A First Total Synthesis of ( L)-erythro -Ceramide C6 from Natural ( L)-Serine

Aurora Sganappa

Dissertação para obtenção do grau de Mestre em

Química

Júri

Presidente: Prof. Armando José Latourette de Oliveira Pombeiro

Orientadores: Profª. Maria Matilde Soares Duarte Marques

Prof. Enrico Marcantoni

Vogal: Prof. Pedro Paulo de Lacerda e Oliveira Santos

Outubro de 2012

SSSCHOOL OF SCIENCE AND TECHNOLOGY CHEMISTRY DIVISION

Master Degree in Chemistry and Advanced Chemical Methodologies (Class LMLM----54)54)54)54)

A First Total Synthesis of (L)-erythro -Ceramide C6 from Natural ( L)-Serine

Experimental Thesis in Organic Chemistry CHIM/06

Candidate Supervisor Sganappa Aurora Prof. Enrico Marcantoni

Co-Supervisors Prof. Matilde Marques Dott.ssa Roberta Properzi

Academic Year 2011-2012

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Acknowledgments

In these few lines I would like to thank all the people who contributed to this project.

The first one is my Supervisor the Professor Enrico Marcantoni who gave me the opportunity to do my thesis in his research group, showing his confidence and availability.

I would also to thank my Co-Supervisors Prof. Matilde Marques and Dr. Roberta Properzi for their help and unlimited patience.

Another thank you is for the other Doctors and Researchers of the work group: Stefano Lancianesi, Matteo Di Nicola, Alessandro Palmieri and Serena Gabrielli.

In the end I would like to thank my family and friends for their immense support. Thank to everyone.

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RESUMO

A Química de Síntese é a ciência que permite construir moléculas complexas a partir de outras mais simples. Uma das vertentes provavelmente mais interessantes desta área científica é a síntese total de moléculas de interesse biológico. Em química orgânica de síntese o termo “síntese total” corresponde ao planeamento, passo a passo, da síntese química de uma molécula a partir de materiais de partida relativamente simples e, desejavelmente, disponíveis comercialmente a baixo custo. Em anos recentes, a investigação tem vindo a ser dirigida para a síntese de moléculas pequenas, devido à sua capacidade de ligação com afinidade elevada a uma grande variedade de biopolímeros, incluindo proteínas, ácidos nucleicos ou polissacáridos, alterando a estrutura destes e, consequentemente, a sua função. As moléculas pequenas podem ter uma grande variedade de funções biológicas, sendo a sua versátil aplicabilidade devida à pequena dimensão e baixo peso molecular (< 2000 Da), que lhes permitem uma difusão rápida através das membranas celulares de modo a atingir alvos intracelulares. O grupo do professor Marcantoni tem explorado ambos os campos da síntese orgânica: a síntese total e o desenvolvimento de novas metodologias sintéticas. Está, na verdade, interessado na síntese total de moléculas pequenas com actividade biológica e de blocos moleculares de construção úteis na síntese de produtos naturais; foca-se também no desenvolvimento de novas metodologias envolvendo o uso do ácido de Lewis CeCl 3 como promotor da formação de ligações carbono-carbono. Dá ainda atenção ao uso mais eficiente dos materiais, tendo em conta que o desenvolvimento de fontes de energia limpas e renováveis representa uma contribuição essencial da comunidade química para um desenvolvimento verdadeiramente sustentável. Nesta dissertação estuda-se a adição de reagentes do tipo organocério a um (R)-aldeído de tipo Garner derivado da (L)-serina, com o objectivo de melhorar a estereosselectividade, minimizar as reacções laterais, e aumentar o rendimento do produto desejado. Esta metodologia tem aplicação na síntese de molécula pequenas importantes, como a (L)- eritro-ceramida, e representa um objectivo de grande relevância por permitir a síntese desta molécula usando um material de partida de baixo custo, e facilmente disponível no conjunto de compostos quirais comuns – o amino ácido natural (L)-serina.

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PALAVRAS CHAVE

Ácidos de Lewis, Aldeído de tipo Garner, Ceramida, Esfingolípidos, Esfingosina, Moléculas biologicamente activas, Moléculas pequenas, Síntese total.

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ABSTRACT

Synthetic chemistry is the science of constructing complex molecules from simpler one. Maybe and one of the most important development in this research area is the total synthesis of biologically interesting molecules. In synthetic organic chemistry, the term total synthesis refers to the step-by-step design of the chemical synthesis of a molecule from relatively simple and hopefully cheap commercially available starting materials. In recent years, research has been directed towards the synthesis of small molecules, because of their capacity of binding with high affinity to a large variety of biopolymers such as proteins, nucleic acids, or polysaccharides, altering their structure and, so, their function. Small molecules can have a variety of biological functions, and their varied applicability is due to their small size and low molecular weight (< 2000 Da), which allow them to rapidly diffuse across cell membranes reaching intracellular sites of action. The Marcantoni's research group has been interested in both fields of organic synthesis: the total synthesis and the development of new synthetic methodologies. In fact, it is interested on the total synthesis of small biologically active molecules, and of useful building blocks for the synthesis of natural products; it also focus on the development of new methodologies involving the use of the Lewis acid CeCl 3 as a promoter in carbon-carbon bond forming reactions. It is aware that the more efficient use of materials and the development of clean and renewable sources of energy represent an essential contribution from the chemical community to true sustainable development. In this thesis project the addition of organocerium reagents to an (R)-Garner-type aldehyde derived from (L)-serine will be studied, in order to improve the stereoselectivity, minimizing side reactions, and increasing the yield of the desired product. This work will find application in the synthesis of an important small molecule such as (L)-erythro - ceramide, and it represents an important goal for organic synthesis, since this molecule would be synthesized from a cheap starting material, easily found in the chirality pool, the natural amino acid (L)-serine. Ceramide, containing a 2-amino-1,3-diol moiety, is an important mammalian lipid which plays a critical role in several important biological and physiological processes, and its role is well established in the apoptosis process. In particular, it has been tested in vitro that the ceramide inhibiting ability for sphingosine kinase and it has emerged that the enzyme is specific for erythro isomers, while threo isomers behave as enzyme inhibitors. The results

- 6 - are the confirmation that the erythro isomers have the right spatial conformation that allow them to behave as substrates for this enzyme. The project of thesis would be the first example of synthetic procedure for obtaining the L-erythro -ceramide from (L)-serine, offering an easier, stereoselective, and sustainable synthetic route to this important target small molecule, as an alternative to the unfavourable synthetic and chemoenzymatic routes reported in the literature.

KEYWORDS

Biologically active molecules, Ceramide, Garner type Aldehyde, Lewis acids, Small molecules, Sphingolipids, Sphingosine, Total Synthesis.

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INDEX

ACKNOWLEDGMENTS 3 RESUMO 4 PALAVRAS CHAVE 5 ABSTRACT 6 KEYWORDS 7 INDEX OF FIGURES 11 INDEX OF SCHEME 13 ABBREVIATION LIST 14

1. TOTAL SYNTHESIS AND SMALL MOLECULES 16

1.1 INTRODUCTION TO ORGANIC SYNTHESIS 16 1.2 A BRIEF HISTORY ON THE TOTAL SYNTHESIS 18 1.3 HOW TO DESIGN A TOTAL SYNTHESIS 23 1.4 SMALL MOLECULES 26

2. CHEMISTRY AND BIOLOGY OF SPHINGOLIPIDS 29

2.1 INTRODUCTION AND BIOLOGICAL IMPORTANCE OF SPHINGOLIPIDS 29 2.2 BIOSYNTHESIS OF SPHINGOLIPIDS 33 2.3 SPHINGOSINES: SYNTHESIS AND BIOLOGICAL ACTIVITY 35 2.3.1 Strategic Pathway for the Synthesis of Sphingosines 41 2.4 SPHINGOMYELINS: STRUCTURE AND THEIR BIOLOGICAL PROPERTIES 44 2.5 CERAMIDES: A KEY INTERMEDIATES IN THE SPHINGOLIPIDS METABOLISM 49

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3. GARNER’S ALDEHYDE AND ORGANOCERIUM REAGENTS 61

3.1 GARNER’S ALDEHYDE AND GARNER-TYPE ALDEHYDE: SYNTHESIS AND REACTIVITY 61 3.2 THE ORGANOCERIUM REAGENTS 63 3.2.1 Cerium 63 3.2.2 Organocerium Compound 63 3.2.3 Addition of Organocerium to Carbonyl compound 64 3.2.4 Organocerium reagent: their importance in Organic Synthesis 67

4. TOTAL SYNTHESIS OF L-ERYTHRO-CERAMIDE C6 70

4.1 INTRODUCTION TO THE EXPERIMENTAL WORK 70 4.2 CERAMIDE C6 71 4.3 DESIGN OF TOTAL SYNTHESIS: INNOVATIVE STRATEGIES IN THIS SYNTHETIC PATHWAY 73 4.4 PART ONE: FORMATION OF GARNER-TYPE ALDEHYDE 76 4.5 PART TWO: SYNTHESIS OF SPHINGOSINE 80 4.6 PART THREE: SYNTHESIS OF C6 L-ERYTHRO-CERAMIDE 85 4.7 TOTAL SYNTHESIS OF L-ERYTHRO-CERAMIDE C6 87 4.8 CONCLUSION AND FUTURE PROJECT 88

5. EXPERIMENTAL SECTION 87

5.1 INSTRUMENTATION 87 5.2 SYNTHETIC PROCEDURES AND CHARACTERIZATION OF PRODUCTS 90 5.2.1 Protection of hydroxyl group 90 5.2.2 Reduction of the ester 92 5.2.3 Protection of amino and hydroxyl group with Cyclohexanone 93 5.2.4 Protection of amino group with Boc 95 5.2.5 Deprotection of the alcoholic group 96 5.2.6 Oxidation of the alcoholic group to aldehyde 97 5.2.7 Oxidation of tetradecanol 99 5.2.8 Takai olefination 100 5.2.9 Formation of organolithium 102

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5.2.10 Addition of the organocerium to aldehyde 102 5.2.11 Acid hydrolysis for the deprotection of sphingosine 105 5.2.12 N-acylation of sphingosine 106

