Organic Chemistry (OC)https://etd.iisc.ac.in/handle/2005/142024-03-29T15:53:17Z2024-03-29T15:53:17Z2,3-Dideoxy Sugars in Glycoconjugations and Cyclic Oligosaccharide SynthesisSarkar, Biswajithttps://etd.iisc.ac.in/handle/2005/59572023-06-08T10:21:47Z2,3-Dideoxy Sugars in Glycoconjugations and Cyclic Oligosaccharide Synthesis
Sarkar, Biswajit
2,3- Dideoxy sugars are versatile synthons for organic synthesis. The
applications are diverse in biological systems and organic synthesis. In the first part of the
thesis, 2,3-unsaturated sugars are used for the glycoconjugation of amino acids, peptides and
proteins. In the later part, 2,3-dideoxy sugar is used to synthesize carbohydrate
macrocycle. Finally, sugar vinyl sulfoxide is used to synthesize substituted pyran via 2,6-
anhydro sugar formation.
Chapter 1 of the thesis is divided into two parts. The first part describes the literature
on unsaturated sugar, especially 2,3-unsaturated sugars, synthesis and their
modifications. Attention is given to their addition reactions with nucleophiles and
conjugation with biomolecules. A brief introduction to glycoconjugation is also reported in
this part. Different glycoconjugation methods are discussed briefly, and the advantages are
compared accordingly. The second part of this chapter elaborates on the synthesis of cyclic
oligosaccharides. The approaches and the difficulties in the respective
approaches are mentioned accordingly. Challenges with synthesising small cyclic
oligosaccharides are cited according to the available literature. Current development
in the field is also covered in the discussion.
Chapter 2 of the thesis deals with the glycoconjugation methods using sugar vinyl
sulfoxide involving Michael addition reaction. Glycoconjugations of amino acids, peptides
and protein, namely lysozyme are demonstrated in benign physiological conditions. The
smaller glycoconjugated molecules are characterized with the help of NMR spectroscopy
and mass spectrometry, while the larger glycoconjugated peptide and protein are
characterized with the help of mass spectrometry. Biophysical studies of glycoconjugated
lysozyme showed increased stability in the presence of trypsin while retaining its antimicrobial
activity. Thus, a benign glycoconjugation method is developed.
Chapter 3 of the thesis unravels further potential of glycoconjugation using sugar vinyl
sulfoxide. PETIM dendrimers of generation zero to three are glycoconjugated with sugar
vinyl sulfoxide. The glycoconjugations of the lower generation dendrimers are confirmed
using NMR spectroscopy and mass spectrometry; for higher generations, only NMR
spectroscopy was employed for the characterization. The first-order reaction rate constant
of the glycoconjugation reaction is also determined using NMR spectroscopy. Further
biological evaluation of the native and glycoconjugated PETIM dendrimer reveals that
PETIM dendrimers show selective antibacterial activity against M. smegmatis, and the
native dendrimers show higher efficacy over the glycoconjugated dendrimer.
Chapter 4 of the thesis describes the synthesis of the cyclic disaccharide molecule
composed of 2,3-dideoxy furanoside monomer units. The synthesis started from protected
glucal molecules and followed a few simple reaction steps, including the Ferrier reaction,
desulfurization reaction, and selective hydroxy group protection and finally,
glycoconjugation reaction. While the formation of the disaccharide is confirmed using NMR
spectroscopy and mass spectrometry, the conformation of the constituting monomeric unit
of the cyclic disaccharide is ascertained through solid state structure determination using
the single crystal X-ray diffraction method. The ring contraction of the pyranoside monomer to
furanoside cyclic disaccharide is explained by two plausible mechanisms involved in the
glycosylation step. Further encapsulation property of the cyclic disaccharide molecule was
evaluated against the 1-aminoadamantane using the ITC method. This experiment allows looking into the thermodynamics of the encapsulation and the encapsulation mode of the
molecule.
Chapter 5 of the thesis shows sugar vinyl sulfoxide's application and potential
as a synthetic intermediate. Intra-molecular Michael addition reaction of the sugar vinyl
sulfoxide in basic condition affords elusive 2,6-anhydro sugar molecules in a single step.
