Synthesis of fragments and one analogue of S. Aureus LTA : investigations toward the synthesis of S. pneumoniae LTA

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Synthesis of fragments and one analogue of S. Aureus LTA. Investigations toward the synthesis of

S. pneumoniae LTA

Dissertation

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften

(Dr. rer. nat.) im Fachbereich Chemie der Universität Konstanz

vorgelegt von

Ignacio Figueroa Pérez

aus Havanna/Kuba 2007

Tag der mündlichen Prüfung: 13/09/2007 Referenten: Richard R. Schmidt

Valentin Wittmann

Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2007/4326/

URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-43267

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A mis padres, a Manolo

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The experimental work presented in this dissertation was carried out at the Universität Konstanz, Fachbereich Chemie, in the research group of Prof. Dr. Richard R. Schmidt from January 2003 to December 2006.

I would like to express my immense gratitude to Prof. Dr. Richard R. Schmidt for providing me very good research facilities and support, for his advices and suggestions.

To Anke Friemel, I gratefully acknowledge for NMR analysis and structural assignments.

I m grateful to all members of the research group, especially to Dr. Jiansong Sun, Dr. Gopal Reddy, Dr. Jianjun Zhang, Dr. Rengarajan Balamurugan, Dr. Kandasamy Pachamuthu, Dr.

Xiang Yang Wu, Dr. Annekatrin Heimann and Dr. Ines Eleuterio for stimulating discussions and support. I´m gatefull also to Christian Draing, Dr Susanne Deininger, Dr. Sigfried Morath, Leonardo Cobianchi, Dr. Sonja von Aulock and Prof. Dr. Thomas Hartung for the support to this project.

Finalmente quisiera agradecer a Remberto Espinosa por su cuidadosa revisión de este manuscrito y por ser más que mi mejor amigo, mi hermano; a toda mi familia por haber confiado en mí siempre. Al Dr. Santiago Figueroa, mi hermano mayor, por ser siempre guía en mi camino de la vida y la química. A Alejandro Figueroa, mi hermano menor y a veces mi jefe, por siempre estar conmigo, aun en la distancia. A Loyda Santiesteban por ser mi segunda madre. A mis padres por haber hecho de mí lo que soy hoy. Por último quisiera agradecer inmensamente a mi esposa Loyda Tejera, por todo su apoyo, desvelo y amor, por Lia y Alejandro.

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Index

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La cie n cia s e com p on e de errores, que a su vez, s on los p a s os h a cia la v e rd a d

Julio Verne

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Index

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Content

1. Introduction and Objectives 1

1.1 Preface 1

1.2 Glycobiology 3

1.2.1 Glycolipids 3

1.3 Cell Wall 4

1.3.1 Lipoteichoic Acids 5

1.3.2 Role of LTAs in infection and inflammation 6 1.3.3 Biosynthesis of Lipoteichoic acids 7

1.4 Cell mediated immunity 9

1.4.1 Cytokines 10

1.5 Objectives 11

1.5.1 Analogue and fragments of S. aureus LTA 11 1.5.2 Studies toward the synthesis of S. pneumoniae LTA 12

2. Theoretical Part 14

2.1 Chemical synthesis of oligosaccharides 14

2.1.1 Koenigs-Knorr method 14

2.1.2 Trichloroacetimidate method 15

2.1.3 ß-N-Phenyl trifluoroacetimidate 16

2.1.4 Thioglycoside method 16

2.1.5 Phosphite method 17

2.2 Fragments of Staphylococcus aureus LTA 17

2.2.1 Retrosynthetic analysis 17

2.2.2 Synthesis of the fragments 18

2.2.2.1 Gentiobiose moiety 18

2.2.2.2 Glycerol moieties 19

2.2.2.3 The phosphoramidite method 20 2.2.2.4 Assembly of the building blocks 21 2.2.2.5 Coupling of the lipid anchor 23

2.2.3 Purification by HIC 26

2.2.3.1 Theory 26

2.2.4 LTA mass spectrometry 29

2.2.5 NMR-Spectroscopy characterization 32

2.2.6 Analogue of S aureus LTA 33

2.2.7 Immunological results 37

2.2.7.1 General 37

2.3 Streptococcus pneumoniae LTA 41

2.3.1 Retrosynthetic analysis 41

2.3.2 Reports in the literature 42

2.3.3 Synthesis of the building blocks 43

2.3.3.1 Ribitol moiety 43

2.3.3.2 D-Galactosamine building blocks 44

2.3.3.3 The rare AATp-Gal 46

2.3.3.3.1 Reports from the literature 46

2.3.3.3.2 Our approach 48

2.3.3.4 D-Glucose building block N 52 2.3.3.5 D-Glucose building block R 53

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Index

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2.3.3.6 Rare AATp-Gal building block T 54 2.3.3.7 D-Glucose building block U 55

2.3.4 Construction of the fragments 56

2.3.4.1 Pseudo-pentasaccharide 56

2.3.4.2 Lipid anchor 62

3. Experimental Part 66

4. NMR Spectra 137

5. Summary 188

Zusammenfassung 203

6. References 199

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Abbreviations

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Abbreviations

Ac acetyl

Ala alanine

Ac₂O acetic anhydride All allyl

Aq acuous

ATT 6-aza-2-thiothymine

BF3·OEt2 borontrifluoride-diethyletherate

Bn benzyl

Bu2BOTf dibutylboryl triflate

nBuOOH n-butyl hydroperoxide

Bz benzoyl

CAN ceric ammonium nitrate Cbz benzyloxycarbonyl

CHCA -cyano-4-hydroxycinnamic acid COSY correlated spectroscopy

CSA (1 R/S) Camphor-10-sulfonic acid

d day(s)

DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DHB 2,5-dihydroxybenzoic acid

DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide eq equivalent

Et ethyl

Et3N triethylamine Et2O diethyl ether EtOAc ethyl acetate EtSH ethanethiol Gal galactose GalNAc galactosamine Glc glucose GlcNAc glucosamine

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Abbreviations

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GroP glycerol phosphate

h hour(s)

HMBC heteronuclear multiple bond connectivity HOAc acetic acid

HSQC heteronuclear single quantum correlation HVL high vacuum line

iPr isopropyl

LPS lipopolysaccharide LTA lipoteichoic acid

MALDI matrix-assisted laser desorption

Me methyl

MeOH methanol

MMTr monomethoxy trityl

MPLC medium pressure chromatography MPM p-methoxybenzyl

Ms mesyl

MS mass spectrometry NBS N-bromosuccinimide NMR nuclear magnetic resonance NOESY nuclear overhauser effect OTf trifluoromethanesulfonate Pd/C palladium on charcoal

Ph phenyl

PMB p-methoxybenzyl PPh3 triphenylphosphine ppm parts per million Pyr pyridine

quant. quantitative yield R protecting group Rf retention factor

ROESY rotation frame overhauser effect correlated spectroscopy R.T. room temperature

TBAF/Br/I tetrabutylammonium fluoride/bromide/iodide TBDMS tert-butyldimethylsilyl

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Abbreviations

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TBDPS tert-butyldiphenylsilyl TDS thexyldimethylsilyl THAP trihydroxy acetophenone THF tetrahydrofuran

TIPS triisopropylsilyl TMS trimethylsilyl

TMSOTf trimethylsilyl trifluoromethanesulfonate TLC thin layer chromatography

Ts tosyl

p-TsOH p-toluenesulphonic acid

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1. Introduction and Objectives

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1

1. Introduction and Objectives

1.1 Preface

In order to improve our knowledge of biological processes such as interaction of cells, cellular differentiation, inflammatory processes, and immunoresponse among others, is necessary to have complex molecules¹. There are only two possibilities to obtain these molecules, they are either isolated from natural material or synthesized. The isolation of complex natural products from biologic material has often proven to be very difficult mainly because of the extremely low concentrations of the target compound and fluctuations in the biological synthetic processes; therefore the synthetic way is the best to obtain a pure and homogeneous structure. Not in every case it is possible to obtain the natural compound but analogues instead, which can offer comparable information of their biological functions. In this work we describe the synthesis of a natural compound, some analogues, and their biological activity.

1.2 Glycobiology.

Carbohydrates, lipids, nucleic acids and proteins, are the most important class of natural macromolecules. Around 200 billion tons of carbohydrates are produced worldwide through photosynthesis every year. The role of carbohydrates in many biological pathways has been better defined in recent years^1,2,3. The traditional view of carbohydrates as solely sources of energy has been augmented by advances in glycobiology that establish oligosaccharides and glycoconjugates as essential components of information transfer in biological systems⁴. Specific oligosaccharides that participate in both beneficial and pathogenic events have been identified⁵. Oligosaccharide components of human milk are known to protect breast-fed infants from a host of bacterial infections⁶. In contrast, the cell-surface glycoconjugates found on protozoan parasites play a role in the infection of human hosts⁵. Finally, particular oligosaccharides such as globo H hexasaccharide are markers of malignant transformation of human breast, prostate, or ovarian cancer cells⁷. Carbohydrates play a particularly important role in the assembly of complex multicellular organs and organisms where interactions between cells as well as surrounding matrix are essential⁴(Fig. 1).

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These carbohydrates are frequently covalently bound to non

different kinds, and the molecules which are derived from the combinations of sugars with other biomolecules are called glycoconjugates. Consequently, glycobiology addresses the research in structure, biosynthesis, and biology of the sugars

nature⁸.

A large variety of modifications of the oligosaccharides enhances the diversity of glycoconjugates found in nature, and this diversity serves to mediate specific biological functions

phosphorylated, sulfated, methylated, acylated or

adjacent hydroxy groups.

Fig. 1 Interactions with glycoconjugates

On the other hand, complex carbohydrates are linked to proteins or lipids producing a large number of different glycoconjugates. These glycoconjugates are named proteoglycans, glycoproteins or glycolipids,

is linked. Thereby the carbohydrate moieties comprise an enormous structural variety which ___________________________________________________________________________

.

A large variety of modifications of the oligosaccharides enhances the diversity of glycoconjugates found in nature, and this diversity serves to mediate specific biological functions⁹. For example, the hydroxy

phosphorylated, sulfated, methylated,

acylated or N-sulfated, and carboxylic groups are occasionally involved in lactonization of adjacent hydroxy groups.

On the other hand, complex carbohydrates are linked to proteins or lipids producing a large number of different glycoconjugates. These glycoconjugates are named proteoglycans, glycoproteins or glycolipids,

is linked. Thereby the carbohydrate moieties comprise an enormous structural variety which ___________________________________________________________________________

A large variety of modifications of the oligosaccharides enhances the diversity of glycoconjugates found in nature, and this diversity serves to mediate specific biological

. For example, the hydroxy phosphorylated, sulfated, methylated,

sulfated, and carboxylic groups are occasionally involved in lactonization of adjacent hydroxy groups.

