth which eventually yielded a single potent antifeedant 
and toxic compound, 2-propyl piperidine (coniine). I have discovered the occurrence 
of this chemical in water hyacinth plant for the first time and also its insect 
controlling activity/ use in pest control on agricultural crops for the first time. A 
patent was filed on isolation of an active insecticidal compound, 2-propylpiperidine 
(coniine) from aquatic weed water hyacinth, Eichhornia crassipes (Mart.). More 
particularly, the compound can be used as a total insect-controlling agent (Toxicant, 
antifeedant and growth regulator) for pest management in agricultural fields. The 
compound 2-propylpiperidine can be used as a substitute for chemical pesticides or as 
a supplement to chemical pesticides to reduce the usage of synthetic pesticide 
concentrations to protect the environment.   
 
 Different isolations techniques and the purification methods lead to isolate an 
antifeedant compound from the P. capensis. The extensive spectroscopic analysis 
confirmed the compounds as plumbagin. The antifeedant activity of the plumbagin 
was enhanced to more than 10 folds on formulating with silica nanoparticles. 
Moreover the nano nature of formulation has an advantage of distributing the active 
compound on the leaf which enhances the bioavailability of the compound.    
129 
 
 Bioactivity guided fractionation of Piper betle L. yielded two bioactive fractions 
which on fractionation yielded total 8 bioactive compounds. After various chemical 
analysis such as, NMR, FTIR, EIMS, UV-VIS and HPLC, the isolated compounds 
were confirmed as Eugenol, α- pinene, Linalool, Limonene, Trans-caryophyllene, 
Nerolidol, Carene and Ocimene. The results reveal that the Eugenol was obtained in 
higher quantities from P. betle. However the rest of the compounds miner 
constituents of the plants and their quantity isolated from was little and are procured 
from Sigma-Aldrich to prepare the formulation and further investigations.   
 
 The isolated compounds were conjugated with silica nanoparticles to increase its 
shelf life, controlled release properties as well as the bio-activity against major 
agricultural pests. All the formulations prepared showed high stability with higher 
zeta potential, controlled release property as well as enhanced shelf life of the isolated 
botanicals. The added advantage of these formulations is their ability to enhance the 
bioactivity of the isolated botanical compounds.  
 
 The preparation of nanobioformulation with Trans-caryophyllene and silica 
nanoparticles showed enhanced efficiency of Trans-caryophyllene in controlling the 
population of two major agricultural pests. A patent was filed on synergistic 
nanobioformulation with Trans-caryophyllene and silica nanoparticles useful as insect 
controlling agent and a method of its formulation. Nano silica formulation protects 
the botanicals against degradation, enhance the bioavailability and create high local 
concentrations of the bioactive chemicals in target cells. The formulation controls the 
release rate and prolongs the duration of antifeedant effect of trans-caryophyllene. 
This formulation offer synergistic effect, which gives added advantage for the 
compounds in enhancing antifeedant activity as well as the duration of protection 
against agricultural pests.  
 
 Result revealed that Trans-caryophyllene and its nano-formulations were directly not 
toxic to A. janata, H.armigera and S. litura. But they inhibit the feeding activity of 
130 
the insects leading to pest’s starvation that ultimately leads to its death. Additionally 
the amorphous silica is considered to be safe for human by world health organization 
and US department of agriculture. Therefore their persistence on plant or soil after 
spraying in the field will not have any adverse effects on the environment.  
 
 Regulation of the particle size, morphology, zeta potential, controlled release and 
shelf life of formulation can be easily done by adjusting the concerned parameters, 
e.g., the time of compound loading type of solvent used in the preparation and 
concentrations of precursor’s i.e. isolated compounds and silica nanoparticles.  
 
 Compositions of the isolated bioactive compounds and silica nanoparticles present in 
the nanobiocomposites are found to be directly influencing the size and bioactivity of 
the nanoformulations. Thus, to get the maximum antifeedant activity optimum 
composition of the botanicals were needed in effective nanoformulation.  
 
 The result of present investigation indicated that, there is tremendous scope for 
developing nano-biocomposites in to future pest management as they are 
environmentally safer than the existing synthetic pesticides.  
 
