Open Access Repository of Indian Theses

Koul, Bhupendra

Abstract

Expression of Insecticidal Toxin Coded by Modified Full length and Truncated Bt-cry1Ac Gene in Transgenic Tomato for Assessment of their Stability and Efficacy Against Target Insects

In spite of several modifications reported to improve expression of the cry1Ac gene in plants achieving high expression of Cry1Ac toxin does not seems to be a routine affair and reflect complexcities for differential expression of insecticidal toxin in plants from full-length and truncated version of the gene (Diehn et al., 1996; Rocher et al., 1998; Burma et al., 2011). In a recent report it has been demonstrated in tobacco and cotton that the expression of Cry1Ac endotoxin has detrimental effect on both in vitro and in vivo growth and development of transgenic plants (Burma et al., 2011). These observations have inspired us to give another dimension to our research objective. Tomato expressing Cry1Ab insecticidal protein from Bt, giving complete protection against H. armigera damage in laboratory, green house and field has been reported earlier (Kumar and Kumar 2004; Srivastava S Ph.D thesis ). The truncated cry1Ab gene bears 80% homology with truncated cry1Ac gene and the results which would be generated regarding truncated and full length cry1Ac transgenic tomato will be compared with that of cry1Ab transgenic tomato. The outcome of this comparative study will reveal the reliability and toxicity of both the truncated variants of the cry1A genes, in tomato. In view of these facts the present problem is identified with following objectives.  
	Development of transgenic population of tomato with modified full-length and truncated version of cry1Ac gene and cry1Ab.
	To assess the stability and efficacy of insecticidal toxin coded by full-length cry1Ac gene in plants compared to 3’ truncated version.
	 Recovery of promising transgenic event in tomato with full-length cry1Ac gene and cry1Ab.
	Molecular characterization of insecticidal toxins coded by two forms of the gene in transgenic tomato plants.
	The stability, efficacy and inheritance of cry1Ac gene shall be compared to that of cry1Ab gene in transgenic tomato. 




English

Expression of Insecticidal Toxin Coded by Modified Full length and Truncated Bt-cry1Ac Gene in Transgenic Tomato for Assessment of their Stability and Efficacy Against Target Insects

Padhye, Sameer Mukund

Ecology and Biodiversity of Branchiopods with special reference to Cladocerans from Pune region