On the other hand, complex carbohydrates are linked to proteins or lipids producing a large number of different glycoconjugates. These glycoconjugates are named proteoglycans, glycoproteins or glycolipids, depending on the nature of molecule to which the carbohydrate is linked. Thereby the carbohydrate moieties comprise an enormous structural variety which

___________________________________________________________________________

A large variety of modifications of the oligosaccharides enhances the diversity of glycoconjugates found in nature, and this diversity serves to mediate specific biological

. For example, the hydroxy phosphorylated, sulfated, methylated, O-

sulfated, and carboxylic groups are occasionally involved in lactonization of

On the other hand, complex carbohydrates are linked to proteins or lipids producing a large number of different glycoconjugates. These glycoconjugates are named proteoglycans, depending on the nature of molecule to which the carbohydrate is linked. Thereby the carbohydrate moieties comprise an enormous structural variety which

___________________________________________________________________________

A large variety of modifications of the oligosaccharides enhances the diversity of glycoconjugates found in nature, and this diversity serves to mediate specific biological . For example, the hydroxy groups of the different monosaccharides can be

-acylated or fatty acylated; amino groups can be sulfated, and carboxylic groups are occasionally involved in lactonization of

___________________________________________________________________________

These carbohydrates are frequently covalently bound to non-carbohydrate natur

different kinds, and the molecules which are derived from the combinations of sugars with other biomolecules are called glycoconjugates. Consequently, glycobiology addresses the research in structure, biosynthesis, and biology of the sugars or glycans that are distributed in

A large variety of modifications of the oligosaccharides enhances the diversity of glycoconjugates found in nature, and this diversity serves to mediate specific biological groups of the different monosaccharides can be acylated or fatty acylated; amino groups can be sulfated, and carboxylic groups are occasionally involved in lactonization of

___________________________________________________________________________

carbohydrate natur

different kinds, and the molecules which are derived from the combinations of sugars with other biomolecules are called glycoconjugates. Consequently, glycobiology addresses the or glycans that are distributed in

On the other hand, complex carbohydrates are linked to proteins or lipids producing a large number of different glycoconjugates. These glycoconjugates are named proteoglycans, depending on the nature of molecule to which the carbohydrate is linked. Thereby the carbohydrate moieties comprise an enormous structural variety which ___________________________________________________________________________

carbohydrate natural products of different kinds, and the molecules which are derived from the combinations of sugars with other biomolecules are called glycoconjugates. Consequently, glycobiology addresses the or glycans that are distributed in

On the other hand, complex carbohydrates are linked to proteins or lipids producing a large number of different glycoconjugates. These glycoconjugates are named proteoglycans, depending on the nature of molecule to which the carbohydrate is linked. Thereby the carbohydrate moieties comprise an enormous structural variety which ___________________________________________________________________________

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al products of different kinds, and the molecules which are derived from the combinations of sugars with other biomolecules are called glycoconjugates. Consequently, glycobiology addresses the or glycans that are distributed in

A large variety of modifications of the oligosaccharides enhances the diversity of glycoconjugates found in nature, and this diversity serves to mediate specific biological groups of the different monosaccharides can be acylated or fatty acylated; amino groups can be N- sulfated, and carboxylic groups are occasionally involved in lactonization of

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is used by nature to store biological information that is crucial for the development, growth, function or survival of organisms.

1.2.1 Glycolipids

The term glycolipid designates any compound containing one or more monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety such as an acylglycerol, a sphingoid, a ceramide (N-acylsphingoid) or a prenyl phosphate. Glycolipids are a wide range of substrates and thus often overlap with other classes of lipids containing common structural motifs, making a classification difficult and confusing. The following classification of glycolipids is based on the origin reflecting the structural complexity of glycolipids from various sources¹⁰.

Animal Glycolipids: The minimal motif that defines the glycosphingolipids is a monosaccharide attached directly to a ceramide unit. In higher-animal cells, this monosaccharide is usually glucose or galactose, giving glucosylceramide or galactosylceramide (Fig.2). More than 200 structurally distinct glycosphingolipids (GSLs) from a wide variety of eukaryotic cells have been reported¹¹. More complex GSLs containing Neu5Ac are called gangliosides. Essentially all of the GSLs are antigenically active, and one of their biological properties is that they act as immunogens. Some of the GSLs serve as cell receptors for bacterial toxins and even possibly for bacteria and viruses.

O

HN

OH O O

OH OH

HO

OH

Ceramide

Sphingosine Fatty acid

-

D

-Galactose

Fig. 2: Structure of -GalCer

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Plant and algal glycolipids: D-Galactosyl diacylglycerols are the major components in plant lipid material. The variations found in the fatty acids and other parts of the lipids are more diverse than those found in the animal glycolipids but the carbohydrate moieties are less variable with D-galactose being the most abundant carbohydrate residue.

Microbial Glycolipids: Lipopolysaccharides (LPS)¹²are the major constituents of the outer cell envelope of Gram-negative bacteria and represent the first line of defense against the complement system and bacteriophages. On the other hand, most Gram- positive bacteria produce lipoteichoic acids (LTAs)¹³. These are, just like LPS, amphiphilic compounds composed of a membrane lipid or glycolipid covalently linked to a polymer of either glycerol or ribitol phosphate with various sugars and amino acids as substituents. Thus LTAs and LPS share many of their pathogenic properties.