***** 
 
  
 


Bioactive compounds from certain plants and their stabilization using silica nanoparticles for effective management of agricultural pests

Wadhwani, Ashish Devidas

                                                     Abstract


Chapter 1: Biotechnological processes for the removal of dissolved sulfides (S2-) and Hydrogen sulfide (H2S) 
Gaseous sulfides (H2S) and Dissolved sulfides (S2-) emits into environment by different industrial activities, which includes paper and pulp industry, viscous rayon, petroleum refineries, petrochemical industries, bulk drug industries, anaerobic treatment of sulfate rich wastewater. Sulfide discharge into environment leads corrosion of sewer pipes, reduction in pH of receiving water bodies, reduction in dissolved oxygen concentration, bad odor. Various methods physcio-chemical methods are available for the treatment of sulfides emitting from different sources. Chapter 1 of this thesis has discussed the sources of sulfides in the environment, their toxic affects and available physical and chemical removal methods. Chapter 1 also discussed the biological sulfur and mediation of sulfur microbiology for the removal sulfides. 
Various sulfur oxidizing bacteria and their metabolism is discussed in detail. Sulfur is the immediate metabolic product in the biological sulfide oxidation process. Sulfate will form if there is a direct contact between elemental sulfur and cells. Autotrophic colorless Thiobacillus cultures are better for the sulfide oxidation process as their metabolism and nutritional requirements are simple when compared with other sulfur oxidizing bacteria. Dissolved sulfides could be treated in reactor using immobilized cells to reduce the formation sulfates. Hydrogen sulfide gas can also be treated efficiently in biofiltration process using sulfur oxidizing bacteria. and a In conclusion to Chapter 1 it was summarized that autotrophic sulfur oxidizing bacteria (SOB) are more useful for the sulfur oxidation in techno-economical perspective and ease of process operation. At the end of chapter 1 it was also concluded that isolation of autotrophic sulfur oxidizing bacteria could be done from different habitats. To reduce the sulfates formation cells could be immobilized using suitable matrix. 
Development of microbial processes for the removal of water dissolved sulfide and gaseous sulfide (H2S) is highly essential in order to gear up the industries to 
i 
operate in the global environment of new regulations. Therefore, in order to fill up the technological gaps in the area of biotechnological treatment of gases and liquid, the following aims and objectives are addressed in the present work. 
Chapter 2: Isolation, identification and characterization of Thiobacillus sp. from aerobic sludge of distillery and dairy effluent treatment plants 
Chapter 2 deals with the isolation of the SOB from different habitats by using standard microbiological methods and their identification. Thiobacillus sp. are extensively used worldwide for removal of both organic and inorganic sulphur compounds in wastewater; however, a little used in India. In the present study, two strains of Thiobacillus sp. were isolated from aerobic sludge of distillery and dairy effluent treatment plant using standard methods of isolation and enrichment. Experiments were conducted using isolated cultures in batch bioreactor with initial sulfide concentration of 75 and 150 mg/l. The effect of initial sulfide concentration on the activity of isolated Thiobacillus sp was studied. Sulfide oxidizing capacity was also determined at above initial concentrations of sulfide. 
The two isolated SRB strains, 5 Days old cultures are viewed under scanning electron microscopy for various characteristics like size and shape. Standard plate count method is used for the colony count at different serial dilution ranging from 10-1 to 10-10 and the cell count is in the range of 7x105 and 4x107 cells/ml for IICT-SOB-DAIRY-201 and IICT-SOB-DIST-210 respectively. The results of gram staining for both the isolated SOB’s from their native sources are negative. 
The results from the study also indicate the possible isolation of Thiobacillus cultures from native source and its application in the full-scale reactor. Two cultures were isolated from dairy and distillery wastewater treatment plant sludge. The two isolated SOB’s were identified by various physiological, morphological and biochemical properties and concluded that the cultures belongs to genera Thiobacilli. Based on the sulfide oxidation and sulfate formation ability, one SOB culture isolated from distillery wastewater treatment plant sludge was selected and further studies were carried out under different conditions to treat sulfides. The 
ii 
culture was also identified by the 16sRNA phylogenetic analysis and based on the comparison with gene bank data it was named as Thiobacillus sp. Growth medium was optimized by using response surface methodology as a statistical tool. 
Chapter 3: Studies on immobilization of Thiobacillus sp. for sulfide oxidation 
Thiobacillus sp cells were immobilized using different immobilizing matrices such as calcium alginate, carrageenan, and agar (entrapment method), activated carbon, and polyurethane (adsorption) were used for the sulfide oxidation. Thiobacillus sp. was immobilized in various matrices and tested for the sulfide oxidation. 