Tripathi, Manish Kumar

MIXED OXIDES AND METAL SUPPORTED APATITE CATALYSTS
FOR ORGANIC TRANSFORMATIONS
The thesis mainly deals with the development of heterogeneous solid catalysts and
their applications in organic transformations particularly C-C bond formation reactions.
Chapter I highlights the importance of heterogeneous catalysis and discuss various options
for the synthesis of heterogeneous catalysts. The need and development of cleaner and
greener alternative technologies using truly heterogeneous catalytic system in the synthesis
of pharmaceuticals and fine chemicals is also described. Chapter II describes the 1,4-
addition of phenylboronic acids to α,β-unsaturated carbonyl compounds by using rhodium
fluorapaptite (RhFAP) catalyst. Chapter III deals with the conversion of azides to N-(tertbutoxycarbonyl)
amines and the synthesis of 1,3-butadienes by using palladium fluorapatite
(PdFAP) catalyst. Chapter IV deals with the synthesis of tetrazoles and propargylamines
by using zinc based catalysts. Chapter V mainly deals with the synthesis of α-diazo-β-
hydroxy and α-diazo-β-amino carbonyl compounds using Mg/La mixed oxide catalyst and
β-keto esters by using and Pd0/Mg-La mixed oxide catalyst.
Chapter-I: Introduction
This chapter describes the various catalyst / process options available for an
industrial chemist to effect different organic transformations. It includes a brief
introduction of homogeneous, heterogeneous catalysis and basic concepts commonly
encountered in catalysis such as selectivity, turnover number, atom economy etc. This
chapter also describes the structure, properties and applications of basic supports such as
apatites, hydrotalcites, mixed oxides and their importance as catalysts in organic reactions.
Abstract ii
The need for the design and development of environmentally cleaner catalytic
methodologies using supported solid catalysts is also presented in this chapter.
Chapter-II: Rhodium fluorapatite catalyzed conjugative addition of phenylboronic
acids to ,-unsaturated carbonyl compounds
Rhodium catalyzed carbon-carbon bond formation via cross-coupling of
organoboron compounds and organic electrophiles is one of the most important reactions
in organic synthesis. Rhodium catalyzed conjugate addition of arylboronic acids to ,-
unsaturated carbonyl compounds has received considerable attention in the field of organic
synthesis, ever since it was reported by Miyaura et al. in 1997. The discovery of such and
important reaction has triggered much research activity in the area of carbon-carbon bond
formation catalyzed rhodium complexes. Despite the advantages of homogeneous metal
complex catalysts, difficulties in recovering the expensive catalyst metals and ligands from
the reaction mixture severely limit their industrial applications. Moreover the use of toxic
and expensive phosphine ligands is undesirable in viewpoint of environment.
Heterogeneous catalysis is particularly attractive as it allows production and ready
separation of large quantities of products with the use of a small amount of catalyst.
The present chapter describes the conjugate addition of phenylboronic acids to ,-
unsaturated carbonyl compounds to yield conjugate addition products (Scheme 1).
R1B(OH)2
RhFAP
Methanol,
1,5-cod, N2, 65 °C
+
O
R2
O
R1 R2
Scheme 1. Conjugate addition of arylboronic acids to α,β-unsaturated carbonyl compounds
catalyzed by RhFAP
Abstract iii
Chapter-III: One pot approach for the synthesis of N-(tert-butoxycarbonyl)amines
and 1,3-butadiene derivatives using highly efficient palladium fluorapatite catalyst
This chapter is divided into two sections.
Section I: This section deals with the chemoselective reduction of azides to N (tbutoxycarbonyl)
amines by using PdFAP catalyst.
Section II: This section deals with the three component coupling of aryl iodides, alkynes
and alkenes by using PdFAP catalyst.
Section I: Palladium fluorapatite catalyzed chemoselective reduction of azides to N-
(tert-butoxycarbonyl)amines
Organic azides are an important class of compounds, particularly for producing
primary amines via reductive transformation, and several reducing agents have been
developed to date. In a closely related field, protection of amines with appropriate
protecting agents also plays an important role in multistep synthesis. Among the various
amine-protecting group, the tert-butoxycarbonyl (Boc) group is frequently used due to its
chemical stability to acids, bases, and hydrogenation. Based on this background, it is
reasonable to consider that the combination of azide reduction and N-Boc-protection
should offer an efficient approach to the manipulation of organic amino compounds.
The present chapter describes one pot method for the transformation of azide to NBoc-
protected amines by using palladium fluorapatite (PdFAP). This catalyst is recovered
and recycled for several times without loss of activity (Scheme 2).
Scheme 2. Chemoselective conversion of azide to Boc-protected amine using PdFAP
R
NH-Cbz
R
PdFAP NH-Cbz
H2 atm, Boc2O
EtOAc, r.t.
R=Phenyl
N3 NH-Boc
Abstract iv
Section II: An efficient palladium fluorapatite catalyzed three component coupling of
aryl iodides, alkynes and alkenes
One of the most attractive synthetic strategies favored by organic chemists is the
multi-component domino coupling reactions for constructing polyfunctional molecules by
linking several organic moieties through carbon-carbon bond formation in one step. In
particular, the transition-metal catalyzed coupling reactions are recognized as versatile
tools for constructing complex molecules from simple starting materials. Among such
reactions, palladium-catalyzed carbon-carbon bond formation reactions are the most
versatile by virtue of their generality and ability to tolerate a wide range of important
organic functional groups. In the present chapter, a three-component coupling of aryl
iodides, alkynes, and alkenes to generate 1,3-butadiene derivatives using palladium
fluorapatite (PdFAP) catalyst is described (Scheme 3).
DMF/H2O (9:1)
+ +
Yield: up to 71 %
+
130 oC
PdFAP
R1I
R1
R4
R2 R3 R4 R2
R1
R3
R4
Scheme 3. Three-component coupling of iodobenzene, diphenylacetylene and butyl
acrylate by using PdFAP catalyst.
Chapter-IV: Synthesis of 5-substituted 1H-tetrazoles and propargylamines by using
zinc based catalysts
This chapter is divided into two sections.
Section I: This section deals with the synthesis 5-substituted 1H-tetrazoles by using zinchydroxyapatite
catalyst
Section II: This section deals with the synthesis of propargylamines using zinc dust.
Section I: Zinc hydroxyapatite catalyzed efficient synthesis of 5-substituted 1Htetrazoles
Abstract v
Tetrazoles, a class of heterocycles, received considerable attention because of their
wide range of applications in pharmaceuticals as lipophilic spacers, in speciality
explosives, photography, and information recording systems. They are important ligands
for many useful transformations and also precursors for a variety of nitrogen containing
heterocycles. The most convenient route for the synthesis 5-substituted 1H-tetrazoles is the
addition of azide ion to nitriles. The earlier reports for this transformation fall in to three
main categories: i) the use of tin or silicon azides, ii) the use strong Lewis acids, and iii)
running the reaction in acidic media. The drawbacks in the earlier methods are, use of
expensive and toxic metals, severe water sensitivity, and the presence of hydrazoic acid,
which is highly toxic and explosive as well as volatile. It is also known that acid-catalyzed
cycloaddition between hydrazoic acid and isocyanides is one of the main routes to 5-
substituted 1H tetrazoles. However, this procedure needs the direct addition of large excess
amounts of dangerous and harmful hydrazoic acid. Therefore, it is desirable to develop a
more efficient and convenient method for the synthesis of 5-substituted 1H- tetrazoles.
In the present chapter, the synthesis of 5-substituted 1H- tetrazoles by addition of
sodium azide with organic nitriles using zinc hydroxyapatite is described. This catalyst is
recovered and recycled for several times without loss of activity (Scheme 4).
R
CN
NaN3
R N N
N
HN
DMF, 120-130 oC
+
ZnHAP
Scheme 4. Synthesis of 5-substituted 1H-tetrazoles using ZnHAP
Section II: Efficient one-pot synthesis of propargylamines using zinc dust
One pot multi-component coupling reaction (MCR) by coupling of several organic
moieties in one step is an attractive synthetic strategy. Propargylamines are important
Abstract vi
synthetic intermediates for biologically active compounds such as β-lactums,
conformationally restricted peptide, isosteres, natural products and therapeutic drug
molecules. The standard practice for synthesis of propargylamines is carried out by
amination of propargylic halides, propargylic phosphates or propargylic triflates.
The present chapter deals with the three-component coupling of aldehydes, amines and
alkynes to generate propargylamines using zinc dust without using any co-catalyst or
additive (Scheme 5).
 Easy separation of the catalytic by simple centrifugation.
 No other co-catalyst or additive is required.
 No pre-activation of Zn dust is required.
Scheme 5. Synthesis of propargylamines by using zinc dust
Chapter-V: Synthesis of α-diazo-β-hydroxy and α-diazo-β-amino carbonyl
compounds using Mg/La mixed oxide catalyst and β-keto esters by using Pd0/Mg-La
mixed oxide catalyst
This chapter is divided into two sections.
Section I: This section deals with the synthesis of α-diazo-β-hydroxy and α-diazo-β-amino
carbonyl compounds using highly basic Mg/La mixed oxide catalyst.
Section II: This section deals with the arylation of Baylis-Hillman adducts with iodo- and
bromoarenes using Pd0-Mg/La mixed oxide
Section I: Synthesis of α-diazo-β-hydroxy and α-diazo-β-amino ester compounds via
aldol type reaction of aldehydes and imines with ethyl diazoacetate using basic Mg/La
mixed oxide catalyst
R1-CHO + R2R3NH +
Zn dust
reflux
NR2R3
R1
R4
R4 H
CH3CN
Abstract vii
α-Diazo carbonyl compounds are potential source of amino alcohols and acids. α-
Diazo carbonyl compounds are generally prepared by the azido transfer reaction of
carbonyl compounds. Condensation of acyldiazomethane to aldehydes and imines yields α-
diazo carbonyl compounds but requires deprotonation of the acyldiazomethane. Some of
the methods reported require low temperature and absolutely anhydrous conditions;
moreover the use of strong bases may not be compatible with functional groups in the
substrates. In recent years, there has been increasing emphasis on the use and design of
environment-friendly solid catalysts to reduce the amount of toxic waste. The use of a solid
catalyst allows easy separation of catalyst from the reaction mixture in large-scale
production to conform to the concept of green technology.
The present chapter addresses the synthesis of β-hydroxy-α-diazo carbonyl
compounds and β-amino-α-diazo carbonyl compounds by condensation of a wide variety
of aldehydes and imines with ethyl diazoacetate using the Mg/La mixed oxide catalyst in
water (Scheme 6). The noticeable merits of Mg/La mixed oxide catalyst in synthesis of β-
hydroxy-α-diazo carbonyl compounds and β-amino-α-diazo carbonyl compounds are:
1) The reaction is performed at room temperature.
2) Low catalytic loading with good reusability.
3) Water is used as a solvent in the reaction, which is safer, cheaper and more
environmentally friendly.
Scheme 6. Synthesis of diazo carbonyl compounds by using Mg/La mixed oxide.
R
X
H
N2
CO2Et
R
N2
CO2Et
XH
Water, rt
+
Mg/La mixed oxide
X= O, N-TS
Abstract viii
Section II: Synthesis of β-keto esters via arylation of Baylis-Hillman adducts with
iodo- and bromo- arenes catalyzed by Pd0/Mg-La mixed oxide catalyst
Baylis-Hillman adducts are interesting class of disubstituted olefins possessing both
allylic alcohol and α,-unsaturated ester moieties in which carbon-carbon double bond is
an integral part of both allyl alcohol and α,-unsaturated ester moieties. Such a highly
desirable olefin arylation is an important area in the synthesis of β-keto esters products, an
important and useful class of synthons. Few reports on such coupling are reported in
literature using homogeneous palladium salts. Despite the synthetic elegance and high
turnover numbers, the non-reusability of the precious palladium precludes wide synthetic
applications in the pharmaceutical industry. Heterogeneous catalysts have been proven to
offer similar levels of activity to their homogeneous counterparts whilst eliminating the
need for an extensive work-up and preventing the metal from being found in the product,
which restricts its use in industry.
In the present chapter, the arylation of di-substituted (Baylis-Hillman adducts)
olefins with iodo- and bromoarenes to afford -keto ester products in good yields via -
hydride elimination in a single pot by using Pd0/Mg-La mixed oxide catalyst (Pd0/Mg-La
MO) (Scheme 7) is described.
Scheme 7. Arylation of Baylis-Hillman adduct with iodo- and bromobenzenes using
Pd(0)/MgLa MO catalyst
R2
CO2CH3
OH
R1
X
Pd0/Mg-La MO
DMF, Base
R2
CO2CH3
O
X = Br , I R1
90o C
R2
CO2CH3
OH
R1
+
R2= Aromatic,
Aliphatic