6. REFERENCES 108

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INDEX OF FIGURES

Figure 1. Scheme of organic synthesis 17 Figure 2. Selected XIX century Landmark total synthesis of natural product 18 Figure 3. Selected syntheses by the Woodward group 19 Figure 4. Longifolene and its retrosynthetic analisis 20 Figure 5. Retrosynthetic analysis of taxol 21 Figure 6. The total synthesis of taxol 22 Figure 7. Scheme of S-Naproxene (anti-inflammatory agent) obtained by two different route: asymmetric and kinetic resolution 24 Figure 8. Scheme of ideal synthesis 25 Figure 9. Example of small molecules with biological activity 26 Figure 10. Structure of Colchicine and Chloropromazine 27 Figure 11. Structure and different stereoisomer of a generic molecule containing 2-amino- 1,3-diol moiety 27 Figure 12. Structure of sphingosine, sphingomyelin, ceramide and cerebroside 30 Figure 13. Some example of sphingoid bases in mammalian cell 31 Figure 14. Example of sphingoid bases found in the other organism 32 Figure 15. Scheme of biosynthesis de novo of sphingolipids 34 Figure 16. The four possible stereoisomer of sphingosine 35 Figure 17. Scheme of interconversion of sphingosine, sphingosine-1-phosphate and ceramide 36 Figure 18. Scheme of reaction of sphingoid base chloroamine and hypochlorous acid 37 Figure 19. Phorbol and phorbol12-myristate 13-acetate 38 Figure 20. Structure of FTY720 and Sphingosine 39 Figure 21. Theoretical binding conformation of D-erythro -S1P in the EDG-1/S1P1 active site. Hydrogen bonds are indicated as dashed lines 40 Figure 22. Structures of L-serine and Garner’s Aldehyde 43 Figure 23. Structure of generic sphingomyelin 45 Figure 24. Molecular structure of phosphatidylcholine (1-stearoyl-2-oleoyl-sn - sphingomyelin) on the right 47 Figure 25. Structure of generic ceramide 49 Figure 26. Scheme of interconversion of ceramide in all other sphingolipids 51

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Figure 27. The four stereoisomers of ceramide 52 Figure 28. Structure of Fumonisin B1 53 Figure 29. Morphological characteristics of hippocampal neurons of representative cells at 12 and 24 h in culture after addition of C6-NBD-D-erythro -Cer immediately after cells were placed in multiwall dishes. 54 Figure 30. Structure of L-buthionine-(S,R)-sulfoximine β-alanyl cysteamine disulphide 56 Figure 31. Structure of Tamoxifen and Raloxifene 56 Figure 32. Structure of Daunorubicin and Etoposide 57 Figure 33. Transition state of product anti and syn . 62 Figure 34. Chiral precursor (L)-serine and (D)-serine. 72 Figure 35. Structure of L-erythro -ceramide C6 and l-serine 73 Figure 36. Principal difference between conventional and our strategy 74

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INDEX OF SCHEMES

Scheme 1. The first synthesis of sphingosines by Shapiro 42 Scheme 2. Synthesis of Garner for D-erythro -sphingosine 43 Scheme 3. Synthesis of ceramide. 58 Scheme 4. Synthesis of Herold. 60 Scheme 5. Synthesis of Garner’s aldehyde 61 Scheme 6. Synthesis of Norcholestane. 65 Scheme 7. Reaction of addition of N-benzil-α,N-dilitihium methanesulfonamide to aldehyde and ketouridines. 66 Scheme 8. Reaction of N,N-Dibenzylserine aldehyde with organocerium. 67 Scheme 9. Synthesis of (±)- Roseophilin 68 Scheme 10. Retrosynthetic analysis. 75 Scheme 11. Synthesis of Garner-type aldehyde 76 Scheme 12. Protection of the hydroxyl group of L-methyl ester serine. 77 Scheme 13. Reduction of the ester and conversion of boronate ester into 86 77 Scheme 14. Protection of amino and hydroxyl groups by ciclohexanone. 78 Scheme 15. Protection of amino group by Boc. 78 Scheme 16. Deprotection of hydroxyl group. 79 Scheme 17. Oxidation of alcoholic functionality to obtain aldehyde. 79 Scheme 18. Synthesis of protected sphingosine. 81 Scheme 19. Oxidation of 1-tetradecanol. 81 Scheme 20. The Takai reaction. 82 Scheme 21. Alternative Takai reaction 82 Scheme 22. Test with Grignard reagent. 83 Scheme 23. Formation of organolithium compound. 83 Scheme 24. Synthesis of protected sphingosines. 84 Scheme 25. Synthesis of L-erythro -ceramide. 85 Scheme 26. Deprotection of sphingosine. 85 Scheme 27. Acylation of amino group 86 Scheme 28. Total synthesis of L-erythro -ceramide C6. 87 Scheme 29. Future project. 88

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ABBREVIATION LIST

Arg – Arginine

Boc 2O – Di-tert -butyl dicarbonate BuLi – Butyllithium C1P – Ceramide-1-phosphate Cer – Ceramide CHOL – cholesterol DHSM – Dihydrosphingomyelin DIBAL-H – Diisobutylaluminium hydride DMSO – Dimethyl sulfoxide EDG – Endothelial Differentiation Gene ER – Endoplasmic Reticulum

Et 2O – diethyl ether EtOAc – ethylacetate FDA – Food and Drug Administration GlcCer – Glucosylceramide Glu – Glutamic acid GSH – Glutathione HL – Human Leukemia cells MAMs – mitochondria-associated-membranes MeOH – Methanol MS – Mass Spectrometry NMR – Nuclear Magnetic Resonance PC – phosphatidylcholine PCC – Piridinium chlorocromate PCK – Protein Kinase C Phe – Phenylalanine RMgX – Grignard reagent RLi – Organolithium reagent ROS – Reactive oxygen species S1P – Sphingosine-1-phosphate SPHK – Sphingosine Kinase

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S1PP – Sphingosine-1-phosphate phosphatise SM – Sphingomyelin SMases – Sphingomyelinases TBAF – Tetrabutylamonium fluoride TBS (or TBDMS) – tert-butyldimethylsilyl THF – tetrahydrofuran TNF – Tumour Necrosis Factor TsOH (or PTSA) – p-Toluenesulfonic acid Tyr – Tyrosine

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1 TOTAL SYNTHESIS AND SMALL MOLECULES

1.1 Introduction to organic synthesis

Synthetic chemistry (from the Greek word synthesis = the process of putting together) is the science of constructing molecules from atoms and/or simpler molecules. This discipline may be subdivided according to the molecules involved, into synthetic organic chemistry and synthetic inorganic chemistry. [1] The higher level of complexity of organic compounds as well as their importance in the biology, medicinal and material sciences, have favoured their study and diffusion with respect to the inorganic compounds, making the synthesis of organic compounds the main branch of organic chemistry. [2] Several of the millions of organic compounds made over the last century and half trough chemical synthesis are directly linked to important application in everyday life: pharmaceuticals that can cure or prevent diseases, insecticides, pesticides, plant an animal hormone to increase food production and nutritional quality, polymers, fabrics, dyes, cosmetics, detergents, photographic and electronic items, and other high-technology materials used in automobile, aircraft, and computer industries, are some examples of such relevant organic compound. Organic synthesis can be divided in two main research areas: total synthesis or target oriented, and methodology or methods-oriented synthesis ( figure1 ).

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Figure 1. Scheme of organic synthesis

The method-oriented synthesis indicates a method used in order to obtain a compound including chemical reaction, reagents, and condition. Methodology research usually involves three main stages: discovery, optimisation, and studies of scope and limitations. The discovery requires extensive knowledge about the chemical reactivity of appropriate reagents. Once discovered a new reaction the following step is the optimisation one. This process consists in the study of the best reaction conditions in terms of solvents, reactants, temperature and pressure, which allow the synthesis of the new target in the highest efficiency. The last step of the methodology research is the study of scope and limitation. In this step, with the scope to demonstrate the applicability and generality of the procedure, the researchers test a wide number of starting materials under the optimized reaction conditions. Moreover, another parameter such as the scalability of the process is usually investigated; in particular it’s crucial from the industrial point of view. The total synthesis is the step by step process of assembling complex organic molecules starting from simple commercially available molecules as well as from natural chiral pool. Generally this process can be (i) a linear synthesis, where each step is performed in sequence and often it used for the synthesis of simple structure; (ii) a convergent synthesis that consists in an individual preparation of several key intermediates which are then

- 17 - combined to form the final target. Sometimes total synthesis are used to showcase the new methodology and demonstrate its value in a real-world application.

1.2 A brief history on the total synthesis

The birth of the total synthesis coincides with the synthesis of urea by Friedrich Wöhler from ammonium cyanate (NH 4CNO) in 1828 ( figure 2) , because this compound is a naturally occurring substance. Besides giving birth to organic synthesis, that landmark event served to ‘‘demystify’’ nature by burying, once and for all, the myth that the synthesis of nature’s molecules is her exclusive domain. [3] The second major achievement in the story of total synthesis is the synthesis of acetic acid from elemental carbon by Kolbe in 1845 (figure 2) . After these, the most spectacular total synthesis of the XIX century was that of (+)-glucose by E. Fischer (figure 3) . This total synthesis is remarkable not only for the complexity of the target, which included for the first time, stereochemical elements, but also for the considerable stereochemical control that accompanied it. [4]

HOH O O H O HO H2N NH2 Me OH HO H H OH H OH urea acetic acid glucose

(Wohler, 1828) (Kolbe, 1845) (Fischer, 1890)

Figure 2. Selected XIX century Landmark total syntheses of natural product.

In the XX century the total synthesis has been a notable development with increasing molecular complexity and sophistication in strategy design. In particular with R. B. Woodward the total synthesis would be elevated to a powerful science and fine art. The following structures are amongst his most spectacular synthetic achievements: quinine (1944), cholesterol and cortisone (1951), strychnine (1954) and erythromycin A (1981)( figure3). In this period the avalanche of new natural products appearing on the scene as a consequence of the advent and development of new analytical techniques demanded a new

- 18 - and more systematic approach to strategy design. A new school of thought was appearing on the horizon which promised to take the field of total synthesis, and that of organic synthesis in general, to its next level of sophistication.

Figure 3. Selected syntheses by the Woodward group.

For this new school a person of considerable importance has been E. J. Corey, who introduced in the total synthesis two distinctive elements: retrosynthetic analysis and the development of new synthetic methods as a part of the synthetic program. In the 1961, with the synthesis of longifolene (figure 4 ), [5] Corey introduced the principles of retrosynthetic analysis that learn how to analyze complex target molecules and devise possible synthetic strategies for their construction. New synthetic methods are often incorporated into the synthetic schemes towards the target and the exercise of the total synthesis becomes an opportunity for the invention and discovery of new chemistry. Combining his systematic and brilliant approaches to total synthesis with the new tools of organic synthesis and analytical chemistry, Corey synthesized hundreds of natural and designed products.

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Longifolene (1961)

Figure 4. Longifolene and its retrosynthtic analisis.

The period after 1950 was an era during which the total synthesis underwent explosive growth as evidenced by inspection of the primary chemical literature. In addition to the Woodward and Corey schools, a number of other research groups contributed notably to this rich period for the total synthesis and some continue to do so today. Indeed, throughout the second half of the twentieth century a number of great synthetic chemists made significant contributions to the field, as natural products became opportunities to initiate and focus major research programs and served as ports of entry for adventures and rewarding voyages. The fruitfully productivity of the 1980s in the total synthesis boded well for the future of this science, and the seeds were already sown for continued breakthroughs and a new explosion of the field. Entirely new types of structures were on the minds of synthetic chemists, challenging and presenting them with new opportunities. An example of total synthesis of this natural small molecules is a synthesis of Taxol by Nicolaou in the 1994 (figure 5) . [6]

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Figure 5: Retrosynthetic analysis of taxol.

