Synthesis and Evaluation of Biological Activity of a Potential Immunomodulatory Zwitterionic Polysaccharide Vikram Basava [email protected]

Synthesis and Evaluation of Biological Activity of a Potential Immunomodulatory Zwitterionic Polysaccharide Vikram Basava Vikram.Basava@Student.Shu.Edu

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Spring 5-14-2016 Synthesis and Evaluation of Biological Activity of a Potential Immunomodulatory Zwitterionic Polysaccharide Vikram Basava [email protected]

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Recommended Citation Basava, Vikram, "Synthesis and Evaluation of Biological Activity of a Potential Immunomodulatory Zwitterionic Polysaccharide" (2016). Seton Hall University Dissertations and Theses (ETDs). 2170. https://scholarship.shu.edu/dissertations/2170 Synthesis and Evaluation of Biological Activity of a Potential

Immunomodulatory Zwitterionic Polysaccharide

A Dissertation

In the Department of Chemistry and Biochemistry submitted to the

faculty of the Graduate School of Arts and Sciences in partial

fulfillment of the requirements for the

degree of

Doctor of Philosophy

In Chemistry

Seton Hall University

Vikram Basava

May 2016

ACKNOWLEDGEMENTS

I am greatly thankful to the support and encouragement that I have received from Dr.

Cecilia H. Marzabadi. Her ideas and guidance have made this dissertation possible.

She always had the time to answer all my questions and let me try reactions with the ideas which I came up with.

I would like to thank the Department of Chemistry and Biochemistry for giving me the opportunity to pursue my PhD at Seton Hall University. I am extremely thankful for Dr.

Sergiu Gorun for helping me in getting the x-ray crystal structure of the anhydro sugar which I synthesized. I would like to acknowledge Dr. Constantine Bitsaktsis for performing biological tests on the PSA 1 analogue which was synthesized in my project.

I would again like to thank Dr. Bitsaktsis along with Dr. James E. Hanson for taking their time to review my dissertation write-up.

I am thankful for the support of Dr. Robert L. Augustine and Dr. Setrak K. Tanielyan at the CAC. I would even like to thank Dr. Mahesh K. Lakshman for his support at the

CCNY, CUNY.

I could have not completed this dissertation without the unlimited support from my family and friends. I particularly would like to thank my mom, Dr. Barnana Vijaya Kumari, as well as my dad, Basava Vijaya Bhaskara Rao, and my sister, Shilpa Basava, for supporting me morally throughout my PhD programme. I would even like to thank Irene,

Kyle, Nada, Sumiea and Emi for their endless support.

Table of Contents

Acknowledgements 4

Abstract 7

Chapter 1 Introduction 8 1.1 General Information 8 1.2 Antigen presenting cells (APCs) 10 1.3 Major Histocompatibility Complex 11 1.4 Stimulation of T-cell activation by dendritic cells 13 1.5 Zwitterionic polysaccharides as T-cell dependent antigens 13 1.6 T-cell dependent immune responses: Peritonitis and abscess formation 15 1.7 Bacteroides fragilis 16 1.8 PSA structure 18 1.9 ZPS-mediated CD4+ T-cell activation 18 1.10 Cytokines involved in T-cell-dependent immune responses 19 1.11 Toll-like receptor (TLR) signaling 20 1.12 Aim of the present study 22

Chapter 2 Synthesis and Biological Evaluation of a Potential Immunomodulatory 23 Zwitterionic Polysaccharide 2.1 Introduction 23 2.2 Results and Discussion 37 2.2.1 Synthesis of the pyruvate containing galactose moiety 37 2.2.2 Synthesis of the phthalimido aduct 44 2.2.3 Synthesis of the mono-silylated 1,4-cylcohexanediol 48 2.2.4 Synthesis of the title compound 49 2.2.5 Biological evaluation 51

Chapter 3 Synthesis of Chemical Reactivity of 3,6-Anhydro-D-glucal 56 3.1 General Information 56 3.2 Background 68 3.3 Results 71 3.4 Summary 75 Chapter 4 4.1 Experimental 76

4.2 NMR Spectra 93 4.3 References 164

ABSTRACT

Synthesis and Evaluation of Biological Activity of a Potential Immunomodulatory Zwitterionic Polysaccharide

Vikram Basava

Doctor of Philosophy in Chemistry

Seton Hall University

Dr. Cecilia H. Marzabadi

The gram-negative anaerobic bacterium Bacteroides fragilis is an integral component of the normal gastrointestinal flora. The bacterium colonizes the intestinal tract of human beings as it has no reservoir other than mammals. An unprecedented proportion of the genomic DNA of B. fragilus is involved in the production of capsular polysaccharides.

These capsular polysaccharides are important virulence factors in most extracellular bacterial pathogens. Eight of these polysaccharides have been identified thus far, out of which two were found to be zwitterionic polymers, PSA1 and PSA2. PSA1 was found to stimulate T-cell lineage of the immune system because of the dual charge structural feature of the molecule.

This dissertation discusses about the synthesis of a PSA1 analogue and evaluating its immunomodulatory activity. Also discussed are the synthesis and the chemical reactivity of 3,6-anhydro-D-glucal which was formed unexpectedly in the synthesis of L-rhamnal.

Chapter 1

Introduction

1.1 General Information

The human immune system utilizes a variety of defense mechanisms to maintain the integrity of the human body. Recognizing, as well as differentiating between self and foreign antigens and defending the body from diverse infectious invaders are the major tasks of the immune system. The defense mechanisms consist of several protective measures such as natural barriers, non-specific (innate) as well as specific (adaptive) immune responses. Natural barriers are the first line of defense and are comprised of both physical, as well as, chemical barriers. Skin, being the toughest layer for a pathogen to overcome, is a physical barrier. Chemical barriers come into action when the integrity of the skin is compromised, often due to injury. Each region of the body has its own strategy and contributes to host defense against pathogens. Mucous membranes are protected by the secretion of anti-microbial immunoglobulins that can be found in saliva or tears. Mucous as well as cilia located in lower respiratory tract prevent colonization from pathogens. Similarly, the acidic pH in the gastrointestinal tract is an inhospitable environment for most microorganisms.1

The second line of defense is the innate immune system, which acts on the antigens that penetrated the first line of defense, i.e., the skin and mucous membranes. Antigens are toxins or other foreign substances that induce an immune response in a body by producing antibodies. Innate immune responses which are found in all living organisms

8 are antigen independent and act immediately on the clearance of pathogens with a maximal response. The response is cell mediated and humoral, but, does not generate a lasting protective immunity because of the lack of immunologic memory.

Microorganisms are detected and destroyed within minutes or hours by the various mechanisms of the innate immunity and occasionally causing perceptible disease in a normal, healthy individual.1

The cells of the immune system originate from the stem cell. The stem cells produce myeloid progenitor cells as well as lymphoid progenitor cells. Myeloid progenitor cells in the bone marrow give rise to neutrophils, eosinophils, basophils, monocytes and dendritic cells. The lymphoid progenitor cells give rise to T-cells and B-cells.

Macrophages and dendritic cells, which play a key role in innate and adaptive immunity, are derived from monocytes. Mast cells, which are fixed in tissues, develop from the same precursor cell as the circulating basophils. B-cells are produced in the bone marrow and are released into the blood the lymphatic systems. B-cells can further develop into plasma cells that secrete antibodies. Precursor T-cells undergo differentiation in the thymus into two distinct types of T-cells, the CD4+ T-helper cells and the CD8+ cytotoxic T-cells. Macrophages and dendritic cells function as bridges between innate immunity and adaptive immunity, since they present antigens to the immunocompetent T-cells which initiate an immunological response. Phagocytes involved in the innate immune system are macrophages, mast cells, dendritic cells and granulocytes. They act upon the antigens by breaking them down with the digestive enzymes in their lysosomes. Only when an infectious organism breaches these early lines of defense will an adaptive immune response ensue.1

The third line of defense is the adaptive immune system which comprises of the antigen-specific B-cell and T-cell lymphocytes. T-cells get involved with the B-cells, which have the ability to rearrange genes of the immunoglobulin family, as soon as the innate immune system signals the presence of a danger to the T-cells of the adaptive immune system. This results in the creation of diverse antigen-specific clones and immunological memory.2 The adaptive immune system then activates antigen-specific lymphocytes to proliferate and to differentiate into effector and memory cells that eliminate pathogens. Memory cells can prevent recurrence of disease caused by the same microbe. This highly sophisticated and potent adaptive immune system needs to be conducted, instructed and regulated by antigen presenting cells (APCs).

1.2 Antigen Presenting Cells (APCs)

Antigen Presenting Cells (APCs) include dendritic cells (DCs), macrophages as well as the B-cells. Bone-marrow derived APCs are present on peripheral blood and lymphatic tissues as well as in other organs to guard the host.1 DCs carry the antigens of the pathogen to the secondary lymphatic organs, the lymph nodes and the spleen. They also help in intracellular engulfing and degrading the antigens.

The activity of the DCs depends upon the extent of their maturation. Immature DCs have a tremendous phagocytic activity to ingest antigens via pinocytosis or receptor- mediated endocytosis. They have a number of receptors that enhance the uptake of antigens, and they are specialized to convert these antigens into Major

Histocompatibility Complex (MHC)-peptide complexes that can be recognized by lymphocytes. Mature DCs lose their phagocytic activity and activate the adaptive immune system response by transforming into highly-effective APCs which have a very

10 short life span with no proliferation capacity.3 Upon antigen-mediated activation, DCs influence both the innate and the adaptive immune responses, thereby determining how the immune system responds to the presence of infectious agents.1

1.3 Antigen processing and Major Histocompatibility Complex (MHC)-dependent antigen presentation in APCs

Activation of the adaptive immune system by DCs is mediated by cytokine secretion and by direct presentation of antigens to T-cells. T-cells, which circulate between blood and peripheral lymphoid tissues, consist of T-helper, T-cytotoxic and T-regulatory cells. The

CD4+ T-helper cells depend on the major histocompatibility complex (MHC) class II peptide-contact to initiate and modulate receptor- and cytokine-mediated functions of the immune system.4 CD8+ T-cytotoxic cells depend on the MHC class I by recognizing the endogenous antigens and mediating the cytotoxic immune system.5 MHC is a membrane glycoprotein encoded in a cluster of genes. It displays peptide antigen to T- cells. T-regulatory cells suppress the immune response and protect the host from aggressive effector mechanisms of the immune system.

Extracellular pathogens and proteins captured by APCs are either recycled to the plasma membrane or transported to the lysosome for degradation. They are then presented to the CD4+ T-cells through MHC class II proteins to induce an immune response.1, 6 Proteases, glycosidases and lipases present in endolysosomes are activated by low pH giving rise to protein degradation into smaller peptide fragments.

The peptide needs to get loaded onto the MHCII molecule for it to present the antigen to a CD4+ T-cell.7 The MHCII molecules are present only on the professional APCs, unlike

11 the MHCI molecules. The MHCII molecule complex consists of two transmembrane glycoproteins, the α and the β, which form a non-covalently bound complex. The open ends on the peptide-binding groove of the MHCII molecules get filled and blocked when the invariant chain prevents uncontrolled early binding thereby targeting it to an acidic endosomal compartment. The activated proteases cleave the MHCII:invariant chain complex when it enters the acidic endosomal pathway leaving a short fragment called class II-associated invariant-chain peptide (CLIP). Intracellular antigen loading of MHCII is dependent on HLA-DM, a MHCII-like molecule in humans (H-2M in mice) which is found predominantly in the MHCII compartment.8 The antigen first binds to the MHCII molecule, catalyzes the release of the CLIP fragment from the MHCII binding groove, stabilizes the MHCII molecule to prevent aggregation and aids in rapid and tight binding of peptides to the empty MHCII molecule. The MHCII:peptide complex is then transported from the lysosome to the cell surface and the peptide presented to CD4+ T- cells.9 Once recognized, the CD4+ T- cells activate other effector cells of the immune system, like macrophages, which kill the intravesicular pathogens they harbor, or B-cells to secrete immunoglobulins against the foreign bodies.

The B-cell antigen receptors are a membrane-bound form of antibodies which will be released upon activation, plasma cell differentiation and proliferation of the B-cell with a specific antigen. The MHCII:peptide complex must be stable at the cell surface. If it isn’t stable, the pathogen in the infected cell could escape detection or be picked by MHC molecules of an infected cell and be wrongfully destructed by cytotoxic T-cells.1

There are about 30,000 antigen-receptor molecules, called T-cell receptors (TCRs) on the T-cells which consist of TCRα and TCRβ polypeptide chains linked by a disulfide

12 bond. The TCR on the cell surface recognizes processed and presented peptide antigens. The α:β heterodimers have a similar structure to the Fab fragment of an immunoglobulin molecule which account for antigen recognition by most T-cells.1

1.4 Stimulation of T-cell activation by Dendritic cells (DCs)

An immunological synapse made up of MHCII:peptide complexes on the DC and the

TCR on the T-cells is formed when the mature DCs interact with the T-cells. This interaction leads to the activation of the T-cells by an intracellular signaling pathway which leads to the induction of differentiation and proliferation. The naïve T-cells proliferate and their progeny adhering to the APCs develop into armed effector T-cells.1

The secretion of cytokines leads to the DCs further directing T-cell development towards Th1 and Th2 phenotypes via cytokine production.10 Interaction of immature or tolerogenic DCs with T-cells can result in T-cell deletion, anergy, or the generation of

Treg cells.11

1.5 Zwitterionic polysaccharides as T-cell-dependent antigens

MHCII proteins were previously only known to present proteins, peptides or superantigens. Most of the known bacterial polysaccharides do not get processed or presented on the APC surface to T-cells, as they fall apart in the lysosomes of the

APCs.6 But, recent in vivo and in vitro studies showed that some polysaccharides of certain symbiotic bacteria get transported from lysosomes to the cell surface to get presented by MHCII protein. This results in the activation of CD4+ T-cells in vitro and the induction of CD4+ T-cell-dependent abscesses in vivo.12,13,14,15,16 Hence, this discovery of the MHCII pathway being responsible for carbohydrate antigen presentation and

13 eventual T-cell recognition alters the previous fundamental model of MHCII-antigen binding.

The CD4+ T-cell activation by bacterial polysaccharides is dependent on the transport of bacterial polysaccharides via MHCII. The functional role of HLA-DM is to help the bacterial polysaccharide to compete with peptides for MHCII binding.13,14

The few polysaccharides which induce the CD4+ T-cell-dependent formation of abscesses in vivo are characterized by the presence of opposite charged motifs on each repeating unit. They are therefore termed as zwitterionic polysaccharides (ZPS).

They include polysaccharides like PS A1 and PS A2, Sp1, and CP5 from the commensal bacteria Bacteroides fragilis, Streptococcus pneumonia and

Staphylococcus aureus, respectively (Fig. 1.1).17 The presence of zwitterionic charges is critical for the antigenic function of the ZPSs. Removal of either of the charges on the

ZPS resulted in compounds that failed to stimulate CD4+ T-cells.18

Fig. 1.1: Subunits of PSA1, PSA2, Sp1 and CP5

1.6 T-cell-dependent immune responses: Peritonitis and abscess formation

Secondary peritonitis, the most common one, is observed because of the loss of integrity in the gastrointestinal tract. This is due to the contamination of the peritoneal space by symbiotic intestinal bacteria.19 Development of intraperitoneal abscesses, a bacterial infection, is followed by colonic leakage of commensal intestinal bacteria. It is seen in patients with inflammatory bowel diseases (IBD) like Crohn’s disease or ulcerative colitis. Almost a third of patients infected with general peritonitis die in spite of improved diagnostic modalities, potent antibiotics, modern intensive care and aggressive surgical treatment.20 The surgical treatment for generalized peritonitis is through peritoneal lavage, drainage of localized abscesses, removal of the contaminating source by repairing, and drainage of the peritoneal cavity. However, even in the presence of improvised therapy, residual abscesses form in patients resulting in substantial morbidity and mortality.21

Abscess formation is known to be a CD4+ T-cell-dependent immune response induced by ZPS.18 The zwitterionic charge promotes CD4+ T-cell-dependent abscess induction

15 in experimental murine and rat models.22 Chemical abrogation of the zwitterionic charge demonstrated that the charged nature of ZPS is critical for its ability to induce abscess formation. No CD4+ T-cell-dependent immune responses are observed for ZPS derivatives that have been made neutral or only possess a single charge (cation or anion).18 In contrast, uncharged polysaccharides induce T-cell-dependent immune responses after they are modified to possess a zwitterionic charge.18,23,24

1.7 Bacteroides fragilis

Bacteroides fragilis is a Gram-negative, anaerobic, bile-resistant, non-spore forming, rod-shaped bacterium whose primary known environmental reservoir is the human lower gastrointestinal tract where it lives as a commensal organism. They are significant clinical pathogens and are found in most anaerobic infections, with an associated mortality of more than 19%. The bacteria maintain a complex and generally beneficial relationship with the host when retained in the gut. They fermentation of carbohydrates in the human intestine results in the formation of a pool of volatile fatty acids which are reabsorbed through the intestine and utilized by the host as an energy source.25 But, when they escape from the gut, they can cause significant pathology including bacteremia and abscess formation in multiple body sites. Though B. fragilis accounts for only 0.5% of the human colonic flora, it is the most commonly isolated anaerobic pathogen because of its potent virulence factors. They have the most antibiotic resistance mechanisms and the highest resistance rates of all anaerobic pathogens.

Clinically, Bacteroides sp. have exhibited increasing resistance to many antibiotics including cefoxitin, clindamycin, metronidazole, carbapenems and fluoroquinolines (e.g., gatifloxacin, levofloxacin and moxifloxacin).