Melatonin, sex steroids, and innate immune responses of splenic macrophages in the fresh-water snake, Natrix piscator

KULKARNI, GIRISH JAYANT

Molecular characterization of Ochrobactrum spp. isolated from human gastric biopsies

REDDY, M. VENKATESWER

		Abstract

The majority of conventional plastics are produced based on fossil fuels. Conventional plastics are durable and degrade slowly and their incineration releases pollutants, they contribute to environmental problems. In response to the world’s growing awareness about environment and limited fossil fuel resources, intensive research have focused on the production of bioplastics as “green” alternatives to synthetic plastics. Bioplastics such as polyhydroxyalkanoates (PHA) have attracted great interest due to their biodegradable nature. PHA are high molecular weight carbonaceous compounds produced as cellular reserve storage products under excess carbon and nutrient deprived conditions to protect bacteria against starvation. PHA has special physical traits, such as the elasticity, high crystallization rate, high degree of polymerization and can be converted to a wide range of finished products for application in industry, agriculture and medicine. 

One of the major drawbacks for commercialization of PHA production is the high production cost, in which feedstock accounts for 50% of the overall price. Hence, there is a big interest in the use of cheap renewable resources. PHA production from renewable resources and their complete biodegradability make them promising from an environmental point of view. A considerable effort has gone in production of PHA using mixed cultures of microorganisms and cheaply available wastewater. The merits of PHA production with mixed culture include an enhanced economy, a simpler process control, non sterile conditions and an improved use of wastes. Mixed culture utilization can lower the input costs by allowing for large scale fermentations to occur without overhead costs of sterilization. It also allows for a greater variety of substrates to be used due to the presence of several PHA producing organisms. As an alternative to sugars, to attain commercial viability as well as to further improve the sustainability profile of PHA production by fermentation, cheap renewable resources or secondary raw materials must be used instead of expensive carbohydrates such as glucose. Utilization of wastewater as substrate and mixed culture as biocatalyst makes these technologies as sustainable for bioplastics production as well as waste management. Waste/wastewater generally contains good amount of organic matter which can be considered as an ideal commodity to produce bioplastics. 

Various studies have already been conducted in the direction of utilizing wastewater as substrate, but many other organic waste streams are available as well and are currently not valorized. However, there still remain certain limitations to overcome these technologies to become alternatives for the traditional energy production processes. Lower substrate conversion efficiency, enhancement of bioplastics production, production of co-polymer instead of homopolymer is the major concerns which should be addressed prior to the up scaling of bioplastics production. In this context present thesis deals with some of the strategies to reduce the substrate cost, and regulate microbial metabolism towards enhancement of bioplastics production. Mixed culture bioplastics production process was understood by optimizing different critical factors and PHA production was enhanced using Taguchi – DOE methodology. PHA was produced using different types of pure substrates and waste streams with pure and mixed type of bacteria. PHA production was enhanced by employing bioaugmentation strategy and by addition of co-factor (molybdenum). Bioelectrochemical behavior of biocatalyst during PHA production was evaluated using cyclic voltammetry. Microbial diversity analysis was done for mixed culture to know the organisms involved in PHA production process by using molecular biology techniques. Bioplastics production process was integrated with acidogenic fermentation to utilize the acid rich effluents generated during hydrogen production process towards reduce production cost along with additional waste remediation. 




PRODUCTIONOF BIOPLASTICS AS POLYHYDROXYALKANOATES FROM WASTE REMEDIATION: REGULATING MICROBIAL METABOLISM TOWARDS PROCESS ENHANCEMENT

Kad, Trupti Dattatray

Gene Expression Analysis of 2 acetyl-1-pyrroline Biosynthesis Gene(s) In Pandanus amaryllifolius Roxb

P, Balamurugan

Microbial fouling, corrosion and their control in the freshwater distribution system of a nuclear test reactor

K., Balaji

Synopsis | 1 
 
The thesis entitled “Culture independent detection and characterization of 
Streptococcus pyogenes and its transcriptional gene regulation upon subinhibitory 
exposure to antibiotics” deals with the use of culture independent approaches for the 
detection of pharyngitis causative bacteria directly from the throat swabs; molecular 
characterization and comparison of S. pyogenes isolated from different infection cases 
and the in vitro and in vivo analysis of S. pyogenes challenged with subinhibitory 
concentrations of antibiotics. The work elements have been divided into five major parts 
viz., 
1. Culture independent analysis of throat swabs from pharyngitis patients and 
healthy subjects by Denaturing Gradient Gel Electrophoresis.  
2. Culture independent analysis of throat swabs from pharyngitis patients and 
healthy subjects by Terminal Restriction Fragment Length Polymorphism. 
3. Development of multiplex PCR assay for the detection of pharyngitis causing 
bacteria directly from the throat swabs of pharyngitis patients. 
4. Comparative molecular analysis of Streptococcus pyogenes isolates from ocular 
infections, pharyngitis and asymptomatic children in south India. 
5. Effect of subinhibitory concentrations of fluoroquinolones on biofilm production 
by clinical isolates of Streptococcus pyogenes. 
6. In vitro and in vivo analysis of genes involved in virulence, biofilm formation and 
antibiotic resistance in Streptococcus pyogenes exposed to subinhibitory 
concentrations of antibiotics. 
 
Streptococcus pyogenes is one of the most common cause of bacterial pharyngitis 
in human, which upon left untreated may result in deadly post-infection sequelae such as 
scarlet fever, rheumatic heart disease and necrotizing fasciitis. Apart from S. pyogenes, a 
number of other bacteria are also involved in the pharyngitis. A greater understanding of 
the bacteria involved is needed to improve our knowledge as well as prescribing right 
method to eliminate the causative pathogen. Increasing knowledge in the field of 
molecular diagnostics opens up new strategies to explore the changes in bacterial 
community structure between the throats of pharyngitis and healthy subjects. In addition, 
PCR based methods allow rapid, accurate and simultaneous detection of more than one 
causative bacterium directly from pharyngitis throat swabs by means of multiplex PCR. It 
also allows molecular comparison of S. pyogenes strains isolated from different infection 
Synopsis | 2 
 