Taxol is one of the most celebrated natural products, it was isolated from the Pacific yew tree and its structure was reported in 1971. Its arduous journey to the clinic took more than 20 years, being approved by the Food and Drug Administration (FDA) in 1992 for the treatment of ovarian cancer. Synthetic chemists were challenged for more than two decades as taxol’s complex molecular architecture resisted multiple strategies toward its construction in the laboratory. All these syntheses, which are characterized by novel strategies and brave tactics, contributed enormously to the advancement of total synthesis and enabled investigations in biology and medicine. Among the various synthetic pathway of taxol, in the total synthesis of Nicolaou, the key strategy used are boron-mediated Diels- Alder reaction to construct the highly functionalized C ring, the application of the Shapiro and McMurry coupling reactions, and the selective manner in which the oxygen functionalities were installed onto the 8-membered ring of the molecule (figure 6 ). Because of the great drama associated with cancer, this and the other syntheses of taxol received headliner publicity. The art and science of total synthesis was once again brought to the attention of the society.

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Figure 6 . The total synthesis of taxol

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At the dawn of the XXI century total synthesis assumed a more serious role in biology and medicine. The more aggressive incorporation of this new dimension to the enterprise was aided and encouraged by combinatorial chemistry and the new challenges posed by discoveries in genomics. Thus, new fields of investigation in chemical biology were established by synthetic chemists taking advantage of the novel molecular architectures and biological action of certain natural products. Besides culminating in the total synthesis of the targeted natural products, some of these new programs expanded into the development of new synthetic methods as in the past, but also into the areas of chemical biology, solid phase chemistry, and combinatorial synthesis. Nowadays the last frontier of the organic synthesis can be considered the flow chemistry (born in 2005) which permits to discover new chemical activity exploiting the reactivity of intermediates species which can not be handled by the traditional batch chemistry. [7]

1.3 How to design a total synthesis

In designing a total synthesis some key points must be taken into account: the first of these is obviously the structure of the target molecule we want to obtain. In fact on the basis of the conformation of the target, that can be a pharmaceutical drug, natural product or other kind of molecules, it is possible to study an efficient synthetic pathway. To know exactly the structure with the right stereochemistry and functional groups of the target molecule is fundamental the retrosynthetic analysis. This analysis begins from the target and go back until the initial reagent we have to use. We can distinguish three concept, which are at the basis of a retrosynthetic analysis and that we can manage to produce an efficient synthetic pathway: (i) disconnection an analytical operation, which breaks a bond and converts a molecule into a possible starting material; it is the reverse of a chemical reaction and the arrow symbol ⇒ and a curved line drawn through the bond being broken. (ii) Synthon is a general fragment, usually an ion, produced by a disconnection; (iii) Synthetic equivalent that consist in a reagent carrying out the function of a synthon, which we cannot use, often because it is an ideal reagent or too unstable. At the end of this analysis the starting materials appears; it should be readily available, low cost and enantiopure (if it is necessary) generally from a chirality pool. When we design a total synthesis we have to

- 23 - pay attention to the stereochemical outcome of reactions and often a biologically active molecule shows its function only if enantiomerically pure. For example if the target molecule is a drug only the right enantiomer shows therapeutic properties but the others stereoisomer could have no effect or negatively sides-effects. In order to avoid this problem it can be planned an asymmetric synthesis where a chiral reagents or catalysts can react differently with an achiral molecule to obtain only one enantiomer. Otherwise the product is in the form of a racemic mixture, it can be performed a kinetic resolution where a chiral substance, such as an enzyme, reacts differently with the two enantiomers (figure 7).

CH3

CH3

CH3O Prochiral Molecule

Asymmetric Synthesis Microbial Oxidation

H CH3 COOH

CH3O

Kinetic Resolution Enzymatic Hydrolysis H CH H C 3 + 3 H COOH COOH

CH3O CH3O (S) (R) Racemic Mixture

Figure 7. Scheme of S-Naproxene (anti-inflammatory agent) obtained by two different route: asymmetric synthesis and kinetic resolution.

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Another key point which is important to keep in mind when we want design a total synthesis is to “propose an ideal synthesis”. It means a synthesis from a readily available, inexpensive, and non toxic starting materials, the target should be easily isolated, quantitative reaction after performed at room temperature (figure 8).

Starting Material Target

- readly available - 100% yield - cheap - easily isolated - non-toxic

Figure 8. Scheme of ideal synthesis

This concept boils down to whether some of the fundamental principles of organic synthesis as: (i) the atom economy, where all reagents used should be integrated into the final product; (ii) achieve synthesis with the least number of steps possible in order to limit the losses of the desired product since each single reaction does not have a yield of 100% because always formed by-products, (iii) perform the reactions at room temperature and leading to the formation of the product easily identified in a way to limit waste in the work up and purification steps. When we design a total synthesis the final point to be considered is the reactions involved, especially their feasibility and reproducibility in order to optimize the synthetic process. For a synthetic chemist the design and implement of a total synthesis is one of the most attractive aim, since it represents a considerable challenge with himself and with his skills and knowledge of organic chemistry. In the next paragraph we will talk more in detail of the more frequent target of total synthesis: small molecules.

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1.4 Small molecules

All the molecules reported in the previous paragraphs can be considered a small molecules, namely highly functionalized molecules having a molecular weight < 1000 Dalton. These peculiarity allow their rapid diffusion across cell membranes making this class of molecules very important from a biological point of view both as drugs, pesticides and herbicides. Moreover a wide number of small molecules exploited for their biological activity haven’t a much complex structure, such as Ibuprofen, L-Dopa (one chiral centre) and Captopril (two chiral centers) (figure 9) . This aspect makes small molecules valuable targets spurring the synthetic chemists to planed innovative synthetic pathways.

Ibuprofen (1961) L -Dopa (1958) Captopril (1975) Anti-inflammatory Precursor of dopamine Ace- inhibitor

Figure 9. Example of small molecules with biological activity

Nowadays small molecules are also used for the study of biology because their small size and the right structural conformation (a target of a great synthesis) allow them to bind to proteins and this results in a perturbation of their function by activation or inhibition of [8] them. An example of this type of application is the study of cytoskeleton of the cell [9] using colchicine like a small molecule to identify the tubulin protein present there. Colchicine inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules. Another example of use of the small molecules to investigate biology is for the ion channels and signaling in the neurosciences. The neurobiology has employed the skill of small molecules, like a chloropromazine , to target the neurotransmitter receptors and ion channels, in order to change the interaction with the receptor modifying the intracellular [8] processes, or rather alteration of signal transduction.

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O O O O N O N Cl HN S O

Colchicine Chloropromazine Anticancer Antipsychotic

Figure 10 . Structure of Colchicine and Chloropromazine

Other interesting highly bioactive and largely studied are the molecules that containing 2- amino-1,3-diol moiety in their skeleton structure (figure 11).

OH OH

2 2 (R) (S) (S) (R) R 3 OH R 3 OH

NH2 NH2

2S,3R D-erytro 2R,3S L-erytro

OH OH

2 2 (R) (S) (R) (S) R 3 OH R 3 OH

NH2 NH2

2S,3S L-treo 2R,3R D-treo

Figure 11. Structure and different stereoisomer of a generic molecule containing 2-amino-1,3-diol moiety.

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Generally these small molecules are constituted by a polar head, that is the 2-amino-1,3- diol group, followed by a long hydrophobic aliphatic chain. For this characteristic composition, these molecules are found in the membrane of mammalian cell. From synthetic point of view these molecule are very interesting for the presence of different substituent at the carbons 2 an 3 (NH 2 and OH group) that can bring at four different stereoisomer of the same molecule. Often the diverse stereoisomer of a molecule have different properties and biological activities and for the synthetic chemist it is indispensable to found the right synthetic strategy in order to obtain the desired product with the right stereochemistry. The most important class that contains the 2-amino-1,3-diol moiety is the sphingolipids family, subdivided into subclasses sphingosine, sphingomyelins and ceramides. These compound will be treated more in detail in the next chapters and we will go deeper inside their chemistry and biology, in order to explain our interest in this kind of small molecules.

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2 CHEMISTRY AND BIOLOGY OF SPHINGOLIPIDS

2.1 Introduction and biological importance of Sphingolipids

The Sphingolipids are a class of lipids that was discovered in 1884 by J. L. W. Thudichum during his studies of the brain. This lipid were isolated from neuronal tissues but can be found in all eukaryotic cells in plasma membrane, cell membrane such as Golgi membranes and lysosomes. He choose the name “sphingosin” for the skeleton structure common in all sphingolipids on the basis of their enigmatic nature like a Sphinx of a Greek mythology. In fact, in ancient Greece and ancient Egypt, the sphinx was a monster with a lion’s body and human head that posed riddle to all encountered her, at the same manner the sphingolipids pose the riddle of their function. For a long time their function has remained a mystery despite the innumerable studies. However in the last decades was discovered that the sphingolipids play a important role in signal transduction pathways, cell’s growth, differentiation, proliferation and apoptosis. Recent studies reported that, disruption of sphingolipids metabolism by food and feed contaminants has been implicated in several animal diseases and possibly human cancer. It also studied the role of sphingolipids as components of the diet that may contribute or protected against [10,11] disease. The structure of sphingolipids ( figure 12 ) consist in a sphingoid base backbone, formed by a long aliphatic chain C18of 2-amino-1,3-diol. If there is a double bond at C4-C5 with E conformation the name is sphingosine. Changing the H of 1-OH group with other substituent group like as phosphodylcholine or various sugar monomer or dimer, and acylation of NH 2 group, it’s possible to obtain the different derivatives of sfingolipidis and the three main subclasses are ceramides, sphingomyelins, cerebrosides and globosides, collectively know as glycosphingolipids.

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O- N+ P O

O Sphingomyelin D-erythro-sphingosine OH OH

OH OH NH2

H O H HO H H OH

H OH

Cerebroside (GalCer) Ceramide (N-Acylsphingosine) O

Figure 12 . Structure of sphingosine, sphingomyelin, ceramide and cerebroside.