Selective opening of the bicyclic ring of the anhydro sugar converts it to a substituted
pyran. The applicability of this two-step, one-pot reaction is also tested on sugar vinyl
sulfoxide derived from galactal.
The thesis describes the achievement of the diversification of 2,3-unsaturated sugar. A
benign glycoconjugation method is developed and adequately characterized. The synthetic
potential of 2,3-dideoxy sugar is demonstrated through the synthesis of cyclic disaccharide
via a ring contraction of pyranoside to a furanoside. And finally, a two-step reaction
protocol converts pyranoside sugar into a substituted pyran.
Achieving Efficiency and Specificity in Multifarious Systems for Anti-cancer Gene TherapeuticsKamra, Mohinihttps://etd.iisc.ac.in/handle/2005/46552020-11-17T09:16:51ZAchieving Efficiency and Specificity in Multifarious Systems for Anti-cancer Gene Therapeutics
Kamra, Mohini
The research work described in this thesis encompasses the syntheses, characterization and bioactivity determination of various novel systems crafted for achieving high efficacy and targeted gene therapeutic effect in cancer cells in vitro and in vivo. Cancer being one of the prominent causes of mortality over the world, has gained tremendous attention of scientists over the past few decades. The latest approach has been to target the disease at the genetic level itself, a field of research known as ‘Gene therapy’. Several favorable results have been obtained in cancer gene therapy, but each accompanied by a set of limitations, the most common being the lack of gene transfection efficiency in vivo. A chemist’s approach to solving this problem is by designing an efficient gene delivery vector that can bind the nucleic acid cargo in a reversible manner to ensure the subsequent release at the target site. It should provide protection from nucleases and should also meet the very important criteria of biocompatibility. In the studies described herein, a range of naturally occurring biomolecules i.e. a pH-sensitive tripeptide, a biodegradable polysaccharide and an essential vitamin (alone and in conjugation with a set of receptor targeted antibodies) have been explored for their ability to deliver therapeutically relevant agents inside cancer cells. These have been derivatized using simple and cost-effective synthetic methods for fine-tuning of their physicochemical properties. The aggregation behavior of these derivatives, in solution, was studied along with their nucleic acid binding abilities. After complete characterization of the systems by various biophysical techniques, their delivery efficacy was gauged by performing various biological assays involving cancer cells in culture. The promising systems were taken for studies in animal model (nude mice) for assessment of in vivo anti-cancer therapeutic potential. Target-specific gene transfection has been attempted using immunoliposomes guided by monoclonal antibodies decorated on the surface. Taken together the thesis presents a potential cisplatin prodrug, a safe and efficient gene transfecting polysaccharide for co-delivery of gene and drug, besides an amphiphilic vitamin for efficacious delivery of short therapeutic oligonucleotides and for cell-specific DNA delivery allowing effective gene transfer in the hard to transfect suspension cells.
Chapter 1: A Concise Overview of Cancer Gene Therapy, the Futuristic Medicine Gene therapy has been proposed to be the future of medicine in case of most genetic disorders like cancer. The scope of this technique has widened over the years and the easiest approach devised has been to alter the regulation patterns of certain oncogenes, tumour suppressor genes or the genes responsible for modulating drug sensitivity. After giving a throwback into the birth and growth of the concept of gene therapy, the first chapter of this thesis covers the different facets of this promising mode of therapy while briefly summarizing the advances made in the field thus far. First, a comprehensive description of the mechanism of action of cisplatin, which is the first line of treatment for cancer, is provided followed by the advantages of a Pt(IV) prodrug over a Pt(II) drug. This section then surveys various cell-cycle dependent drugs based on a Platinum core, some of them even being clinically approved. The readers are also introduced to the Nobel Prize winning technique of RNA interference (RNAi) along with its implications in cancer gene therapy. The utilization of small interfering RNA (siRNA) for downregulation of oncogenes, and for identification of putative gene targets for effectively countering malignancy, is described therein. Putting the siRNA-based strategies to effect requires an efficient vector system for intracellular transfer of the exogenous short oligonucleotides. The designing of such vectors needs a comprehensive understanding of the process of nucleic acid delivery and the various impedances encountered in its pathway to bring about the desired therapeutic effect. The most important ones among these are: nucleic acid complexation, cellular binding, cellular uptake, endosomal escape, intracellular trafficking, nuclear entry and release of the complexed nucleic acid. These have also been highlighted along with the steps that have been taken to overcome the limitations of non-viral gene delivery and development of newer systems for enhancement of delivery efficacy. Some of the most promising non-viral transfect ants belong to the category of cationic polymers that condense genes into compact polyelectrolyte complexes, by virtue of formation of which, they offer protection against nuclease induced damage. A plethora of natural and synthetic polymers that have demonstrated gene transfection abilities have been described along with an important class of amphiphilic gene vectors i.e. cationic lipids. The way their self-assembly drives the formation of nano-sized vesicles in aqueous medium and their strong nucleic acid binding and release properties make them attractive candidates for highly effective gene transfection. Since vectors of synthetic origin exert immunostimulatory effects, the focus of this thesis is to utilize naturally occurring unique systems for attaining safe, effective and target specific gene transfection.