Based on the results obtained by the sulfide oxidation calcium-alginate immobilization method was used for further studies. Different parameters like alginate concentration, CaCl2 concentration, bead diameter, and curing time were studied for optimal immobilization conditions with calcium alginate matrix. Different concentrations of Na-Alginate in the levels of 1, 2, 3, 4 and 5% were tested to determine the optimal concentration for increased sulfide oxidation. The concentration of the cationic solution was one the most significant factor affecting bead stability. Different CaCl2 concentrations/ curing were studied to see the effect of bead hardening/strength which directly influences on the diffusability of substrates and products thus influences sulfide oxidation. To study influence of different CaCl2 concentrations on sulfide oxidation, the immobilization procedure was carried out with different concentrations of CaCl2 in the levels of 1, 2, 3, 4 and 5 %. Cell leakage and sulfide oxidation were determined. The effect of bead diameter on sulfide oxidation by different bead sizes in the range of 1, 2, 3, 4 and 5 (≈ mm diameter) was tested. The effect of curing time of alginate beads on sulfide oxidation and cell leakage also tested. The beads were cured in CaCl2 at optimum molar concentration for different time periods in the levels of 1, 3, 6, 12, and 18 h. 
These results indicated that the type of the entrapping matrix significantly affects the sulfide oxidation. Sodium alginate resulted in relatively high sulfide oxidation 
iii 
when compared to other carriers. Maximum sulfide oxidation of 96 % was achieved with alginate matrix followed by k-carrageenan which produces 88%. However the adsorption methods resulted in complete oxidation of sulfide but resulted in more sulfate and thiosulfate formation. Hence further studies were carried out using ca-alginate immobilization method. Repeated batch experiments were carried out to test the reusability of ca-alginate immobilized beads for the sulfide oxidation. Further sulfide oxidation was tested in batch STR and FBR at different sulfide concentrations. 
Chapter 4: Fluidized bed bioreactor operation using immobilized Thiobacillus sp for sulfide oxidation 
4.1 Continuous operation of Fluidized Bed Bioreactor for biogenic sulfide oxidation using immobilized cells of Thiobacillus sp. 
In the present studies obligate autotrophic Thiobacillus sp. was isolated from aerobic sludge distillery effluent treatment plant and the experiments were conducted in a laboratory scale fluidized bed bioreactor for the biological oxidation of sulfide using Ca-alginate immobilized Thiobacillus sp. All the experiments were conducted in continuous mode at different sulfide loading rates of 0.018, 0.02475, 0.03375, 0.03825 and 0.054 and different hydraulic retention times of 5, 3.67, 2.67, 2.35 and 1.67 hrs by varying flow rates 2.4x10-4, 3.3 x10-4, 4.5 x10-4, 5.1 x10-4 and 7.2 x10-4. Sulfide conversions higher than 90% were obtained at almost all sulfide loading rates and hydraulic retention times. All the experiments were conducted at constant pH of around 6 and temperature of 30+50C. The sulfate production rate decreased and sulfur production rate increased while increasing the sulfide-loading rate to the reactor. 
4.2: Biological treatment of toxic petroleum spent caustic in fluidized bed bioreactor using immobilized cells of Thiobacillus RAI01 
Synthetic sulfide spent caustic effluent was prepared in the lab and at different concentrations of Sulfide by using sodium sulfide. Synthetic sulfide caustic effluent was mixed with 50 ppm of phenol to test the effect of its presence on the sulfide oxidation activity of the Thiobacillus sp. Further the caustic effluent was 
iv 
treated in a fluidized bed reactor using Ca-alginate immobilized Thiobacillus sp. cells for sulfide oxidation under different loading rates. Varying caustic effluent inlet flow rates without changing initial sulfide concentration in the effluent varied the sulfide loading rates to the reactor. The reactor was operated at three different inlet flow rates, viz., 4.17x10-5, 5.56 x10-5 and 8.33 x10-5 m3/hr which resulted in HRT’s of 24, 18 and 12 hrs. This corresponds to the respective sulfide loading rates of 0.020833, 0.027778 and 0.041667 g-S/m3.hr. The samples are drawn at 24 hours frequency and analyzed for pH, Temperature, DO, sulfate sulfide, and thiosulfate. 
Chapter 5: Treatment of H2S gas in biofilter by immobilization of Thiobacillus sp on mixed agricultural residue 
Microbial removal of H2S, by using biofilter is gaining interest because of its obvious advantages besides being competitive. However, most of the installed biofilters were failing, as the operators were not aware of process parameters, which are important for appropriate operation of the biofilter. Furthermore, biofilters packed with natural materials were subjected to operating difficulties such as compacting or channeling. Therefore there is a need to identify new natural filter material which can overcome the aforesaid difficulties. 
In the present work, a new filter material from mixed agricultural residue was studied for the removal of H2S. This material is a combination of sawdust, bagasse, rice husk and coconut coir pith inoculated with Thiobacillus sp. This material was studied for various process parameters that are important for performance evaluation; specifically, for the effects of humidification and inlet concentration of H2S on the performance of biofilter. The study showed that mixed agricultural residue could be one of the best alternate biofilter bed materials for biofiltration of H2S. A suitable combination of four materials was worked out in order to retain the maximum moisture content. 
In the present study, the RE was around 99% at 1416 ppmv of inlet H2S concentration without mixing the filter media. It indicates that the new filter 
v 
material and their combination played a crucial role in retaining the MC during the operation and also lower pressure drop with good growth of Thiobacillus sp across the filter bed. Estimation of kinetic parameters is important and valuable tool in analyzing the biofilter process for H2S removal. In the present study, kinetic constants were estimated using three different plots i.e Lineweaver-Burk (LB), Hanse-Woolf (HW) and Eadie-Hofstee (EH). The SEM analysis has shown the good growth of Thiobacillus sp in the filter bed and hence the new material is good support for the immobilization.