For their typical structure the sphingolipids have a hydrophilic head group, that can be more or less substituted, and the hydrophobic long chain that permit the classical array for the membrane lipids, due to the van der Waals interaction between the alkyl chain, in order to have an amphipathic behaviour that it’s much important for the life of cell. The sphingosine isn’t a only type of sphingoid base backbone in fact in the mammalian tissue were found sphinganine C18 ( 1 also called dihydrosphingosine). S mall amounts of sphingoid bases with other chain lengths of 12 to 26 carbons where the most common chain length is eisosasphingosine 2 (2S,3R,4E-d20:1), which has been found in substantial amounts in gangliosides from brain and human stomach and intestinal mucosa. 4E,14Z- diene 3 was found in plasma, brain, and human aorta, 6-hydroxysphingosine 4 in skin sphingolipids and 5-hydroxy-3-E-sphingosine 5 in acid-hydrolyzed brain extracts. There are also sphingoid base with branched chain 6,7 in bovine milk and kidney, atherosclerotic human aorta and pig harderian gland. [12]

- 30 -

OH OH 2S,3R-d18:0

1 NH2

OH OH 2S,3R,4E- d20:1

2 NH2

OH OH 4E,14Z-Sphingadiene 2S,3R,4E,14Z-d18:2 3 NH2

OH OH 6-hydroxysphingosine (6- t18:1) OH NH2 4

OH OH 5-hydroxy-3-E-sphingosine (5OH,3E-d18:1)

5 NH2

OH OH

n Iso-branched sphingoid bases

6 NH2

OH OH Anteiso-branched sphingoid bases n

7 NH2

Figure 13. Some example of sphingoid bases in mammalian cell

Sphingolipids were also found in others species like as fungi, plants, insects, and aquatic organisms such as sponges, corals, starfish, anemones and these lipids have different structure and properties. For example compound 8 was found in insects and compound 9 in plants. The ceramide 10 was extract from sponge of red sea and has antiepileptic properties, and ceramide 11 was isolated from coral of the Indian ocean and has antibacterial properties. [13,14]

- 31 -

OH OH OH OH

8 NH 9 2 NH2 4E-d14:1 4E,6E-d14:2

OH OH OH OH 13 13 OH NH NH 11 19 10 O O 9

Figure 14. Example of sphingoid bases found in other organism.

There are over 300 different known sphingolipids with distinct headgroup and various length of alkyl chain though the most common sphingoid base is the D-erythro - sphingosine. This molecule is an important intermediate in the de novo biosynthesis of all sphingolipids in the mammalian cell and in the inverse route namely the metabolic process. The metabolism of sphingolipidis, and consequently malfunction of the enzymes involved, is the principal cause of some diseases known as sphingolipidoses. Any examples are: (i) Niemann-Pick disease that consist in an accumulation of sphingomyelins in brain and red blood cells; (ii) Gaucher disease when glucocerebrosides increase in and red blood cells (RBCs), liver and spleen, (iii) Fabry disease that involves in accumulation in glycosphingolipids in brain, heart and kidney and (iv) Sjögren-Larsson syndrome that is associated at a deficiency of the enzyme “fatty aldehyde dehydrogenase” in metabolic degradation of sphingoid base.

- 32 -

2.2 Biosynthesis of Sphingolipids

Sphingolipid biosynthetic pathways have been extensively studied for more than three decades and showed that there are 2 major synthetic pathways: the synthesis de novo and the degradation of membrane lipids. [15] The de novo biosynthesis of sphingolipid is required because, although sphingolipids are present in most foods (such as pork, beef and chicken that containing 0,3-0,5 mol/g, milk, butter and cheese have 0,5-1 mol/g while in fruit and vegetables contain < 0,1 mol/g) and then may be introduced into exogenously way, the sphingoid bases are largely degraded in the mammalian intestine. In the de novo biosynthesis, there are so many compounds that can be modify the cell behaviour if only one enzyme or reaction does not work in the correct way. [16] The de novo biosynthesis of Sphingolipids (Figure 15) starts with the condensation of (L)- serine and palmitoyl-CoA by serine palmitoyltransferase (which occurs in the endoplasmic reticulum) to produce 3-oxosphinganine, that is rapidly reduced to dihydrosphingosine (sphinganine) by an NAD(P)H-dependent reductase that is stereospecific for the D-isomer. Subsequently, dihydrosphingosine is N-acylated to dihydroceramide, catalyzed by dihydroceramide synthase. Dihydroceramide desaturase synthase catalyses the subsequent introduction of a trans double bond at C-4–C-5 to produce ceramide. At this point from ceramide can be originate the different sphingolipids following diverse paths. Ceramidase catalyzes the deacylation of ceramide to produce nonesterified fatty acid and sphingosine, which might be act as the substrate for sphingosine kinase (SPHK) to produce sphingosine- 1-phosphate (S1P), which is converted by S1P lyase in palmitaldehyde and phosphoethanolamine. The glycosphingolipids can be generated from ceramide by glucosylceramide synthase. By sphingomyelin synthase, that add phosphocoline head group, ceramide can be converted in sphingomyelin. Then agin ceramide can be phosphorilated by a calcium-activated ceramide kinase to produce ceramide-1-phosphate.

- 33 -

serine O palmitoyl-CoA palmitoyltransferase HO C13H27 L-serine NH2 3-oxosphinganine

NADPH + H+ 3-oxosphinganine reductase NADP

OH

HO C13H27 NH2 dihydrosphingosine (sphinganine)

acil-CoA dihiceramide synthase OH O CoASH (CH3)3N(CH2)2O P O C13H27 OH O HN C15H31

HO C13H27 O HN C15H31 sphingostlphosphorylcholine (SPC) O dihydroceramide sphingomyelin dihydroceramide desaturase synthase deacylase

OH OH OH O sphingomyelin ceramide synthase (CH3)3N(CH2)2O PO C13H27 HO C13H27 HO C13H27 synthase ceramidase sphingomyelinase O HN C15H31 NH2 HN C15H31 D-eritro-sphingosine O ceramide O ceramide kinase sphingomyelin OH S1P sphingosine kinase glucosylceramide phosphatase synthase H2O3PO C13H27 HN C15H31 O OH OH ceramide-1-phosphate (C1P) sugar gangliosides H2O3PO C13H27 O C13H27 NH2 HN C15H31 sphingosine-1-phosphate (S1P) O glycosphingolipids S1P lyase

palmitaldehyde phosphoethanolammine

Figure 15. Scheme of biosynthesis de novo of sphingolipids.

The key enzyme which is part of this synthetic process, can be inhibited by naturally or synthetic inhibitor. For instance serine palmitoyltransferase was inhibited by ( L)- cycloserine or by the fungal metabolite myriocin; ceramide synthetase and ceramidase were inhibited respectively by fumonisim B 1 and (D)-erythro -2-(N-myristoylamino)-1- phenylpropanol (D-MAPP) and the sphingosine kinase was repressed by (DL)-threo - sphinganine.

- 34 -

2.3 Sphingosines: synthesis and biological activity

Sphingosines or (2S,3R, 4E )-2-aminooctadec-4-ene-1,3-diols, (also called trans-4- sphingenine) are the main constituent of sphingoid base backbone of sphingolipids. It is a molecule with 18 carbon atom, that present an double bond at C4-C5, bind two OH group at C1 and C3 and in C2 there is a NH 2 group. For the presence of these different groups, the molecule has two stereogenic centres and it’s possible to have four different stereo isomers that have similar or dissimilar properties (figure 16). The single long chain is responsible for its sufficient solubility in water and this explain its ability to move between membranes and to flip-flop across membrane. Study conducted at physiological pH demostrad that 70% of sphingosine remains in membrane while the 30 % is in water. [17]

OH

HO (2S,3R)-D-erythro-sphingosine

NH2 12

OH

HO (2S,3S)-L-threo-sphingosine NH 2 13

OH

HO (2R,3S)-L-erythro-sphingosine NH2 14

OH

HO (2R,3R)-D-threo-sphingosine NH 2 15

Figure 16. The four possible stereoisomer of sphingosine .

- 35 -

Generally when we talk about a sphingosines we refer at molecule 12 which is the natural one. Its enantiomer has similar biological activity but the other two threo-stereoisomer don’t enclose biological activity. How we can see in the biosynthesis, the presence of sphingosine is controlled by different enzyme: ceramide synthase and ceramidase that regulate the balance from sphingosine to ceramide and the inverse; sphingosine kinase (SphK) and sphingosine-1-phosphate phospathase (S1PP) that control the switch between sphingosine and sphingosine-1- phosphate (S1P) (figure 17).

OH OH OH ceramide synthase H O PO C H S1P phosphatase HO C H 2 3 13 27 HO C13H27 13 27 NH sphingosine kinase ceramidase HN C H 2 NH2 15 31 sphingosine-1-phosphate (S1P) ceramide D-eritro-sphingosine O

Figure 17. Scheme of interconversion of sphingosine, sphingosine-1-phosphate and ceramide.

Further the biosynthesis, it is also important the metabolism of sphingosine because some structural changes occur, that lead to different properties. An example of this is the product 17 (sphingoid base chloroamine) of the reaction between the free amino group of sphingosyphosphorylcholine 16 and with hypochlorous acid and hypochlorite, which are produced in some biological system. [12] The product 17 is used by neutrophils to kill bacteria. [18] If this compound react with HCl the results is 1-cyanomethanophosphocholine 19 and fatty aldehydes (hexanaldecenalhyde 18 ) which are responsible for the Sjögren- Larsson syndrome, an inherited neurocutaneous disorder caused by mutation in the enzyme that catalyzes the oxidation of fatty aldehydes to fatty acids (figure 18) . [19]

- 36 -

O

OH O P O CH2CH2N(CH3)3 O

16 NH2 Cl OH

O H O O P O CH2CH2N(CH3)3 O

17 NH Cl HCl

O

O O P O CH2CH2N(CH3)3 H O H 18 NH 19

Figure 18. scheme of reaction of sphingoid base chloroamine and hypochlorous acid

For nimble conversion of sphingosine to sphingosine-1-phosphate and vice versa in vivo, by SphK and S1PP, it is difficult to identify the specific function of individual sphingolipids. However, we know that the main role of this class of compound is signalling, intracellular second messenger and extracellular ligand for G-protein receptor (EDG1). In particular sphingosines have vital roles in membrane microdomains, and for these reasons they are so-called ‘lipid rafts’. [20] These lipid rafts are important for the life of the cell because, they allow the transport of molecules between the endoplasmic reticulum membranes and those of the Golgi, the major sites of biosynthesis of sphingolipids, in order to activate the turnover of complex sphingolipids.[21] The most studied function of sphingosine is the inhibitory effect on several enzymes. One of the first to be studied is protein kinase C (PCK), an enzyme that plays important roles in several signal transduction cascades. The D-erythro-sphingosine has been demonstrated that, this enzyme acts by promoting the increase in the invasive potential of several types of tumor cells, and for this has become a target in the development of therapy against cancer. The activation of PKC is performed by tumor promoters, such as phorbol 20 , esters such as

- 37 - phorbol 12-myristate 13-acetate 21 (PMA) ( figure 19). PKC in fact works by intracellular receptor for this type of promoters.

O OH O OH O H3C CH3 O H H3C H CH3 H3C CH3 H H H OH H3C CH3 OH H O HO O HO 20 OH 21 OH

Figure 19 . Phorbol and phorbol 12-myristate 13-acetate

Sphingosine has been shown to be a competitive inhibitor, potent and reversible, for enzymes in different cellular systems. The advantage of sphingosine compared to other known inhibitors lies in the fact that, it is a component of course available within the cell. This observation suggests that, the Sphingosine produced in a context intracellular, through the biosynthetic pathways seen previously, can also serve as a negative effector of the activity of PKC. Probably then, using this in vitro study has also revealed another biological activity of sphingosine in cellular context.[22,23,24] Sphingosine inhibit also the enzyme ICRAC (stands for Calcium-Release Activated Calcium Current) that control the slowly replenish the level of calcium ions (Ca 2+ ) in the endoplasmic reticulum. [25] An other important function is the regulation of growth, differentiation and apoptosis of cell; in particular sphingosine enhance the apoptosis, while the sphingosine-1-phosphato suppress it. Many studies were conducted in order to know how this complex mechanism work and was found that regulation of concentration of sphingosine and sphingosine-1- phosphato, by SphK and S1PP, decide the life or the die of the cell. This mechanism is very interesting from medicinal point if view. In fact diseases like as tumor consist of a cell proliferation without differentiation and find molecules (agonist or antagonist), able to control the enzymes that can regulate this process is the object of medicinal chemistry. An example of this is FTY270, an analogue of sphingosine (agonist); this molecule demonstrate its ability to alters the activity of S1P (figure 20). FTY270 was further verified in clinical tests to have roles in immune modulation, such as that on multiple sclerosis.