Chapter 2: New Cisplatin Prodrugs Originating from the pH-Sensitive Self-Assembling Natural Tripeptide In this chapter, a pH-sensitive self-assembling tripeptide, KFG (Lysine-Phenylalanine-Glycine) is used for preparation of a Pt(II) core based cisplatin analogue and a Pt (IV) core based cisplatin prodrug, both of which were completely characterized. They demonstrated higher potency than the native drug in cell lines that are resistant to clinically relevant concentrations of cisplatin. Additionally, when used in combination with Doxorubicin encapsulated tripeptide vesicles, Pep- DOX, they were found to lower the effective dosage of the anti-cancer drug in vitro exhibiting synergistic enhancement in cytotoxicity. The more active Pep-Pt(IV) complex was taken for in vivo studies where the combinatorial therapy with Pep-DOX was able to cause complete flattening of xenograft tumours within a week of treatment in nude mice model (Figure I). Although found quite potent, the cisplatin analogues have been reported to exhibit several side effects at the medicinal stage, which may be attributed to the heavy load of renal clearance and accumulation of metal-ion based by-products. Figure I. Molecular structure of the Pep-Pt (II) and Pep-Pt(IV) complexes used for preparation of dual drug delivery systems that exhibit synergistically enhanced drug internalization in vitro and tumour regression in vivo.
Chapter 3: Exploration of Derivatives of Chitosan, a Naturally Occurring Polymer, as a Biocompatible, Potential Vector for Sustained Gene Delivery Chapter 3A: Chitosan Imine Derivative for Sustained Gene Transfection The common concern of biocompatibility of a gene delivery vector was met by designing a water-soluble derivative of a naturally abundant carbohydrate polymer for safe and efficient gene delivery. Chitosan, a partially deacetylated derivative of the second most abundant natural polymer, chitin that is found in the shells of crustaceans was chosen due its biodegradability and ability to condense DNA by virtue of proton able amine functionalities (pKa = 6.5) running along the length of the polymer backbone. It was solubilized in water by O-carboxymethylation and then used for imine formation with pyridine-4-carboxaldehyde. The pyridine imine derivative, (py)CS(CH2COOH) was observed to form nano-sized spherical polyplexes with pDNA having positive surface charge in aqueous medium. The gene transfection ability of this derivative was found to be comparable to that of commercial standard, Lipofectamine 2000. However, it displayed sustained transfection over longer durations with minimal loss of cell viability as opposed to the commercial standards which became toxic with increased dosage or time of incubation (Figure II). This chapter presents an in-depth study of the biocompatible, non-toxic, chitosan derivative for efficient and sustained gene transfection owing to the formation of nuclease tolerant and serum stable polyplexes with pDNA. Figure II. Molecular structure of chitosan imine derivative, (py)CS(CH2COOH) and its application as a safe and efficient gene delivery carrier.