The exact biological function of glycolipids is still a subject of much speculation. These types of molecules are associated with several processes such as the biosynthesis of glycoproteins and complex polysaccharides, the mechanism of control for proteoglycans biosynthesis, and the inhibition of biological activities of toxins and antiviral agents¹⁴.

1.3 Cell wall

A great pressure is generated inside the bacteria cell due to the concentration of solutes. In the case of Escherichia coli this pressure has been estimated in 2 atmospheres, comparable to the pressure inside a car tire. To resist this pressure the bacteria need a cell wall, which is also responsible for the rigidity and form of the cell.

Bacteria can be divided into two big groups depending on the result of the Gram staining test¹⁵: Gram-positive if the bacteria develop a purple color and Gram-negative if the bacteria give a red color. The reason for this difference is in the cell wall, Gram negative bacteria have a complicated multilayer cell wall while Gram positive bacteria are mainly made of one type of compound called peptidoglycan.

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Fig. 2 Cell wall of bacteria

In Gram-positive bacteria, up to 90% of the cell wall can be formed by peptidoglycan and small amounts of teichoic acids can also be present. One could find from only one layer of peptidoglycan up to twenty five in some Gram-positive bacteria. On the other hand, in Gram- negative bacteria, peptidoglycan only forms 10% of the cell wall and the rest is a complex mixture of other macromolecules.

1.3.1 Lipoteichoic Acids

Acidic polysaccharides called teichoic acids (Greek: teichos means wall) and linked to the cell wall can be found in Gram-positive bacteria. The term teichoic acid includes the wall, the membrane or capsular polymers that contain glycerophosphates or ribitol phosphate residues^16,17. These polyalcohols are linked through phosphate esters and usually other sugars;

D-alanine also forms part of the molecule. The negative charge of the cell surface is due to the teichoic acids; it serves to the ion movements through the cell wall. In Gram-positive bacteria some of these acids are linked to the lipids of the membrane, and therefore are called lipoteichoic acids.

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Fig. 3 Cell wall structure of Gram positive bacteria

1.3.2 Role of lipoteichoic acid in infection and inflammation.

Sepsis occurs in 1-2% of hospital admissions and is secondary to the appearance of bacterial toxins including the Gram-negative lipopolysaccaride (LPS) or the Gram-positive lipoteichoic acid (LTA) in the circulation¹⁸. The frequent progression of sepsis to septic shock results in hypotension and inadequate tissue perfusion. Septic shock carries a 45% risk of mortality, making it the most common cause of death in intensive care units¹⁹. Consequently, over 30 pharmaceutical products are in development for this condition although none has reached the market yet ²⁰. Since the process of sepsis involves multiple mediators, many of these target specific inflammatory mediators have been, in general, unsuccessful^21,22. More promising strategies include the targeting of the end stage of sepsis, altered coagulation or mediators such as LPS¹³. There are some works focused on opportunities surrounding the therapeutic targeting of the Gram-positive counterpart of LPS, LTA¹³. This toxin is released by Gram- positive bacteria following exposure to antibiotics or leukocytic mediators and plays an important role in both the colonization of bacteria and the consequent release of cytotoxic mediators in colonized organs. LTA binds to CD-14 and Toll-like receptors, thus initiating numerous events associated with sepsis including respiratory burst and release of leukocytic mediators²³, production of growth factors, arachidonic acid metabolites, reactive oxygen species, NO, cytokines and stimulators²³of chemotaxis and phagocytosis. In addition, LTA inhibits platelet aggregation²⁴, which may contribute in part to bleeding diathesis, a characteristic of Gram-positive septicemia patients. One important aspect of septicemia is that

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due to synergistic interaction of LPS and LTA, Gram-positive and Gram-negative infections often co-exist and it produces a clinical situation of even greater severity than infection with a single pathogen. Thus LTA sits at the heart of septicemia and blockade of its binding could be of significant clinical importance. Phase I studies support this since chimeric anti-LTA antibodies were able to reduce bacterial levels by 96%¹³. Development of further LTA blockers represents a novel approach to a serious and largely unmet market.

1.3.3 Biosynthesis of lipoteichoic acids.

Usually lipoteichoic and teichoic acids are structurally and metabolically distinct entities²⁵. In streptococci, the lipoteichoic acid has an unusual complex structure and its hydrophilic chain is identical to the chain of the teichoic acid. The identical chain structures suggest common steps in the biosynthesis of the two polymers²⁶. A unique feature of the biosynthesis of polyglycerolphosphate lipoteichoic acids is the lipid nature of all reactants. This observation suggests a transmembrane process with the polymerization occurring in the outer leaflet of the cytoplasmatic membrane, the location of completed lipoteichoic acid. (Figs. 4 and 5)

P

P P

P

ATP ADP CDP P

CTP PP_i P

P

GroP GMP H₂O Pi

Cytosol Membrane MembraneSurface

UDP

UDP ^Glc

Glc P PP_i ATP UTP

ADP P Glc P Glc

P

Glycosylation Glc

P P

P

P Ala

Ala

Alanylation

AlaDcp Dcp

AMP PP_i ATP

Ala

Realanylation

Start

Diglucosyldiacylglycerol Phosphatidylglycerol

P

Fig 4. LTA-Biosynthesis. In the symbols used for the structures, oxygen atom were omitted

P P

, Glycerolphosphate; , Diacylglycerol; , Undecaprenolphosphate

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The polymeric chain is initiated by glycerophosphate (GroP) transfer from phosphatidylglycerol to the glycolipid which serves as the definite lipid anchor.