Investigation of Antiviral Properties of Medicinal Plant Extracts

MAISNAM, MANORAMA DEVI

Studies on distribution and biology of an ornamental fish, Puntius shalynius Yazdani and Talukdar in Meghalaya

J, SELVARAJ

Undetermined

EXCESS ALDOSTERONE-INDUCED CHANGES IN INSULIN SIGNALING MOLECULES AND GLUCOSE OXIDATION IN GASTROCNEMIUS MUSCLE AND LIVER OF ADULT MALE RAT

Saxena, Roopali

Role of Membrane Organization and Dynamics in the Function of Serotonin1A Receptors

Jain, Manish Kumar

The thesis entitled “Studies directed towards the total synthesis of polypropionate
natural products (–)-Ebelactone-A and (–)-Maurenone” is divided into three
chapters.
CHAPTER I: This chapter deals with the introduction and earlier synthetic
approaches of (–)-Ebelactone-A and a small review on
desymmetrization.
CHAPTER II: This chapter describes the stereoselective synthesis of C1-C6 and C7-
C14 fragments of (–)-Ebelactone A.
CHAPTER III: This chapter divided into two sections.
Section A: This section deals with the introduction and earlier synthetic approaches of
(–)-Maurenone.
Section B: This section describes the formal stereoselective synthesis of (–)-
Maurenone.
This chapter describes the β-lactones which are having enzymatic inhibitory
activity and also describes the introduction and previous synthetic approaches of
ebelactones, having proven esterase inhibitor activity. Also a brief literature survey on
“desymmetrization technique” a strategy used for introducing chirality into the target
(–)-Ebelactone-A is described.
This chapter describes the stereoselective synthesis of C1-C6 and C7-C14
fragments of (–)-Ebelactone A.
CHAPTER-I
CHAPTER-II
SYNOPSIS
15
In 1980 Umezawa and co-workers first reported the isolation of (–)-Ebelactone
A 1, a β-lactone enzyme inhibitor from the cultured strain of soil actinomycetes (mG7-
G1 related to streptomyces aburaviensis). The Ebelactones show structural
characteristics in common with macrolide antibiotics and butyrate precursors indicate
that they are like polyketide in origin. The Ebelactones act as potent inhibitors of
esterases, lipases and N-formylmethionine aminopeptidases located on the cellular
membrane of the various kinds of animal cells and they have shown to produce enhance
immune responses. They are also reported to inhibit cutinases produced by fungal
pathogens. Due to its biological activity combined with unique and challenging
structure have made this compound an exciting target for total synthesis (Figure 1).
Some of the synthetic approaches for the synthesis of (–)-Ebelactone A have
been disclosed in literature. As part of our program towards the synthesis of Ebelactone
A, we chose to adopt a highly convergent strategy, disconnecting the carbon back bone
at C6-C7 trans alkene, thus dividing the target into two key fragments 2 and 3. The
synthesis of fragments 2 and 3 were envisaged from common precursor lactone 6 which
was generated from bicyclic ketone 7 (Scheme 1).
O O OH
O
Figure 1
(−)-Ebelactone A
1
16
Synthesis of lactone intermediate 6:
The synthesis of lactone intermediate 6 was achieved from bicyclic ketone 7,
which was inturn synthesized from furan and 2,4-dibromo-3-pentanone. Accordingly,
the acid catalyzed dibromination of 3-pentanone 8 afforded the dibromo compound 9.
The dibromo compound 9 when treated with furan in the presence of Zn-Cu couple