cases. Apart from diagnostics and molecular characterization, the present study is focused 
on the in vitro and in vivo effects of subinhibitory concentrations of various antibiotics 
against S. pyogenes isolates. 
      The section “Culture independent analysis of throat swabs from pharyngitis 
patients and healthy subjects by Denaturing Gradient Gel Electrophoresis” deals 
with the comparative analysis of throat metagenomes of pharyngitis patients and healthy 
subjects using nucleic acid based culture independent method Denaturing Gradient Gel 
Electrophoresis (DGGE). In this study, 40 throat metagenomes each from healthy and 
pharyngitis patients were taken. The 16S rRNA gene of the metagenomes was amplified 
through two rounds of PCR. In the first round PCR, ~1500 bp 16S rRNA gene was 
amplified using universal 16S primers. The second round PCR is a nested PCR in which 
first round PCR amplicons were used as templates and V3 region of 16S rDNA (~250 bp) 
was amplified using GC-clamped primers. DGGE reference marker for inter-gel 
comparison of DGGE gels was prepared using Streptococcus pyogenes, Staphylococcus 
aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Haemophilus influenzae. 
DGGE banding patterns generated directly from the throat swabs showed considerable 
difference between healthy and pharyngitis samples. The number of separate bands in the 
studied samples ranged from 3 to 26 in healthy subjects and 2 to 29 in the case of 
pharyngitis samples. Comparative band analysis showed the presence of more pathogens 
in pharyngitis samples than the healthy subjects. S. pyogenes in 8 (20%); S. aureus in 9 
(22.5%); P. aeruginosa in 4 (10%); H. influenzae and K. pneumoniae in 7 (17.5%) in 
healthy subjects’ throat swabs and S. pyogenes in 20 (50%); S. aureus in 16 (40%); P. 
aeruginosa in 9 (22.5%); H. influenzae in 13 (32.5%) and K. pneumoniae in 19 (47.5%) 
in pharyngitis samples. Sequencing of dominant bands revealed the presence of 
uncultured Capnocytophaga sp., Enterobcter sp., Serratia sp. and Aeromonas sp. in the 
throat metagenomes.  
The section “Culture independent analysis of throat swabs from pharyngitis 
patients and healthy subjects by Terminal Restriction Fragment Length 
Polymorphism” deals with the comparative analysis of throat metagenomes of 
pharyngitis patients and healthy subjects using terminal restriction fragment length 
polymorphism (T-RFLP). For T-RFLP analysis, metagenomic DNA was extracted from 
10 throat swabs each from healthy and pharyngitis patients. The 16S rRNA genes of 
Synopsis | 3 
 
bacteria present in throat metagenomes were amplified by PCR with 6-carboxy-
fluorescein (Fluorophore) - labeled universal forward primer (6-FAM - 27F) and a 
universal reverse primer (1513R). The 16S rDNAs were digested with restriction 
enzymes with 4bp recognition sites such as MspI or RsaI and analyzed by using an 
automated DNA sequencer. T-RFLP patterns were numerically analyzed using computer 
programs. From analysis of the throat bacterial community, patterns derived from MspI 
and RsaI digested samples of healthy subjects and pharyngitis patients were grouped into 
different clusters, though RsaI digested samples showed some uncertainty. Pharyngitis 
throats generated an average species richness of 9 (2.1 [SD]) and 10 (2.9) for MspI and 
RsaI digests respectively, whereas healthy throats generated 6.3 (1.2) and 6.1 (1.5) in 
MspI and RsaI digests, respectively. These results suggest that samples from pharyngitis 
patients contain an unexpected diversity of causative bacteria.  The pharyngitis throats 
were colonized with a rich diversity of bacterial species than that of healthy throats. In T-
RFLP analysis, the reference strain, Streptococcus pyogenes SF370 was used for T-RF 
comparison. The T-RF patterns of the reference strain coincides with the Streptococcus 
pyogenes T-RFs generated from the metagenomes.  
 The section “Development of multiplex PCR assay for the detection of 
pharyngitis causing bacteria directly from the throat swabs of pharyngitis patients” 
deals with the development of a pentaplex PCR assay for rapid and simultaneous 
detection of five oral pathogens namely S. pyogenes, S. aureus, P. aeruginosa, N. 
gonorrhoeae and K. pneumoniae directly from the throat swabs of pharyngitis patients. 
The designed species-specific primers detected the five targeted pathogens without any 
cross reaction. Multiplex PCR amplification of the five species specific genes was 
achieved by optimizing the PCR conditions. 16S rRNA gene was taken as internal 
control. Annealing temperature of 58°C for 1 min was found to be the optimum 
temperature for the pentaplex PCR amplification. The sensitivity checking using serially 
diluted DNA (20ng to 1.25ng) showed unambiguous amplification up to 2.5ng of DNA 
and slight amplification at 1.25ng. Metagenomic DNA (n=39) isolated from throat swabs 
of pharyngitis patients were used to evaluate the developed assay. The multiplex PCR 
assay revealed the presence of P. aeruginosa, S. aureus and S. pyogenes in 2 throat swab 
samples each. None of the metagenomes showed the presence of N. gonorrhoeae and K. 
pneumoniae or the combination of P. aeruginosa, S. aureus and S. pyogenes. This study 
Synopsis | 4 
 
once again proves that multiplex PCR based pathogen detection is an effective tool in the 
field of clinical diagnostics. 
 The section “Comparative molecular analysis of Streptococcus pyogenes 
isolates from ocular infections, pharyngitis and asymptomatic children in south 
India” deals with the molecular characterization of S. pyogenes isolates from ocular, 
pharyngitis and asymptomatic carrier cases. In the present study, emm type surveillance 
as well as its association with toxin gene profiles was analyzed. S. pyogenes was detected 
in 20 isolates of the ocular infected cases such as lacrimal abscess, corneal ulcers and 
mucocoele. For noninvasive isolates, 370 pharyngitis cases and 400 asymptomatic school 
children were screened and recovered 33 pharyngitis and 14 carrier isolates respectively.  
Fourteen emm type distributions were observed in ocular isolates whereas 11 emm types 
each were noticed in pharyngitis and asymptomatic carrier isolates. The two dominant 
emm types, emm49 and emm63 were accounted for 33% of the total S. pyogenes isolates. 
Among ocular isolates, slo, smeZ, speB and speG were found in >50% of isolates and in 
pharyngitis smeZ (48%), speB (45%) and speG (42%) genes were found to be prevalent. 
Alarmingly, carrier isolates showed more prevalence to virulence genes than the ocular 
and pharyngitis isolates with speF (79%), speB, speG (64%), slo and sil (64%). Among 
the three groups, pharyngitis isolates harbored more prtF1 (33%) and prtF2 (94%) genes 
than the asymptomatic carriers (28% and 71%) and the ocular isolates (45% and 40%). 
450 bp size band in prtF1 and 350 bp size band in prtF2 showed dominance. Among the 
three groups tested, the distribution of ermB and mefA was high in pharyngitis isolates 
(30%) where 10 isolates showed the presence of both genes. None of the isolates showed 
the presence of ermA and tetO genes. Dendrogram generated based on the virulence and 
antibiotic resistance gene profiles revealed that the isolates are intermingled (except few 
clusters), irrespective of their emm types and isolation source. 
 The section “Effect of subinhibitory concentrations of fluoroquinolones on 
biofilm production by clinical isolates of Streptococcus pyogenes” deals with revealing 
the effects of sub-MICs of fluoroquinolones (FQs) on S.  pyogenes biofilm formation. In 
this study, biofilm forming six M serotypes M56, st38, M89, M65, M100 and M74 of S. 
pyogenes strains were challenged against four FQs namely, ciprofloxacin, ofloxacin, 
levofloxacin and norfloxacin. The antibiofilm potential of these FQs were analyzed at 
their subinhibitory concentrations (1/2-1/64 MIC) using biofilm assay, XTT reduction 
Synopsis | 5 
 