Phosphatidylglycerol is also a glycerophosphate-donor for chain elongation and proceeds distal to the lipid anchor. Presumably two glycerophosphate transferases are involved, one that recognizes the glycolipid and the other one for chain elongation. For each glycerophosphate transferred in chain synthesis, one diacylglycerol is formed and recycled to phosphatidylglycerol on the inner membrane leaflet where the four enzymes involved have access to their water soluble substrates. Part of the diacylglycerol is used in glycolipid synthesis. Inhibitors of phosphatidylglycerol block both lipid and lipoteichoic acid synthesis.^27,28

Fig. 5. LTA biosynthesis and its relationship to membrane lipid metabolism in S. aureus²⁹. Reactions on the cytosolic site are marked with asterisks. For the lipoteichoic acid to occur

on the outer leaflet of the membrane, transmembrane movements of phosphatidylglycerol, glycolipid and diacylglycerol are required.

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1.4 Cell-mediated Immunity

Adaptive (acquired) immunity refers to antigen-specific defense mechanisms that take several days to become protective and are designed to remove a specific antigen. This is the immunity one develops throughout life. There are two major branches of the adaptive immune responses³⁰: humoral immunity and cell-mediated immunity.

1. Humoral immunity: humoral immunity involves the production of antibody molecules in response to an antigen and is mediated by B-lymphocytes.

2. Cell-mediated immunity: Cell-mediated immunity involves the production of cytotoxic T- lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigen and is mediated by T-lymphocytes.

Cell-mediated immunity (CMI) is an immune response that does not involve antibodies but rather involves the activation of macrophages and NK-cells, the production of antigen- specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. Cellular immunity protects the body by:

1. Activating antigen-specific cytotoxic T-lymphocytes (CTLs) that are able to lyse body cells displaying epitopes of foreign antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens.

2. Activating macrophages and NK cells, enabling them to destroy intracellular pathogens.

3. Stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.

Cell-mediated immunity is directed primarily to microbes that survive phagocytes and microbes that infect non-phagocytic cells. It is most effective in removing virus-infected cells, but also participates in defending against fungi, protozoans, cancers, and intracellular bacteria.

It also plays a major role in transplant rejection.

As mentioned earlier, the immune system of the body has no idea as to what antigens it may eventually encounter. Therefore, it has evolved to be a system that possesses the capability of responding to any conceivable antigen. The immune system can do this because both B- lymphocytes and T-lymphocytes have evolved to a unique system of gene-splicing called

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gene translocation; a type of gene-shuffling process where various different genes along a chromosome move and join with other genes from the chromosome. In a manner similar to B- lymphocytes, T-lymphocytes are able to cut out and splice together different combinations of V, D, and J genes along their chromosomes to produce unique shaped T-cell receptors (TCRs), capable of reacting with complementary-shaped peptides bound to MHC molecules.

1.4.1 Cytokines

Cytokines are small secreted proteins that mediate and regulate immunity, inflammation, and hematopoiesis³¹. They must be produced de novo in response to an immune stimulus. They generally, although not always, act over short distances and short time spans and at very low concentration. They act by binding to specific membrane receptors, which then signal the cell via second messengers, often tyrosine kinases, to alter its behavior (gene expression).

Responses to cytokines include increasing or decreasing expression of membrane proteins (including cytokine receptors), proliferation, and secretion of effector molecules.Although cytokine is used as a general term³²; other names include lymphokine (cytokines made by lymphocytes), monokine (cytokines made by monocytes), chemokine (cytokines with chemotactic activities), and interleukin (cytokines made by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or in some instances on distant cells (endocrine action).

It is common for different cell types to secrete the same cytokine or for a single cytokine to act on several different cell types (pleiotropy; see table below.) Cytokines are redundant in their activity, meaning that similar functions can be stimulated by different cytokines.

Cytokines are often produced in a cascade, as one cytokine stimulates its target cells to make additional cytokines. Cytokines can also act synergistically (two or more cytokines acting together) or antagonistically (cytokines causing opposing activities). Their short half life, low plasma concentrations, pleiotropy, and redundancy make the isolation and characterization of cytokines a complicated process. Search for new cytokines are now conducted at the DNA level, identifying genes similar to known cytokine genes.The largest group of cytokines stimulates immune cell proliferation and differentiation. This group includes Interleukin 1 (IL-1), which activates T cells; IL-2, which stimulates proliferation of antigen-activated T and B cells; IL-4, IL-5, and IL-6, which stimulate proliferation and differentiation of B cells;

Interferon gamma (IFNg), which activates macrophages; and IL-3, IL-7 and Granulocyte Monocyte Colony-Stimulating Factor (GM-CSF), which stimulate hematopoiesis.

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1.5 Objectives.

From what has previously been discussed, it is clear that the presence of the carbohydrate unit in natural occurring structures results in dramatic effects in their physical, chemical and biological properties. Therefore, in this work we describe the synthesis of some important analogues and fragments of two different LTAs, from Streptococcus pneumoniae and Staphylococcus aureus, in order to understand the function of this molecules and the influence of some substituents that are conforming them.