O O OH
O
O OH
O
O
O
O
OBn
O
OBn OH OH
BnO
MeO
OBn
O
O
O
(−)-Ebelactone A
1
2 6
7
14
C1-C6 Fragment C7-C14 Fragment
7
6 14
1
6
1
7 14
1
2 3
4
5
6
7
Scheme 1: Retrosynthetic Analysis
17
underwent a (3+4) cycloaddition reaction to afford the compounds 7, 10 and 11 in the
ratio 8:1:1. These bicyclic ketones on selective reduction with DIBAL-H gave the
corresponding alcohols (Scheme 2).
The required alcohol 12 was isolated from the other isomers using column
chromatography and the structure was confirmed from spectral studies. The hydroxyl
group of compound 12 was protected as its benzylether 13 using NaH and
benzylbromide. Asymmetric hydroboration of olefin 13 using (–)-
diisopinocampheylborane (Ipc2BH) proceeded smoothly gave the alcohol 14 with high
enantiomeric purity. The alcohol 14 was converted to the lactone 16 by a two step
sequence, PCC oxidation of alcohol 14 followed by Baeyer-Villiger oxidation afforded
the lactone 16. The lactone 16 was subjected regioslective methylation using LDA and
methyl iodide to afford the methylated lactone 6 (Scheme 3). Thus the lactone
compound 6 was employed as a common precursor for both the key fragments.
O O
Br Br
Br O 2 / AcOH
Zn-Cu couple
O
O
O
O
O
O
O
OH
DME, -10 OC
DiBAL-H
THF, -10 oC
+ +
+
8 9
10 11
12
Scheme 2
7
mixture of isomers
18
Synthesis of C1-C6 fragment:
The synthesis of C1-C6 fragment 2 was started from lactone intermediate 6
which is having three stereogenic functionalized carbons which serves as the C-2, C-3
and C-4 carbons of the (–)-Ebelactone A. Accordingly, reductive opening of bicyclic
lactone 6 with LiAlH4 afforded the triol 17, which was further treated with 2,2-DMP
and PTSA (cat.) to give acetonide compound 18 (Scheme 4).
O
OH
O
OBn
(-) -Ipc2BH
O
OBn
HO
O
O OBn
PCC / CH2Cl2
O
O
O
OBn
O
O
O
OBn
NaH / BnBr
THF, reflux
12 13
(+)-α-pinene
14
r.t,
15
m-CPBA, NaHCO3
DCM, 25 0C
16
Scheme 3
6
LDA/MeI
90% 95%
90%
90%
92%
THF, -78 oC.
O
O
OBn
O OH OBn OH OH
OH OBn O O
6
LiAlH4, THF
0 0C-25 0C.
17
2,2-Dimethoxypropane
p-TSA (cat), acetone
18
Scheme 4
85%
81%
19
The hydroxyl group of compound 18 was protected as benzyl ether 19,
followed by cleavage of the acetonide group in 19 using 2N HCl in THF/H2O (1:1)
afforded diol 4. Selective protection of primary hydroxyl group was achieved in diol 4
as its tert-butyldiphenylsilyl ether with TBDPSCl and imidazole to give 20 (Scheme
5).
Next aim was deoxygenation of the hydroxyl group at C-5 carbon of compound
20. Accordingly the secondary hydroxyl group of compound 20 was converted as its
xanthateester derivative 21 using NaH, CS2 and MeI in dry THF, followed by
deoxygenation using n-Bu3SnH and cat. AIBN as a radical initiator in toluene to give
deoxygenated product 22. Deprotection of TBDPS group in 22 with TBAF in THF
afforded corresponding primary alcohol 23 (Scheme 6).
OH OBn O O OBn O O
BnO
OBn OH OH
BnO
OBn OH OTBDPS
BnO
NaH, Bn-Br
THF, reflux, 85%
2N HCl, THF/H2O
25 0C, 90%
TBDPS-Cl
18 19
4 20
Scheme 5
imidazole, 88%
OBn OH OTBDPS
BnO
OBn O OTBDPS
BnO
S
SMe
OBn OTBDPS
BnO
OBn OH
BnO
CS2, MeI, NaH
nBu3SnH, AIBN
THF, reflux, 80%
Toluene, reflux,
75%
TBAF, THF
0 oC-25 0C,
90%
20 21
22 23
Scheme 6
20
The hydroxyl group of compound 23 was treated with tosylchloride,
triethylamine and cat. amount of DMAP in DCM to afford the tosylate compound 24,