A distinctive feature of B. fragilis is the large proportion of the genome devoted to carbohydrate metabolism, including the degradation of dietary polysaccharides and the production of surface polysaccharides.26,27,28 The ability to express multiple capsular polysaccharides has been shown to enhance intestinal colonization by B. fragilis.29 Thus far, eight different capsular polysaccharides, each synthesized from different genomic loci, have been identified. Two of the capsular polysaccharides of B-fragilis, polysaccharide A (PSA) and polysaccharide B (PSB) belong to a class of carbohydrates known as zwitterionic polysaccharides (ZPSs). Previously, carbohydrates had been characterized as classic T-cell-independent antigens that induce a specific IgM response but fail to evoke an IgG or memory response.30,31 However, it is now known that ZPSs are capable of inducing CD4+ T-cell-dependent immune responses. All of the known ZPSs have immunomodulatory properties that depend on the presence of both positively and negatively charged groups within the repeating units.18,22,32 Of all the known ZPSs, PSA is the best characterized.

PSA is a unique and potent antigen. Among carbohydrates, this polysaccharide stands out as a T-cell dependent rather than a T-cell-independent antigen. Although it is processed and presented along the same pathways as conventional peptide antigens,

PSA modulates the host immune system more extensively than many other known T- cell-dependent antigens.

1.8 PSA Structure

Fig 1.2: Structure of Polysaccharide A (PSA)

PSA is a zwitterionic polysaccharide, with an average molecular mass of 100 kDa, comprising of repeating tetrasaccharide units. The positive charge is situated at the free amino group of the rare 2-acetamido-4-aminofucose moiety and the negative charge resides on the carboxylate of the pyruvate ketal spanning C4 and C6 in the galactopyranosyl residue.

1.9 ZPS-mediated CD4+ T-cell activation

Activation of CD4+ T-cells requires the specific interaction of the α-β T-cell receptor

(TCR) on the T-cell with the antigen presented in the context of MHCII on a professional

APC.33 To induce an immune response, PSA must be processed in an endocytic pathway and presented on an MHCII molecule in a manner analogous to that by which peptide antigens are presented. The notable difference is that, in the endosome, the degradation of PSA to a molecule size small enough for presentation by the MHCII

18 molecule is a non-enzymatic chemical process. Whereas, protein degradation occurs enzymatically.33,34 Once taken up into an APC, PSA follows the same processing pathway as peptide antigens. Upon entry into the endosome of an APC, PSA is directed to a MHCII compartment. The up-regulation of inducible nitric oxide synthetase, which is associated with the oxidative burst, results in increased nitric oxide (NO) production. NO breaks down PSA through deamination, a chemical reaction in which PSA is oxidized and then depolymerized to a much smaller molecular size while retaining the zwitterionic motif and the overall structure of the repeating unit. Finally, co-localization of

PSA, MHCII, and α-β TCRs can be detected on the cell surface of murine splenocytes by confocal microscopy. However, no PSA is found on the cells lacking MHCII.34 In the absence of direct contact between APCs and T-cells, ZPSs cannot stimulate T-cell proliferation or induce cytokine production in vitro.16

1.10 Cytokines involved in T-cell-dependent immune responses

Cytokines are of great importance in CD4+ T-cell-induced abscess formation. It has been suggested that cytokines may be responsible for triggering the migration of immune cells into the peritoneal cavity following contamination with B. fragilis.35

Cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, IL-12, IL-

15 and IL-18 are involved in T-cell activation as well as proliferation.36 It was demonstrated that ZPS PSA induces the production of TNF-α and IL-1α on peritoneal cells which act as significant immune mediators for abscess formation.37 When TNF-α is blocked, expression of the ICAM-1 (intercellular adhesion molecule-1) is significantly reduced leading to the accumulation of polymorphonuclear leukocytes (PMNLs) within the abdominal cavity. This then inhibits the formation of a peritoneal sepsis.

Furthermore, PSA stimulates the secretion of chemokines such as IL-8 which stimulate

T-cells.

IL-10, one of the most potent anti-inflammatory cytokines in the immune system, is required for PSA suppression of the inflammatory immune response. It plays a critical role in protection against inflammation in several animal models.

During sepsis and peritonitis, IL-6 is considered as an import mediator of immunologic alterations in the host, along with TNF-α and IL-1.38 From the results observed, IL-6 is the most potent proinflammatory cytokines because it stimulates the expression of the broadest spectrum of acute phase proteins in inflammation and infection.39

IL-6 is a pleiotropic inflammatory cytokine which is produced by a variety of cells. It acts on a wide range of tissues by stimulating the activation, survival and proliferation of

CD4+ T-cells and acts on T-cells as an anti-apoptotic factor.40 IL-6 prevents apoptosis of normal and resting T-cells in the absence of additional cytokines. It has also been identified as a migration factor for human primary T-cells.41 In vitro experiments showed chemotactic activity of IL-6 on peripheral blood lymphocytes, lymphokine-activated killer cells, and CD4+ and CD8+ T-lymphocytes.42 IL-6 was also found to be linked to T-cell- dependent immune diseases and autoimmune diseases such as the inflammatory bowel diseases, ulcerative colitis and Crohn’s disease.43

1.11 Toll-like receptor (TLR) signaling

Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single, membrane spanning, non-catalytic receptors usually expressed in cells such as macrophages and dendritic cells. TLRs play a critical role in

20 host defense by sensing the presence of microbes. They mediate inflammatory responses, coordinate innate and adaptive immunity, prime naïve T-cells, and induce memory to facilitate the elimination of pathogens, upon activation.44,45 The TLRs include

TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. TLR12 and TLR13 are not found in humans. TLRs recognize certain highly conserved microbial components known as pathogen-associated molecular patterns

(PAMPs). They even recognize other molecules like bacterial lipopolysaccharides, flagellin, bacterial DNA, and viral double-stranded RNA.46 Nevertheless, both pathogenic and commensal microbes produce the microbial ligands which are recognized by TLRs. Although TLR signaling was traditionally associated with the sensing of the pathogens, it is now appreciated that commensal bacteria are also recognized by TLRs, and that this interaction is critical for protection against epithelial injury and intestinal homeostasis.47

The triggering of TLRs on DCs leads to the induction of co-stimulatory signals (e.g.

MHCII) and to the production of cytokines that orchestrate T-cell recruitment, activation, differentiation and survival and are essential for the induction of adaptive immune responses.48 Likewise, the decrease in processing and presentation of PSA combined with the decreased production of IL-12 by dendritic cells activated by PSA in the absence of TLR2 signaling demonstrates that in addition to the stimulation of a specific

CD4+ T-cell immune response, innate immune pathways are required for PSA to establish a Th1/Th2 balance.

1.12 Aim of the present study

From what was discussed above, it is quite evident that ZPSs like PSA have the potential to induce CD4+ T-cell-dependent immune responses in vitro and are also capable of inducing abscess formation in vivo in a CD4+ T-cell-dependent manner. But, not much work has been done to date to study the potential to induce CD4+ T-cell- dependent immune responses of simplified, zwitterionic polysaccharides which are analogous to PSA.

We propose to study the synthesis of an immunomodulatory zwitterionic polysaccharide as well as its interactions with APCs and T-cells. The molecule synthesized will be evaluated for its ability to produce IL-6 and TNF-α in APCs. IFN-γ (T-cell response) and

IgM (B-cell response) production will be studied using mouse spleen cells in co-culture with APCs. Finally, a cell proliferation assay will be done on the spleen cells of mouse.

The initiation of both innate and adaptive responses by the ligands could prove to be a major step forward in the development of a carbohydrate-based vaccine.49

Chapter 2

Synthesis and Biological Evaluation of a Potential Immunomodulatory

Zwitterionic Polysaccharide

2.1 General Information

A considerable amount of work has been done for the synthesis of various zwitterionic molecules. The compounds synthesized were found to be of incredible importance in the field of science. Kowalczky50 reported the synthesis of alkylammonium zwitterionic amino acid derivatives (Fig 2.1). The molecules synthesized were characterized by

FTIR, NMR spectroscopy and DFT calculations. The pharmacotherapeutic potential of the molecules synthesized was estimated by Prediction of Activity Spectra for

Substances (PASS) which showed that the compounds could be used for as creatinase inhibitors, polyamine-transporting ATPase inhibitors, hydrogen dehydrogenase inhibitors and alkylacetylglycero-phosphatase inhibitors. They were even predicted to be useful as antiviral, antitoxic and antimutagenic agents.

Fig. 2.1 Synthesis of 3-(3-dimethylamonio)propylammonio propanoate bromide (a), 4- (3-dimethylamonio)propylammonio butanoate bromide (b), 5-(3- dimethylamonio)propylammonio pentanoate bromide (c)

Similarly, Szoka, et al.,51 reported and synthesized a novel class of zwitterionic lipids with head groups containing a 3o or 4o amine and a carboxylate group. These lipids were found to form stable liposomes that exhibit head group dependent, pH-responsive biophysical characteristics which could make them suitable for drug delivery applications.

Zwitterionic structure monomers such as phoshorylcholine52, sulfobetaine53 and carboxybetaine54 have been found to reduce protein absorption and platelet adhesion.

Xu, et al.,55 incorporated these charged groups into polyurethanes (Fig 2.2) by synthesizing them from a polycarbonatediol with alkyne groups and 3-((azidoethyl)

24 dimethylammonio)propane-1-sulfonate using the copper catalyzed 1,3-dipolar cycloaddition (click) reaction.

Fig. 2.2 Polyurethane

Likewise, Bergstrom56 and others described the synthesis and the structure–activity relationships of zwitterionic spirocyclic compounds which are used as potent cysteine- cysteine chemokine receptor-1 (CCR1) antagonists (Fig 2.3). A spirocyclic amine was treated with 3-bromo-2,2-dimethyl-propan-1-ol in the presence of NaI and Et3N in DMF to give the desired adduct which upon coupling with 4-fluoro-2-hydroxybenzoate in a

Mitsunobu type reaction gave the ester adduct. The ester adduct was then treated with the racemate of methyl pyrollidine-3-carboxylate in the presence of carbonyl di- imidazole (CDI) in N, N-dimethylformamide (DMF) to give a mixture of the diastereomers.

o Fig. 2.3 (a) NaI, Et3N, DMF, 78 C 60 h; (b) methyl 4-fluoro-2-hydroxybenzoate, Ph3P, DEAD, THF, rt 20 h; (c) (R)-methyl pyrrolidine-3-carboxylate/(S)-methyl pyrrolidine-3-carboxylate, CDI, DMF, rt, 20 h

Of the few known zwitterionic capsular polysaccharides, the structures of only a handful of them (PS A1 and PS A2, Sp1, and CP5 from the commensal bacteria Bacteroides fragilis, Streptococcus pneumonia and Staphylococcus aureus, respectively) have been characterized thus far (Fig 2.4).

Fig. 2.4 Zwitterionic polysaccharides (Subunits)

Bundle, et al.,57 first synthesized the hexasaccharide representing two repeating units of the zwitterionic capsular polysaccharide from Streptococcus pneumoniae type 1 (Sp1).

The synthesis involved the stereoselective construction of 1,4-cis-α-galactose linkages based on a reactive trichloroacetimidate donor that incorporates a 6-O-acetyl group which has been proposed to account for the α-selectivity in glycosylation.

Later, Codee, et al.,58 came up with a modular approach towards the synthesis of all possible trimer repeating units of the Sp1 polysaccharide. For efficiently introducing α- galacturonic acid bonds stereoselectively, galacturonic-[3,6]-lactone building blocks

27 were used. The building blocks not only acted as donor galactosides, but they were also functional as reactive acceptor glycosides when equipped with a free hydroxyl group

(Fig 2.5).

Fig. 2.5 Three frame-shifted trimer repeats of Sp1 polysaccharide

Similarly, Bundle, et al., were successful in synthesizing a trisaccharide repeat of the zwitterionic Sp1 polysaccharide. 2-Azido-4-benzylamino-4N,3-O-carbonyl-2,4,6- trideoxy-D-galactopyranosyl trichloroacetimidate (B), was synthesized in six steps from

6-deoxy-D-glucal. The glucal was glycosylated with selectively protected α-1,3-linked methyl galabioside (C, D) to produce the trisaccharide skeleton of Sp1 (Fig 2.6).

Fig. 2.6 Trisaccharide repeat of zwitterionic Sp1 (A) and the monosaccharide synthons (B, C, D)

PS A1, one of the zwitterionic capsular polysaccharides of Bacteroides fragilis, was first synthesized successfully by van der Marel, et al.,59 in its fully protected form (Path I, Fig

2.7). One of the drawbacks of this synthesis is that the overall yield was low because of very low nucleophilicity of molecule B.

Seeberger, et al.,60 later synthesized the same molecule by the [3+1] glycosylation technique, in its unprotected form (Path II, Fig 2.7). Glycosylation in this process took place between the highly electro deficient trisaccharide glycosylating agent C, and the pyruvated galactose nucleophile D. The –OH group on C3 of the galactose residue acted as a very good nucleophile.

Fig. 2.7 Retrosynthesis of PS A1

For the de novo synthesis of the 2-acetamido-2-amino-2,4,6-trideoxy-D-galactose

(AAT), N-Cbz-L-threonine (i) was used as the starting material (Fig 2.8). It was first converted to a methyl ester followed by acetylation to give (ii). Treatment of (ii) with lithium bis(trimethylsilylamide) (LHMDS) resulted in a Dieckmann cyclization61, 62 to give enoate (iii). The treatment of (iii) with 1,2-diisobutylaluminium hydride (DIBAL)63 gave

(iv) which upon reduction under Luche64 conditions gave allylic alcohol (v). Acetylation of

(v) followed by azidonitration65 gave (vii). Cleavage of the anomeric nitrate in (v) with p-

TolSH/DIPEA66 followed by reaction with 2,2,2-triflouroacetamidoyl chloride resulted in the formation of the N-phenyl trifluoroacetimidate sugar (A).

Fig. 2.8 Synthesis of the AAT building block (A)

Reagents and conditions (a) i., FmocCl, py., 0 °C, 79%; ii., BzCl, py., 0 °C, 82%; iii., TsOH, MeOH, 40 °C; iv., CH3C(O)CO2Me, BF3·OEt2, CH3CN, 23 °C, 39%, two steps; (b) i., i-PrOH, NIS, AgOTf, CH2Cl2, 0 °C; ii., NEt3, CH2Cl2, 23 °C, 49%, two steps; (c) LevOH, DIPC, DMAP, CH2Cl2, 0 to 23 °C, 90%; (d) i., NIS, THF, H2O, 23 °C; ii., F3CC(NPh)Cl, Cs2CO3, CH2Cl2, 0 °C, 83%, two steps; (e) i., NBS, EtOAc, H2O, 23 °C, 91%; ii., F3CC(NPh)Cl, Cs2CO3, CH2Cl2, 23 °C, 87%

The galactosamine derivative was earlier synthesized (Fig 2.9) by the initial treatment of

(viii) with naphthyl bromide and NaH followed by cleavage of the benzylidene ring with

67 TES and TFA in CH2Cl2 to give the 6-O-benzylated galactosamine derivative, (ix).

Fig. 2.9 Synthesis of the galactosamine derivative (ix)

o o o Reagents and conditions (a) NapBr, NaH, THF, 0 C to 23 C, 84%; (b) TES, TFA, CH2Cl2, 0 C to 23 oC, 81%

The galactosamine derivative obtained, (ix), was then glycosylated (Fig 2.10) with the

o AAT, (A), in the presence of TMSOTf in CH2Cl2 at 0 C to give the disaccharide (x).

Fig. 2.10 Glycosylation of the AAT building block (x)

Reagents and conditions (a) TMSOTf, CH2Cl2, 0 °C, 74%

Similarly, the galactofuranose building block (xii), was synthesized (Fig 2.11) by reacting the known thiol68 (xi) with N-bromosuccinimide (NBS) to form the lactol, followed by the formation of the N-phenyl trifluoroacetimidate derivative (xii).

Fig. 2.11 Synthesis of the galactofuranose building block (xii)

Reagents and conditions (a) i., NBS, EtOAc, H2O, 23 °C, 91%; ii., F3CC(NPh)Cl, Cs2CO3, CH2Cl2, 23 °C, 87%

The last building block, the pyruvated galactose sugar (D), was synthesized from the benzylidene galactoside (xiii).69 The C3 and C2 hydroxyl groups were initially protected with Fmoc70 and Bz groups respectively (Fig 2.12). Cleavage of the benzylidene acetal

71 was followed by the formation of the pyruvate ketal (xiv) using BF3. OEt2. Finally, glycosylation of (xiv) with isopropanol in the presence of NIS/AgOTf followed by removal of the Fmoc protecting group gave the pyruvated galactose nucleophile (D).

Fig. 2.12 Synthesis of the pyruvated galactose nucleophile (D)

In the synthesis of the trisaccharide (C), the first step involved the removal of the naphthyl ether group using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (Fig

2.13). The resulting alcohol (xvi) was glycosylated with galactofuranose N-phenyl trifluoroacetimidate (xii) to give the trisaccharide adduct (xvii) as the β-anomer. The anomeric tert-butyl-dimethylsilyl (TBDMS) group was removed in the presence of tetrabutylammonium fluoride (TBAF) to give a lactol that upon treatment with 2,2,2- triflouroacetamidoyl chloride in the presence of Cs2CO3 resulted in the formation of the

N-phenyl trifluoroacetimidate adduct (xviii). Thioethyl glycoside (C) was later synthesized

33 from glycosyl imidate (xviii) by the reaction of the latter with ethanethiol (EtSH) in the presence of TMSOTf at 0 oC in the presence of 4 Ao molecular sieves.

Fig. 2.13 Synthesis of the trisaccharide, C

Reagents and conditions (a) DDQ, MeOH, CH2Cl2, 23 °C, 86%; (b) TMSOTf, CH2Cl2, −30 °C, 90%; (c) i., TBAF, AcOH, THF, 0 °C; ii., F3CC(NPh)Cl, Cs2CO3, CH2Cl2, 23 °C, 82%, two steps; (d) EtSH, TMSOTf, o 4 A MS, CH2Cl2, 0 °C, 96%

The resulting trisaccharide (C) formed was made to react with pyruvated galactose moiety (D) in the presence of dimethyl (methylthio) sulfonium triflate (DMTST)72 and

2,4,6-tri-tert-butylpyrimidine (TTBP) at 0 oC to give the totally protected PS A1 adduct

(xix) in 58% yield (Fig 2.14).