We describe the synthesis of:

Fragments of Staphylococcus aureus LTA Analogue of Staphylococcus aureus LTA Fragment of Streptococcus pneumniae LTA

1.5.1 Analogues and fragments of Staphylococcus Aureus LTA

Staphylococcus aureus is the most frequently isolated Gram-positive bacterium that causes infections³³, and the synthesis of the LTA of these bacteria has already been achieved in our group³⁴. The alanine residues present in this LTA are cleaved when the molecule is in a pH=8.5 medium.

Our first objective has been to design and synthesize analogues of Staphylococcus aureus LTA in order to gain in stability without loosing activity in a significant degree (Fig 6). An interesting point was also to know which effect would cause the inversion of configuration at C-2 of the glycerol residues in the backbone. It was also interesting to investigate which is the smallest fragment capable to activate the immune system in terms of cytokine release by human blood leukocytes.

P

-O O

P O

-O O O

O

NH₃⁺ O

P

-O O

P O

-O NH O

O

NH₃⁺ O

Fig 6 Ester and amide type compounds

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The following compounds were synthesized (Schemes 1 and 2)

O OH

HO O

O O

OH

HOHO O

O O P

OH HOHO

OH O

O O

C₁₃H₂₇ C₁₃H₂₇ O

O P

-O O O

O

NH₃⁺ O

n A, n=2 B, n=3 C, n=4 D, n=5 Scheme 1. Fragments of S. Aureus LTA

O OH

HO O P O

OH

O O O

C₁₃H₂₇ C₁₃H₂₇ O

O P

-O NH O

O

NH₃⁺ O

n

F Scheme 2. Analogue of S. Aureus LTA.

1.5.2 Total Synthesis of Streptococcus pneumoniae LTA.

Pneumonia is a disease of the lung that is caused by a variety of bacteria including Streptococcus, Staphylococcus, Pseudomonas, Haemophilus, Chlamydia and Mycoplasma, several viruses, certain fungi and protozoans. The disease may be divided into two forms:

bronchial pneumonia and lobar pneumonia. Bronchial pneumonia is most prevalent in infants, young children and aged adults. It is caused by various bacteria, including Streptococcus pneumoniae. Bronchial pneumonia involves the alveoli contiguous to the larger bronchioles of the bronchial tree. Lobar pneumonia is more prone to occur in younger adults. More than 80% of lobar pneumonia cases are caused by Streptococcus pneumoniae. Lobar pneumonia involves all the single lobes of the lungs (although more than one lobe may be involved), wherein the entire area of involvement tends to become a consolidated mass, in contrast to the spongy texture of normal lung tissue. Streptococcus pneumoniae is known in medical microbiology as the pneumococcus, referring to its morphology and its consistent involvement in pneumonia.

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13

The cell wall of S. pneumoniae is roughly six layers thick and is composed of peptidoglycan with teichoic acid attached to approximately every third N-acetylmuramic acid. Lipoteichoic acid is chemically identical to the teichoic acid but is attached to the cell membrane by a lipid moiety. Both the teichoic acid and the lipoteichoic acid contain phosphorylcholine; two choline residues that are covalently linked to each pentasaccharide repeating unit. This is an essential element in the biology of S. pneumoniae since the choline specifically adheres to choline-binding receptors that are located on virtually all human cells.

This LTA is in some way untypical because it is one of the very few LTAs in which the repeating unit is not formed basically by glycerophosphate or ribitol phosphate backbones;

instead it has a pentasaccharide with two phosphorylcholine residues linked to the N-acetyl galactosamine moieties. In the natural molecule the number of repeating units is between 2 and 8 (Scheme 3).

HO O HO

OH O

O H₃N⁺

O

AcNH O

O

HO AcNH OP O

-O O Me₃N⁺

O O

HO AcNH OP O

-O O Me₃N⁺

O OH OH OH

HO O HO

OH O

O H₃N⁺

O AcNH

HO O O

HO OH

O P

O

-OO n

O O C₁₃^H₂₇

O

C₁₃H₂₇ O H

G, n=1

Scheme 3: Structure of S. Pneumoniae LTA

In this work we present some investigations toward the synthesis of a fragment of Streptococcus pneumoniae where n=1.

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5. Summary

___________________________________________________________________________

2.

Theoretical Part

2.1 Chemical synthesis of oligosaccharides.

Chemical and biological studies of oligosaccharides are of crucial importance to our understanding of living organisms and to biomedical research. The explosion of glycoscience research that has taken place during the last decades has fostered research in the field and has paid particular attention to the difficulties associated with chemical syntheses of oligosaccharides. The synthetic methods available for glycosidic bond formation are becoming increasingly powerful and efficient as well evidenced by the ever increasing complexity of targeted oligosaccharides. However, even more effective methods are required.

The most important aspects for comparing the different existing methods are generation, accessibility, and reactivity of the key reactive intermediates in glycoside couplings, i.e. the cyclic oxacarbenium ions (Scheme 4).

O L

O⁺ ROH

HL

O OR L-leaving group Oxocarbenium ion

Scheme 4: Chemical O-glycoside synthesis.

Much effort has been properly devoted to the development of new glycosidic donors, particularly of those where the anomeric leaving groups are halides, trichloroacetimidate, ß- N-Phenyltrifluoroacetimidate, alkyl or arylthio and phosphite glycosides. These five types have been recently used in the synthesis of complex oligosaccharides consisting of at least five monosaccharide units.