- 38 -

This highlights the importance of S1P in the regulation of lymphocyte function and immunity. Most of the studies on S1P are used to further understand diseases such as cancer, arthritis and inflammation, diabetes, immune function and neurodegenerative disorders.

HO OH HO HO NH2 NH2 D-erythro-sphingosine FTY720

Figure 20. Structure of FTY720 and Sphingosine

For a molecule with biological activity, in order to work the best, it is extremely important the interaction with the receptor; in fact, by changing the interactions have different biological responses, that lead to pathological conditions such as angiogenesis, inflammation and tumor cells. [26,27] The S1P works in this field in the extracellular environment as a second messenger binding to receptors EDG (Endothelial Differentiation Gene). In particular way five member belong to the family of EDG, EDG-1, EDG-3, EDG- 5, EDG-6 and EDG-8 show a preference of S1P and have been studied interactions necessary to ensure a good affinity between the S1P and the receptor EDG-1. [28] In the figure 21 we show that D-erythro form of S1P has higher affinities than the other stereoisomers, indicating that 3D spatial orientations of key functionalities, that is, the C2- amino group that binds Glu 121 , C3-hydroxyl groups that binds Phe 296 and Tyr 98 , and the polar head that interact with Arg 120 and Arg 292 .

- 39 -

Phe296

Tyr98

Arg120 OH O

HO P O C13H27 O NH Arg292 3 Hydrophobic Interaction Glu121

Figure 21. Theoretical binding conformation of D-erythro S1P in the EDG-1/S1P1 active site. Hydrogen bonds are indicated as dashed lines.

This 3-D theoretical model was developed based on data accumulated by experimental evidence involving S1P analogs. The results obtained show that the double bond of S1P allows a greater binding affinity than the 4,5-dihydro-S1P, synthesized and tested on the receptor; the same way as the amine functionality must have at least one hydrogen available to create a hydrogen bond with the portion Glu 121 . The computational model also suggests that it is possible to modulate the length of the aliphatic chain and its characteristics in order to obtain different selectivity and affinity. A precise and detailed understanding of ligand-receptor interactions is therefore essential for the further design of molecules, that can bind also in the active site of the receptor, functioning as agonists or antagonists and thus exerting their biological and pharmaceutical function.

- 40 -

2.3.1 Strategic pathway for the synthesis of Sphingosines

In view of the multiple biological activities carried out by Sphingosine, it is obvious to think that only the natural biosynthesis is not sufficient to be able to study extensively all its roles. For this reason in the last 50 years, synthetic chemists have developed several synthetic strategies to achieve this multifunctional molecule. The first synthesis of D-erythro -sphingosine reported in 1958 by Shapiro and collaborators (scheme 1).[29] In their strategy, they used a diazonium salt that reacted with an substituted ester acetoacetic 22 in the presence of an ammonium salt. The reduction of the phenylhydrazone 24 with Zn / acetic acid / acetic anhydride and subsequent treatment with

NaBH 4, originated the mixture of diastereoisomers 25 a and 25 b in a ratio 3:2 erythro- threo . The mixture was purified by crystallization, the compound erythro deacetylated and reduced with LiAlH 4 to finally give the D-erythro -sphingosine .

- 41 -

O O NH Cl-/PhN Cl EtO2C 4 2 H COC C13H27 3 C13H27 EtO2C N O N 22 23 Ph

O 1) Zn/AcOH/Ac2O OH 2) NABH EtO C 4 2 C H EtO C cristallization 13 27 2 C H N 13 27 HN NHAc 24 Ph 25b

OH EtO2C 12 C13H27 NHAc 25a

Scheme 1. T he first synthesis of sphingosines by Shapiro

Since them, a large variety of synthetic strategies have been developed for the preparation of sphingosines and their derivatives. The most used for its interesting synthetic point of view, is the synthesis that utilizes the Garner’s aldehyde for the innovative use of (L)- serine 26 (figure 22) such as starting materials. In fact, the amino acid (L)-serine, that belongs to the class of natutal chiral compound is really important. (L)-serine is used as a source of chirality for the synthesis of D-erythro-sphingosine because has the right configuration at C2 and hydroxyl and acid group in correct orientation which can be easy manipulated. Some of the reaction used to covert the serine in the sphingosine is able to modify the chirality of the molecule or can produce diastereoisomer. For these reasons Garner and co-workers [30] synthesized an useful and enantiopure aldehyde, namely the Garner’s Aldehyde 27 (figure 22).

- 42 -

O O H HO OH O N NH2 Boc 26 27

Figure 22. Structures of (L)-serine and Garner’s Aldehyde

The use of this aldehyde open new pathway for the syntesis of sphingosine and one of the first synthesis was carried out was the synthesis of Garner. [31] The synthesis of Garner develops according to the scheme 2 . The first step is the addition of the organolithium to the aldehyde 27 , which proceeds with 8:1 stereoselectivity for the compound erythro 28 a. The second step is the reduction of the triple bond, which occurs under the Benkeser’s condition, using lithium in ethylamine at -78 ° C forming the compounds 29 a - b. The last step is a treatment with HCl which allows the opening of the oxazolidine system, generating the desired product with a total yield of 40%.

O OH OH

LiC C(CH2)12CH3 Li/EtNH2 O H O O C13H27 THF -23°C -78°C N N C13H27 N Boc a:b=8:1 Boc Boc 27 28 a-b 29a-b

Li/EtNH2 a erythro or b threo HCl 1N

OR

RO C13H27 NHR

30a-b R=H Ac2O/pyr 31a-b R=Ac

Scheme 2 . Synthesis of Garner for D-erythro -sphingosine

- 43 -

In the following years, synthetic chemists have developed other approaches to achieve the synthesis of Sphingosine. But many synthetic schemes have as an intermediate step in the formation of the aldehyde of Garner or aldehyde-type Garner. This is because of its stereoselectivity, in the formation of the adduct anti , for the presence of bulky protecting group on the nitrogen atom. This strategic pathway of synthesis is used also for the synthesis of other sphingolipids changing only the characteristic reaction that bring at the specific lipid desired such as spgingomyelin, sphingosine-1-phospato, ceramide, glycosphingolipids and sphingosylphorylcholine.

- 44 -

2.4 Sphingomyelins: structure and their biological properties

Sphingomyelins (SM) or Sphingophospholipids are the major constituent of the mammalian cell lipids. They were found in brain and in the tissue sheath of nerve cell axons, myelin (25%), erythrocytes (18%) and ocular lenses from most mammalian species (from 30% in rabbits to 70% in nuclear lens membranes of elephants). In human lenses SM accounts for only 10 to 15% of the phospholipid content. It’s interesting and strange that the human lens membranes contain high levels of dihydrosphingomyelin (DHSM), in which the double bond between carbons 4 and 5 is not present. [32,33] The first structure of SM, in particular the Nacyl-sphingosine-1-phosphorylcholine or ceramide-1-phosphorylcholine was reported in the 1927 and the composition shows the presence of phosphocholine and the ceramide (that is composed by a sphingosine back bone and acylation at N)(figure 23).

N-acyl-sphingosine-1-phosphorylcholine

OH O- N+ O P O O NH R O

Figure 23 . Structure of generic sphingomyelin

In the figure 23, is possible to see: the polar head group of posphorylcholine, the interfacial part, formed by OH and NH hydrogen bond donor and acceptor and trans-double bond, and the hydrophobic region constituted from fatty acid and sphingoind chain. It is thanks to this conformation that the sphingomyelins are constituents of cell membranes. In fact, have the typical disposition of the phospholipid bilayer of cells. This is permitted by the van der Waals interactions, that are established between the polar, interfacial and hydrofobic region and cholesterol (CHOL) molecules, another important constituent of membrane cell.[34,35] In the mammalian SM the most common sphingoid base backbone is the D- erytro-sphingosine (1,3–dihydroxy-2-amino-4-octadecene) with a trans -double bond between C4 and C5. This double bond is responsible for the interaction of hydrogen bond affecting the strength of its, and has the ability to induce dipoles in the interfacial

- 45 - region.[36] Also the saturated derivative sphinganine (1,3 dihydroxy-2-amino-octadecane) can be found, especially in the human lens membranes (see above). The amide-linked acyl chain can be has a variable number of carbon atom; there are chain with 16,18,20,22 and 24 carbon atom and these chain can have none or various double bond. Generally the double bond is trans but in some cases there are chains with cis -double bond and the most common in mammalian SM is the nervonic acid that has 24 carbon atom and one cis - double bond at C15, or in other type of lipidis such as phosphatidylcholine (PC) (figure 24).

- 46 -

Figure 24. Molecular structure of posphatidylcholine (1-stearoyl-2-oleoyl-sn -glycero-3-phosphocholine) on the left and sphingomyelin (N-nervosyl-sphingomyelin) on the right.

Such as the other sphingolipids, sphingomyelins have an enigmatic biological function. In fact is difficult to assign to each individual sphingolipid its specific function in that, as can be seen in the biosynthesis, are all connected to each other. Even today, studies are underway to solve the riddle of these lipids, but we already know enough about their

- 47 - biological activities, because they work in regulating growth, differentiation and apoptosis of cells, in signal transduction and as inhibitors of carcinogenesis. In particular, in some studies conducted in HL-60 human leukemia cells, it’s possible to see that an defect in the activation of sphingomyelinase, the enzyme that control the hydrolysis of sphingomyelin in ceramide, grades in the loss of induction of apoptosis by [37] tumour necrosis factor-α (TNF-α) mimicking the effect of vitamin D 3. Disfunctions of sphingomyelinase are also responsible for rare inherited diseases such as Niemann-Pick disease that consist in an accumulation of sphingomyelins in the spleen, liver, lungs, bone marrow, and brain. Affected cells become enlarged, sometimes up to 90 micrometres in diameter, secondary to the distention of lysosomes with sphingomyelin and cholesterol. Histology demonstrates lipid laden macrophages in the marrow, as well as "sea-blue histiocytes" on pathology. Numerous small vacuoles of relatively uniform size are created, imparting a foamy appearance to the cytoplasm. This process bring the cell to the death. [38]

- 48 -

2.5 Ceramides: a key intermediates in the sphingolipids metabolism

Ceramides or N-acyl-sphingosine are an sub-class of compound that belong to the family of sphingolipids. They are found in high concentrations within the cell membrane of cells, because they are one of the component lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer. Ceramides are constituted by sphingosine and different type of fatty acid linked by amide bond, that formed ceramide short-chain, from Cer C2 to cer C6 and ceramide long-chain, that can be reach 24 carbon atom or more ( figure 25 ).