assay, Scanning Electron Microscopy and Confocal Laser Scanning Microscopy. From 
the four FQs tested, ofloxacin and levofloxacin at 1/2 MIC showed the maximum 
inhibition (92%) of biofilm formation against M56 and M74 serotypes.  FQs effectively 
interfered in the microcolony formation of S. pyogenes strains at 1/2-1/8 sub-MICs. 
Interestingly, inhibition of biofilm formation was greatly reduced beyond 1/16 MICs and 
allowed biofilm formation. XTT reduction assay revealed the increase in metabolic 
activity of S. pyogenes biofilm against the decrease in FQs concentration. In addition, 
SEM and CLSM validated the potential of sub-MICs of FQs against the six S. pyogenes. 
These results confirm that all the four FQs are concentration dependent in inhibiting S. 
pyogenes biofilm formation. FQs at proper dosage can be effective against S. pyogenes 
and lower dosage concentrations may allow the bacteria to form barriers against the 
antibiotic in the form of biofilm.  
Finally, the section “In vitro and in vivo analysis of genes involved in virulence, 
biofilm formation and antibiotic resistance in Streptococcus pyogenes exposed to 
subinhibitory concentrations of antibiotics” deals with the in vivo and in vitro effects 
of sub-MICs of antibiotics on biofilm and non-biofilm forming S. pyogenes strains. MIC 
determination by broth microdilution method showed the highest MIC value of 1 µg/ml 
for SP7 and SF370 isolates against ofloxacin. SF370 showed the lowest MIC value of 
0.03 µg/ml against erythromycin. Sub-MICs of 1/2 to 1/64 MICs were utilized to 
challenge S. pyogenes strains. Sub-MICs treated S. pyogenes were assessed for 
autoaggregation ability and found that it varies between the strains.  In biofilm forming 
stains SP7and SF370, the average autoaggregation was observed to be 0.25 and 0.7nm. 
Whereas, the non-biofilm former SP24 showed an average of 0.03nm of autoaggregation. 
mRNA isolated from the sub-MIC treated bacterial cells were utilized for analyzing the  
virulence, biofilm and antibiotic resistance associated genes. Real time PCR analysis 
showed gene regulation in concentration dependent manner. The analysis showed 
increase in expression at the higher and lowest sub-MICs and showed a decrease in gene 
expression at the middle sub-MICs. These results have correlation with the 
autoaggregation assay results where, most of the antibiotics showed autoaggregation in 
concentration dependent manner. In vivo analyses using C. elegans through nematode 
liquid killing assay shows biofilm forming strains SP7 and SF370 extending its survival 
up to six days, whereas complete mortality occurred within four days in the case of non 
Synopsis | 6 
 
biofilm former SP24. In addition, lower sub-MICs such as 1/8 to 1/64 MICs showed 
lethal effects within 48h whereas the control showed less lethal effect. The results of the 
present study substantiate that the lower sub-MICs of antibiotics enhance the virulence of 
S. pyogenes. Hence, from the present study, it is evident that usage of antibiotics at 
proper concentration is very crucial to protect the mankind from life threatening 
infections.   


Culture independent detection and characterization of Streptococcus pyogenes and its transcriptional gene regulation upon subinhibitory exposure to antibiotics.

Marbaniang, Carmelita Nora

SYNOPSIS
The thesis entitled “Synthetic approaches for pyran containing bioactive natural
products: Spirastrellolide B, Passifloricins, Aculeatins and 6-Epiprelactone-V” has been
divided into five chapters.
CHAPTER-I : Chapter I describes a brief introduction to pyran containing bioactive
natural products.
CHAPTER-II : Chapter II describes the “Synthetic studies towards the Spirastrellolide
B”.
CHAPTER-III : Chapter III describes the “Asymmetric synthesis of (+)-Passifloricin A
and its 6-epimer”.
CHAPTER-IV : Chapter IV describes the “Total synthesis of Aculeatins A and B”.
CHAPTER-V : Chapter V describes the “Total synthesis of 6-Epiprelactone-V”.
CHAPTER I
This Chapter describes the introduction to “Pyran containing bioactive natural
products”.
CHAPTER II
This chapter describes the “Synthetic studies towards the Spirastrellolide B”.
Spirastrellolide A and B are two closely related polyketides that were isolated by Anderson
and coworkers from the marine sponge Spirastrella coccinea. The key elements of the structure
of spirastrellolide A (1) were first disclosed in 2003 and were followed by a report describing a
structure revision and the inhibition of protein phosphatase PP2A (IC50 = 1 nM for PP2A and 50
nM for PP1). Subsequent cleavage of the Δ40,41 olefin and derivitization of spirastrellolide B (2)
produced a compound suitable for X-ray analysis and revealed the complete relative and absolute
stereochemistry of the macrolide core. Recently, Anderson and coworkers have reported that the
C-46 alcohol is of (R)-configuration and also described the isolation of further congeners
(spirastrellolides C to G).
Present work
Scheme 1: Retrosynthetic analysis of the spirastrellolides.
46
The challenging architecture, in conjunction with the novel biological profile of
spirastrellolides, we disclose an effective construction of the C9-C25 southern fragment 5 of
spirastrellolide B. The retrosynthetic analysis of spirastrellolide is shown in scheme 1. From the
retrosynthetic perspective, disconnection of 2 at the macrocylic lactone and C25-C26 trans double
bond yield the two fragments 3 & 4. The target fragment 5 involves disconnection at the C8-C9
bond affording two subtargets 6 and 7 respectively. The subtarget 6 as represented obviously
was derived from D-glucose 8 whereas subtarget 7 was realized from commercially available
homoallyl alcohol 9. The BC spiroketal was constructed by selective deprotection of acetonide
and silylether using 0.8% H2SO4. The crucial reactions involved in the synthesis of the
individual fragments are Sharpless asymmetric dihydroxylartion, Jacobsen asymmetric
epoxidation, Sharpless asymmetric epoxidation, Barbier allylation and alkyne coupling.
Synthesis of subtarget 6
1,2-5,6-Di-O-isopropylidene-α-glucofuranose 10 was prepared from D-glucose 8 by well
documented procedure. Diacetone-D-glucose 10 was successively subjected to oxidation (PDC,
4 Ao MS, Ac2O, CH2Cl2) to 3-ulose followed by Wittig reaction with Ph3P=CH2 to give the exomethylene
derivative 11 in good yield. Catalytic hydrogenation of 11 afforded 3-C-methyl-3-
deoxy derivative 12 in 95% yield. The primary acetonide in 12 was selecetively deprotected in
the presence of 60% acetic acid to give diol 13 in 75% yield. This diol 13, on treatment with
NaIO4 followed by two-carbon homologation using ethoxycarbonylmethylene
triphenylphosphorane in benzene to furnish the (E)-α,β-unsaturated ester 14 in excellent yield
(Scheme 2).
Scheme 2