compound 24 was treated with DBU and NaI in glyme to afford the terminal olefin 25
(Scheme 7).
Primary and secondary benzyl protecting groups of compound 25 was removed
using Li-Naphthalenide in dry THF to provide the 1,3-diol 26. Compound 26 was
subjected to chemoselective oxidation using TEMPO/BAIB and subsequently
converted to β-hydroxy carboxylic acid 27 under Pinnik’s conditions. β-hydroxy
carboxylic acid 27 was treated with benzenesulphonylchloride in dry pyridine to afford
the β-lactone fragment 2 (C1-C6 fragment) (Scheme 8).
OBn
BnO
OBn
BnO OH
OBn
BnO OTs
25
DBU, NaI
glyme, reflux
Scheme 7
23
TsCl, Et3N
CH2Cl2, 25 0C
24
95%
90%
OBn
BnO
OH
HO
OH
HO
O O
O
25 26
Li-Naphthalenide
THF, -30 oC
Scheme 8
27
2) NaClO2, NaH2PO4
t-BuOH-H2O
PhSO2Cl
Pyridine, -20 oC.
2
78%
70% for 2 steps
85%
1) TEMPO, BAIB
CH2Cl2
21
Synthesis of C7-C14 fragment:
The lactone intermediate 6 contained four stereoselectively functionalized
carbons to serve as the C8, C10, C11 and C12 carbons of the (–)-Ebelactone A and
further functionalisations were carried out on the compound 6 to give corresponding
fragment 3. Accordingly hydrolysis of the bicyclic lactone 6 with catalytic amount of
sulphuric acid in methanol afforded acetal 28 along with a minor amount of the α-
isomer (at C-1 center). The compound 28 was treated with LiAlH4 in dry THF to give
the alcohol 29. The alcohol 29 was converted to methylated product 5 by a two step
sequence, tosylation of the alcohol 29 followed by alkylation with
dimethyllithiumcuperate of the resulting tosylated compound 30 (Scheme 9).
Hydrolysis of acetal 5 in AcOH/water (2:1) at 50-55 °C afforded the lactol 31
which was further subjected to reduction with sodiumborohydride in methanol to give
diol 32. The diol 32 was converted to alcohol 35 by a three step sequence, initially
primary hydroxyl group of the compound 32 was selectively protected as its pivalate
ester 33 and then secondary hydroxyl group of 33 protected as its triisopropyl silyl
ether 34 followed by reduction with DIBAL-H to give alcohol 35 (Scheme 10).
MeO OH
OBn
O
MeO OTs
OBn
O MeO
OBn
O
O
O
O
OBn
O
COOMe
MeO
OBn
LiAlH4, THF
0 0C-25 0C
29
TsCl, Et3N
DMAP/CH2Cl2
0 0C-25 0C,
30
Scheme 9
Me2LiCu, Ether
-30 0C-0 0C
5
6
cat. H2SO4
MeOH
28
86% 90%
95%
92%
22
The primary hydroxyl group of compound 35 was oxidized using Dess-Martin
periodinane to corresponding aldehyde, which was subsequently converted to olefin 36
by Wittig olefination. Compound 36 was treated with Li-Naphthalenide to provide the
hydroxy compound 37. Dess-Martin periodinane mediated oxidation of compound 37
provided the keto compound 38. Silyl protecting group of compound 38 was removed
using aqueous-hydrofluoric acid (40 %) in acetonitrile at ambient temperature to furnish
the C7-C14 fragment 3 in good yield (Scheme 11).
MeO
OBn
O HO
OBn
O
OBn
HO
OH OBn
PivO
OH
OBn
PivO
OTIPS OBn OTIPS
HO
5 31
AcOH/H2O (2:1)
50-55 0C
NaBH4
MeOH, 0 0C
32
Piv-Cl, DMAP
pyridine:CH2Cl2
0 0C-r.t.
33
CH2Cl2, 0 0C
34
DIBAL-H, CH2Cl2
-78 0C
35
TIPSOTf,
2,6-lutidine
Scheme 10
60% 85%
95%
90%
90%
O OTIPS
O OH
OBn OTIPS
HO
OBn OTIPS
OH OTIPS
DMP
DCM, 90%
HF-aqueous (40%)
38
3 Scheme 11
35
1. DessMartin Reagent,
DCM
2. LiHMDS, PPh3=CH2
-78 oC - r.t
36
Li-Naphthalenide
THF, - 35 0C
37
78% for 2 steps
78%
Acetonitrile, 20 oC
95%
23