2.1.1 Koenigs-Knorr method.

As outlined in Scheme 5, the classical Koenigs-Knorr method³⁵of oligosaccharide synthesis, among with its modern variations, achieves activations through the use of glycosyl halides (bromides, chlorides) in the presence of heavy metal salts, generally of silver and mercury.

Advanced modifications make the use of fluorides as glycosyl donors³⁶. This method has been comprehensively reviewed³⁷.

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2. Theoretical Part.

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15

O O

OR R´OH

O O R

X

O O R O

O O

R Ag⁺/Hg⁺

Scheme 5: Koenigs-Knorr method.

Despite the efficiency of several of the variants of the Koenigs-Knorr method, some disadvantages still remain:

The glycosyl halide generation requires harsh conditions.

The glycosyl halides are relative unstable intermediates.

The glycosyl halides are high sensitive to aqueous hydrolysis.

The heavy metal salts used in the activation are expensive and very toxic.

2.1.2 Trichloroacetimidate Method.

Glycosyl trichloroacetimidates were first developed by Schmidt and Michel in 1980³⁸(Scheme 6). These are stable, frequently isolable intermediates that can be activated through acid catalysis without the use of heavy metal salts. Nowadays this is the widest used method for glycosylation reactions.

O O

O ROH OR

NH CCl₃

+ Lewis Acid

O NH₂ CCl₃ Scheme 6: Trichloroacetimidate method

The coupling method generally leads to inversion of the configuration of the anomeric site;

however, the presence of 2-acyl substituents invariably generates the product of neighbouring group participation, i.e. the 1,2-trans compound. Furthermore, in the case of mannose and rhamnose, generally the product is formed owing to the strong anomeric effect in these systems. An additional advantage of this method is the control of the anomeric configuration of the product by selecting the required donor precursor.³⁹

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16

2.1.3 ß-N-Phenyl-trifluoroacetoimidate Method.

N-Phenyl-trifluoroacetimidates were first developed by Huchel and Schmidt⁴⁰and later by Yu and Tao⁴¹(Scheme 7). This is a variation of Schmidt donor; these donors are also stable and isolable; generally they are a little bit more stable than trichloroacetimidates and therefore a little bit less reactive, what requires a greater amount of Lewis acid catalyst for the activation.

This method has also been used as leaving group for sialic acid glycosylation reactions⁴².

O O

O ROH OR

NPh CF₃

+ Lewis Acid

O NPhH CF₃ Scheme 7: Phenyl trifluoroacetimidate method.

A new application of this leaving group has recently appeared in the literature⁴³. Takahashi and collaborators reported a glycosylation reaction for the synthesis of N-glycosyl peptides by N-glycosylation of non activated primary amides.

The disadvantage of this method compared to the trichloroacetimidate s is that the reagent to generate the imidate is not commercially available as the trichloroacetonitrile and a complicated reaction must be carried out⁴⁴.

2.1.4 Thioglycoside Method.

Thioglycosides are highly useful and versatile intermediates in oligosaccharide synthesis. A distinct advantage of these derivatives is that their thio group can be used as temporary protection, and then employed for any type of coupling procedure currently in use⁴⁵.

The activation mechanism of this reaction is similar to the Koenigs-Knorr procedure as a thiophile activates the sulphur first, then it is released to give the corresponding oxonium ion.

Glycosylation then follows by trapping this species with a hydroxyl group of the acceptor as shown in the Scheme 8.

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17

O SR

O S⁺

O OR´

R´OH

-RSX thiophile

X⁺ R

X Scheme 8: Thiogycoside method.

More effective activating agents introduced recently include methyl triflate, dimethyl(methylthio)sulfonium triflate (DMTST) and iodosuccinimide-perchlorate (IDCP).

However, all these thiophilic promoters are required at least in equimolar amounts, which can also lead to undesired reactions.

2.1.5 Phosphite method.

The glycosyl-donating abilities of phosphites as intermediates in the synthesis of oligosaccharides were discovered by Martin and Schmidt in 1992⁴⁶. These intermediates can be synthesized from free anomeric sugars and a phosphitylating agent in presence of Hünig´s base. This procedure is quite general since the activation of glycosyl phosphites can be achieved by treatment with catalytic amounts of TMSOTf^46,47. The most interesting examples reported so far are those using sialic acid phosphites as sialylating agent⁴⁸.

O O AcHNAcO

AcO OAc

AcO CO₂Me

P X

Y

O OR AcHNAcO

AcO OAc AcO

CO₂Me HOR

O P X

Y H Scheme 9: Phosphite method.

2.2 Fragments of Staphylococcus Aureus LTA

2.2.1 Retrosynthetic analysis

The synthesis of these fragments follows the previous work done in our group^49,50, therefore the synthetic strategy used is almost the same as reported^49,50and only a brief overview will be described.

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18

It is necessary to explain that in none of these molecules the GlcNAc residue is present, and this is due to the fact that in our previous work it was demonstrated that this residue is not decisive to obtain good levels of cytokine release in human blood leukocytes.

HO O HO

OH O O

HO O HO

OH

O O

O

O C13H27 O

C13H27 P

O O O O

R P O

O O O

R P O

O O O

R P O

O O O

R P O

O O O

R P O

O

O RO

R

n

R =

O CH3 O

NH3 OH

OH O

R P O

OR¹ O O

R P O

OR¹ O O

R P O

OR¹ O O

R P O

OR¹ O O

R P O

O

OR¹ R¹O

R

n

R¹O O R¹O

OR¹ O O

R¹O O R¹O

OR¹

O O

O

O C₁₃H₂₇ O

C13H27 P

R¹O iPr2N

D

K H

1

OH R¹O

OR¹

OTBDPS O

OR² P R¹O

iPr2N

I

J

R¹= Bn R²= MPM

Scheme 10: Rretrosynthetical analysis 2.2.2 Synthesis of the fragments 2.2.2.1 Gentiobiose moiety.