8
The α,β-unsaturated ester 14 was exposed to the Sharpless asymmetric dihydroxylation using
ADmix-α to give diol 15 (92% yield) which was followed by the acetonide protection to give the
compound 16 in 96% yield. Reduction of the ester functionality in 16 was achieved with LiAlH4
to give the alcohol 17 in 98% yield. The alcohol was oxidized to the aldehyde followed by
Barbier allylation (Zn, allyl bromide, sat. NH4Cl) gave inseparable diastereomeric mixture
(80:20 by HPLC) of homoallyl alcohol 18 in 92% yields. The two diastereomers were separated
as their TBS-derivatives 19a and 19b (anti:syn = 80:20) using simple column chromatography
(Scheme 3).
Scheme 3
The major anti-TBS ether 19a was desilylated with TBAF to give alcohol 18a, which was
again protected as methyl ether 20 using MeI and sodium hydride in quantitative yield. The
hydroboration of the terminal olefin of methyl ether 20 was accomplished with 9-BBN to get the
alcohol 21 in 92% yield. The primary alcohol was further oxidized using IBX to realize the key
fragment 6 in 96% yield (Scheme 4).
Scheme 4

18a 20
21 6
Synthesis of subtarget 7
Synthesis of the chiral fragment 7 was started with the kinetic resolution of 2-
(benzyloxyethyl)oxirane 23 (obtained from 3-butenol 9 in two steps as shown scheme 5) using
(S,S’)-(-)-N-N’-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt (II) to afford the
chiral epoxide 23a and diol 23b.
Scheme 5
Chiral epoxide 23a was subjected to Yamaguchi’s protocol with THP protected propargyl
alcohol to afford secondary alcohol 24 in 91% yield. The benzylation of alcohol in 24 was
realized using NaH as base and benzyl bromide which on further hydrolysis with p-TSA in
MeOH provided 25. The LiAlH4 reduction of propargyl alcohol to allyl alcohol 26 set the
platform of introducing two more chiral centers via Sharpless asymmetric epoxidation. Thus the
allyl alcohol 25 on treatment with (-)-DET, titanium(IV)isopropoxide and tert-butyl
hydroperoxide in dichloromethane in anhydrous conditions to yield epoxy alcohol 27 in 92%
yield (Scheme 6).
Scheme 6

9 22 23
23a 23b
The regioselective opening of Sharpless epoxy alcohol 27 using Me2CuLi gave 1,3-diol 28 in
65% yield. 1,3-Diol was disilylated as 29 using TBDMSCl in dichloromethane and selective
primary desilylation was achieved successfully using catalytic p-TSA in methanol at 0 oC to give
primary alcohol 30 in 87% yield. The alcohol was oxidized to aldehyde in good yield using IBX
and the aldehyde was subjected to Corey-Fuchs protocol (CBr4, TPP) to afford the dibromo
olefin 31 in overall yield of 80% for two steps. The dibromo olefin was converted into alkyne 7
under Grignard reaction condition by treating of EtMgBr at 0 oC in 91% yield (Scheme 7).
Scheme 7
Synthesis of southern (C9-C25) region
Coupling of aldehyde 6 with 7 was achieved via alkyne lithiation using n-BuLi, followed by
addition of 6 to furnish the desired coupled products 32 as a diastereomeric mixture (1:1
epimeric mixture at C17) in 82% yield. The seconday alcohol 32 was further oxidized using
Dess-Martin periodinane gave alkynone 33 in 98% yield which is the common precursor of
spirastrellolides. Pd-CaCO3 catalyzed hydrogenation of 33 smoothly delivered the
corresponding saturated ketone 34 in 95% yield without any deprotection of benzyl ethers. The
crucial spiroacetalization reaction was achieved by the treatment of 0.8% H2SO4 in methanol led
to complete desilylation, and selective acetonide removal followed by the formation of a single
spiroacetal product 5 in 69% yield (Scheme 8). Slight amount of over hydrolysis products 35a
and 35b were noticed with the aid of mass spectrum during the reaction. The diol 35a can be
converted back to 5 in the presence of 2,2-DMP and CSA.
Scheme 8
In conclusion C9-C25 southern fragment of Spirastrellolide B was achieved starting from Dglucose.
The key steps involved in this synthesis are Sharpless asymmetric dihydroxylation,
Jacobsen asymmetric epoxidation, Sharpless asymmetric epoxidation, Barbier allylation and
alkyne coupling, spiroacetalization reactions.
CHAPTER-III
This chapter describes the “Asymmetric synthesis of (+)-passifloricin A and its 6-epimer”.
The δ-lactone ring is an important structural feature of a good number of natural products. A
few of these natural products also encompass non-polar lipophilic tail, which give these
compounds an amphiphilic nature. Passifloricin A is one such natural product isolated from
Passiflora foetida. This compound has shown interesting antiprotozoal and antifungal properties.
OMe Me
OHC
OBn OTBS
Me
O
DMP, CH2Cl2
rt, 30 min, 98%
Pd-CaCO3, EtOAc
rt, 1 h, 95%
Present work
Having a general interest in total synthesis of bioactives and in particular six membered
oxygen heterocycles using intra-molecular oxa-Michael reaction of Evans et al. and Grubbs
olefin metathesis as key steps, passifloricin A was chosen as a target. The retrosynthetic analysis
of this natural product based on this strategy is shown below in Scheme 1.
Scheme 1
Initially, the cobalt-based chiral salen complex mediated resolution of racemic epoxide 4a
was obtained in 95% yield from commercially available olefin 5. The racemic epoxide 4a was
efficiently resolved with Jacobsen catalyst A and the chiral oxirane 4 was recovered with high
enantiomeric excess and 45% yield.
Scheme 2

1a or 1b
RCM
Evans oxa-michael
addition
2a or 2b
4 3
Jacobson resolution
COOEt
12 12 O 12
m-CPBA, CH2Cl2
0 oC, 1 h, 95%
A, 0.55 eq, H2O
THF, 36 h, 45%
allylmagnesium chloride
Et2O, 0 oC to rt, 1 h
92%
MOMCl, iPr2EtN, CH2Cl2
0 oC to rt, 2 h, 95%
Ethylacrylate, B (2 mol%)
CH2Cl2, reflux, 12 h, 99%
5 4a 94% ee 4
6 7
8
)