Fig. 2.14 Formation of the tetrasaccharide, (xix)

Reagents and conditions (a) dimethyl (methylthio) sulfonium triflate (DMTST), 2,4,6-tri-tert- o o butylpyrimidine (TTBP), 4 A MS, 0 C, CH2Cl2

Finally, the azide groups of fully-protected PS A1 (xix) were converted to acetamides using ethanethioic acid (AcSH) and pyridine (Fig 2.15). Global deprotection of the acetamide adduct of PS A1 was performed in two different steps. Initially, the compound was hydrogenated in the presence of Pearlman’s catalyst73 which removed the benzyl as well as the CBz groups. Next, NaOMe in MeOH/H2O was added dropwise to form the final PS A1 tetrasaccharide repeating unit in 46% yield.

Fig. 2.15 Deprotection and formation of PS A1 tetrasaccharide repeating unit

Reagents and conditions (a) AcSH, py., 23 °C, 67%; (d) i., H2, Pd(OH)2/C, MeOH, 23 °C; ii., THF, then 0.5 M NaOMe in 1:1 MeOH/H2O, 23 °C, 46%, two steps

Earlier studies revealed that peptides synthesized to mimic the zwitterionic charge motif associated with zwitterionic polysaccharides exhibited biologic properties. Lysine- aspartic acid (KD) peptides with more than 15 repeating units stimulated CD4+ T-cells in vitro and gave protection against abscesses induced by bacteria such as Bacteroides fragilis and Staphylococcus aureus.74

Alterations made in the charge motifs of the zwitterionic polysaccharides were shown to have either partially or totally removed the stimulatory properties of the sugars.75 It was

36 also proved that the non-charged portions of the saccharide chain are not essential for the stimulation by PS A1. Modifications made to the non-charged sugars failed to diminish the production of cytokines.

Discussed hereafter is the synthesis of an analogue of the PS A1 zwitterionic polysaccharide that could be of utmost importance in investigating its biological activity.

Since only the charged sugar residues have been implicated in binding and processing of PS A1, the synthesis has been carried out using a commercially-available cycloalkane diol as the bridging moiety. As the galactofuranose moiety has zero charge on it, and as it doesn’t contribute to the size of the PS A1 moiety in increasing the distance between the charges (Calculated: 13 Ao; Observed: 15 Ao), we decided to not include it in the synthesis of the analogue.

2.2 Results and Discussions

2.2.1 Synthesis of the pyruvate-containing galactose moiety

The synthesis of the pyruvated sugar moiety started with the commercially available β-

D-galactose pentaacetate (1). It was first made to react with 1.2 equivalents of thiophenol (PhSH) in the presence of BF3.OEt2 to give predominantly the anomeric β- substituted thiophenol adduct (2) in 97% yield (Scheme 2.1).76

Scheme 2.1 Synthesis of β-thiophenyl-2,3,4,6-tetra-O-acetyl-D-galactopyranose

The thiophenyl adduct obtained was then de-acetylated using Zemplen’s conditions to yield 98% of the galactopyranose sugar (3) in 98% yield (Scheme 2.2).77

Scheme 2.2 De-acetylation using Zemplen’s conditions

A series of reactions were then set up to selectively protect the C4 and C6 hydroxyl functional groups. The best possible route chosen involved first forming of the 4, 6-O- benzylidene protecting group. As the benzylidene ring is preferentially six membered, there is a greater chance of ring formation between the C4 and the C6 hydroxyl groups.

The de-acetylated thiophenyl adduct (3) was treated with benzaldehyde dimethylacetal in the presence of camphor sulfonic acid in DMF at 65 oC (Scheme 2.3).78 However,

TLC analysis of the reaction mixture showed that under these conditions esentially no reaction took place. A similar reaction was also carried out using the dimethyl acetal in the presence of p-toluenesulfonic acid. But, again there was no reactivity observed when monitoring by TLC.79 Reactions were set up separately with (3) using benzaldehyde and benzaldehyde dimethyacetal in the presence of pyridinium p- toluenesulfonic acid. These reactions also did not show the formation of product.

Scheme 2.3 Various attempts in synthesizing the benzylidene sugar moiety

When the reaction when set up in the presence of ZnI2 and benzaldehyde, none of the desired product was obtained (Scheme 2.3). But, the same reaction (Scheme 2.4) when repeated in the presence of ZnCl2 did give the desired benzylidene sugar (4) in

65% yield.80 The formation of product was supported by the 1H NMR spectrum that showed a singlet at 5.53 ppm which represents the axial hydrogen on the benzylidene ring.

Scheme 2.4 Synthesis of the benzylidene sugar moiety

Compound (4) was subsequently reacted with dibutyltin oxide and benzoyl chloride in the presence of 4 Ao molecular sieves in toluene to give a mixture of 2-O-benzoyl and 3-

O-benzoyl sugar derivatives (Scheme 2.5). The latter, being the major product, was easily separated by flash column chromatography yielding 50% of the 3-O-benzoyl adduct (5a).81

Scheme 2.5 Selective benzoylation using organo tin chemistry

Various attempts were made to protect the hydroxyl group at the C-2 position. A benzylation reaction was initially carried out by reacting compound (5a) with benzyl

40 chloride in the presence of NaH and 15-crown-5 in DMF (Scheme 2.6).82 As the reaction failed to occur, the benzylation reaction was attempted by using benzyltrichloroacetimidate and trifluoromethane sulfonic acid.83 But, again there was no product formation. Reaction of (5a) with p-nitro-benzyl bromide in the presence of NaH and DMF did not show the addition of the benzyl group at the C-2 position. Reaction of p-methoxylbenzyl chloride with (5a) in the presence of NaH and 15-crown-5-ether did not show the benzylation reaction happening, but instead resulted in debenzoylation giving (4) as the final product.84 This offside reaction could have occurred because of the formation of NaOH when NaH reacted with trace amounts of water in the solution.

Scheme 2.6 Attempted benzylation reactions at the C-2 hydroxyl group

The reason for the lack of reactivity of the C-2 was likely due to the bulky nature of the protecting group used and the sterically crowded environment at that position. Hence, a smaller protecting group was used. A reaction was carried out between (5a) and acetyl chloride in the presence of triethylamine and N, N-dimethylaminopyridine which resulted in the formation of the 2-O-acetyl adduct (6) in 69% yield (Scheme 2.7).

Scheme 2.7 Successful acetylation at the C-2 position

The next step was to introduce a pyruvate moiety into the sugar. This could be done by cleaving the benzylidene ring and subsequently introducing the pyruvate functional group into the sugar. The benzylidene ring can be cleaved in different ways. Depending on the reagents used, there may form a 4-O-benzyl-6-hydroxy sugar or a 4-hydroxy-6-

O-benzyl sugar. But, for introduction of the pyruvate ring into the sugar, formation of the

4,6-diol is imperative. Therefore, compound (6) was treated with 20% trifluoroacetic acid

85 in CH2Cl2 to give a 4,6-diol sugar (7a, 45% yield). The diol sugar upon treatment with methyl pyruvate in the presence of BF3-Et2O gave the desired pyruvate sugar (7) in

80% yield (Scheme 2.8).86

Scheme 2.8 Addition of the pyruvate group to the sugar moiety

2.2.2 Synthesis of the phthalimido adduct

Various attempts were made in synthesizing the positive charged moiety having the amino group on it. First, glucosamine was made to react with acetyl chloride which gave the per-acetylated-2-acetamido α-glucopyranosyl chloride with a very good yield.87 The product was then reacted with thiophenol in the presence of Et3N in acetonitrile which gave the desired β-thiophenyl adduct.88 The obtained product showed no reactivity any further probably because of the formation of the stable oxazoline ring with the acetamido group at the C-2 position. Attempts in reacting the glucopyranosyl chloride with 1, 4-cyclohexanediol in the presence of N-iodosuccinimide and Yb(Otf)3 to yield the desired O-glycoside were not fruitful (Scheme 2.9).89

Scheme 2.9 Earlier attempts in synthesizing the thioglycoside

Subsequently, glucosamine first was per-acetylated, and the product was treated with

BF3. Et2O to give the oxazoline derivative. The oxazoline derivative when treated with thiophenol and camphor sulfonic acid (CSA) showed no reactivity.90 This can be attributed to the stability of the oxazoline ring. One-pot reaction for the addition of the thiophenyl moiety to the per-acetylated glucosamine in the presence of BF3. Et2O upon sonication did give the desired thiophenyl adduct.91 The reaction was found to have not reached completion (Scheme 2.9).

Having observed problems with the 2-acetamido group because of its formation of a stable oxazoline ring, plans were made to incorporate a phthalimido group as it doesn’t take part in the neighbouring group participation. Glucosamine HCl was first reacted with NaOMe in methanol to get glucosamine which was further reacted with phthalic

92 anhydride in the presence of Et3N. The product (8) was then acetylated by reacting it with acetic anhydride and di-azo-bicyclooctane (DABCO) to give the desired per- acetylated 2-phthalimido adduct (9) in 30% overall yield (Scheme 2.10).93

Scheme 2.10 Synthesis of the 2-phthalimido adduct

The 2-phthalimido adduct (9) was then made to react with thiophenol in the presence of a mild base, Et3N. TLC of the reaction did not show the formation of the desired product. This was observed because of the bulkiness of the thiophenol group (Scheme

2.11).

Scheme 2.11 Substitution of thiophenol at the anomeric carbon atom

Therefore, a smaller thiol, like ethanethiol, was chosen as the leaving group. Reaction of (9) with ethanethiol and BF3. Et2O in anh. CH2Cl2 gave the desired ethanethiol adduct

(10) in 79% yield (Scheme 2.12).94

Scheme 2.12 Substitution of ethanethiol at the anomeric carbon atom

2.2.3 Synthesis of the monosilylated 1, 4-cyclohexanediol

Synthesis of the of one mono-protected hydroxyl group of 1, 4-cyclohexanediol selectively was not as challenging as the synthesis of the other subunits. First, the cis/trans mixture of 1, 4-cyclohexanediol was reacted with 1.2 equivalents of benzyl chloride and NaH in anh. DMF which gave a separable cis/trans mixture of mono- benzylated product (11a, 11b) with an overall yield of 88% (Scheme 2.13).

Scheme 2.13 Synthesis of the mono-benzylated adduct of 1, 4-cyclohexanediol

But, from the earlier experiences, it was quite evident that debenzylation occurs when the reactions were carried out at elevated temperatures. Hence, attempts were made in substituting the benzyl group with a more robust silyl moiety. Reaction of 1, 4- cyclohexanediol with 1.2 equivalents of tert-butyldimethylsilyl chloride and imidazole in anh. DMF gave the desired mono-silylated product (12a, 12b) in 50% yield as a white fluffy solid (Scheme 2.14).95

Scheme 2.14 Synthesis of the mono-silylated adduct of 1, 4-cyclohexanediol

2.2.4 Synthesis of the final product

Addition of thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose (10) to the cis- mono-silylated adduct of 1,4-cyclohexanediol (12a) in the presence of trimethylsilyl

o trifluoromethanesulfonate (TMSOTf) and NIS in CH2Cl2 at -78 C gave the desired product (13) in 1.5 h. The same reaction when allowed to warm up to room temperature gave the desilylated product (14) in 7 h (Scheme 2.15).96

Scheme 2.16 Reaction of compound (10) with (12) in the presence of TMSOTf and NIS

Further reaction (Scheme 2.17) of the product (14) with the earlier obtained pyruvate adduct (7) in the presence of TMSOTf and NIS at -40 oC gave the final desired polysaccharide subunit (15). As the yields were incredibly low, no further steps were taken in deprotecting the final compound, and the obtained product was sent for biological evaluation.

Scheme 2.17 Synthesis of the PSA1 analogue

2.3 Biological evaluation

The protected PSA1 analogue (15) was evaluated using ELISA assays for the production of proinflammatory cytokines TNF-α and IL-6 (innate immunity). The cells used were the RAW 264.7 and the mouse peritoneal macrophages (C57, BL/6, Jackson

Laboratories, Maine, US). Initial results showed (Fig. 16) that the cytokines produced by

15 were negligible in comparison to those produced by the lipopolysaccharides, when analyzed on the RAW264.7 and the mouse peritoneal macrophages.

Fig. 16 Innate Immunity: Production of TNFα and IL-6 by RAW264.7 and the mouse peritoneal

macrophages

The compound (15) was further evaluated for adaptive immune responses on the spleen cells. The responses which were analyzed were for the T- and B-cell proliferation, levels of INF-γ in the supernatants which indicates a T-cell response, and polyclonal IgM and IgG levels in the supernatants which signifies a B-cell response. It was found that neither the T-cell nor the B-cell responses were observed with (15).

To elucidate the toll like receptor responsible for cytokine production, the peritoneal macrophages from TLR2 and TLR4 knockout mice (female, 4-6 weeks old, C57BL/6) were obtained. The cells were cultured with variable concentrations of the compound

(15) and, the TNF-α and IL-6 levels in the supernatants were measured at different time points (Fig. 17).

Fig. 17 Innate Immunity: Production of TNFα and IL-6 by the peritoneal macrophages from TLR2 and

TLR4 knockout mice

It was observed that the production of both TNF-α and IL-6 was reduced in only the

TLR4 knockout mice suggesting that signaling of (15) occurs only through TLR4.

Furthermore, the graphical representation showed that the magnitude of TLR4 signaling with (15) was greater than the known TLR4 agonist, E. coli LPS (lipopolysaccharide).

Similarly, the extent of binding of compound (15) to the TLR4 receptors was illustrated by treating the peritoneal exudate cells (PECs) with different concentrations of the compound. The cells were pre-incubated with the compound, after which they were washed. To the washed cells were then added the antibody followed by the phytoerythrin dye (PE) and, the number of the PE positive cells was observed by flow cytometry (Fig. 18).

PECs 80 ** N.S.* 60

0 % TLR-4% expression

g/ml g/ml g/ml    control 1 5 10

Fig. 18 Results from flow cytometry

The graphs in the figure above show that the increase in the concentration of the compound (15) results in an increased binding of the TLR-4 receptors to the compound.

The graphs with TLR-2 show that the presence of the compound (15) doesn’t interfere with the cell count. This clearly depicts that the PSA1 analogue doesn’t bind to the TLR-

2 receptors.

Chapter 3

Synthesis and Chemical Reactivity of 3,6-Anhydro-D-glucal

3.1 General Information

Anhydrosugars (or intramolecular anhydrides) are the derivatives of monosaccharides obtained by the intramolecular elimination of a water molecule resulting in the formation of a new three-, four-, five-, six- or seven-membered heterocyclic rings. Their advantage of easy removal has led to the development of numerous derivatives, the synthesis and properties of which are described in the literature.97, 98, 99, 100, 101 These anhydrosugars are an important class of sugars which are used for the preparation of modified carbohydrates including the C-glycosides102, nucleosides103 and heterocyclic compounds104. They are also used in the synthesis of complex enantiomerically pure products where in the chirality of the parent sugar is transferred to the target via the chiron approach.105 They constitute very versatile starting materials not only in carbohydrate chemistry but also for the synthesis of non-carbohydrate and non-natural products. Anhydrosugars are the suitable monomers for preparing stereoregular polysaccharides and their specifically substituted derivatives.1

For the synthesis of the anhydro skeleton in a normal sugar molecule, one of the hydroxyl groups of the diol which is taking part in the anhydro ring formation is converted into a leaving group such as a halogen, tosylate or triflate. The other hydroxyl group acts as a nucleophile resulting in the formation of the anhydro ring by an intramolecular SN2 approach (Fig. 3.1).

Formation of the anhydrosugar by elimination of the water molecule

Mechanism for the formation of the anhydrosugar

Fig. 3.1 Synthesis of anhydrosugar

The reaction protocols proposed by Mitsunobu106 and Castro107 for the synthesis of the anhydrosugars involve activation of the hydroxyl groups under milder conditions (Fig.

3.2).

Fig. 3.2 Proposed protocols of Mitsunobu and Castro

The formation of anhydrosugars, may involve various carbon atoms of the sugar, thus leading to their categorization into different classes. They are divided into two main categories: 1. the anhydrosugars which involve the participation of the anomeric carbon atom, called glycosanes, which resemble the intramolecular glycosides; 2. the anhydrosugars which do not involve the anomeric carbon atom, called simply as anhydrosugars.1, 108, 109 The latter group comprises of purely synthetic materials which are exclusively used in synthetic carbohydrate chemistry.

Classification of the anhydrosugars is also based on the heterocyclic ring formed upon dehydration. Oxirane (sugar epoxide), oxetane (oxacyclobutane), oxolane

(tetrahydrofuran) and oxane (tetrahydropyran) are the various heterocyclic anhydrides known. Oxirane and oxetane rings show higher reactivity, whereas oxane and oxolane rings show comparatively low reactivity. The position of the anhydro ring, the steric arrangement as well as the conformation of the molecule also play a major role in determining the reactivity of the heterocyclic anhydrides.

3.1.1 Glycosanes

Compounds which are formed by the dehydration of the anomeric hydroxyl group and the terminal alcohol of a sugar molecule (1,6-anhydropyranoses, 1,6-anhydrofuranoses) or with any other alcohol on the sugar ring (1,2-, 1,3-, 1,4-anhydrosugars, etc) come under the class of anomeric anhydrosugars or glycosanes. The most common of these are the 1,6- and 1,2-anhydrosugars, while the others are less commonly seen in carbohydrate chemistry. They resemble conventional glycosides in their sensitivity to

58 acids by undergoing acid hydrolysis. But, highly strained anhydrosugars tend to undergo hydrolysis even in alkaline solutions.