D-erythro-sphingosine

OH OH

NH R O

Fatty acid

Ceramide or N-acyl-sphingosine

Figure 25 . Structure of generic ceramide

This structure shows the hydrophobic region formed by long acyl chain, and the small polar head group constituted by the OH group at C1. For this feature ceramide is a cone- shaped molecule that is not very soluble in water and differently from sphingosines. While sphingosine is able to diffuse rapidly between cell membranes, ceramides remain associated with the membrane and participate in raft formation. Ceramides are generated within rafts by the action of acid sphingomyelinase. This cause small rafts to merge into larger units and modifying the membrane structure, in a manner that is believed to permit oligomerization of specific proteins. Through the medium of these modified rafts, they are able to function in signal transduction. The polar head group is the crucial point of this

- 49 - molecule, because changing the polar group are obtained different sphingolipid. For example esterifing the C1 hydroxyl group with phosphocholine moiety, we have sphingomyelin or substituting the OH group with a various carbohydrates, we have the glycosphingolipids. The presence of nitrogen make ceramide different from the other glycerol ester lipid because, it allows inter- and intramolecular hydrogen bonding in which the CONH group can serve as donor and acceptor H-bond. From structural and biological point of view, the existence of double bond at C4-C5 is very important. In fact comparison of ceramide and dihydroceramide, despite their structural similarity, removal of the double bond induces changes in interfacial conformation and compressibility. That regulate the packing behaviour of dihydroceramide in membrane. This is a consequence of the van der Waals interaction, that are established between the double bonds of aliphatic chains in order to pack together in an ordered arrangement to form the cell membranes. [15,39] Given the considerable importance of ceramide, it plays important roles in many physiological events of the cell, small modifications to its structure have a profound impact on its biological activity. One example is that D-erythro -ceramide induces apoptosis of cell but its equivalent without double bond (D-erythro -dihydroceramide) is inactive. [40] Another example is that the double bond and the OH group at C3 are both indispensable for the fusion of Semilki Forest virus with target membrane. [41] Ceramides (Cer) is the key intermediate for the synthesis and degradation metabolism of sphingolipids (figure 26).

- 50 -

OH

HO C13H27 HN C15H31 O dihydroceramide

dihydroceramide desaturase synthase

OH OH OH O sphingomyelin ceramide synthase synthase (CH3)3N(CH2)2O PO C13H27 HO C13H27 HO C13H27 ceramidase sphingomyelinase O HN C15H31 NH2 HN C15H31 D-eritro-sphingosine O ceramide O ceramide kinase sphingomyelin

glucosylceramide OH synthase H O PO C H gangliosides 2 3 13 27 HN C15H31 OH O sugar ceramide-1-phosphate (C1P) O C13H27 HN C15H31

O glycosphingolipids

Figure 26 . Scheme of interconversion of ceramide in all other sphingolipids

In de novo biosynthesis of sphingolipids, ceramide derived from dihydroceramide by the dihydroceramide desaturase synthase, in organelles such as the endoplasmic reticulum (ER) and possibly, in the mitochondria-associated membranes (MAMs) and the perinuclear membranes. Subsequently it can be generated by the breakdown of sphingomyelin (SM) by sphingomyelinases (SMases), which are enzymes that hydrolyze the phosphocholine group from the sphingosine backbone. As in the figure 26 this process is reversible and sphingomyelin synthase forms sphingomyelins accepting a phosphocholine head group. The same type of relatioship existe between ceramide and sphingosines by the enzymes ceramidase, that brekdown ceramide, and ceramide synthase, that forming ceramide starting to sphingosine. A sugar can be attached to ceramide (glycosylation) through the action of the enzymes, glucosyl or galactosyl ceramide synthases. Moreover, ceramide can be attaced by a phosphate group (phosphorylation) by the enzyme, ceramide kinase and it brings at the formation of ceramide-1-phosphate. [42,43,44,45] From the biosynthetic pathway the ceramide that is formed is only in the D-erythro configuration because is the natural product, but there are other three stereoisomer L-erythro , D-threo and L-threo (figure 27).

- 51 -

OH OH (E) (E) (S) (R) (R) (S) HO C13H27 HO C13H27 HN (CH2)nCH3 HN (CH2)nCH3

O ceramide O ceramide 2S,3R D-erythro 2R,3S L-erythro

OH OH (E) (E) (S) (R) (S) (R) HO C13H27 HO C13H27 HN (CH2)nCH3 HN (CH2)nCH3

O ceramide O ceramide

2S,3S L-threo 2R,3R D-threo

Figure 27. The four stereoisomer of ceramide.

There are various studies on the ability of the different stereoisomer, to interact with the enzyme present on the metabolic process of cell. One of these was performed testing the response of the four stereoisomer of ceramides with three different enzymes: the dihydroceramide synthase, sphingomyelinase and glycosphingolipids synthase. The result of this study is that the L-erythro enantiomers of sphinganine, dihydroCer and Cer do not work as substrates for any of the three enzymes. Conversely, the diastereoisomer, L-threo- sphinganine, is acylated by dihydroCer synthase, and L-threo -dihydroceramide and L- threo -ceramide are both metabolized to dihydrosphingomyelin synthase, and sphingomyelin synthase, but not to dihydroglucosylceramide and glucosylceramide. No significant difference was distinguished in the ability of sphingomyelin synthase to metabolize ceramide containing a short versus long acyl chain. Demonstrating that, short- acyl chain ceramides mimic their natural counterparts, at least in the sphingolipid biosynthetic pathway. [46] Another interesting study was executed on the influence of the isomers of ceramides and glucosylceramide to growth of hippocampal neurons at different

- 52 - stages. There are three diverse stage in the neuronal growth: stage 1 or the initial stage of growth where hippocampal neurons are characterized by the presence of many lamellipodia around the cell body. Stage 2 or minor process, is the loss of lamellipodia and extension of a number of short processes. Stage 3 is the rapidly growth of axon. From previous studies was demonstrated that inhibiting the synthesis of ceramides, by fumonisin B1 (32) a specific inhibitor of N-acilation of the spingoid long chain bases, disrupt axon growth in the early stage 3.

O COOH COOH O OH OH

OH NH2 O COOH O COOH

32 Fumonisin B1

Figure 28. Structure of Fumonisin B1

The results of this study are: (i) In the stage 1 and 2 of neuronal development, the GlcCer synthesis is not required while incubation with high concentrations of ceramide or sphingomyelinase, but not dihydroceramide, induces apoptosis. (ii) During axon growth, ceramide must be metabolized to glucosylceramide (GlcCer) to maintain growth and, while D-erythro -ceramide, which is metabolized to GlcCer, is able to antagonize the disruptive effects of fumonisin B1 on axon growth, L-threo -ceramide, which is not metabolized to GlcCer, is ineffective (figure 29). [47]

- 53 -

Figure 29. Morphological characteristics of hippocampal neurons of representative cells at 12 and 24 h in culture after addition of C6-NBD-D-erythro -Cer immediately after cells were placed in multiwall dishes.

From biological point of view, ceramide has been implicated in a variety of physiological functions including stuctural, signaling pathway, apoptosis, cell growth arrest, differentiation, cell senescence, cell migration and adhesion. Roles for ceramide and its downstream metabolites have also been suggested in a number of pathological states including cancer, neurodegeneration, diabetes, microbial pathogenesis, obesity, and inflammation. [48] There are more than 5000 publication on the biochemical activity of

- 54 - ceramides and this different activities are still under study in order to understand the mechanisms and how this sphingolipid works. Remains the fact that the biological activity most studied for the ceramide is apoptosis, in particular the induction of tumor cell death. This is very interesting for medicinal chemistry in the design of new anticancer drugs. How we see in the previous paragraph the balance between apoptosis and cell proliferation is regulated by the relationships from ceramide/sphingosine-1-phosphate that inter converge on each other. In particular sphingolipids such as sphingosine-1-phosphate and glucosphingolipids stimulate tumor growth, proliferation, angiogenesis, and resistance to attack by the patient’s immune system. The mechanisms by which ceramide produces apoptosis are understood, but they appear to involve the allylic alcohol group in ceramide at C-3, since dihydroCer (a precursor of Cer) has rarely shown biological effects. Oxidation of the C-3 hydroxy group in mitochondria, generating reactive oxygen species (ROS). Glutathione (GSH), the major reducing agent in cells, normally reduces the ROS and thus blocks the spiraling generation of ceramide. Inhibitor of GSH that lowers its concentration in cell, increases the rate of ceramide formation from SM. Antioxidants or GSH precursors ( N-acetylcysteine) tend to elevate the GSH concentration and prevent apoptosis, while inhibitors of GSH synthesis (buthionine sulphoximine) promote apoptosis.

ROS (H 2O2, nitric oxide, etc.) and ROS-producing substances destroy GSH and speed Cer formation and apoptosis. Some anti-cancer drugs react with GSH via a Michael condensation reaction or produce ROS, and may apply their beneficial effects via these mechanisms; an example of this is (L)-buthionine-(S,R)-sulfoximine ( 33 ) that lead to loss of GSH and accumulation of ceramide that enhance the tumor apoptosis. Other example is Betathine ( 34 ) ( β-alanyl cysteamine disulphide), has been producing good effects in patients with myeloma, melanoma and breast cancer. It oxidizes GSH and improved the surface expression of tumour necrosis factor α (TNF-α) in T-cells and monocytes, spuring the ceramides synthesis. [49,50]

- 55 -

H2N OH O - H2N S+ O NH O - S SH S NH β-Alanyl cysteamine disulphide 33 34 (L)-Buthionine-(S,R)-sulfoximine

Figure 30 . Structure of L-buthionine-(S,R)-sulfoximine β-alanyl cysteamine disulphide

There are other commercially drugs that involve in the control of the production of ceramides, but in different pathway; for example taxol, an anticancer drug, produce apoptosis in a leukaemia T cell line incentive a faster de novo biosynthesis by serine palmitoyltransferase (SPT) and then Sphingomyelinase. Moreover Protein kinase C seems to be essential for ceramide formation and the ceramide itself promotes activation and transfer of the kinase into the mitochondria. Another example is Tamoxifen ( 36 ) and Raloxifene ( 37 ), anti-neoplastic drugs, that behave as an anti-estrogen useful for the prevent breast cancer, which inhibit glucosylceramide synthase. Raloxifene also prevent or repairs osteoporosis and may act like as ceramide in terms of bine protection as well as tumor apoptosis.