Grubbs catalyst (2nd generation)
Mes N N Mes
PCy3
(S,S)-(salen)CoIII(OAc)
(A) (B)
The regioselective opening of epoxide 4 with allylmagnesium chloride resulted in alcohol 6 in
92% yield. Protection of the alcohol as MOM ether 7 was achieved using MOMCl and
diisopropylethylamine which followed cross metathesis reaction with ethyl acrylate in presence
of 2 mol% of Grubbs’ catalyst B (2nd generation) to realize the conjugated ester 8 in
quantitative yield (Scheme 2).
Reduction of the ester functionality in 8 was achieved with DIBAL-H at -15 oC to room
temperature to give 9 in 96% isolated yield. The stage was then set for introduction of the
stereogenic hydroxy functionality under Sharpless asymmetric epoxidation conditions using (+)-
DET to give chiral epoxy alcohol 10 in 85% isolated yield. The reductive opening of this
epoxide was achieved with Red-Al to realize the 1,3-diol. This diol 11, on selective oxidation in
the presence of bis(acetoxy)iodobenzene (BAIB) and 2,2,6,6-tetramethylpiperidine-N-oxide
(TEMPO), followed by exposure of the crude β-hydroxy aldehyde to ethoxycarbonylmethylene
triphenylphosphorane furnished δ-hydroxy-α,β-unsaturated ester 12 in 80% overall yield for the
two steps. This substrate has all the functionalities necessary to perform the tethered Evans’
intramolecular oxa-Michael syn-addition reaction which was executed easily using benzaldehyde
and potassium tert-butoxide at 0 oC in anhydrous THF to furnish benzylidene acetal 3 in 72%
isolated yield. The diastereoselectivity was greater than 95% favoring the more stable synisomer.
The ester group in 3 was reduced to aldehyde 13 using DIBAL-H at -78 oC in 90% yield
(Scheme 3).
Scheme 3
12 OMOM
COOEt
 (+)-DET, Ti(OiPr)4, tBuOOH
CH2Cl2, 4 Ao MS, -20 oC
8 h, 85%
DIBAl-H, CH2Cl2
-15 oC to rt, 3 h
96%
Red-Al, CH2Cl2
0 oC, 6 h, 90%
BAIB, TEMPO, CH2Cl2, rt
1 h then Ph3P=CHCO2Et
80%
PhCHO, KOtBu, THF
0 oC, 45 min., 72%
DIBAL-H, CH2Cl2
-78 oC, 1 h, 90%
8 9
10 11
12 3
13
The allylation on aldehyde 3 gave a diastereomeric mixture of 2a and 2b in variable yields
and ratios (Table 1). The chiral BINOL-mediated Maruoka allylation (Table 1, entry 4) was
most efficiently providing a syn-selectivity of 90% albeit with a low yield of product. Column
chromatography allowed us to separate the diastereomers 2a and 2b, which were independently
into (+)-passifloricin A (1a) (Scheme 5) and its 6-epimer 1b (Scheme 5a) respectively.
Scheme 4
Table 1. Metal catalyzed allylation of aldehyde 13
Synthesis of passifloricin A (1a)
Diastereomer 2a was subjected to acrylation with acrolyl chloride in the presence of
diisopropylethylamine to generate the diene ester 14a setting the stage for the Grubbs’ ringclosing
metathesis (RCM) reaction. The diene ester 14a in CH2Cl2 underwent a smooth
intramolecular metathesis reaction in the presence of 5 mol % of Grubbs’ catalyst C (1st
generation) to produce the α,β-unsaturated lactone 15a in 92% yield. Treatment of lactone 15a
with 4 N HCl in THF resulted in cleavage of the MOM-ether and benzylidene acetal to afford
passifloricin A (1a) in 89% yield.
13 2a 2b
aIsolated yield after column chromatography purification
bRatio based on the yields of both isomers after seperation by silica
gel chromatography
Entry Reagents and conditions Yielda (%) syn:antib
1 Allyl bromide, Mg, dry ether,
0 oC, 1 h.
2 Allyl bromide, Zn, THF/sat.NH4Cl
-15 oC, 1 h.
3 Allyl bromide, In, THF/H2O
-15 oC, 1 h.
4 Allyltri-n-butyltin, Ti(OiPr)4, TiCl4,
Ag2O, (R)-BINOL, CH2Cl2, 24 h
Scheme 5
Synthesis of 6-epi-passifloricin A (1b)
The other diastereomer 2b was also subjected to the same sequence of reactions to realize the
6-epimer 1b. The spectroscopic data of both compounds 1a and 1b were in full agreement with
those reported in the literature.
Scheme 5a
In conclusion, asymmetric total syntheses of (+)-passifloricin A and its 6-epimer have been
achieved using Jacobsen asymmetric epoxidation, Grubbs’ metathesis, an Evans’ intramolecular
oxa-Michael and a Maruoka allylation as key steps.
CHAPTER-IV
This chapter describes the “Total synthesis of Aculeatins A and B”.
The aculeatins A (1) and B (2) are two epimeric spiroacetals isolated from the terrestrial plant
species Amomum aculeatum Roxb. (fam. Zingiberaceae). These compounds were found to
 (C)
display antiprotozoal activity against some Plasmodium and Trypanosoma species. In addition,
they showed antibacterial activity and were cytotoxic against the KB cell line.
Present work
In continuation of strategies towards pyran-containing natural products has been culminated
in a practical synthesis of both aculeatins A and B in good yields (overall yield of about 15%).
Retrosynthesis revealed tetradecanal 6 and the known 4-benzyloxyphenyl acetylene 5 as starting
materials. (Scheme 1)
Scheme 1
Synthesis of Weinreb amide
The aldehyde 6 was subjected to an enantioselective Maruoka allylation using titanium
complex (S,S)-I and allyltri-n-butyltin to furnish the homoallylic alcohol 8 in 86% yield with
excellent an enantioselectivity of 98% ee (determined by chiral HPLC). A one-pot ozonolysis
followed by two-carbon homologation using ethoxycarbonylmethylene triphenylphosphorane
produced the δ-hydroxy-α,β-unsaturated ester 9, which is set up for the tethered intramoleclar
oxa-Michael reaction to install the second stereocenter. As anticipated, treatment of 9 with
benzaldehyde and potassium tert-butoxide at 0 0C in anhydrous THF furnished benzylidene
acetal 10 in good yield. The diastereoselectivity was greater than 95% favoring the more stable
syn-isomer. The conversion of the ester functionality in 10 to Weinreb amide 4 via acid 11 was
uneventful (Scheme 2).