The formation of the carbocation at the anomeric carbon atom controls the regioselectivity of the opening of the ring.110 Nucleophilic attack occurs preferentially at the anomeric carbon atom resulting in the formation of various glycosyl derivatives or glycosides. The anhydrosugars act as monomers in polymerization reactions to form stereoregular polysaccharides, as the configuration of the corresponding anomeric, as well as the nonanomeric carbon atoms, remain the same.

3.1.1.1 1,2-Anhydroaldoses

1,2-Anhydroaldopyranoses have the 2,7-dioxabicyclo[4.1.0]heptanes skeleton which

4 5 111,112 preferentially adopts H5 and H4 conformations. 1,2-anhydrohexofuranoses have the more flexible 2,6-dioxabicyclo[3.1.0]hexane skeleton which adopts an E conformation. (Fig. 3.3).

Fig. 3.3 1,2-Anhydroaldoses

Brigl was the first to synthesize the 1,2-anhydrosugar (Fig. 3.4).113 1,2-anhydro-3,4,6-tri-

O-acetyl-α-D-glucopyranose or ‘Brigl’s anhydride’ was prepared from β-D-glucopyranose

114 pentaacetate by sequential treatment with PCl5 and NH3. The product was used in the synthesis of glycosides115 and α-D-linked disaccharides, like sucrose.116

Fig. 3.4 Synthesis of Brigl’s anhydride

Subsequently, Danishefsky’s research group synthesized the 1,2-anhydrosugar by treating a glycal with dimethyldioxirane (Fig. 3.5).117 The product obtained upon treatment with a nucleophile gave the 2-hydroxy-β-D-glycoside.

Fig. 3.5 Synthesis of 1,2-anhydride using dimethyldioxirane

Similarly, Marzabadi, et al. synthesized the 1,2-anhydro glucal by making a halohydrin undergo a base induced cyclization (Fig. 3.6).118 Many other 1,2-anhydrides were synthesized later on.

Fig. 3.6 Synthesis of 1,2-anhydride from a halohydrin

3.1.1.2 1,3-Anhydroaldoses

1,3-Anhydrosugars have the 2,6-dioxabicyclo[3.1.1]heptane skeleton and may adopt the

5 C2, B2,5 or E2 conformation of the pyranose ring. The presence of axial substituents at

5 C-2, C-4 and C-5 positions destabilize the C2 conformation (Fig. 3.7). The oxetane ring of 1,3-anhydrosugars is less reactive towards acids in comparison to the sensitivity of the oxirane ring in both acidic, as well as, in alkaline aqueous solutions.

Fig. 3.7 1,3-Anhydroaldoses

Many of the 1,3-anhydrohexopyranoses of gluco-,119, 120 manno-,121, 122 galacto-,123 talo-

,124 and 6-deoxy derivatives125, 126, 127 were earlier synthesized via analogous procedures. They were obtained by reacting partially-protected glycosyl halides with strong bases like NaH and potassium tert-butoxide (Fig. 3.8).

Fig. 3.8 Synthesis of a 1,3-anhydrohexose derivative

3.1.1.3 1,4-Anhydroaldoses

The 1,4-anhydroaldoses have the 2,7-dioxabicyclo[2.2.1]heptane skeleton and adopt

1,4 two non-interconvertible conformations, B1,4 and B (Fig. 3.9). They tend to be very stable in alkaline conditions, but get readily hydrolyzed in the presence of acids. The synthesis is similar to that of 1,3-anhydrosugars, and many compounds in the D- gluco,128, 129, 130 D-galacto,131, 132 6-deoxy-L-manno, L-talo,133 L-arabino, D-ribo, D-xylo and D-lyxo configurations have been synthesized.134, 135, 136, 137, 138

Fig. 3.9 1,4-Anhydroaldoses

3.1.1.4 1,6-Anhydroaldoses

These compounds have the 6,8-dioxabicyclo[3.2.1]octane skeleton and have limited

1 steric flexibility. Most of them adopt themselves to C4 conformation, while some of them are more flattened in B3,0 conformation. An inversion in the orientation at all the

4 1 stereocenters is observed in comparison to the C1(D) and C4(L) conformations of the parent compounds. These anhydrosugars resemble methyl β-D-hexopyranosides in their chemical activity. They are very stable in alkaline conditions, but are very sensitive to acids (Fig. 3.10).

Fig. 3.10 1,6-Anhydroaldoses

Their synthesis includes cyclization of the hexopyranosyl derivatives having a good leaving group like halides, azides,139, 140, 141 tosylates142 or 2,4,6-trimethylbenzoates143 at the C-1 position and alkali-sensitive glycosides, like phenyl glycosides,144, 145 by the action of strong bases.

3.1.2 Anhydrosugars

Compounds which are formed by the dehydration of any two hydroxyl groups on the sugar molecule, other than the hydroxyl group on the anomeric carbon atom, are the anhydrosugars. These heterocyclic anhydrides include the oxirane (sugar epoxides, 3- membered), oxetane (oxacyclobutane, 4-membered), oxolane (tetrahydrofuran, 5- membered) and oxane (tetrahydropyran, 6-membered) derivatives.

3.1.2.1 Oxirane

Oxiranes are the anhydro sugars having a fused oxirane ring to a pyranose or a furanose moiety. These have the 3,7-dioxabicyclo[4.1.0]heptane and 3,6-

5 1 dioxabicyclo[3.1.0]hexane basic skeletons that adopt either H0 or H0 conformations in

63 the ground state (Fig. 3.11). Generally the 2,3- and 3,4-anhydrohexopyranoses adopt the flexible half-chair conformation (H) depending on the presence or the absence of additional fused rings.

Fig. 3.11 Oxiranes

Though there exist many synthetic procedures for oxiranes, the most common procedure followed is the intramolecular SN2 nucleophilic displacement reaction.

Usually, a tosyloxy or a mesyloxy group gets displaced by a vicinyl hydroxyl group that has been deprotonated by a base such as sodium methoxide.146, 147, 148

These epoxy sugars are generally used in the synthesis of various derivatives of sugars like halo-, amino-, thio-, azido-, deoxy- and branched-chain derivatives. The oxirane ring, which is more reactive than oxetane and oxolane rings, opens in the presence of nucleophiles both in acidic and in basic conditions. But, it tends to be stable to catalytic debenzylation reactions (only palladium), in acylations and alkylations in basic media, and in detosylation reactions in the presence of nucleophiles.

3.1.2.2 Oxetane

Anhydrosugars containing a fused oxetane ring in the 2,6-dioxabicyclo[3.2.0]heptane or the 2,7-dioxabicyclo[4.2.0]octane skeleton are called oxetanes (Fig. 3.12).

Fig. 3.12 Oxetanes

3.1.2.3 Oxolane

These include the bicyclic 2,5-anhydro- as well as the 3,6-anhydro-aldose derivatives.

Their general skeleton is either 2,5-dioxabicyclo[2.2.1]heptane, 2,6- dioxabicylco[3.2.1]octane or 2,6-dioxabicyclo[3.3.0]octane (Fig. 3.13). They exist even as monocyclic oxolane derivatives possessing a free aldehyde group. As they are quite stable in acidic and basic conditions, reactivity of these anhydrosugars is dependent on the presence or the absence of a free aldehyde group.

Fig. 3.13 Oxolanes

From the conformational analysis, it can be inferred that the relatively strained 2,6- dioxabicylco[3.2.1]octane skeletons of 3,6-anhydro-D-gluco- and –D-manno-pyranose tend to rapidly recyclize into the less strained 2,6-dioxabicyclo[3.3.0]octane skeletons of the 3,6-anhydrohexofuranose in the presence of methanolic HCl. Such a rearrangement was not observed in the galacto- and talo- configurations.149, 150

Many 2,5-anhydropentoses have been described so far, of L-arabino,151 D-lyxo, D-ribo and D-xylo sugars.152 Chitose or 2,5-anhydro-D-mannose is the most common 2,5- anhydrohexose derivative.153, 154, 155 It can be prepared by the deamination of 2-amino-

2-deoxy-D-glucose. Other isomers include the derivatives of D-allose,156, 157 D-altrose, L- idose,158, 159 D-glucose160, 161 and L-talose162.

Among oxolanes, 3,6-anhydroaldohexose derivatives form the major class. As per the configuration and conformation of all the eight possible 3,6-anhydroaldohexoses described, some of them exist as bicyclic pyranose, while others exist as bicyclic

66 furanose forms.163, 164 Hence, 3,6-anhydro derivatives of glucose and mannose adopt both the pyranose and the cis-fused forms, while those of galactose and talose adopt the pyranose form only. Gulose and idose can adopt only the cis-fused form. Likewise, free 3,6-anhydro-D-allose and –D-altrose can only exist in the acyclic form.165, 166

3.1.2.4 Oxane

Pyranose and furanose derivatives adopting either the rigid 2,5-

2,5 dioxabicyclo[2.2.2]octane skeleton with the pyranose ring in B or B2,5 conformation or

4 the less flexible 2,6-dioxabicyclo[3.2.1]octane skeleton in C0 conformation are categorized as oxanes (Fig. 3.14). They comprise of 2,6-anhydrohexoses and 1,5- anhydroketoses. Among the 2,6-anhydrohexoses, pyranoid derivatives in configurations of altro,167 ido,168 manno169 and talo170 configurations have been reported. Of the furanose derivatives, 2,6-anhydro-D-mannofuranose and its derivatives are known.171

Fig. 3.14 Oxanes

They can be synthesized by the reaction of sodium methoxide on the corresponding 6- tosylate sugar (Fig. 3.15).172 1,5-anhydro-D-fructose is one of the 1,5-anhydroketose

67 sugars which is of particular interest.173 It is the central intermediate formed in the alternative glycogen catabolic pathway, also called as the Anhydrofructose (AF) pathway.174

Fig. 3.15 Synthesis of a 2,6-anhydropyranose derivative

3.2 Background

The most widely known compounds from the tetrahedral class of anhydrosugars are

3,6-anhydrofuranoses.175 Furanodictine A and B [Fig. 3.16] are good examples of this class that show neuronal differentiation activity.176 3,6-Anhydro-D- and L-galactoses and their partially methylated derivatives are typical constituents of polysaccharides of red algae.177 3,6-Anhydro sugars forming bicyclic nucleosides have shown antiviral activity.178 The puckering in the bicyclic carbohydrate moiety is believed to cause the molecule to be locked in the proper conformation for biological activity to be observed.179, 180

Fig. 3.16 3,6-Anhydrofuranoses

3,6-anhydrohexoses are considered to be of more importance as they are used to synthesize new nucleoside analogues.181 Reinhold et al. (1974) first synthesized 3,6- anhydrohexose sugars by selective tosylation at the C-6 carbon atom followed by treatment of the intermediate tosylates with a strong base (sodium methoxide) (Fig.

3.17). 182 Kreimeier et al. and Gross et al. substituted the hydroxyl group at C-6 by

(Me2N)3P and halogens, upon which the tosylated sugars were treated with a strong base, as mentioned earlier.183, 184 Liptak et al. later confirmed that the presence of benzyloxy and methoxy groups at the C-3 position would help in the formation of the

3,6-anhydride bond as they act as good internal nucleophiles. 185

Fig. 3.17 Synthesis of 3,6-anhydropyranose

Similarly, Shibayama, et al. and Santoyo-Gonzales et al. described the synthesis of 3,6- anhydrohexofuranose derivatives by the basic solvolysis of 5,6-thionocarbonates, sulfates and sulfites.186, 187 A cyclic 3,5,6-orthoester was shown to have reacted likewise by basic solvolysis.188 Ishido et al. described the photochemical cyclization of a

69 substituted oxolane ring (Fig. 3.18).189 Yamaguchi et al. showed that the mercaptolysis of agar as a convenient method for the synthesis of 3,6-anhydro-L-galactose diethyl dithioacetal.190

Fig. 3.18 Solvolysis

Very little research has been done on the preparation of anhydro-D-glycal sugars, which are of great importance in the synthesis of complex new derivatives. 3,6-Anhydro-D- glucal was earlier synthesized by Tucker et al. from 3,4-di-O-acetyl-6-O-tosyl-D-glucal using Deacidite FF IP (OH-) resin in 66% yield (Fig. 3.19).191 4,6-O-anhydro-D-glucal was previously reported as a byproduct (40% yield) when an equimolar amount of lithium aluminum hydride in THF was reacted with 6-O-tosyl-D-glucal, though the structure of this molecule was not fully characterized.192

Fig. 3.19 Synthesis of 3,6-anhydro-D-glucal

The results presented describe the synthesis of 3,6-anhydro-D-glucal from 6-O-tosyl-D- glucal using excess LiAlH4 in THF as the reagent. With this combination of reagents, an

82% yield of anhydro-D-glycal was realized.193 The unusual chemical reactivity of the resulting anhydro glycal was also investigated. Also described is the synthesis of an unusual 1,4:3,6-dianhydrosugar via N-iodosuccinimide (NIS) addition to the 3,6- anhydro-D-glucal.

3.3 Results: Synthesis of 3,6-anhydro-D-glucal and a study of its chemical reactivity

3,4,6-Tri-O-acetyl-D-glucal was deacetylated using Zemplen conditions to give D-glucal

16 in 98% yield.194 The resulting D-glucal 16 was selectively tosylated at the primary hydroxyl using p-toluenesulfonyl chloride in a mixture of pyridine and dichloromethane.

The 6-O-tosylated-D-glucal 17 was obtained in 56% isolated yield. Treatment of 17 with three equivalents of LiAlH4 in THF did not produce the anticipated 6-deoxy-D-glycal, as previously reported, but instead gave the 3,6-anhydro sugar 18 in 82% yield (Scheme

3.1).195

Scheme 3.1 Synthesis of 3,6-anhydro-D-glucal

Structural assignments for 18 were based on 1D and 2D 1H and 13C-NMR experiments.

The proposed bridged structure was supported by the observation of long range

71 coupling between the vinylic proton at C2 and the bridgehead proton at C4 (W-coupling) in the proton NMR. In the 1H-1H-COSY (Appendix), coupling between H4 and H6 and

H6’ was also seen, while in the 1H-NMR the H6 and H6’ peaks only appeared as broad doublets of doublets. X-ray quality crystals were obtained and the structure of the molecule established unambiguously (Fig. 3.18), detailed information of which is given in the Appendix.196

Fig. 3.18 ORTEP representation of compound 18

Compound 18 was subjected to a series of different glycal addition reactions. Treatment of compound 18 with NIS in the presence of methanol led to the formation of a mixture of β-gluco-, -manno- and -manno-2-deoxy-2-iodo-O-methylglycosides (19a, 19b, 19c) in a ratio of 5.2: 1.5: 1 with an overall yield of 93% (Scheme 3.2).197 None of the -gluco diastereomer was detected in the mixture.

Scheme 3.2 Synthesis of 2-deoxy-2-iodo-O-methylglycosides Treatment of 18 with thiophenol in the presence of a catalytic amount of ceric (IV) ammonium nitrate (CAN) gave a mixture of α- and β-2-deoxy sugars (20a, 20b) in a ratio 1.85:1 with a 65% yield. Also formed in the reaction was an α-, β-mixture of the

Ferrier rearranged adducts (21a, 21b) with a 35% yield in a ratio of 2.5:1, formed upon the breakdown of the anhydride ring (Scheme 3.3).198

Scheme 3.3 Addition of thiophenol to 18 in the presence and absence of CAN

When the anhydro sugar 18 was reacted with thiophenol and a catalytic amount of ceric

(IV) ammonium nitrate in the presence of sodium iodide, a reagent known to enhance - selectivity, a mixture of α- and β-2-deoxy sugars (20a, 20b) was again obtained in this reaction (34%), but only the α-adduct 21a from the Ferrier rearrangement was isolated

(65%) (Scheme 3.3).199

Because of the success of the NIS addition with methanol, attempts were made to synthesize disaccharides from the anhydro sugar donor. Compound 18 was treated with an equimolar amount of 1,2:3,4-di-O-isopropylidine-D-galactopyranose (DIPG) in the presence of N-iodosuccinimide to give 2-deoxy-2-iodo-1,4:3,6-dianhydride 22 as the major product (48%). An inseparable mixture of 2-deoxy-2-iodo sugars (23a, 23b, 23c) was also formed in low yield, and residual starting materials were isolated (~20%)

(Scheme 3.4). When the same reaction was repeated in the absence of 1,2:3,4-di-O- isopropylidine-D-galactopyranose and in the presence of 4 Ǻ molecular sieves, 22 was obtained in a much higher yield of 76% (Scheme 3.5).

Scheme 3.4 Synthesis of 1,4:3,6-dianhydride 22 from 18 in the presence of DIPG

Scheme 3.5 Synthesis of 1,4:3,6-dianhydride 22 from 18 in the absence of DIPG

3.4 Summary

In summary, good yields of 3,6-anhydro-D-glucal 18 have been obtained from 6-O- tosyl-D-glucal 17 via reaction with excess lithium aluminum hydride. The resulting anhydro glycal was studied under a variety of reaction conditions. Iodoglycosylation of

18 with methanol gave good yields of a mixture of 2-deoxy-2-iodomethylglycosides 19a- c, but attempts to use the primary hydroxyl sugar donor 1,2:3,4-diisopropylidine-D- galactopyranose gave the dianhydro sugar 22 and iodohydrins 23a-c. These observed products may result due to the steric hindrance present in the anhydro glycal reactant or in the transition state for the reaction. Alternatively, unfavorable dipole-dipole interactions in the transition state for the sugar nucleophile approaching the glycal double bond may be responsible for the observed products. β-Anomers were favored over α-anomers by a ratio of ~ 4:1. Furthermore, the formation of 22 is favored because the intramolecular attack by the bridgehead hydroxyl group is anticipated to be faster entropically than is the attack by the solvated nucleophiles. The small methanol nucleophile and adventitious water can more easily access the double bond and mixtures of product resulted from their attack. In those cases where the DIPG was excluded and freshly powdered and activated 4 Ao molecular sieves were added, only the dianhydro sugar 22 was obtained. Further reactions with 18 are ongoing.