HO

O S

N 36 OH O O Tamoxifen N 37

Raloxifene

Figure 31 . Structure of Tamoxifen and Raloxifene

- 56 -

The other biological activity of the ceramide are related to the de novo synthesis. In fact chemotherapeutic agent, such as daunorubicin and etoposide, enhance the synthesis on mammalian cell inducing apoptosis by stimulation of particular receptors of lymphocytes called B-Cells. Instead regulation of de novo synthesis by palmitate may have a key role in diabete and metabolic syndrome. [51,52]

O O HO H O OH O H O CH HO O OH 3 H O O H OH O OH O O O CH3

OH O OH O O 38 Daunorubicin NH2 39 Etoposide

Figure 32. Structure of Daunorubicin and Etoposide.

As we have seen so far, ceramide is implicated in many important physiological event in the cell and for this reason that it is largely studied from chemist and biologist. Indeed it’s very interesting discover all the skill of this sphingolipid in that it offers countless prospects such as the design of new drugs and study the structure-interaction with the receptor. For this purpose is very useful the organic synthesis in order to obtain not only ceramides of natural origin (D-erythro conformation), but also non-natural stereoisomers which, however, prove to be interesting from the biological point of view. Generally the synthetic route to obtain ceramide proceeds from Garner’s aldehyde to sphingosine and subsequently N-acylation by nitro compound. Recently, this synthetic route have been reviewed, trying to improve yield, reactivity and type of reaction used. In scheme 3, it is possible see an type of synthesis for ceramide performed by Bittman ad co-workers. [53] The synthesis starts from 40 (L)-serine derived methyl ester that condense with methyl phenyl sulfone 41 at – 78°C and gave the β-ketosulfone 42 in 71% yield. The second step is the alkylation of 42 with allylic bromide 43 in presence of base giving the

- 57 - copound 44 in 79%. The compund 44 and 45 were separated by column chromatographic and the β-ketosulfone 44 is desulfunylated forming the ketone 46 in high yield. The reduction of 46 with NaBH 4 or DIBAL-H gave the alcohol 47 and the fifth step is the acid hydrolysis (HCl 1 M) of 47 in the sphingosine 48 . Last step is the N-acylation of sphingosine with p-nitrophenyl octanoate that lead at two diastereoisomer of the ceramide 49 . The erythro /threo ratio is about 6:1.

O O O O R Br 1 n-BuLi 43 R O OCH3 SO2Ph 1 + MeSO2Ph R R O DBU O + O N 41 THF -78°C Boc N Benzene rt N S(O)2Ph N S(O)2Ph Boc Boc Boc 45 40 (71%) 42 44 1 R= C11H23-n R = CH2CH=CHC11H23-n (79%) (9%)

Al(Hg) THF/H2O rt

OH OH O 1 M HCl NaBH4 HO R O R O R H2N THF 70°C N MeOH N 48 Boc Boc

46 (86%) 47 (88%)

p-O2NC6H4CO2C7H15-n OH

HO R THF, rt HN C7H15 -n O 49 (63%)

Scheme 3. Synthesis of ceramide.

Also the prevoius reactions see in the paragraph of synthesis of sphingosines (Shapiro and Garner Synthesis), are useful for the synthesis of ceramide doing an final acylation on the

NH 2 group of sphingosines. Another valuable synthetic pathway for ceramide is the synthesis of Harold [54[] for D-erythro and D-threo , Z or E, sphingosine (scheme 4), followed by acylation. This synthesis shows, how careful selection of reaction conditions allows to drive the reaction to synthesize selectively four different isomers. Herold in fact able to synthesize selectively and with good yields, ranging from 60% to

- 58 -

92%, isomers 54 ,55 and 56 . Its products are D-erythro -sphingosine 57 , D-threo - sphingosine 55 with an E geometry of the double bond geometry and their isomers with Z 56 and 55 . The first selectivity occurs in step which provides for the addition of organolithium to the Garner’s aldehyde. This is achieved through the use or not of a Lewis acid, which, as discussed in Chapter 3, affects the type of addition to aldehyde and then on the stereochemical outcome. The second opportunity to induce two different selectivity, is the reduction of the triple bond of compounds 51 and 52 . Using respectively the reducing compound of aluminum Red-Al and H 2 in the presence of Lindlar catalyst are obtained isomers D-erythro -(57 ), D-threo -sphingosine (55 ) with E geometry of the double bond and 54 , 56 with geometry Z. At this point for each isomer of sphingosine by first deprotonation in acid condition, second acylation of free amino group, is possible obtain the four different ceramide.

- 59 -

O O DIBAL-H toluene O OCH3 O H N N Boc Boc 40 27 LiC C(CH2)12CH3 LiC C(CH2)12CH3 THF ZnBr2, Et2O

OH OH

O O N C H N C13H27 13 27 Boc Boc 50 51

Amberlyst 15/ Amberlyst 15/ MeOH MeOH

Red-Al Et O OH 2 OH

HO HO HN C H HN C13H27 13 27 Boc Boc 53 52

H2/Lindlar cat./ Red-Al H2/Lindlar cat./ Red-Al EtOAc Et2O EtOAc Et2O

C H OH OH C13H27 OH OH 13 27

HO HO C13H27 HO HO C13H27 HN HN HN HN Boc Boc Boc Boc 54 55 56 57

Scheme 4. Synthesis of Herold.

There are a lot of synthetic methods for the formation of D-erythro adduct starting from (L)-serine or its derivatives, but a few synthetic methods to obtain the L-erythro - sphingolipids. Intended to overcome this lack we wanted to develop a synthesis that will be discussed in Chapter 4, and that use of a versatile and valuable ally: the Garner-type aldehyde. The next chapter will help us recall some concepts of this type of aldehyde that will be useful in explaining and understanding the strategy developed and the organocerium reagent.

- 60 -

3 GARNER’S ALDEHYDE AND ORGANOCERIUM REAGENTS

3.1 Garner’s aldehyde and Garner-type aldehyde: synthesis and reactivity

In the chapther two we see the importance of the Garner’s aldehyde like as versatile staring material for a wide number of synthesis. The first appearance of this aldehyde was in the 1984, when Garner published a synthesis of (S)-tert -butyl 4-formyl-2,2- dimethyloxazolidine-3-carboxylate (scheme 5). [30] The first reaction is the protection of amino group to the (L)-serine ( 26 ) with Boc and subsequently the 58 was converted in compound 59 by MeI. At this point the reaction of 59 with 2,2-dimethoxypropane and TsOH gave the compound 40 , then the reduction of oxazolinidic ester forms the aldehyde 27 .

O O O

Boc2O CH3I HO OH HO OH HO OCH3 NaOH K2CO3 NH2 NHBoc NHBoc 26 58 59

(CH3)2C(OCH3)2 TsOH

O O

O H DIBAL O OCH3 N toluene N Boc Boc 27 40

Scheme 5. Synthesis of Garner’s aldehyde

- 61 -

The great utility of the Garner and Garner-type aldehyde, is not only due to the fact that is adaptable at different reaction, but also for its diastereoselectivity in a wide number of molecule target. In effect, a hindered protecting group on the nitrogen atom allows a highly diastereoselectivity for the anti adduct. The nucleophilic addition to the Garner’s aldehyde lead at the formation of γ-hydroxy-β-amino alcohols, that are a important sctructural moiety of bioactive naturally product [55] and generally the diastereoisomer anti is much more favoured. The formation of this type of reaction products is explained by the model of Felkin-Ahn, which involves a non-chelated transition state and leads to the anti product, and using a model that provides for the formation of the adduct syn , passing through a transition state chelate (Figure 33). Without the use of chelation the syn product seems to be unfavorable.

Mn+ t O CO2Bu t CO2Bu N O H N H anti H syn O O face H O face Si chelated Re Felkin-Ahn transition state transition state

Figure 33. Transition state of product anti and syn .

For understand this stereoselectivity of the Garner’s aldehyde was performed an study using n-BuLi , acetylides and THF at different temperature. [56] From this study emerged that: at low temperature the anti product is favoured because is under kinetic control, while at high temperature the termodinamic produc syn is in a greater amount. For some substrate the stereoselectivity is non so highly defined and in order to improve the selectivity and yield are used an Lewis acid.

- 62 -

3.2 The organocerium reagents

3.2.1 Cerium

Cerium is a metallic element that belongs to the family of the lanthanides, the fifteen elements that are part of the third group and of the sixth period of the periodic table. These elements are also called elements of block f, except lanthanum, because have electrons in the 4f orbitals. The most stable oxidation state for these element is +3, although it’s possible encounter in the state +2 and +4. The Ce 3+ is hard cation according with HSAB terminology and so show affinity to the hard bases such as oxygen and nitrogen ligands. In the organic chemistry CeCl 3 is considered a mild Lewis acid. The most common source 3+ commercially available of Ce is the cerium trichloride heptahydrate (CeCl 37H 2O) that can be used in hydrated and anhydrous form. Despite its name rare earths, these elements are found in great abundance, at a good price, and their toxicity is comparable to that of sodium chloride. For these reasons, the cerium appears to be a desirable promoter to be used in organic synthesis.

3.2.2 Organocerium compounds

One of the most important methods in organic chemistry for the formation of new C-C bonds, is the nucleophilic addition of organometallic reagents to multiple bonds. A very useful compound for this scope are the Grignard (RMgX) or organolithium (RLi) reagents, but the high reactivity of this two reactive can be lead at byproduct during the reaction. If in these compound was added the cerium chloride, due to the Lewis acidity, were obtained the organocerium compounds that have high versatility and low toxicity of byproduct. [57] These compounds also show limited basicity, nucleophilicity and high potentiality that generally exceed those of organolithium and Grignard reagents from which they originates. Their widespread use in the addition nucleophilic it is possible, thanks to the easy preparation derived from RMgX and RLi using CeCl 3 anhydrous. The latter can be easily prepared by dehydrating under vacuum CeCl 37H 2O, given the remarkable stability of the salt during the thermal dehydration. [58] The compounds that are derived from RLi are stable

- 63 - only at low temperatures and are generally used at -78°C, while those prepared from RMgX are stable even at temperatures that reach 0°C; organocerium reagents however, can not be stored at long, on the contrary are preferably prepared just before use. Generally, the reactions are carried out by adding a solution of THF or ether with the electrophilic substrate to a suspension of the organocerium freshly prepared in THF. The alternative is the addition of the electrophile substrate dissolved in a solution of THF to a suspension of anhydrous CeCl 3 at room temperature. It is interesting to note that the course of the reaction is controlled among other factors also by the choice of the solvent. Generally the use of THF allows to improve yields and to reduce the reaction times compared to Et 2O. The crystal structure of anhydrous cerium chloride in THF revealed a polymeric nature in which the incorporation of the solvent within the crystal structure is the basis of the best performances for the reactions carried out in THF. The THF is in fact more basic than ether and coordinates much more effectively than the metal, especially if is oxyphilic as the cerium. [59] Although the nature of the reagents organocerium is not yet well explicated, is a common opinion that the compounds arising from RLi have a structure of the type

RCeCl 2, while for the compounds resulting from the Grignard reagents are assumed a frame with RMgX CeCl 3. The most persuasive evidence that support the existence of two different structures, is the best nucleophilic character and weaker base of the RLi/CeCl 3, [60] than the complex RMgX/CeCl 3.