Coupling of C1-C6 fragment with C7-C14 fragment:
Finally fragments 2 and 3 were planned to couple using olefin cross metathesis
approach29 to construct tri-substituted olefin as well as finish the total synthesis of
target molecule (–)-Ebelactone-A 1. For this crucial transformation we selected the
Grubbs second generation catalyst 39 according to literature procedures. Initially we
tried the cross metathesis reaction between fragments 2 and 3, the cross-metathesis
reaction was not succeeded (Scheme 12).
The cross-metathesis reaction was performed between compounds 2 and silyl
protected compound 38 with Grubbs second generation catalyst 39 in dichloromethane
at reflux temperature about 15 h, the reaction was not proceeded both starting materials
were recovered completely (Scheme 13).
O
O
O OH
O
O
O OH
MesN NMes
PCy Ph 3
Cl
Cl
+
Grubbs 2nd generation
Catalyst, 15 mol%
(−)-Ebelactone-A
CH2Cl2, 40 oC
X
Scheme 12
2nd generation
Ru Grubbs catalyst
2
3
1
39
O
O
O OTIPS
Grubbs 2nd generation
Catalyst
X No Product
2 38
39
+
Scheme 13
24
By the observation of above results we assumed that homoallylic keto group of
C7-C14 fragment 3 is responsible for failure of cross metathesis, then we planned the
cross metathesis reaction between compound 2 and homo-allylalcohol 37 with the same
Grubbs catalyst 39, in this reaction also no cross metathesis product was observed
(Scheme 14).
In conclusion synthesis of fully functionalized C1-C6 Fragment and C7-C14
fragments of (–)-Ebelactone-A has been achieved. This involved a novel strategy in
which bicyclic intermediate has been elaborated in a stereo controlled manner by using
desymmetrization of meso-bicyclic compound by using asymmetric hydroboration.
O
O
OH OTIPS
Grubbs 2nd generation
Catalyst
X No Product
2 37
39
+
Scheme 14
25
This chapter divided into two sections.
Section A: This section deals with the introduction and earlier synthetic approaches of
(–)-Maurenone.
The natural product maurenone was isolated by Faulkner et al. in 1986 from
specimens of the pulmonate mollusc Siphonaria maura, collected from Jaco Beach,
Costa Rica. In this section briefly discussed about the diverse polyketide natural
products from marine pulmonates of the genus Siphonaria.
Section B: This section describes the formal stereoselective synthesis of (–)-Maurenone.
The natural product maurenone was isolated by Faulkner et al. in 1986 from
specimens of the pulmonate mollusc Siphonaria maura, collected from Jaco Beach,
Costa Rica (Figure 1).
Marine pulmonates of the genus Siphonaria are rich sources of diverse
polyketide-derived natural products. There are some examples on polyketide natural
products which include siphonarin A and B, muamvatin, denticulatin A and B,
membrenone A–C, and vallartanones A and B. All species examined are the
metabolites of polypropionate origin that appear to share a common biosynthesis with
macrolides and polyether antibiotics.
O
OH
O
1
(−)-Maurenone
Figure 1
CHAPTER-III
26
In continuation of our interest on the total synthesis of polypropionate natural
products, and extreme scarcity of the natural material together with the novel structure
prompted us to attempt the total synthesis of (–)-maurenone.
The formal synthesis of (–)-Maurenone 1 was achieved via stereoselective
construction advanced intermediate 2, which was synthesized by coupling of fragments