4.1 Experimental

General Methods.

Melting points (mp) were recorded on a Mel-Temp melting point apparatus (Laboratory Devices,

Inc., USA) and are uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian Inova (500 MHz) spectrometer. NMR samples were dissolved in CDCl3, and chemical shifts were reported in ppm relative to the residual non-deuterated solvent (CHCl3 as δ = 7.26 ppm). 1H NMR data are reported as follows: chemical shift, integration, multiplicity

(s = singlet; d = doublet; d, d = doublet of a doublet; d, d, d = doublet of a doublet of a doublet; m = multiplet), coupling constant, and assignment. 13C NMR spectra were recorded on a Varian

Inova (125 MHz) spectrometer. The samples were dissolved in CDCl3, and chemical shifts are

1 13 reported in ppm relative to the solvent (CDCl3 as δ = 77.0 ppm). H and C NMR spectra were measured at room temperature and the chemical shifts are reported in parts per million, unless otherwise noted. 1H NMR assignments were done using gCOSY and 13C NMR assignments were done using gHMQC. Elemental analysis was performed at Robertson Microlit Laboratories Inc.,

Ledgewood, NJ. High resolution and low resolution mass spectra (HRMS and LRMS) were obtained at the Mass Spectrometry Lab, University of Illinois, Champaign-Urbana, IL and are reported in m/z (electrospray ionization). X-ray crystallographic data for compound 2 was deposited at the Cambridge Crystallographic Data Center (CCDC); deposition number CCDC

988697.20 Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel coated aluminum sheet 60 F254 (Analtech, # 157017). Silica gel (particle size 40-63 µm, 230-400 mesh Silicycle Siliflash P60) was used for flash column chromatography. Preparative thin-layer chromatographic separations were carried out on 2000 µm silica gel coated glass plate 60 F254

(Analtech). Rotary evaporation was performed using a Buchi R-114 rotary evaporator. Unless

76 otherwise noted, non-aqueous reactions were carried out in oven-dried (125 oC) glassware under nitrogen. Dry CH3CN, THF and methanol were purchased from Pharmco-Aaper products, Inc.

All other commercially available reagents were used as received.

Thiophenyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (2): To a solution of penta-O-acetyl-

β-D-galactopyranose (9 g, 23.056 mmol) in 30 mL of anhydrous CHCl3 in a 100 mL r. b. f. was added 2.84 mL of thiophenol (3.048 g, 1.2 eq, 27.667 mmol) under an atmosphere of N2. To the mixture was then added 7.3 mL of BF3. Et2O and stirred at room temperature for 3 days. The reaction was then terminated by diluting with excess CHCl3, followed by washings with saturated NaHCO3 soln. (2 x 50 mL) and 10% w/v NaOH soln. (2 x 50 mL). The organic extract was dried over anhydrous Na2SO4 and concentrated in vacuo to yield the crude product. The product was purified by flash column chromatography using 30%, 40% and 50% EtOAc in hexanes to give 9.809 g (97%) of the desired compound as a white solid. Rf (1:1 EtOAc/

1 hexanes): 0.66; H NMR (500 MHz, CDCl3) δ 7.49-7.51 (m, 2H, Ar), 7.26-7.31 (m, 3H, Ar),

5.41 (dd, 1H, J3,4 = 3.4 Hz, J4,5 = 1 Hz, H-4), 5.23 (dd, 1H, J1,2 = 10 Hz, J2,3 = 9.8 Hz, H-2), 5.04

(dd, J2,3 = 10.2 Hz, J3,4 = 3.4 Hz, H-3), 4.71 (d, 1H, J1,2 = 10.3, H-1), 4.18 (dd, 1H, J6,6’ = 11.45,

13 J5,6’ = 6.8, H-6’), 4.1 (dd, 1H, J5,6 = 5.8, J6,6’ = 12.1, H-6), 3.93-3.95 (m, 1H, H-5); C NMR (125

MHz, CDCl3) δ 170.36, 170.19, 170.04, 169.42 (C=O, OAc), 132.57, 132.47, 128.89, 128.16

(CH, Ar), 86.6 (C-1), 74.42 (C-5), 72 (C-2), 67.26, 67.22 (C-3, C-4), 61.63 (C-6), 20.85, 20.66,

20.63, 20.58 (CH, OAc).

Thiophenyl-β-D-galactopyranose (3): To a solution of the above obtained thiophenyl adduct

(8.33 g, 18.93 mmol) in 30 mL of anhydrous CH3OH was added 0.1 eq of 5% NaOCH3 in

CH3OH solution. The mixture was stirred at room temperature for 4 h under an atmosphere of

N2. The reaction upon reaching completion was rotary evaporated to get rid of CH3OH. The

77 resulting syrupy product was then dried under high vacuum to give 5.1589 g (100%) of the

1 desired deacetylated product as a yellowish-white powder. Rf (5% CH3OH/CHCl3): 0.03; H

NMR (500 MHz, CDCl3) δ 7.43-7.45 (d, 2H, JAr,Ar = 6.3 Hz, Ar), 7.27-7.31 (m, 2H, Ar), 7.18-

7.21 (m, 2H, Ar), 4.72 (s, 4H, -OH), 4.57 (d, 1H, J1,2 = 9.8 Hz, H-1), 3.71 (d, 1H, J3,4 = 3.4 Hz,

H-3), 3.46-3.53 (m, 3H, H-5, 6, 6’), 3.42 (d, 1H, J1,2 = 9.3 Hz, H-2), 3.35 (dd, 1H, J3,4 = 3.4 Hz,

13 J4,5 = 9 Hz, H-4); C NMR (125 MHz, CDCl3) δ 140.76, 134.49, 134, 131.3 (CH, Ar), 92.95 (C-

1), 84.37 (C-5), 79.9 (C-2), 74.46 (C-6), 73.54 (C-3), 65.77 (C-4).

Thiophenyl-4,6-O-benzylidene-β-D-galactopyranose (4): Into an oven-dried 250 mL r. b. f. was taken ZnCl2 (14.27 g, 5 eq) and flame fused under an atmosphere of N2 to make it totally anhydrous. To the cooled r. b. f. containing fused ZnCl2 was added benzaldehyde (12.21 g, 5.5 eq) and thiophenyl-β-D-galactopyranose (5.69 g, 20.945 mmol) and the mixture was stirred under an atmosphere of N2 overnight. Additionally, 15 mL of benzaldehyde was added to the mixture, for it being the only solvent used in this reaction. When the reaction was found to have almost reached completion, it was diluted with excess CHCl3 and washed with saturated

NaHCO3 solution (2 x 50 mL). The organic layer was separated and dried over anhydrous

Na2SO4, and evaporated in vacuo to get a crude viscous oily mass. The crude product was purified by flash column chromatography using gradient dilution technique (40%, 50%, 60%,

70%, 80% and 90% EtOAc in hexanes) to result in 5.2 g (70%) of the desired benzylidene

1 adduct as a solid white powder. Rf (80% EtOAc/hexanes): 0.245; H NMR (500 MHz, CDCl3) δ

8.12-8.14 (m, 2H, Ar), 7.69-7.71 (m, 2H, Ar), 7.61-7.65 (m, 1H, Ar), 7.48-7.51 (m, 1H, Ar),

7.41-7.43 (m, 2H, Ar), 7.37-7.4 (m, 2H, Ar), 5.53 (s, 1H, Bn-Ar), 4.53 (dd, 1H, J1,2 = 4.4 Hz, J1,3

= 2.2 Hz, H-1), 4.4 (dd, 1H, J6,6’ = 12.7 Hz, J5,6’ = 1.5 Hz, H-6), 4.23 (d, 1H, J3,4 = 1.5 Hz, H-4),

4.04 (dd, 1H, J6,6’ = 12.45 Hz, J5,6’ = 2 Hz, H-6’), 3.71-3.75 (m, 2H, H-2, 5), 3.55 (d, 1H, J3,4 = 1

13 Hz); C NMR (125 MHz, CDCl3) δ 137.6, 133.7, 133.69, 130.18, 129.34, 128.93, 128.48,

128.23, 128.18, 126.54 (CH, Ar), 101.4 (Bn-Ar), 87 (C-1), 75.4 (C-4), 73.8 (C-5), 70.06 (C-3),

69.28 (C-6), 68.8 (C-2).

Thiophenyl-3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranose (5a): 4.834 g (13.4 eq) of thiophenyl-4,6-O-benzylidene-β-D-galactopyranose was taken into an oven-dried 250 mL screw- cap tube and to it was added 60 mL of anhydrous toluene. The sealed tube was heated to 117 oC upon thorough stirring for three days in the presence of 4 Ao molecular sieves. After three days, the tube was cooled to r.t., filtered and evaporated in vacuo to yield a syrupy liquid. To the syrupy liquid was added 15 mL of anhydrous CHCl3 and an ice-cooled solution of benzoyl chloride (1.977 g, 1.05 eq) in 15 mL of anhydrous CHCl3. The mixture was stirred for 2 h at r. t. after which it was evaporated in vacuo to yield 7.838 g of the crude product as a yellowish- orange solid. Gradient dilution technique (30%, 40%, 50% and 60% EtOAc in hexanes) was used to purify the crude by flash column chromatography to yield 1.928 g (31%) of the desired product as an oily colourless liquid. 0.71 g of a mixture of the 2-O-benzyl- and the 3-O-benzyl- adducts was also obtained. Rf (30% EtOAc/hexanes): 0.327 (Rf of the 2-O-benzyl adduct: 0.39);

1 H NMR (500 MHz, CDCl3) δ 8.03-8.05 (m. 2H, Ar), 7.72-7.7 (m, 2H, Ar), 7.53-7.56 (m, 1H,

Ar), 7.38-7.42 (m, 5H, Ar), 7.31-7.38 (m, 4H, Ar), 7.25-7.28 (m, 1H, Ar), 5.51 (s, 1H, Bn-Ar),

5.19 (dd, 1H, J2,3 = 11.3 Hz, J3,4 = 3.1 Hz, H-3), 4.68 (d, 1H, J1,2 = 9.5 Hz, H-1), 4.51 (s, 1H, H-

13 4), 4.42 (d, 1H, J6,6’ = 12.3 Hz, H-6), 4.06-4.18 (m, 2H, H-2, 6’), 3.7 (s, 1H, H-5); C NMR (125

MHz, CDCl3) δ 165.9 (-CO-Ar), 137.3, 133, 130, 129.9, 128, 127.8, 126.3 (CH, Ar), 108.9 (Bn-

Ar), 93.1 (C-1), 79.3 (C-4), 79.2 (C-5), 74.4 (C-3), 71.2 (C-2), 67.8 (C-6); LRMS (ESI): Calcd

+ C26H24O6S: 464.1. Found for (M+Na) : 487.1.

Thiophenyl-2-O-acetyl-3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranose (6): To a solution of the above obtained thiophenyl-3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranose

(2 g, 4.31 mmol) in 10 mL of N, N, N-triethylamine was added N, N-dimethylaminopyridine

(38.67 mg, 0.08 eq) and stirred at 0 oC for 30 min. To the ice-cold solution was added 507.54 mg

(1.5 eq) of acetyl chloride drop-wise and the reaction was vigorously stirred at 0 oC initially and at r.t. for 2 days. The reaction was terminated by diluting with CH2Cl2 (50 mL) and, by washing with 2 N HCl (2 x 25 mL) and saturated NaHCO3 solution (2 x 25 mL). The organic layer was dried over anhydrous MgSO4 and evaporate in vacuo to give 2.44 g of the crude product as an orange fluffy solid. Purification was carried out by flash column chromatography using 30%

EtOAc in hexanes as the eluent. 1.3 g (60%) of the desired product was obtained as a yellow oily

1 liquid. Rf (30% EtOAc/hexanes): 0.44; H NMR (500 MHz, CDCl3) δ 7.99-7.97 (m, 2H, Ar),

7.63-7.64 (m, 2H, Ar), 7.52-7.55 (m, 1H, Ar), 7.31-7.41 (m, 8H, Ar), 5,56 (d, 1H, J2,3 = 9.9 Hz,

H-2), 5.48 (s, 1H, Bn-Ar), 5.2 (dd, 1H, J1,2 = 9.9 Hz, J2,3 = 9.8 Hz, H-3), 4.79 (d, 1H, J1,2 = 9.8

Hz, H-1), 4.54 (s, 1H, H-4), 4.42 (d, 1H, J6,6’ = 12.4 Hz, H-6), 4.06 (d, 1H, J6,6’ = 12.4 Hz, H-6’),

13 3.69 (s, 1H, H-5), 2.01 (s, 3H, -CO-CH3); C NMR (125 MHz, CDCl3) δ 170.1 (-CO-CH3),

165.9 (-CO-Ar), 137.3, 133.9, 133, 130, 129.3, 128.6, 127.8, 125 (CH, Ar), 108.9 (Bn-Ar), 89.8

(C-1), 79.6 (C-4), 78.9 (C-5), 73.8 (C-3), 70.4 (C-2), 67.8 (C-6), 21.0 (CH3); LRMS (ESI): Calcd

+ C28H26O7S: 506.1. Found for (M+NH4) : 524.2.

Thiophenyl-2-O-acetyl-3-O-benzoyl-β-D-galactopyranose (7a): To a solution of thiophenyl-2-

O-acetyl-3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranose in anhydrous dichloromethane

(20 mL) at -15 oC was added drop-wise a mixture of 20% v/v trifluoroacetic acid (880 mg, 6 eq) in CH2Cl2. The mixture was brought to r.t. and stirred overnight in an atmosphere of N2. The reaction was terminated by the addition of excess CH2Cl2 (30 mL), and washed with saturated

NaHCO3 solution (2 x 25 mL) and water (2 x 25 mL). The organic layer was separated and dried over anhydrous Na2SO4, and evaporated in vacuo to yield 820 mg of the crude 4,6-diol sugar.

The compound was purified by flash column chromatography by gradient dilution method (30%,

40%, 50% and 60% EtOAc in hexanes) to yield 244 mg (45%) of the pure product. Rf (30%

1 EtOAc/hexanes): 0.137; H NMR (500 MHz, CDCl3) δ 7.98-8.0 (m, 3H, Ar), 7.93-7.95 (m, 2H,

Ar), 7.52-7.58 (m, 3H, Ar), 7.44-7.45 (m, 2H, Ar), 5.58 (d, 1H, J1,2 = 9.9 Hz, H-2), 5.22 (dd, 1H,

J2,3 = 9.9 Hz, J3,4 = 3.1 Hz, H-3), 5.07 (d, 1H, J1,2 = 10.1 Hz, H-1), 4.42 (d, 1H, J3,4 = 3.1 Hz, H-

13 4), 4.06 (m, 1H, H-5), 3.84 (m, 2H, H-6, 6’), 1.97 (s, 3H, -CO-CH3); C NMR (125 MHz,

CDCl3) δ 170.1 (-CO-CH3), 165.9 (-CO-Ar),133.9, 133, 129.3, 129.3, 129.9, 128, 125.1 (CH,

Ar), 89.8 (C-1), 86.3 (C-5), 73.3 (C-3), 70.1 (C-2), 67.7 (C-4), 61.2 (C-6), 21.0 (CH3); HRMS

+ (ESI): Calcd C21H23O7S for (M+H) : 419.1165. Found: 419.1173.

Thiophenyl-2-O-acetyl-3-O-benzoyl-4,6-O-pyruvate-β-D-galactopyranose (7): To a suspension of thiophenyl-2-O-acetyl-3-O-benzoyl-β-D-galactopyranose (10 mg, 0.024 mmol) in methyl pyruvate (57.5 mg, 23.5 eq) was added BF3-Etherate drop-wise. The mixture was allowed to stir at r.t. overnight in an atmosphere of N2. The reaction was terminated by the addition of 1 mL of N, N, N-triethylamine to neutralize the acid. The mixture was poured into 25 mL of saturate NaHCO3 solution and extracted with CH2Cl2 (2 x 50 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated in vivo to give the desired pyruvate adduct in its crude form. The crude product was purified by preparatory TLC using 50% EtOAc in hexanes mixture as the eluent (with trace amounts of N, N, N-triethylamine) to give 10 mg (80%) of the pyruvate

1 adduct. Rf (50% EtOAc/hexanes): 0.77; H NMR (500 MHz, CDCl3) δ 7.97-8.01 (m, 3H, Ar),

7.92-7.95 (m, 2H, Ar), 7.5-7.54 (m, 2H, Ar), 7.44-7.49 (m, 3H, Ar), 5.45-5.47 (m, 1H, H-2),

5.29-5.34 (m, 1H, H-3), 5.08 (dd, 1H, J1,2 = 10 Hz, J1,3 = 2.8 Hz, H-1), 4.63 (dd, 1H, J3,4 = 7.8 Hz,

J4,5 = 3.4, H-4), 4.09-4.1 (m, 1H, H-6), 4.06-4.07 (m, 1H, H-6’), 3.89-3.9 (m, 1H, H-5), 3.27 (s,

13 3H, -OCH3), 2.01 (s, 3H, -CO-CH3), 1.63 (s, 3H, CH3); C NMR (125 MHz, CDCl3) δ 170.2 (-

CO-CH3), 168.9 (-CO-OCH3), 165.9 (-CO-Ar) 133.9, 133, 130.1, 129.3, 128.6, 125.1 (Ar),

114.9 (Pyr), 89.8 (C-1), 79.1 (C-4), 78.9 (C-5), 73.8 (C-3), 70.4 (C-2), 65.1 (C-6), 52.5 (-CO-

+ OCH3), 25.5 (CH3), 21.0 (-CO-CH3); HRMS (ESI): Calcd C25H26O9NaS for (M+Na) : 525.1195.

Found: 525.1205.