Chapter 3B: Chitosan Oxime Ether with Doxorubicin for Gene and Drug Co-Delivery Having succeeded in developing a safe, effective and biocompatible polymeric gene delivery carrier, we embarked upon using it in combination with a drug bearing polymer for co-delivery of gene and drug. A novel oxyamine derivative of chitosan, CS(ONH2) was first synthesized which was then used for preparation of oxime ether with pendant drug entities anchored on to a DNA complexing polymer backbone. The oxime ether linkage, being stable under biologically relevant conditions and hydrolysable at the endosomal pH, conferred pH sensitive drug release properties to the Doxorubicin tethered polymer, CS(Dox). When mixed with pDNA carrying (py)CS(CH2COOH) polyplexes, enhanced internalization of both, the gene and drug. The extent of cell death caused by the polymer-drug conjugate was improved when used in combination with (py)CS(CH2COOH) polyplexes carrying the tumour suppressor gene, p53. The improvement in potency relative to the free drug pointed to the advantageous role of the oxime ether linkage. Fluorescence based cytometry and microscopy studies have shown that pH-sensitive linkage indeed elevated the cellular internalization of the anti-cancer drug. The extent of co-delivery is demonstrated to increase as compared to the use of free drug with the polyplexes (Figure III). Figure III. Molecular structures of CS(ONH2) and CS(Dox) along with a schematic representation of novel nanocomposites for gene and drug co-delivery.
This chapter, thus, evaluates a versatile delivery vector capable of efficiently delivering both, a drug and a gene to bring about an enhanced anti-cancer therapeutic response. Polymeric vectors are known to deliver polynucleotides by virtue of formation of polyelectrolyte complexes. A vector for adept delivery of short oligonucleotides requires a vector of non-polymeric origin. Chapter 4: Assessment of siRNA Delivery using Tocopherol Gemini Lipids Terminated with Hydroxyethyl Head Groups Chapter 4A: Efficacy of siRNA Transfection by Hydroxyethyl Terminated Tocopherol Gemini Lipids in vitro and its use for Induction of Apoptosis in Cancer Cells Owing to high positive surface charge, cationic lipids are known to efficiently condense small oligonucleotides into compact nano-aggregates called lipoplexes. Since synthetic lipid vectors are usually associated with immunogenic responses, a vector molecule that is essential to the target cells and does not possess a biosynthetic pathway inside the cell, was chosen for the study. Vitamin E or α-Tocopherol was taken for construction of a hydroxyethyl terminated Gemini lipid, TH8S that has proven its ability to deliver pDNA effectively in combination with a helper lipid, DOPE, at a molar ratio of 2:1. Chapter 4A discusses the use of this efficient co-liposomal formulation for delivery of short interfering RNA (siRNA) of therapeutic importance. Selection of an appropriate target gene is crucial for achieving a therapeutically relevant gene knockdown. Since surviving is one such gene that is essential for the survival of cancer cells and inhibition of their apoptotic pathway, the TH8S-DOPE co-liposomes were used for transfection of anti-surviving siRNA in HepG2 cells. Effect of hydroxyl functionality, at the head group, on the efficiency of siRNA delivery was explored using MTT assay and apoptosis detection assay in HepG2 cells. Validation of the same was obtained by RT-PCR and Western blot studies where a mechanistic insight into the route of apoptosis induction by surviving downregulation was obtained (Figure IV). Downregulation of surviving by the interfering nucleotide was also found to sensitize the transfected cells towards the chemotherapeutic agent, Doxorubicin. The details of the in vitro studies have been described in this chapter.
Figure IV. Molecular structures of TH8S Gemini and DOPE helper lipids used for the formation of co-liposomes that demonstrate GFP knockdown and induction of apoptosis by surviving downregulation in vitro along with tumour regression by intratumoral delivery of anti-surviving siRNA leading to chemo sensitization. Chapter 4B: Transfection of siRNA Mediated by Hydroxyethyl Terminated Tocopherol Gemini Lipids for Tumour Regression in vivo This chapter describes the study assessing the ability of afore mentioned tocopherol Gemini lipids with hydroxyethyl terminated head groups, to regress xenograft tumours in nude mice model system. For any anti-cancer gene therapeutic to be clinically relevant, it must be exhibit high efficiency of gene transfection in vivo. Tumorigenic cells, HepG2 explant cells were used for generating xenografts in athymic nude mice. After validating the TH8S/DOPE co-liposome mediated internalization of anti-surviving siRNA in the cells of the tumour tissue by RT-PCR, the ability of lipoplexes to lower the rate of tumour growth was determined. The effect of inhibition of tumour growth, by way of post-transcriptional gene silencing of surviving in the tumour cells, by the interfering oligonucleotide was further supplemented by a sub-optimal concentration of Doxorubicin (Figure IV). The improvement of responsiveness of the tumour cells towards anti-
apoptotic agents by downregulation of surviving was, thus, used to achieve complete inhibition of tumour progression in the animal model studied.