Scheme 2
Synthesis of alkyne
4-Hydroxy benzaldehyde 7 was protected as benzyl ether 12 using BnBr and K2CO3 in 95%
yield. Aldehyde was subsequently treated with CBr4 and TPP to afford the dibromo olefin 13 in
87% yield. The dibromo olefin was converted into alkyne 5 under Grignard reaction condition
by treating with EtMgBr at 0 oC in 92% yield.
Scheme 3
Synthesis of aculeatins A and B
The addition of lithiated 4-benzyloxyphenyl acetylene 5 to Weinreb amide 4 furnished
alkynone 3. Catalytic hydrogenolysis of the benzylidene, benzyloxy and acetylene groups gave
14 (not isolated) and further treatment with phenyliodonium(III) bis(trifluoroacetate) (PIFA) in
acetone–H2O (9:1) yielded a mixture of aculeatins A (1) and B (2) in a ratio of 5:2, which were
separated by column chromatography (Scheme 4).
7 12 13
5
Scheme 4
In conclusion, a stereo-controlled total synthesis of aculeatins A and B has been achieved in
eight steps and in 15% overall yield. The key feature of this synthetic approach is the application
of Maruoka allylation and tethered intramolecular oxa-Michael reaction.
CHAPTER-V
This chapter describes the “Total synthesis of 6-epiprelactone-V”.
Prelactones are examples of δ-lactones isolated from bafilomycin producing microorganisms
and other polyketide macrolide producing organisms. The structures of these compounds have
been assigned on the basis of the proposed biosynthesis of macrolides. Biologically, these
lactones exhibit properties such as ATpase inhibitor and antibacterial, antifungal and
immunosuppressive activities.
Present work
In continuation of practical synthetic approaches towards the substituted pyran scaffold in
chiral form, a total synthesis of the non-natural enantiomer of prelactone, (-)-6-epiprelactone-V
(1) was achieved employing a highly chemo-regio- and stereoselective tandem ester reduction,
epoxide formation-reductive epoxide opening reaction protocol and a base catalyzed oxygen
tethered intramolecular Michael addition as key steps (Scheme 1). The highlight of the synthesis
2.5:1 mixture of Aculeatins A and B
Aculeatin A Aculeatin B
4 5 3
14
1 2
is the utilization of the chiral centre in commercially available (S)-malic acid 4 and creation of
the syn-1,3-diol functionality through an intramolecular Michael addition of a benzyloxy tether.
Scheme 1
Commercially available (S)-malic acid 4 was subjected to esterification using BF3.OEt2 in
methanol to give dimethyl malate 5 in 98% yield. Diester 5 was treated with LHMDS and
methyl iodide in THF at -78 oC to yield α-alkylated dimethyl malate 6 in good yield with high
diasteroselectivity (12:1). However, the diastereomers could not be separated by
chromatography. Tosylation of 6 gave a separable mixture of 7 and its syn-diastereoisomers in
90% yield. Treatment of the anti-isomer 7 with lithium aluminium hydride (6 equiv) in THF
afforded the desired diol 3 in 79% yield (Scheme 2).
Scheme 2
1,3-Diol 3 was disilylated using TBDMSCl in dichloromethane and selective primary
desilylation was achieved successfully using catalytic p-TSA in methanol at 0 oC to give primary
alcohol 9 in 75% yield. To install the requisite α,β-unsaturated ester for the subsequent base
catalyzed intramolecular oxygen tethered Michael reaction, the 10 alcohol group in 9 was
oxidized with IBX to the corresponding aldehyde, followed by two-carbon homologation using
ethoxycarbonylmethylene triphenylphosphorane in benzene to furnish the (E)-α,β-unsaturated
6-epiprelactone-V 1
syn:anti = 1:12
ester 10 in good yield. Deprotection of the hydroxyl group in 10 was achieved using p-TSA in
methanol to provide the key synthon 2 in high yield (Scheme 3).
Scheme 3
Alternatively, δ-hydroxy-α,β-unsaturated ester 2 was obtained by selective oxidation and
Wittig olefination of 3. Thus, diol 3 was selectively oxidized with IBX, followed by two-carbon
homologation with the stable Wittig ylide ethoxycarbonylmethylene triphenylphosphorane to
furnish 2 in 64% yield (Scheme 4).
Scheme 4
The δ-hydroxy-α,β-unsaturated ester 2 was subjected to a base catalyzed intramolecular
Michael addition using 1.1 equiv of benzaldehyde in the presence of 0.1 equiv of potassium tertbutoxide
at 0 oC in THF to furnish benzylidene acetal 11 in good yield. The diastereoselectivity
was greater than 95% favoring the more stable syn-isomer. Finally, hydrolysis of the benzylidene
acetal and cyclization were successfully achieved in one pot using catalytic conc. H2SO4 in
methanol to furnish the target molecule 1 in 95% yield (Scheme5).
Scheme 5
In conclusion, the intramolecular protective group assisted syn-1,3-diol synthesis has been
efficiently utilized in as synthesis of 6-epiprelactone-V starting from (S)-malic acid.




ArgP protein of Escherichia coli: roles in osmoregulation, gene regulation and inter-relationship with LysG of Corynebacterium glutamicum

D, Bakkiyaraj

Abstract
Identification and characterization of anti-pathogenic agents from marine bacteria

The thesis entitled ‘Identification and characterization of anti-pathogenic agents from marine bacteria’ documents the hidden potentials of marine bacteria like the antibiofilm, anti-quorum sensing and anti-virulence properties. Since resistance to antibiotics is the striking factor that predisposes therapeutic breakdown and subsequent increase in morbidity and mortality, novel targets like biofilms, cell to cell communication (quorum sensing, QS) and production of virulence factors are of prime interest and are targeted for the development of novel drugs.  These phenotypes were shown crucial for the virulence and pathogenesis of the bacterial or fungal pathogens, besides it is widely believed that targeting these phenotypes will not let the pathogen to develop resistance. Epibiotic bacteria were explicitly studied knowing their defensive role in protecting the host from invading pathogens.  Bacteria associated with marine ecosystem are reported to play such a defensive role since the ecosystem is highly populated and a constant rivalry between the organisms takes place for nutrients, space, etc.  The potential of such bacteria to inhibit other bacteria were exploited to inhibit human bacterial and fungal pathogens.
	Marine samples like sediment, mangrove rhizosphere soil, sea water, sea grass and sea weed were collected from the Karankadu mangrove forest region in Palk Bay and 149 morphologically distinct bacteria were isolated from them.  Nineteen morphologically different bacteria isolated from coral mucus of Acropora digitifera in an earlier study were taken from the laboratory’s culture collection repository, Biobank.  All the marine bacteria were primarily screened for their ability to inhibit QS in an indicator bacterium, Chromobacterium violaceum ATCC12472.  Nine coral associated bacteria and 37 other bacteria from Karankadu region were found to possess anti-QS activity and are further studied against Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Serratia marcescens, Candida albicans and C. tropicalis which play a crucial role in nosocomial and device associated infections in human.
	A coral associated actinomycete CAA-3, identified as Streptomyces akiyoshiensis, displayed potential antibiofilm activity against S. aureus, its clinical isolates and its drug resistant counterpart, Methicillin Resistant S. aureus (MRSA).  Another actinomycete (G9d) isolated from the seaweed Gracilaria gracilis, identified as Kocuria marina significantly inhibited the production of extracellular polymeric substance (EPS) production and biofilm formation in S. epidermidis. Two other coral associated bacteria called Delftia tsuruhatensis (CAB23) and Stenotrophomonas maltophilia (CAB41) were found to inhibit the biofilms
P. aeruginosa and S. marcescens.  Apart from inhibiting the biofilms, the extracts of CAB23 and CAB41 effectively inhibited the production of QS regulated virulence factors like LasA protease, LasB elastase, protease, pyocyanin and pyoverdin in 
P. aeruginosa and prodigiosin, total protease, lipase and swarming motility in S. marcescens.  K. marina was also found to inhibit the biofilms of C. albicans and C. tropicalis.  The extract of K. marina inhibited the production of secreted aspartyl proteinase (SAP), a major virulence trait in C. tropicalis that plays a crucial role in host tissue damage and immune evasion.  Purification and structural characterization of the active principles revealed the presence of Perfluoroeicosane in a fraction active against S. epidermidis and Gancidin W in a fraction active against C. tropicalis.
	On the whole, the study explains the potential of marine bacteria with special reference to actinomycetes as a potential source for anti-pathogenic agents which could replace the antibiotics in treatment of infectious diseases.  Further, this study unveils various leads for future research which will deliver fruitful products for use in the field of medicine.




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