4-O-tert-butyldimethylsilyl-cyclohexanol (12a): To a solution of cis/trans mixture of 1, 4- cyclohexanediol (200 mg, 1.72 mmol) in N, N-dimethylformamide (5 mL) was added 2.5 eq of imidazole (292. 7 mg) and the mixture was stirred at r.t. 30 min. To the solution was added tert- butyldimethylsilyl chloride (1.25 eq, 292.7 mg) and the mixture was stirred for 3 days in an atmosphere of N2. At the end of the third day, the mixture was diluted with excess CH2Cl2 (30 mL), washed with water (5 x 25 mL) and saturated NaCl (aq) solution (1 x 25 mL). The organic layer was dried over anhydrous Na2SO4 and then evaporated in vacuo to give 276 mg of the crude product. Purification was done by flash column chromatography using 50% EtOAc in hexanes as the eluent which gave 196 mg (50%) of the product. Rf (50% EtOAc/hexanes): 0.87;

1 H NMR (500 MHz, CDCl3) δ 3.79-3.83 (m, 1H, H-1), 3.65-3.70 (m, 1H, H-4), 1.6-1.77 (m, 6H,

H-2a, 2e, 3e, 5e, 6a, 6e), 1.46-1.5 (m, 1H, H-3a), 1.28-1.32 (m, 1H, H-5a), 0.89 (s, 9H, t-butyl),

13 0.04 (s, 6H, CH3); C NMR (125 MHz, CDCl3) δ 78.1 (C-4), 69.7 (C-1), 32.1 (C-2, 6), 31.3 (C-

3, 5), 30.9 (-CCH3, t-butyl), 25.9 (-CCH3, t-butyl), -2.0 (CH3).

2-Phthalimido-per-O-acetyl-β-D-glucopyranose (9): To a solution of NaOCH3 (2 g, 37.037 mmol) in CH3OH (60 mL) in an oven dried 250 mL r. b. f. was added glucosamine HCl (10 g,

46.3 mmol) and the mixture was stirred at r. t. in an atmosphere of N2 for 30 min. 6.852 g of phthalic anhydride (1 eq) was added to the mixture and was stirred at r. t. for 20 h. The reaction

82 was terminated by evaporating the solvent in vacuo. To the solid residue obtained was added 60 mL of acetic anhydride and 50 mL of pyridine and the mixture was stirred at r. t. for 42 h for per- acetylating the sugar. The reaction was terminated by adding 100 g of ice and stirring the mixture for 30 min. The mixture was extracted with CH2Cl2 (2 x 150 mL). The combined organic extracts were washed with 3M H2SO4 solution (4 x 100 mL), water (2 x 150 mL), saturated NaHCO3 and water (2 x 150 mL) again sequentially. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to dryness in vacuo to give 18.42 g of a yellow fluffy solid. Flash column chromatography of the crude product by gradient dilution method

(40%, 50% and 60% EtOAc/hexanes) gave 6.54 g (30%) of the desired pure product. Rf (40%

1 EtOAc/hexanes): 0.32; H NMR (500 MHz, CDCl3) δ 7.85-7.87 (m, 2H, Ar), 7.73-7.76 (m, 2H,

Ar), 6.51 (d, 1H, J1,2 = 8.9 Hz, H-1), 5.88 (dd, 1H, J2,3 = 10.6 Hz, J3,4 = 9.1 Hz, H-3), 5.21 (d, 1H,

J3,4 = 10 Hz, H-4), 4.46 (dd, 1H, J2,3 = 10.6 Hz, J1,2 = 8.9 Hz, H-2), 4.36 (dd, 1H, J6,6’ = 12.4 Hz,

J5,6 = 4.4 Hz, H-6), 4.11-4.14 (m, 1H, H-6’), 4.01-4.04 (m, 1H, H-5), 2.11 (s, 3H, -CO-CH3),

13 2.04 (s, 3H, -CO-CH3), 2.00 (s, 3H, -CO-CH3), 1.86 (s, 3H, -CO-CH3); C NMR (125 MHz,

CDCl3) δ 172.9, 170.2 (-CO-CH3), 167.6 (-CO-N-), 132.2, 123.7 (Ar), 93.4 (C-1), 74.7 (C-5),

70.8 (C-3), 69.9 (C-4), 61.9 (C-2), 62.4 (C-6), 21.0 (-CO-CH3).

Thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose (10): To a solution of 2- phthalimido-per-O-acetyl-β-D-glucopyranose (1.5 g, 3.14 mmol) in 30 mL of CHCl3 in a clean oven dried 100 mL r. b. f. was added ethanethiol (1.2 eq, 234 mg) and the mixture was stirred at r. t. for 15 min in an atmosphere of N2. To the mixture was added BF3. Et2O drop-wise and the mixture was stirred at r. t. for 4 days. When TLC showed that the reaction reached completion, the mixture was diluted with 100 mL of CH2Cl2, washed with saturated NaHCO3 solution (2 x

150 mL) and 10% w/v NaOH (aq) solution (1 x 100 mL). The combined organic layers were

83 dried over anhydrous Na2SO4 and concentrated to dryness in vacuo to give 1.35 g of a red fluffy solid. The crude product was purified by flash column chromatography, using 50% EtOAc in hexanes as the eluent, to yield 1.188 g (79%) of the pure product. Rf (50% EtOAc/hexanes):

1 0.49; H NMR (500 MHz, CDCl3) δ 7.85-7.86 (m, 2H, Ar), 7.73-7.76 (m, 2H, Ar), 5.82 (dd, 1H,

J2,3 = 10.3 Hz, J3,4 = 9.2 Hz, H-3), 5.48 (d, 1H, J1,2 = 10.7 Hz, H-1), 5.17 (dd, 1H, J4,5 = 10.1 Hz,

J3,4 = 9.2 Hz, H-4), 4.39 (d, 1H, J1,2 = 10.4 Hz, H-2), 4.3 (dd, 1H, J6,6’ = 12.4 Hz, J5,6 = 4.9 Hz, H-

6), 4.17 (dd, 1H, J6,6’ = 12.3 Hz, J5,6’ = 2.3 Hz, H-6’), 3.89 (ddd, 1H, J4,5 = 10.25 Hz, J5,6 = 4.85

Hz, J5,6’ = 2.4 Hz, H-5), 2.62-2.71 (m, 2H, -S-CH2-CH3), 2.10 (s, 3H, -CO-CH3), 2.03 (s, 3H, -

13 CO-CH3), 1.86 (s, 3H, -CO-CH3), 1.21 (t, 3H, JCH2,CH3 = 7.3 Hz, -S-CH2-CH3); C NMR (125

MHz, CDCl3) δ 170.2 (-CO-CH3), 169.6 (-CO-N-), 132.2, 123.8 (Ar), 81.3 (C-1), 76.12 (C-5),

71.7 (C-3), 69.1 (C-4), 62.48 (C-6), 53.86 (C-2), 24.52 (-S-CH2-CH3), 20.91, 20.77, 20.6 (-CO-

CH3), 15.04 (-S-CH2-CH3).

(4-tert-butyldimethylsilyl-cyclohexyl)-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose

(13): Into a clean oven dried 25 mL r. b. f. was taken 575 mg (1.2 mmol) of thioethyl-2- phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose, To it was added 15 mL anhydrous CH2Cl2 and the mixture was cooled to -78 oC (acetone/dry ice) upon stirring in an inert atmosphere. To the mixture was added N-iodosuccinimide (2.5 eq, 675 mg) and stirred at -78 oC for 30 min.

Keeping the temperature constant, 10 µL (0.05 eq) of trimethylsilyl trifluoromethanesulfonate

(TMSOTf) was added drop-wise to the mixture upon vigorous stirring. To the mixture was added drop-wise a solution of 4-O-tert-butyldimethylsilyl-cyclohexanol (1.2 eq, 331.2 mg) in CH2Cl2

(2 mL) and the reaction was stirred at -78 oC for 90 min. The reaction was terminated and the mixture was diluted with excess CH2Cl2 (100 mL) and washed with saturated NaHCO3 solution

(1 x 50 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated to

84 dryness in vacuo to yield a gram of the crude product as a dark brown oily syrup. Purification was done by flash column chromatography using gradient dilution (30%, 40%, 50%, 60%, 70%,

80%, 90% EtOAc in hexanes) which yielded 503 mg (65%) of the product as a pale yellow oily

1 syrup. Rf (60% EtOAc/hexanes): 0.83; H NMR (500 MHz, CDCl3) δ 7.84-7.87 (m, 2H, Ar),

7.71-7.75 (m, 2H, Ar), 5.76-5.83 (m, 1H, H-3), 5.44 (dd, 1H, J1,2 = 8.75 Hz, J1,3 = 2.4 Hz, H-1),

5.13-5.18 (m, 1H, H-4), 4.28-4.35 (m, 2H, H-2, 6), 4.16 (d, 1H, J6,6’ = 12.2 Hz, H-6’) 3.84-3.87

(m, 1H, H-5), 3.59-3.68 (m, 1H, Cyclohexyl-H-1), 3.49-3.53 (m, 1H, Cyclohexyl-H-4), 2.11 (s,

3H, -CO-CH3), 2.03 (s, 3H, -CO-CH3), 1.86 (s, 3H, -CO-CH3), 1.68-1.8 (m, 2H, Cyclohexyl-H-

2e, 6e), 1.24-1.40 (m, 4H, Cyclohexyl-H-3e, 2a, 5e, 6a), 1.19-1.40 (m, 1H, Cyclohexyl-H-3a), 0.83

(s, 5H, H-t-butyl), 0.74 (s, 4H, H-t-butyl), -0.04 (s, 3H, CH3), -0.08 (s, 3H, CH3), -0.12 (s, 3H,

13 CH3); C NMR (125 MHz, CDCl3) δ 172.96, 171.06 (-CO-CH3), 169.84 (-CO-N-), 134.57,

123.92 (Ar), 97.05 (C-1), 72.11 (C-5), 71.21 (C-3), 69.59 (C-4), 62.60 (C-6), 49.66 (C-2), 31.13

(-CCH3, t-butyl), 26.15, 26.02 (-CCH3, t-butyl), 21.13, 21, 20.82 (-CO-CH3), -4.43 (CH3);

+ LRMS (ESI): Calcd C32H45NO11Si: 647.3. Found for (M+H) : 648.3.

Cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose (14): Into a clean oven dried

10 mL r. b. f. was taken 25 mg (0.052 mmol) of thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-

o glucopyranose, To it was added 5 mL anhydrous CH2Cl2 and the mixture was cooled to -78 C upon stirring in an inert atmosphere. To the mixture was added N-iodosuccinimide (2.5 eq, 29.34 mg) and stirred at -78 oC (acetone/dry ice) for 30 min. Keeping the temperature constant, 0.46

µL (0.05 eq) of trimethylsilyl trifluoromethanesulfonate (TMSOTf) was added drop-wise to the mixture upon vigorous stirring. To the mixture was added drop-wise a solution of 4-O-tert- butyldimethylsilyl-cyclohexanol (1.2 eq, 14.35 mg) in CH2Cl2 (1 mL) and the reaction was

o stirred at -78 C for 7 h. The reaction was terminated and the mixture was diluted with CH2Cl2

(25 mL) and washed with saturated NaHCO3 solution (1 x 25 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated to dryness in vacuo to yield the crude product as a orange-brown oily syrup. Purification was done by preparatory TLC technique using 80%

EtOAc in hexanes as the solvent which yielded 32 mg (95%) of the product as a yellowish oily

1 syrup. Rf (60% EtOAc/hexanes): 0.157; H NMR (500 MHz, CDCl3) δ 7.84-7.86 (m, 2H, Ar),

7.72-7.76 (m, 2H, Ar), 5.75-5.82 (m, 1H, H-3), 5.44 (dd, 1H, J1,2 = 8.4 Hz, J1,3 = 6.4 Hz, H-1),

5.15-5.19 (m, 1H, H-4), 4.28-4.36 (m, 2H, H-2, 6), 4.16 (m, 1H, J6,6’ = 10.8 Hz, H-6’), 3.85-3.88

(m, 1H, H-5), 3.73-3.77 (m, 1H, Cyclohexyl-H-1), 3.56-3.65 (m, 1H, Cyclohexyl-H-4), 2.11 (s,

e 3H, -CO-CH3), 2.03 (s, 3H, -CO-CH3), 1.95-1.98 (m, 1H, Cyclohexyl-H-2 ), 1.86 (s, 3H, -CO-

e e e CH3), 1.69-1.76 (m, 1H, Cyclohexyl-H-6 ), 1.33-1.43 (m, 2H, Cyclohexyl-H-3 , 5 ), 1.17-1.31

a a 13 (m, 2H, Cyclohexyl-H-3 , 5 ); C NMR (125 MHz, CDCl3) δ 170.73, 170.22 (-CO-CH3),

169.51, 165.52 (-CO-N-), 134.35, 131.38, 123.6 (Ar), 96.85 (C-1), 76.86 (Cyclohexyl-C-1),

71.78 (C-3, 5), 70.88 (C-4), 69.19 (Cyclohexyl-C-4), 62.23 (C-6), 54.86 (C-2), 30.47, 30.06

(Cyclohexyl-C-2, 3, 5, 6), 20.78, 20.65, 20.46 (-CO-CH3). LRMS (ESI): Calcd C26H31NO11:

533.2. Found for (M+H)+: 534.2.

(Cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranosyl)-2-O-acetyl-3-O-benzoyl-

4,6-O-pyruvate-β-D-galactopyranose (15): 61.279 mg (1.3 eq) of thiophenyl-2-O-acetyl-3-O- benzoyl-4,6-O-pyruvate-β-D-galactopyranose and 5 mL of anhydrous CH2Cl2 were taken in a

o clean oven tried 10 mL r. b. f. and the mixture was stirred at -42 C (CH3CN/dry ice) in an inert atmosphere for 15 min. 68.625 mg (2.5 eq) of N-iodosuccinimide was added to the mixture and stirred for 30 min at -42 oC. Keeping the temperature constant, 1.3 µL of TMSOTf (0.05 eq) was added to the mixture drop-wise upon vigorous stirring. To the mixture was added a solution of cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose (50 mg, 0.0939 mmol) in

o anhydrous CH2Cl2 (1 mL) and the reaction was run at -42 C for 3 h upon continuous stirring.

The reaction was terminated by adding excess CH2Cl2 (40 mL), washed with saturated NaHCO3 solution (1 x 50 mL) and 10% w/v Na2S2O3 solution (1 x 50 mL). The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated to dryness in vacuo to yield

135 mg of the crude product as an orange oily syrup. Purification was done by preparatory TLC technique using 60 % EtOAc in hexanes as the solvent, which gave 50 mg (57.6%) of the

1 product as an off white coloured solid. H NMR (500 MHz, CDCl3) δ 7.99-8.04 (m, 2H, Ar),

7.91-7.96 (m, 1H, Ar), 7.83-7.88 (m, 1H, Ar), 7.73-7.77 (m, 1H, Ar), 7.49-7.54 (m, 1H, Ar),

7.36-7.41 (m, 1H, Ar), 7.35-7.41 (m, 2H, Ar), 5.73-5.80 (m, 1H, Hglu-1), 5.62-5.66 (m, 1H,

Hgal-1), 5.36-5.46 (m, 2H, Hglu-3, Hgal-2), 5.13-5.19 (m, 1H, Hgal-3), 4.48-4.60 (m, 1H, Hglu-4),

4.26-4.34 (m, 2H, Hgal-4, Hglu-2), 4.10-4.27 (m, 2H, Hglu-5, 6), 3.93-4.08 (m, 2H, Hglu-6’, Hgal-6),

3.80-3.90 (m, 2H, Hgal-6’, Cyclohexyl-H-1), 3.73-3.78 (m, 1H, Hgal-5), 3.61-3.7 (m, 3H, -OCH3),

3.41-3.53 (m, 1H, Cyclohexyl-H-4), 2.11 (s, 1H, -CO-CH3), 2.10 (s, 1H, -CO-CH3), 2.06 (s, 1H,

-CO-CH3), 1.87 (s, 1H, -CO-CH3), 1.86 (s, 1H, -CO-CH3), 1.85 (s, 1H, -CO-CH3), 1.65-1.83 (m,

a e a e 4H, Cyclohexyl-H-2 , 2 , 6 , 6 ), 1.57 (s, 3H, -CO-CH3), 1.37-1.42 (m, 1H, Cyclohexyl-H-

e e a 3 ),1.27-1.31 (m, 1H, Cyclohexyl-H-5 ) 1.25 (s, 3H, CH3), 0.86-0.92 (m, 1H, Cyclohexyl-H-3 );

13 C NMR (125 MHz, CDCl3) δ 170.70, 170.20 (-CO-CH3), 169.49 (-CO-OCH3), 165.89 (-CO-

N-), 134.37, 133.16, 131.31, 129.88, 129.77, 129.67, 128.42, 128.36, 127.47, 123.61 (Ar),

100.36 (pyr), 98.69 (Cgal-1), 96.65 (Cglu-1), 96.12 (Cyclohexyl-C-1), 95.91 (Cyclohexyl-C-4),

72.04 (Cgal-4), 71.93 (Cgal-5), 71.79 (Cglu-5), 70.82 (Cgal-3), 69.22 (Cglu-3), 69.13 (Cgal-4), 66.7

(Cgal-2), 65.34 (Cgal-6), 64.01 (Cglu-6), 62.17 (Cglu-2), 54.8 (CO-OCH3), 31.93, 29.70, 29.36,

28.98 (Cyclohexyl-C-2, 3, 5, 6), 25.72 (CH3), 20.78, 20.65, 20.47 (-CO-CH3); HRMS (ESI):

+ Calcd C45H51NO20Na for (M+Na) : 948.2902. Found: 948.2899.

4.2. 6-O-p-toluenesulfonyl-D-arabino-hex-1-enitol (17).