3 and 4. The two key intermediates 3 and 4 were synthesized by silyl triflate mediated
opening of epoxy alcohol 5 and desymmetrization of the meso-bicyclic dihydrofuran 7
by an asymmetric hydroboration (Scheme 1).
O
OH
O
OTBSO
H
O OTES
O
OBn
O
OH O
O
O
OBn
OTBSOH O OTES
+
1
2
3 4
5 6 7
(−)-Maurenone
Scheme 1: Retrosynthetic strategy.
27
The synthesis of fragment 3 began with α,β-unsaturated ester 9, which was
synthesized from n-propanal 8 using stabilized three-carbon Wittig olefination to give
exclusively the E-isomer in 70% yield. Reduction of 9 with DIBAL-H furnished the
allylic alcohol 10 in 80% yield, which on Sharpless asymmetric epoxidation gave the
chiral epoxy alcohol 5. Upon treatment of epoxy alcohol 5 with tert-butyldimethylsilyl
triflate (TBSOTf) at –42 °C gave the syn-aldol product 3 in 85% yield (Scheme 2).
Synthesis of fragment 4 began with bicyclic olefin 7. Asymmetric
hydroboration of olefin 7 using (+)-diisopinocampheylborane (Ipc2BH) proceeded
smoothly to give the alcohol 11 with high enantiomeric purity. The alcohol 11 was
converted to the lactone 6 by a two step sequence, PCC oxidation of alcohol 11
followed by Baeyer-Villiger oxidation afforded the lactone 6. Triol compound 12 was
prepared by the reductive opening of lactone 6 with LiAlH4. Compound 12 was
protected as its acetonide 13 using 2,2-DMP/CSA(cat.). The primary hydroxyl group of
13 was tosylated with TsCl/Et3N and then treated with dimethyllithiumcuprate to afford
the compound 14 in good yield. Acetonide deprotection of compound 14 gave the 1,3-
diol, which was further converted into mono tosylated compound 15 in 85% yield.
Reductive elimination of tosyl group of 15 with LAH gave the secondary alcohol 16
(Scheme 3).
H
O O
OEt
OH
OH
O OTBS
H
O
OTBSO
H
CH2Cl2, 0-25 oC.
DIBAL-H
CH2Cl2, 0- 25 oC.
(-)-DET, TBHP,
CH2Cl2, -23 oC, CH2Cl2, MS oA, -42 oC,
85%.
TBSOTf, EtNiPr2
Ph3P=C(Me)CO2Et
8 9 10
5 3
=
Scheme 2
28
Compound 16 was then subjected to IBX oxidation to furnish keto compound
17 in good yield. Hydrogenolysis of benzyl ether 17 followed by TES protection with
TESOTf afforded the fragment 4 in 92% yield (Scheme 4).
O
OBn
O
O
OH OH OBn OH
O O OBn OH O O OBn
OTs OH OBn OH OBn
O
OBn
(+) -Ipc2BH
O
OBn
HO PCC / CH2Cl2
LiAlH4, THF
0 0C-25 0C, 4h, 85%
2,2-Dimethoxypropane
CSA (cat), DCM, 80%
2. Me2CuLi, Ether
1. TsCl, Et3N,
00C-250C, CH2Cl2
0 0C to -25 0C, 90%
2. TsCl, Et3NH,
0 0C-25 0C, CH2Cl2
1. 2N HCl, THF-H2O
LiAlH4, Ether
0 0C-25 0C, 90%
6 12
13 14
15
Scheme 3
16
7
(-)α-pinene
11
r.t
m-CPBA, NaHCO3
96% DCM, 25 0C
1)
2)
O OBn
O OTES
OH OBn
IBX
DMSO -THF, 88%
1. Pd/C,H2
Ethylacetate,95%
TESOTf
2,6-Lutidine
-78 0C, 92%
2.
17
4
Scheme 4
16
29
The coupling of compounds 3 and 4 gave the key fragment 2 as diastereomeric
mixtures which was consistent with literature (Scheme 5).
In conclusion, the formal synthesis of (–)-Maurenone has been achieved in a
stereocontrolled manner by silyl triflate mediated opening of epoxy alcohol and
desymmetrization of the meso-bicyclic dihydrofuran using an asymmetric
hydroboration. This synthetic sequence provides an easy access to the construction of
the key fragments of maurenone.
O OTES OTBSOH O OTES
O
OH
O
LiHMDS, Et2O
-78 to -50 oC
Fragment-3, -78 oC
(−)-Maurenone
Scheme 5
4 2
1



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