Chapter 5: Targeted Gene Delivery Mediated by Receptor-Guided Immunoliposomes
Chapter 5A: Targeting Hepatocellular Carcinoma using Anti-LDLr mAb Tagged Gemini
Tocopherol Liposomes
A promising strategy to enhance the therapeutic potential of liposome mediated gene delivery is
by directing the liposomes to the target cells. This strategy was employed making use of a very
strong and specific interaction, the antigen-antibody interaction. For targeting hepatocellular
carcinoma, liposomes prepared from the highly efficient Tocopherol Gemini lipids with
hydroxyethyl headgroups were decorated with anti-LDLr antibody which recognizes and binds
to the Low-Density Lipoprotein receptor (LDLr) that is particularly abundant on the surface of
Figure V. Molecular structures of TH8S and TME used for construction of immunoliposome by
conjugation with anti-LDLr mAb achieving p53 delivery specifically in liver cancer cells.
liver cancer cells. Its involvement in cholesterol homeostasis and metabolism of hepatic tumour tissue allows it to possess a special mechanism for intracellular transport of LDL, called ‘receptor-mediated endocytosis’. Making use of the thiol-maleimide addition chemistry, anti- LDLr monoclonal antibody (mAb) was conjugated to monomeric tocopherol lipid (TME) that was incorporated in TH8S liposomes (Figure V). Detailed in this chapter are the methods of purification of the mAb, its conjugation to TME and construction of immunoliposomes. The immunoliposomes, upon complexation with the pCEP4-p53, have been demonstrated to effect p53 inhibition of cancer cell proliferation and induction of apoptosis by reinstatement of the tumour suppressor gene, p53. While being able to selectively transfect liver cancer cells, these tocopherol based immunoliposomes offer an additional advantage due to the presence of the liver cytosolic protein, α-TTP (α-Tocopherol transfer protein) that possesses a specific binding affinity for tocopherol, facilitating vector bioavailability.
Chapter 5B: Efficient and Targeted Gene Transfection in Suspension Cells using Anti- CD4 mAb Directed Immunoliposomes As opposed to adherent mammalian cells in culture, suspension cells are known for being hard to transfect using the traditional non-viral agents like cationic polymers or lipids. Since the difficulty in transfection arises out of the inability of the transfection complex to attach to the surface of the cells floating in the culture medium, the strategy of mAb guided targeting was utilized to facilitate this binding interaction (Figure VI).
Figure VI. CD4 targeted gene transfection in suspension cells using OKT4 tagged TH8S immunoliposomes. A well-established suspension cell culture system was used as a model, with the T-cell receptor, CD4 being the guidance cue for cellular binding and subsequent internalization of p53 gene carrying TH8S immunolipoplexes tagged with OKT4 (anti-CD4 mAb). The chapter goes through a series of assays for understanding the T-cell specific gene delivery by the constructed immunoliposomes, while throwing light on their capacity as being among the few in the league of efficacious suspension cell transfect ants.
Alkynones Derived from Tartaric Acid : Efficient Building Blocks for the Synthesis of Macrolactone Natural ProductsBali, Amit Khttps://etd.iisc.ac.in/handle/2005/37902019-09-13T11:18:43Z2018-07-05T00:00:00ZAlkynones Derived from Tartaric Acid : Efficient Building Blocks for the Synthesis of Macrolactone Natural Products
Bali, Amit K
The thesis describes the synthesis and application of various alkynones derived from the bis-Weinreb amide of tartaric acid in the total synthesis of macrolactone natural products. The thesis is divided into three sections.
First section of the thesis describes the optimization and generalization of the procedure for the addition of alkynyl Grignard / lithium reagents to the bis-Weinreb amide derived from tartaric acid to yield the mono alkynyl ketones was developed. Application of the formed γ-oxo amides was demonstrated in the synthesis of polyols with varied substitutions particularly the synthesis of 1,2,4-triols was accomplished using Ley’s dithianylation as the key step.
Scheme 1: Synthesis of polyols from the bis-weinreb amide of tartaric acid.