To a solution of D-glucal (7.6 g, 52.5 mmol) in anhydrous pyridine was added dropwise a

o solution of p-toluenesulfonyl chloride (15.02 g, 78.8 mmol) in anhydrous CH2Cl2 at 0 C upon vigorous stirring. The reaction was terminated after 3 h by the addition of water, followed by washing with saturated CuSO4 (3 x 30 mL), water (5 x 30 mL) and brine (1 x 30 mL). The organic extracts were dried under anhydrous MgSO4 and concentrated in vacuo to yield crude

17. Although the compound was synthesized earlier,13 no spectroscopic data was reported. The product was purified by flash chromatography using 3%, 4% and 5% CH3OH in CHCl3 mixtures to give 8.18 g (56% yield) of 17 as a colorless oily mass which crystallized upon freezing: Rf

o 1 (1:20 CH3OH/CHCl3): 0.46; mp 50–51 C; H NMR (500 MHz, CDCl3) δ 7.81 (d, 2H, J = 6.3

Hz, Ar), 7.35 (d, 2H, J = 7.9 Hz, Ar), 6.24 (d, 1H, J1,2 = 6.1, J1,3 = 1.8 Hz, H-1), 4.74 (dd, 1H, J2,1

= 6.3, J2,3 = 2.2 Hz, H-2), 4.47 (dd, 1H, J6,6’ = 11.25, J6,5 = 4Hz, H-6), 4.28 (dd, 1H, J6,6’ = 11.45,

J6’,5 = 2.5 Hz, H-6’), 4.26 (d, 1H, J3,4 = 6.8 Hz, H-3), 3.91 (ddd, 1H, J5,4 = 9.75, J5,6 = 3.9, J5,6’ = 2

Hz, H-5), 3.76 (dd, 1H, J4,3 = 8.3, J4,5 =8.3 Hz, H-4), 3.09 (s, 1H, 4-OH), 2.45 (s, 3H, PhCH3),

13 2.23 (s, 1H, 3-OH); C NMR (125 MHz, CDCl3) δ 145.19 (CH, Ar), 143.99 (C-1), 132.61,

129.93 , 128.02 (CH, Ar), 103.04 (C-2), 75.7 (C-5), 69.57 (C-3), 69.35 (C-4), 67.92 (C-6), 21.66

(PhCH3).

4.3. 3,6-Anhydro-2-deoxy-D-arabino-hex-1-enitol (18).

To a solution of 17 (200 mg, 0.33 mmol) in 20 mL of tetrahydrofuran (THF) was added

o o dropwise 0.83 mL of 2.4 M LiAlH4 in THF (2 mmol) at 0 C. The mixture was refluxed at 73 C for two days. It was then cooled to 0 oC and neutralized with 15% w/v NaOH and water, and filtered through a Celite@ bed upon dilution with ethyl acetate (EtOAc). The mixture was concentrated in vacuo and the product was purified by flash chromatography using 1:1 mixture

88 of EtOAc/Hexanes to give 73 mg (84%) of 18 as a white solid: Rf (1:20 CH3OH/CHCl3): 0.42;

22 1 D] -24.17 (c 0.58, CHCl3); H NMR (500 MHz, CDCl3) δ 6.54 (d, 1H, J1,2 = 5.0 Hz, H-1),

5.02 (ddd, 1H, J2,1 = 5.25, J2,3 = 5.5, J2,4 = 1.5 Hz, H-2), 4.36 (br dd, 1H, J5,6 =3.65, J5,6’ =3.65

Hz, H-5), 4.27 (d, 1H, J6’,6 =11.2 Hz, H-6’), 4.23-4.2 (m, 1H, H-4), 4.18 (dd, 1H, J6,6’ = 11.3, J5,6

= 4.5 Hz, H-6), 4.03 (br dd, 1H, J3,4 =4.5, J3,2= 5.5 Hz, H-3), 2.09 (d, 1H, J4,-OH = 10.7 Hz, 4-OH);

13 C NMR (125 MHz, CDCl3) δ 146.53 (C-1), 100.41 (C-2), 76.56 (C-5), 74.71 (C-6), 68.76 (C-

3), 66.26 (C-4); Anal. Calcd. for C6H8O3: C, 56.24; H, 6.29. Found: C, 56.15; H, 6.42.

4.4 . General procedure for the synthesis of 3,6-anhydro-O-glycosides.

o To a solution of 50 mg (0.39 mmol) of 18 in 5 mL of anhydrous CH3CN at 0 C was added

105.5 mg (0.47mmol) of N-iodosuccinimide. To the solution was then added 1.2 equivalents of the desired nucleophile and the reaction mixture was stirred at r. t. overnight. The solvent was evaporated in vacuo and the crude product was purified by preparatory thin-layer chromatography (TLC) using 1:50 CH3OH/CHCl3 as the solvent.

4.4.1. Methyl-3,6-anhydro-2-deoxy-2-iodo-gluco- and manno-pyranosides (19a, 19b, 19c).

1 Data for the mixture of diastereomers: H NMR (500 MHz, CDCl3) δ 5.31 (s, 1H), 5.03 (d,

1H, J = 7.8 Hz), 4.72 (s, 1H), 4.47 (dd, 1H, J = 4.4 Hz, J = 3.4 Hz), 4.42-4.44 (m, 1H), 4.39-4.36

(m, 2H), 4.28-4.32 (m, 2H), 4.24 (d, 2H, J = 10.7 Hz), 4.15-4.12 (m, 1H), 4.09-4.06 (m, 3H),

3.96 (dd, 1H, J = 10 Hz, J = 5 Hz), 3.91 (dd, 1H, J = 5 Hz, J = 10 Hz), 3.54 (s, 1H), 3.48 (s, 1H),

3.46 (s, 1H), 2.64 (d, 1H, J = 8.8 Hz), 2.44 (d, 1H, J = 5 Hz), 2.41 (d, 1H, J = 8.8 Hz), 2.04 (d,

13 1H, J = 5.3 Hz); C NMR (125 MHz, CDCl3) δ 80.43, 72.81, 69.06, 29.57, 26.60, 20.28; HRMS

+ (ESI): Calcd C7H11O4NaI for (M+Na) : 308.9611. Found: 308.9600.

4.5. General procedure for the synthesis of 2-deoxythioglycosides.

To a stirred solution of 50 mg (0.39 mmol) of 18 and 21 mg (0.039 mmol) of Ce(NH4)2(NO3)6 in anhydrous CH3CN (5 mL) was added a solution of 0.2 mL (1.95 mmol) of PhSH in anhydrous

o CH3CN (5 mL) at 0 C and allowed to stir overnight at r.t.. The solvent was evaporated in vacuo and the residue was diluted with excess CH2Cl2, washed with 10% NaOH, saturated NaHCO3 and saturated NaCl and, dried over anhydrous Na2SO4. The organic solvent was evaporated in vacuo and the crude (150 mg) obtained was purified by preparatory TLC using 1:50

CH3OH/CHCl3 as the solvent to give mixtures of compounds 20a, 20b (60 mg) and 21a, 21b (33 mg).

4.5.1. Thiophenyl-3,6-anhydro-2-deoxy-D-glucopyranoside (20a, 20b).

1 Data for α-anomer (20b): Rf (1:50 CH3OH/CHCl3): 0.84; H NMR (500 MHz, CDCl3) δ 7.54-

7.50 (m, 2H, Ar), 7.33-7.24 (m, 3H, Ar), 5.73 (dd, 1H, J1,2 = 8.0, J1,2’ = 3.2 Hz, H-1), 4.69-4.67

(m, 1H, H-5), 4.59 (dd, 1H, J2,3 = 5.4, J3,4 = 1.2 Hz, H-3), 4.29-4.26 (m, 1H, H-4), 3.99 (dd, 1H,

J6,6’ = 9.7, J5,6 = 4.4 Hz, H-6), 3.84 (dd, 1H, J6,6’ = 9, J5,6’ = 5.5 Hz, H-6’), 3.06 (d, 1H, J4,-OH =

10.7 Hz, 4-OH), 2.63 (ddd, 1H, J2,2’ = 14.15, J1,2 = 8.55, J2,3 = 6.3 Hz, H-2), 2.35 (ddd, 1H, J2,2’ =

13 14.1, J1,2’ = 3.15, J2’,3 = 1.3 Hz, H-2’); C NMR (125 MHz, CDCl3) δ 132.3, 130.8, 129.1 (CH,

Ar), 89.8 (C-1), 85.17 (C-5), 82.9 (C-3), 74.86 (C-6), 71.83 (C-4), 40.6 (C-2); HRMS (ESI):

+ Calcd C12H14O3NaS for (M+Na) : 261.0570. Found: 261.0561. Data for β-anomer (20a): Rf

1 (1:50 CH3OH/CHCl3): 0.76; H NMR (500 MHz, CDCl3) δ 7.54-7.50 (m, 2H, Ar), 7.33-7.24 (m,

3H, Ar), 5.66 (dd, 1H, J1,2 = 6.8, J1,2’ = 1.3 Hz, H-1), 4.69-4.67 (m, 1H, H-5), 4.64 (dd, 1H, J3,4 =

5.4, J2,3 = 1.2 Hz, H-3), 4.26-4.25 (m, 1H, H-4), 3.82 (dd, 1H, J6,6’ = 9.55, J5,6’ = 5.7 Hz, H-6’),

3.63 (dd, 1H, J6,6’ = 9.5, J5,6 = 6.2 Hz, H-6), 2.58 (d, 1H, J4,-OH = 7.4 Hz, 4-OH), 2.5 (ddd, 1H,

13 J2,2’ = 14.1, J1,2 = 6.35, J2,3 = 2 Hz, H-2), 2.17-2.22 (m, 1H, H-2’); C NMR (125 MHz, CDCl3) δ

129, 127.78, 127.35 (CH, Ar), 88.41 (C-1), 82.52 (C-5), 82.39 (C-3), 73.16 (C-6), 72.12 (C-4),

+ 40.77 (C-2); HRMS (ESI): Calcd C12H14O3NaS for (M+Na) : 261.0570. Found: 261.0561.

4.5.2. Thiophenyl-2,3-dideoxy-D-glucopyranoside (21a, 21b).

1 Data for α-anomer (21a): Rf (1:50 CH3OH/CHCl3): 0.19; H NMR (500 MHz, CDCl3) δ

7.51-7.53 (m, 2H, Ar), 7.26-7.33 (m, 3H, Ar), 6.0 (dd, 1H, J1,2 = 10.2 Hz, J2,3 = 2 Hz, H-2), 5.98

(d, 1H, J1,2 = 10.2 Hz, H-1), 5.74 (dd, 1H, J3,4 = 1.2 Hz, J2,3 = 2.7 Hz, H-3), 4.32 (dd, 1H, J3,4 = 2

Hz, J4,5 = 8.75 Hz, H-4), 4.03-4.07 (m, 1H, H-5), 3.86-3.92 (m, 2H, H-6, 6’), 1.85 (d, 1H, J4,-OH =

13 7.3 Hz, 4-OH), 1.82 (dd, 1H, J6,-OH = 6.35 Hz, J6’,-OH = 6.4 Hz, 6-OH); C NMR (125 MHz,

CDCl3) δ 131.77, 129.03, 127.32 (CH, Ar), 131.72 (C-1), 127.12 (C-2), 83.61 (C-3), 71.88 (C-5),

+ 64.29 (C-4), 62.91 (C-6); HRMS (ESI): Calcd C12H14O3NaS for (M+Na) : 261.0561. Found:

1 261.0561. Data for β-anomer (21b): Rf (1:50 CH3OH/CHCl3): 0.19; H NMR (500 MHz,

CDCl3) δ 7.51-7.53 (m, 2H, Ar), 7.26-7.33 (m, 3H, Ar), 5.88 (dd, 1H, J1,2 = 2 Hz, J2,3 = 1.9 Hz,

H-2), 5.86 (dd, 1H, J1,2 = 1.5 Hz, J2,3 = 1.4 Hz, H-1), 4.72-4.78 (m, 1H, H-4), 4.54-4.60 (m, 1H,

13 H-5), 4.11-4.16 (m, 2H, H-6, 6’), 2.18-2.24 (m, 4-OH, 6-OH); C NMR (125 MHz, CDCl3) δ

135.10, 132.5, 131.69 (CH, Ar), 131.62 (C-1), 128.84 (C-2), 81.74 (C-3), 79.74 (C-5), 63.40 (C-

+ 4), 62.75 (C-6); HRMS (ESI): Calcd C12H14O3NaS for (M+Na) : 261.0561. Found: 261.0561.

4.6. General procedure for the preparation of the dianhydrosugar.

To 100 mg (0.78 mmol) of 18 in 10 mL of CH3CN was added 210 mg (0.94 mmol) of N- iodosuccinimide in the presence of 4 Ǻ molecular sieves, and allowed to stir overnight at r.t. The solvent was removed in vacuo and the residue was diluted with excess CH2Cl2, washed with 10%

Na2S2O3, saturated NaCl and dried over anhydrous Na2SO4. The organic filtrate was evaporated in vacuo and the crude product (170 mg) was purified by flash chromatography using 2:3

EtOAc/Hexanes to give 150 mg (0.61 mmol, 76 %) of 22. In the absence of activated sieves, a mixture of 2-deoxy-2-iodo-hexopyranoses (23a-c), were also obtained (30%).

4.6.1. 1,4:3,6-Dianhydro-2-deoxy-2-iodo-D-glucopyranose (22).

1 Data for the dianhydrosugar: Rf (1:50 CH3OH/CHCl3): 0.9; H NMR (500 MHz, CDCl3) δ

5.51 (br s, 1H, H-1), 5.09 (dd, 1H, J4,3= 4.25, J4,5 = 4.5 Hz, H-4), 4.27 (dd, 1H, J5,4 = 3.5, J5,6 =

3.25 Hz, H-5), 4.18 (dd, 1H, J3,2 = 7.0, J3,4 = 5.0 Hz, H-3), 4.15 (d, 1H, J6,6’ = 10.8 Hz, H-6’), 4.0

13 (dd, 1H, J2,3 = 7.0, J2,1 = 2.0 Hz, H-2), 3.94 (dd, 1H, J6,6’ =10.7, J5,6 = 3.0 Hz, H-6); C NMR

(125 MHz, CDCl3) δ 100.20 (C-1), 79.87 (C-4), 77.02 (C-5), 73.28 (C-3), 71.30 (C-6), 32.85 (C-

2); LRMS (ESI) m/Z (rel. intensity) 254.9 (M+H)+ (79).

4.6.2. 3,6-anhydro-2-deoxy-2-iodo-D-hexopyranose (23a, 23b, 23c).

1 Data for mixture of diastereomers: Rf 1:50 CH3OH/CHCl3): 0.12; H NMR (500 MHz,

CDCl3) δ 5.76 (s, 1H), 5.68 (d, 1H, J = 5 Hz), 5.38 (s, 1H), 5.02 (dd, 1H, J = 5.4 Hz, J = 4.9 Hz),

4.89 (d, 1H, J =), 4.83 (ddd, 1H, J = 15 Hz, J = 5 Hz, J = 5 Hz), 4.73-4.70 (m, 2H), 4.48 (d, 1H, J

= 1.4 Hz), 4.44 (d, 1H, J = 4.9 Hz), 4.40-4.37 (m, 2H), 4.19 (dd, 1H, J = 4.9 Hz, J = 4.4 Hz), 4.14

(dd, 1H, J =), 4.09-4.06 (m, 2H), 4.04-3.98 (m, 2H), 3.79-3.76 (m, 2H), 3.62 (dd, 1H, J = 9.5 Hz,

13 J = 6.6 Hz); C NMR (125 MHz, CDCl3) δ 107.69, 99.48, 96.06, 90.68, 84.04, 83.32, 82.26,

76.37, 75.55, 71.75, 70.55, 46.57, 46.56, 26.66, 25.12, 23.61, 8.72, 8.71; LRMS (ESI): Calcd

+ C6H9IO4: 271.9. Found for (M+Na) : 294.8.