The gentiobiose moiety K (13) was obtained in 11 steps from gentiobiose in a 14 % overall yield.

Perbenzoylation of the starting material, selective removal of the anomeric benzoyl group with hydrazinium acetate and introduction of trichloroacetimidate gave the donor 5 for the glycosylation reaction with the 1,2-O-isopropylidene-sn-glycerol 1 to give the disaccharide 6.

This glycosylation reaction was performed in DCM using BF₃^.OEt₂complex as Lewis acid catalyst because when TMSOTf was used, a racemization of the 1,2-O-isopropylidene-sn- glycerol moiety was observed. After debenzoylation, selective O-silylation of the 6´ position with TBDPSCl and benzylation of all free hydroxy groups, compound 9 was obtained. At this

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19

point the isopropylidene protecting group was removed from the glycerol ( 10) and the free hydroxy groups were acylated with myristoyl chloride ( 11). Finally, removal of TBDPS ( 12) and introduction of the N,N-diisopropylbenzyloxy phosphite amide gave the gentiobiose building block 13 (Scheme 11).

HO O HO

OH O OH

HO O HO

OH OH

BzO O BzO

OBz O OBz

BzO O BzO

OBz O

HO O

O

BzO O BzO

OBz O OBz

BzO O BzO

OBz

O O

O CCl₃ NH BzCl, Pyr., 40°C,

Overnight

(^98%3)

DMF, 50°C, 3.5 h, N₂H₅OAc 78% ( 4)

CCl₃CN, DBU, 2 h, CH₂Cl₂

97%

0.2 eq BF₃OEt_2,

3Å-MS, -30°C, 20 min., CH₂Cl₂

2 5

6

1 a

b

HO O HO

OH O OTBDPS

HO O HO

OH

O O

O

BnO O BnO

OBn O BnO O

BnO OBn

O O

O P

O N

O C₁₃H₂₇

C₁₃H₂₇ BnO

BnO O BnO

OBn O BnO O

BnO

OBn

O O

O OR

O C₁₃H₂₇

O C₁₃H₂₇ BnO O

BnO OBn

O OTBDPS

BnO O BnO

OBn

O OH

OH

1. NaOMe, MeOH, 24h, RT, quant.

2. TBDPS-Cl, Pyr., -15°C, 2d, 91%

BnBr, NaH, DMF, RT, 24h 66%

75% CH₃COOH/THF, 80°C, 1.5h 78%

( 9) 8

13 10

NEt3, THF, 50°C, 4h, Myristoyl chloride

81%

11: R = TBDPS 12: R = H

TBAF, CH₃COOH, THF, 40°C, 3d, 76%

Tetrazol, CH₂Cl₂, RT, Phosphite diamide

79%

( 7)

80%

Scheme 11: Synthesis of gentiobiose building block

2.2.2.2 Glycerol moieties.

Building block I (14) was obtained following a known procedure⁵¹. For synthesizing glycerol moiety J⁵²(19), 3-O-allyl-sn-glycerol⁵³was selectively silylated with TBDPSCl ( 16), the secondary position was p-methoxybenzylated ( 17) and the allyl group was removed using

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20

Wilkinson catalyst ( 18); after this, the free hydroxyl group was phosphitylated with N,N- diisopropyl bennzyloxy phosphite amide to obtain building block J (19).(Scheme 12)

HO O

O

BnO OH

OBn

AllylO OTBDPS OH

RO OTBDPS

OMPM

O OTBDPS

OMPM P

BnO N 1. BnBr, NaH, DMF, RT, 96%

2. 70% CH3COOH, 80°C, 50 min., 77%

3. TrCl, Pyr., 75°C, 26h, 86%

4. BnBr, NaH, DMF, RT, 91%

5. CSA, MeOH, 65°C, 20 min., 96%

19 14

1

Tetrazol, CH2Cl2, RT, 30 min.

81%

17: R=Allyl 18 R´=H MPMCl, NaH,

DMF, RT 70%

a

a: 1. (PPh3)3RhCl, DBU, EtOH, 90°C 2. 1M HCl/Aceton 1:9

71%

16

Scheme 12: Synthesis of glycerol moieties

2.2.2.3 The phosphoramidite method.

Because many natural compounds exist in form of diester phosphates, some phosphorylating methods were developed in order to obtain these molecules efficiently. Beaucage and Caruthers⁵⁴developed the phosphite amide method which has an immense application in oligonucleotide synthesis. Trzeciak and Bannwarth⁵⁵developed a proper orthogonal synthesis to obtain the phosphite amide activating agent with the desired oxygen protecting groups. The methods based on the phosphorous (III) intermediates are used in this work. The starting material is the properly protected bis-(diisopropylamino)-phosphane, which under mild acid tetrazole catalysis reacts with any desired alcohol. A further activation with tetrazole and a second alcohol leads to the phosphite triester and it is then easily converted into the phosphotriester using I₂/H₂O, t-BuOOH or MCPBA. The product obtained at this stage is a mixture of enantiomers that disappears when the phosphotriester is converted into a phosphodiester (Scheme 13).