Application of the strategy to the total synthesis of decanolactone natural products achaetolide and (Z)-isomer of (6S,7R,9R)-6,7-dihydroxy-9-propylnon-4-eno-9-lactone was featured.
Section B of the thesis deals with the enantiospecific total synthesis of 14-membered macrolactone Sch 725674.
Key reactions in the synthesis include the synthesis of the 1,2,4-triol unit from tartaric acid, olefin cross metathesis and ring closing metathesis.
Scheme 4: Enantiospecific total synthesis of Sch 725674.
Section C of the thesis describes the enantiospecific synthesis of the C9-C22 fragment of the 28-membered polyene polyol macrolide pentamycin. Although isolated in 1958, total synthesis of this antifungal compound was not reported.
In application of the methodology developed, the alkynone prepared from the bis-Weinreb amide was elaborated to the required fragment.
2018-07-05T00:00:00ZAllylic Halogenation Route to Latent-Active Trans-Glycosylation of Allyl Glycoside DonorsDas, Anupamahttps://etd.iisc.ac.in/handle/2005/56262022-03-15T09:44:02ZAllylic Halogenation Route to Latent-Active Trans-Glycosylation of Allyl Glycoside Donors
Das, Anupama
Allylic halogenation of allyl glycosides as a new route to allyl glycoside donors in glycosylations is investigated in this thesis. Allyl functionality is one of the commonly adopted protecting groups to hydroxyl groups in sugar chemistry. In addition, allyl glycosides act as glycosyl donors, through isomerization to the corresponding vinyl glycosides. Facile conversion of allyl moiety to other functionalities, as well as, stabilities under acidic and basic conditions offer rich possibilities of this moiety in sugar chemistry. Chapter 1 provides a succinct overview of glycosylation reactions and mechanisms.
An area of intense interest is to transform a latent allyl moiety to an active glycosyl donor. In this effort, allylic halogenation reaction is considered appealing, due to the expected reactivity of the mixed halo-acetal of allyl glycoside towards an electrophile and the subsequent transformation to a glycosylation-active intermediate, suitable as an active glycosyl donor. Early experiments show that allylic bromination of allyl glycosides, using N-bromosuccinimide (NBS)/azo-bis-isobutyronitrile (AIBN) in CCl4 generates mixed halo-allyl glycoside intermediate, the reaction of which with an acceptor in the presence of Ag(I) or triflic acid (TfOH) affords the corresponding trans-glycoside in a good yield. The reaction is verified with a number of glycoside acceptors, including allyl glycoside acceptors. In the case of allyl glycoside acceptors, the resulting trans-glycoside possesses allyl moiety at the reducing end, which, in turn, is subjected allylic activation and subsequent glycosylation. Di-, tri- and tetrasaccharide syntheses are accomplished in good yields by this new route. Chapter 2 describes the development of this new method.
Radical halogenations in CCl4 warranted a replacement to the solvent, as well as, further optimizations of the reaction. In these efforts, diethylcarbonate (Et2O)2CO) is identified as a suitable solvent to conduct (i) radical halogenation and (ii) the subsequent glycosylation. The glycosylation is promoted either by TfOH or trimethylsilyl triflate (TMSOTf). A one-pot methodology is developed and method is verified with the synthesis of xyloyranoside, mono-, di- and trisaccharides. Chapter 3 provides the details of these developments.
Halo-allyl mixed acetal of allyl glycoside is found to undergo S¬N2 and S¬¬¬¬N2’ reactions with thiolate nucleophiles. The SN2’ reaction leads to 3-thiocresylpropenyl (TCP) glycoside, as a stable vinyl glycoside, which can be stored for longer duration, unlike, vinyl glycosides that are quite unstable due to faster hydrolysis. TCP glycoside is subjected to remote activation using iodonium reagent and activation leads to the formation of glycosylation active intermediate. Glycosylations with aglycosyl and glycosyl acceptors are conducted facile and the corresponding trans-glycosides are obtained in excellent yields. Chapter 4 describes the development of this new, stable TCP-based vinyl glycoside methodology in glycosylations.
Overall, the thesis illustrates establishing allyl glycosides as glycosyl donors as allylic halogenations and subsequent glycosylations. The new method merits in the repertoire of contemporary glycosylation techniques of remote activation-based glycosylations.