4.2 NMR Spectra

vb_276_pure_1H.esp

2.03

2.11 2.09

1.0 1.96

0.9

0.8

0.7

0.6

0.5

0.4 NormalizedIntensity

0.3

7.30

4.72 4.70

0.2 7.31

4.11

5.23

7.51

7.50

7.51

5.05

4.16

5.41 5.05

5.41

4.18

5.21

4.10 3.93

7.26

4.09

4.10

4.19 1.96

0.1 7.32

3.95 2.13

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

vb_276_pure_13C.esp

132.56 128.89 1.0

0.9 77.06

0.8 77.31 76.80

0.7

67.22 74.42

0.6 86.60

72.00

128.16 61.63

0.5 20.63

20.58 NormalizedIntensity

0.4 20.85

0.3

170.18

170.03 169.41 0.2 170.35

0.1

200 180 160 140 120 100 80 60 40 20 Chemical Shift (ppm)

6 F1 Chemical Shift (ppm) Shift Chemical F1

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shift (ppm)

100

120 (ppm) Shift Chemical F1

8 7 6 5 4 3 2 1 0 F2 Chemical Shift (ppm)

vb_277_pure_1H.esp 7.29 1.0

0.9 7.44

4.57

4.58

7.45 4.72

0.8 3.47

0.7 3.49

3.43

7.46 7.28

0.6

3.71 3.72

0.5 7.20

3.36

3.41 3.36

0.4 3.52

NormalizedIntensity

3.34 3.34

0.3 2.50

3.53

2.50

2.50 7.18

0.2

2.49

2.51 3.54 0.1

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

1.0 vb_277_pure_13C.esp

0.9

0.8

0.7 128.74

0.6 129.21

0.5

0.4 NormalizedIntensity

0.3

87.68

79.10 126.03

0.2

60.44

69.14

68.20

74.58 135.51 0.1

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 Chemical Shift (ppm)

6 F1 Chemical Shift (ppm) Shift Chemical F1 8

8 7 6 5 4 3 2 1 0 F2 Chemical Shift (ppm)

100

120 (ppm) Shift Chemical F1

9 8 7 6 5 4 3 2 1 0 F2 Chemical Shift (ppm)

101

vb_285_B_1H.esp 7.39

1.0 5.53

0.9 7.39

0.8 3.72

0.7 3.73

7.50

7.32 7.30

0.6

7.70

7.29

8.12

7.72

8.14 3.55

0.5 3.56

8.14

7.70

8.12 4.23

0.4 4.23

NormalizedIntensity

4.05

4.39

4.06

4.52

3.74

4.54

7.63 4.41 0.3 4.41

0.2 3.71 3.75 0.1

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 Chemical Shift (ppm)

102

vb_285_B_13C.esp

77.04 77.29

1.0 76.79

0.9

0.8

133.71

128.47

130.18 126.53

0.7 128.23

0.6

0.5

101.39

NormalizedIntensity

87.00

75.39 70.06

0.4 73.82

68.80 69.28 0.3

0.2 137.62

0.1

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 Chemical Shift (ppm)

103

8 (ppm) Shift Chemical F1

9 8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm)

104

100

120 F1 Chemical Shift (ppm) Shift Chemical F1 140

9 8 7 6 5 4 3 2 F2 Chemical Shift (ppm)

105

1.0 vb_153_B.esp 7.27

0.9 7.26 7.41

0.8 1.26 1.27

0.7

0.6 5.51

0.5

8.03

7.70

7.42

8.05

3.70

7.72 1.25

0.4 1.28

4.13

4.12

NormalizedIntensity

4.51

4.67 4.51

0.3 4.69

7.25

4.08

7.25 4.41

7.54

4.06

5.18

5.19 4.15

0.2 5.20

5.21

1.81 0.95

0.96 0.94 0.1

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm)

106

6 F1 Chemical Shift (ppm)Chemical Shift F1

8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm)

107

vb_162_A.esp

1.0 2.01

0.9

0.8

0.7 7.26

0.6 7.35

0.5

0.4 7.37

7.39

5.48

NormalizedIntensity

7.97

7.63 3.69

0.3 7.99

4.54

7.41

4.80

4.78 4.07

0.2 4.40

5.56

4.05

4.43

5.21

5.19 7.52

0.1 5.58

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm)

108

6 F1 Chemical (ppm)Chemical Shift F1

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

109

vb_169_B 1.97

0.25

0.20 1.97 0.15

M02(dd) M06(m) NormalizedIntensity 0.10 M03(d) M07(m)

7.98 M01(t)

8.00 7.45

5.08 M04(d)

3.84

5.06

7.44

3.84 8.00

0.05 3.83

5.58

7.93

7.98

7.95

7.47

4.07

4.05

4.42

5.23

4.42

5.56

7.42

5.60

1.96

7.64

4.04

7.42 7.66

1.37 1.81 1.85 1.73 1.234.00

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Chemical Shift (ppm)

110

6 F1 Chemical Shift (ppm) Shift Chemical F1 8

8 7 6 5 4 3 2 F2 Chemical Shift (ppm)

111

vb_171_pure

3.27 1.63 0.9

0.8

0.7

0.6

0.5 2.00

0.4 NormalizedIntensity

0.3

0.2

7.99

7.45

7.52

7.98

7.47

3.27

7.52

5.10

7.93

7.98

7.94

7.50 5.08

1.62

5.32 4.09

0.1 5.09

5.47

5.07

5.30 4.07

1.63

4.07

5.31

3.90

4.07

5.47

5.45

3.26 3.25

4.63

1.59

1.99 1.62

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm)

112

6 F1 Chemical Shift (ppm) Shift Chemical F1 8

8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm)

113

vb_259_pure_1H.esp

2.04

1.86 2.00 1.0 2.11

0.9

0.8

0.7

0.6 7.26 0.5

0.4 NormalizedIntensity

0.3 2.12

7.76

7.86

7.75

6.50

6.52 7.74

0.2 7.75

7.86

4.13

7.87

4.11

4.35

5.21

5.89 5.88 4.36 4.14

4.47 4.47

5.87 4.37 4.16

4.49

5.91

5.23 7.74

0.1 7.73 4.01

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

114

6 F1 Chemical Shift (ppm) Shift Chemical F1 8

8 7 6 5 4 3 2 F2 Chemical Shift (ppm)

115

vb_266_pure_1H.esp

7.26 2.11 2.03 1.86

0.7

0.6 1.22 0.5

0.4

0.3

1.23

NormalizedIntensity 1.20

0.2

5.48

7.75

5.50

4.40

7.74

2.11

7.86

7.74

5.83

5.18

4.19

4.30 4.29

5.81

2.65

2.71 2.69

4.42 4.32 2.67

2.13

5.85 5.20

0.1 5.16

3.89

3.91 3.90

2.72 1.26

9 8 7 6 5 4 3 2 1 Chemical Shift (ppm)

116

vb_266_pure_13C.esp

77.26 77.01 76.76

0.30

0.25

0.20

0.15 NormalizedIntensity

0.10

71.59

75.97

68.95

81.22

53.71

123.72

20.63

20.77

62.33

20.45 24.37

0.05 14.90

169.47 170.70

180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

117

6 F1 Chemical Shift (ppm) Shift Chemical F1 8

8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm)

118

100 F1 Chemical Shift (ppm) Shift Chemical F1

8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm)

119

vb_238_A_1H TMS

0.9

0.8 0.89

0.7

0.6

0.5 0.04

0.4 NormalizedIntensity 0.3

0.2

1.71

1.70

1.65 1.48 1.30

0.1 1.66

1.50

1.30

1.73

3.81

1.46

3.67 3.67

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

120

vb_268_pure_1H

7.26 0.83

0.40 0.74

0.35

0.30

2.11 2.03

0.25 1.52

0.20 1.86

0.15

NormalizedIntensity

-0.03 -0.04

0.10

0.88

7.85

7.86

7.85

1.25

0.05

7.75 1.26

0.05 7.84

4.33

5.43

5.44

4.31 4.31 0.73

5.43 4.30

4.17

5.45

5.15

1.54

5.17

4.32 1.25

7.25 4.34

5.78 5.78

5.76 5.17

7.25 3.87 3.86

3.66 3.63

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

121

vb_268_pure_13C

77.61 77.35 77.10

0.08 26.15

0.07 26.07

0.06 123.92

0.05

72.11

21.00 21.13

0.04

169.84 -4.43

62.60 20.82

0.03 134.57

97.05

172.96

NormalizedIntensity

71.21

69.59 49.66 0.02

0.01

-0.01

180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

122

6 F1 Chemical Shift (ppm)Chemical Shift F1

8 7 6 5 4 3 2 1 0 F2 Chemical Shift (ppm)

123

100 F1 Chemical Shift (ppm)Chemical Shift F1

8 7 6 5 4 3 2 1 0 F2 Chemical Shift (ppm)

124

vb_269_B_1H

7.26 2.11

0.7 2.03

0.6

0.5

1.87 1.87

0.4 1.86 1.86

0.3 NormalizedIntensity

0.2

7.75

7.85

7.75

7.86

7.87

4.32

4.34

4.34 4.17

4.32

5.45 5.45

0.1 5.43

4.18

5.17

7.26

5.44

5.46

4.15

5.80

5.16

4.30

5.78

1.38

7.27

3.87

5.81 5.79 3.85 1.26 1.25

1.39

1.74 1.72

1.37

7.26 3.75

7.27 1.95

3.60 2.12

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm)

125

vb_269_B_13C

77.26 77.01 76.76

0.030

0.025

62.20

71.77 123.62

0.020 134.37 20.66

0.015

70.83

20.79

54.81

NormalizedIntensity

96.84

96.46

20.47

28.78 30.45

0.010 170.24

69.22

68.87

169.51

27.07

131.36 98.66

0.005

180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

126

6 F1 Chemical Shift (ppm)Chemical Shift F1

8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm)

127

100 F1 Chemical Shift (ppm)Chemical Shift F1 120

8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm)

128

vb_272_A_1H.esp

7.26 2.03

0.8

0.7 1.25

0.6

1.85 2.11

0.5 1.57

0.4

3.64 NormalizedIntensity

0.3 3.67

3.68

3.84

7.75

2.11

7.75 1.61

7.38

1.54 7.74

0.2 7.85

8.02

8.00

3.62

4.30

4.06

7.52

0.87

4.31

1.41

4.28

3.48

1.75

8.04

4.31

5.41

5.39

5.16

2.12

1.23

5.75

5.77

5.64

4.32

5.15

0.06

3.85

0.86 1.21 0.1 5.13

8 7 6 5 4 3 2 1 Chemical Shift (ppm)

129

vb_272_pure_13C

77.28 77.03 76.78 128.36 0.09

0.08 20.65

0.07 128.42 134.37

0.06 129.67

29.70

20.47 20.79

0.05 129.88 123.61

NormalizedIntensity 0.04

70.82

62.17 170.20

0.03 71.79

54.80

25.72

169.49

133.16

69.13

69.22 96.65

0.02 170.70

54.72

52.40

71.93

22.69

98.69

29.36

31.93

165.89 66.70

14.12

100.36

127.47

95.35 55.83

96.12

95.91 95.03

91.37 147.56 0.01 163.34

180 160 140 120 100 80 60 40 20 Chemical Shift (ppm)

130

6 F1 Chemical Shift (ppm)Chemical Shift F1

8 7 6 5 4 3 2 1 F2 Chemical Shift (ppm)

131

vb_196_A_1H 2.45 1.0

0.9

0.8

0.7

0.6

0.5

0.4

NormalizedIntensity

7.81

7.26 7.82 0.3

7.37 4.27

0.2 4.28

4.30

4.75 4.75

4.46

4.74 4.47

4.73

6.24

6.25

6.23

3.90

3.91

4.26

3.76

3.89 3.10 3.10 0.1 2.23

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm)

132

vb_196_A_13C

77.00 76.75

1.0 77.26

0.9

0.8

0.7

0.6

0.5

NormalizedIntensity 0.4 128.01

129.93 0.3

67.92

69.57

75.70 69.35

0.2 103.03 143.99

21.66 145.18

0.1 132.60

180 160 140 120 100 80 60 40 20 Chemical Shift (ppm)

133

F1 Chemical Shift (ppm) Shift Chemical F1 7

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 F2 Chemical Shift (ppm)

134

100

120 (ppm) Shift Chemical F1

140

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 F2 Chemical Shift (ppm)

135

1.0 VB_188_A_1H.esp

0.9

0.8

0.7

0.6

0.5

0.4 NormalizedIntensity

0.3

0.2

0.1

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

136

vb_188_A_13C

77.27 76.76

77.02 1.0

0.9

0.8

0.7

0.6

0.5

NormalizedIntensity 0.4

0.3 74.71

66.26

146.52

100.41 68.76 0.2

0.1

152 144 136 128 120 112 104 96 88 80 72 64 Chemical Shift (ppm)

137

F1 Chemical Shift (ppm)Chemical Shift F1 6

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 F2 Chemical Shift (ppm)

138

100

120

F1 Chemical Shift (ppm) Shift Chemical F1 140

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

139

vb_190_pure_1H

3.55

0.15

0.10

3.46

1.25 NormalizedIntensity

0.05 4.38 4.23

4.25

4.47

5.03

4.37

4.09

5.04

4.09

4.48 2.65

2.64 4.07

4.06

5.31

4.29

4.72

2.04

2.03

2.41

2.40

3.92 1.27 3.93 0

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

140

vb_190_13C

77.25 77.00 76.75

0.10

29.57

0.05 NormalizedIntensity

26.60

80.43

20.28 72.81 69.06

96 88 80 72 64 56 48 40 32 24 16 8 Chemical Shift (ppm)

141

F1 Chemical Shift (ppm) Shift Chemical F1

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shift (ppm)

142

64 (ppm) Shift Chemical F1

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

143

vb_195_B_1H 7.32

1.0 7.52

0.9 7.32

0.8 7.30 7.53

0.7

0.6 7.26 3.83

4.68

3.08 3.06

0.5 7.33

3.82

4.59

7.50 NormalizedIntensity

0.4 3.98 4.69

3.81

2.64

5.73

4.27

5.74 3.80

5.72

4.69

5.74

2.04 4.00

5.66

2.37 2.37

0.3 4.28

3.84

4.26

2.62

2.63

2.34

4.58

3.65

3.85

3.64 2.65

2.66

2.67

3.63

5.64 3.62

0.2 4.69

7.25

7.24

4.25

2.22

4.25 2.49

2.49

7.49 2.18 0.1 4.31

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Chemical Shift (ppm)

144

vb_195_B_13C 130.80

1.0 129.15

0.9

0.8

0.7

82.90

89.80

85.17 40.60

0.6 74.85 127.35

71.83 132.32 0.5

NormalizedIntensity 0.4

40.77

73.19

82.52 82.38

0.3 88.41

0.2 135.35

0.1

144 136 128 120 112 104 96 88 80 72 64 56 48 40 Chemical Shift (ppm)

145

(ppm) Shift Chemical F1 7

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

146

100

120 (ppm)Chemical Shift F1

140

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

147

vb_199_B_1H

7.26 7.32

0.10 7.53 7.51

7.51

1.84 7.53

1.86 5.95

1.83

0.05 5.98

5.75

5.74

7.33

NormalizedIntensity 3.87

5.99

3.88

4.06

3.89

5.95

3.91

3.92

4.32

1.81 4.06

7.34

6.00

4.33

6.01

5.74

4.04

5.93

3.86

3.86 7.51 3.85

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Chemical Shift (ppm)

148

vb_199_B_13C

77.26 77.00 76.75

0.15

0.10

NormalizedIntensity

131.77 71.88 64.29

62.91

0.05 83.61

129.03 127.12

127.56

136 128 120 112 104 96 88 80 72 64 56 Chemical Shift (ppm)

149

6 F1 Chemical Shift (ppm) Shift Chemical F1

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 F2 Chemical Shift (ppm)

150

104

112

120 (ppm) Shift Chemical F1

128

136

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 F2 Chemical Shift (ppm)

151

vb_195_C_1H

7.26 7.51

0.50 7.31 1.26

7.53 0.45

0.40

0.35 7.53 0.30

5.94

0.25 3.88

5.74

3.87

5.74

1.71

5.97 NormalizedIntensity

5.97 7.33

0.20 3.89 5.98

7.26

3.90

2.19

5.94

4.05

3.86 2.18 7.50

0.15 4.05

4.04

5.73

2.03

4.32 5.93

3.85

5.62

5.63

7.53

3.57 2.20

5.99

2.22 2.04

5.99

1.27

3.57

6.00 4.06

0.10 3.84 3.59

3.56

5.89

4.33 3.85

2.31 2.02

5.62

2.30

4.57

2.23

4.63

4.64

3.00

5.30

4.58 1.30

2.98 2.33

3.83

5.42

4.24

5.43

7.25 5.42

7.49

5.82

7.24 4.56 7.24

0.05 1.38 1.64 7.25

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

152

vb_195_C_13C

77.27 77.01 76.76

0.35

0.30

0.25

131.74 129.02

0.20

71.87

62.82 127.00

NormalizedIntensity 0.15

83.65

127.53 64.18 131.68 132.48 0.10

81.74

62.74 79.73

0.05 131.52 64.56 135.09

135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 Chemical Shift (ppm)

153

6 F1 Chemical Shift (ppm) Shift Chemical F1

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shift (ppm)

154

104

112 (ppm) Shift Chemical F1 120

128

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shift (ppm)

155

vb_197_A_1H

4.15 1.0

0.9 4.17

0.8 4.27 5.52

0.7

3.96 3.95 5.10

0.6 4.02

4.02 3.94

3.93

4.01 4.27 0.5 4.28

5.09

5.11

4.17 NormalizedIntensity 0.4 4.18

4.20 0.3

0.2

0.1

5.5 5.0 4.5 4.0 Chemical Shift (ppm)

156

vb_197_A_13C 77.02 1.0

0.9

0.8 76.77

0.7 77.28

0.6

0.5 71.30

NormalizedIntensity 0.4

73.28

100.20

79.87 32.85 0.3

0.2

0.1

105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 Chemical Shift (ppm)

157

3.0

3.5

4.0

4.5

5.0

(ppm) Shift Chemical F1

5.5

5.5 5.0 4.5 4.0 3.5 3.0 F2 Chemical Shift (ppm)

158

40 48

88 (ppm) Shift Chemical F1

104

5.5 5.0 4.5 4.0 3.5 3.0 F2 Chemical Shift (ppm)

159

vb_197_B_1H

0.25

4.30

0.20

0.15

4.74

4.73

3.78

3.79

4.19

3.77 NormalizedIntensity 0.10 3.76

4.39

4.07

4.09

4.38 5.02

5.02

4.20

5.76

4.74

5.01

4.18

4.06

4.75

3.76

5.03

4.06

3.63

4.44

3.61 4.14

0.05 3.62

4.02 4.01

4.04

4.83

4.45

4.79

4.48 4.48

4.89 4.47

4.37

4.89

5.69 5.68 5.41

5.5 5.0 4.5 4.0 Chemical Shift (ppm)

160

vb_197_B_13C

77.26 77.00 76.75

0.13

0.12

0.11

0.10

0.09

0.08

0.07 8.73

46.58

0.06 NormalizedIntensity 0.05

0.04 76.36

90.68

84.04

107.69 26.66

0.03 70.55

71.76

82.26

75.55

25.12

83.32 76.26 0.02 99.47

0.01

104 96 88 80 72 64 56 48 40 32 24 16 8 Chemical Shift (ppm)

161

3.0

3.5

4.0

4.5

5.0 F1 Chemical Shift (ppm) Shift Chemical F1

5.5

5.5 5.0 4.5 4.0 3.5 3.0

F2 Chemical Shift (ppm)

162

F1 Chemical Shift (ppm) Shift Chemical F1 90

100

5.5 5.0 4.5 4.0 3.5 3.0 2.5 F2 Chemical Shift (ppm)

163

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