3.2.3 Addition of Organocerium to Carbonyl compound

The addition of organocerium at carbon-oxygen double bond is one of the most significant reaction to form new carbon-carbon bond. It is even very important for the consequently formation of alcohols in high yield also in substrate susceptible to enolization or that have stereogenic centers for the synthesis of small molecules. An example is the synthesis of Norcholestane, a derivative of steroid, by Cristoffers (scheme 6), [60,61] where the first step is the transmetallation of organolithium ( 61 ) to organocerium reagent by the CeCl 3 anhydrous and that the nucleophilic attach at the ketone 60 . The tertiary alcohol is obtained with a yield of 80% and without byproduct controversy to the simple addition of the only

- 64 - alkyllithium. The second step is an dehydratation and the third is an hydrogenation, that leave at the target steroid.

O HO

61 CeCl3 Li OMe THF,-78°C, 5h 60 80%, d.e.95% OMe 62

POCl3,Py 0-23°C 3d, 72%

H2, Pd/C

EtOH-EtOAc OMe OMe 64 99% 63

Scheme 6 . Synthesis of Norcholestane.

[62] Another example of the skills of the CeCl 3 is the methodology used by Widlanski, for the addition of dianions to the aldehydes and ketones that belong at molecule biologically active such as ketonucleosides. This type of molecules have a modest yield using Grignard, organolithium and organoaluminium reagent, but if CeCl 3 was added the basicity of dianion N-benzil-α,N-dilitihium methanesulfonamide ( 67 ) is lowered and the addition to the keto group is eased.

- 65 -

O O NH NH TBSO N O O TBSO N O O

O OTBS HO 65a O OTBS S O NHBn 66a (99%)

O O O NH Li S N O TBSO N O 67 Li O NH

CeCl , THF TBSO N O O 3 O 65b HO O S O O 66b (67%) NHBn NH O N O O H O

O O NH O OH O N O S O BnHN 65c O O 66c (70%)

Scheme 7. Reaction of addition of N-benzil-α,N-dilitihium methanesulfonamide to aldehyde and ketouridines.

The last example to show is the addition at the double bond C=O to form γ-hydroxy-β- amino alcohols that, has we already seen in the previous paragraph, are a key moiety for a molecule with biological activity. The addition of an organolithium using CeCl 3 to the Garner’s aldehyde or its derivatives lead at this important group in good chemical yield and optimum diastereoselectivity for the anti compound. This study was performed by Zhu [63,64] and co-workers starting from N,N-Dibenzylserine aldehyde 70 ,(derived from (L)- serine) and through the organocerium reagent form an γ-hydroxy-β-amino alcohol in anti coformation. The stereochemistry can be verified do the conversion in the oxazolindone

- 66 -

72 . In this synthetic strategy was chosen for the protection of OH group, TBDMS group, the choice was dictated by the ease of introduction and non-chelating properties of the resulting ether. This type of aldehyde is part of our discussion regarding the reactivity of the compounds organocerium since an improvement of the reaction conditions is achieved with the addition of CeCl 3.

HO O TBDMSO TBDMSO O Swern Ox. OCH3 OH H NH2 NBn2 NBn2 68 69 70

RLi/CeCl3

THF o Et2O

TBDMSO R TBDMSO OH O HN R O NBn2 71 72

Scheme 8. Reaction of N,N-Dibenzylserine aldehyde with organocerium.

3.2.4 Organocerium reagents: their importance in organic synthesis

The advantage of the use of organocerium reagents in organic synthesis is due to their pivotal role as intermediate in the total synthesis of target molecule. In the last decade several total synthesis were published, where the promotion of the addition of organometalic species to carbonyl by CeCl 3 is the key step. One representation of this specific case, is the total synthesis of a (±)- Roseophilin ( 76 ), an antibiotic, done by Fürstner [65] ( scheme 9).

- 67 -

H3CO Li

O

CeCl3 i N THF, -78°C Cl N SiPr 3 O SEM H3CO N 73 74 OH SEM O

i Cl N SiPr 3

75 1) TBAF 2 HCl aq.

H3CO N HCl O

Cl NH

76

Scheme 9 . Synthesis of (±)- Roseophilin

Furstner has chosen a organometallic strategy for the synthesis of this antibiotic, looking for promote the condensation of fragments 73 and 74 . The deprotonation of the precursor of fragment 73 takes place exclusively with n-BuLi at low temperatures on the furan ring. Then the following transmetallation of the resulting lithium compound with anhydrous

CeCl 3 generates a highly nucleophilic organocerium species. Although the product obtained 75 can be isolated, but isn’t too stable; for this reason the addition of aqueous HCl and the subsequent loss of water gives the chromophore 76 , which is manifested by an intense orange-red fluorescence. This characteristic feature of organocerium to add its self at carbonyl group, has also been used in the total synthesis studied by the research group where I worked. In Precise, we

- 68 - tested the reactivity of different organocerium derived from Grignard and organolithium on Garner-type aldehyde for the diastereoselective synthesis of L-erythro ceramide. The next chapter is entirely devoted to the project that I worked, the total synthesis of L- erythro ceramide C6 referring to the concepts expressed until now.

- 69 -

4 TOTAL SYNTHESIS OF L-ERYTHRO-CERAMIDE C6

4.1 Introduction to the experimental work

In previous chapters we highlighted a great work behind the design and subsequent construction of a total synthesis. Each reaction step in fact has to be studied, tested and finally optimized in order to obtain valid results, but also for the reproducibility of the synthesis itself. Often, to find the perfect agreement between the different factors involved, we must pursue different paths that can also prove futile. The ultimate goal remains the improvement in finding the most efficient and convenient synthetic route. Our experimental work is based on the total synthesis of L-erythro -ceramide C6, the enantiomer of the natural one, starting from the natural aminoacid (L)-serine and passing through the Garner-type aldehyde. The key step of this synthesis is the diastereoselective addition of the organocerium reagent to the aldehyde. This team has yet studied the Garner-type aldehyde testing and choosing the best protecting group in order to improve the tield and the stereoselectivity. In this chapter we will go deeper inside the scope and the strategic synthetic pathway for the synthesis of L-erythro -ceramide, keeping in mind some of the previous concept and the importance of synthesizing a target molecule with high biological activity.

- 70 -

4.2 Ceramide C6

Referring to Chapter 2 in the section devoted to ceramides, we know that these are biologically active molecules by multiple features. The D-erythro -ceramide C6 is the natural short chain sphingolipids, that can be founded in cell membranes. Many publications refer to the various functions performed by this basic lipid and among their function we remember the most relevant: (i) C6 ceramide induces Cytochrome c release from isolated mitochondria; [66] (ii) [67] ceramide C6 mimics the effect of H 2O2 inducing apopstosis in TBE cell; (iii) it induces the inhibition of potassium channel metiated by tyrosine kinases; [68] (iv) Retinoblastoma is a downstream target for ceramide C6 and may function in a growth suppressor pathway resulting in cell cycle arrest. [69] All this function make the ceramide C6 an important target for the synthetic chemist and useful molecule in the field of bioactive studies. Over the natural D-erythro -ceramide C6 (2S,3R), there are other three stereoisomer: L- erythro (2R,3S), D-threo (2R,3R) e L-threo (2S,3S). Even for this isomer the biological activity was studied. In particular it has been studied the metabolism to GluCer and the results of this study is that the L-erythro-ceramide C6 has the same activity of its natural enantiomer D-erythro, but the two isomer threo are inactive. [48] From synthetical point of view there aren’t much publication about the synthesis of L- erythro (2R,3S) isomer because the cheep starting material (L)-serine has the sterogenic centre in the inverse configuration and its enatiomer, D-serine, with the right conformation, is seven time more expensive (figure 34) . For this two different reasons we choose to design a first total synthesis of L-erythro -ceramide C6.

- 71 -

OH OH (S) (R) HO (R) HO (S) HN HN

O 106 O 77

(2S,3R)-D-erythro-ceramide (2R,3S)-L-erythro-ceramide

O O (R) (S) HO OH HO OH NH2 NH2 26 107 L-serine D-serine

Figure 34. Chiral precursor (L)-serine and D-serine.

- 72 -

4.3 Design of total synthesis: innovative strategies in this synthetic pathway

The first aim of this work is the synthesis of L-erythro -C6 ceramide or N-[(E, 2R, 3S, 4E)- 1,3-dihydroxyoctadec-4-en-2-yl] hexanamide ( 77 ). For this purpose, we initially studied how to design our total synthesis. Via a retrosynthetic analysis, always keeping in mind (L)-serine as starting material.

OH O (E) (R) (S) (S) OH HO OH NH NH2 77 O 25 L-erythro-ceramide C6 L-Serine N-((2R,3S,4E)-1,3-dihydroxyoctadec-4-en-2- (S)-2-amino-3-hydroxypropanoic acid yl)hexanamide

Figure 35. Structure of L-erythro -ceramide C6 and (L)-serine.

The first innovative strategy that we introduced in our synthesis is the modification of the protecting group of the (L)-serine in order to reach the right configuration of our sphingosine backbone. To do this, we built the oxazolidine ring, like as protection for the amino and hydroxyl groups, in the right part of the (L)-serine (not in the left, as in other synthesis) reducing the carboxylic acid to alcoholic group in order to obtain the R configuration at the C2. Subsequently the oxidation of the free hydroxyl group to aldehyde, lead at the Garner-type aldehyde, that it used for the second innovative strategy: the stereoselective addition of organocerium reagent. This novel synthetic pathway is different among the other conventional strategy because: (i) for the synthesis of L-erythro -ceramide, the conventional strategy [30] generally use the enantiomer D-serine, that is economically and environmentally unattractive; (ii) with the other strategy starting from the (L)-serine the product obtained is the D-erythro -ceramide (figure 36).

- 73 -

O Conventional Our Strategy (S) HO OH Strategy

NH2 25 O O

O (S) H H (R) O N N R R P P R R 78 79

OH OH (E) (E) (R) (S) (S) (R) C13H27 OH HO C13H27 NH2 NH2 81 80

OH OH (E) (S) (E) (R) (R) (S) HO C13H27 C13H27 OH HN (CH2)nCH3 H3Cn(H2C) NH O O 82 83

Figure 36. Principal difference between conventional and our strategy.

Taking into account this new stratagem to produce our target molecule we thought at this type of retrosynthetic analysis, that is showed in the scheme 9. In this pathway there are the three principal intermediate: the L-erythro -sphingosine 12 , the Garner-type aldehyde 79 and the alcoholic derivative 83 from the (L)-serine.

- 74 -

OH OH

C13H27 OH C13H27 OH NH NH2 77 O 12

O O

HO OH HO OP H OP NH2 NP NP 25 83 79

Scheme 10 . Retrosynthetic analysis.

In the follow paragraph we will discuss the three major parts that are the heart of this total synthesis: the synthesis of Garner-type aldehyde, the addition of organocerium reagent to obtain sphingosine and the synthesis of C6 ceramide.

- 75 -

4.4 Part one: formation of Garner-type aldehyde

The first part of the total synthesis interested the formation of the Garner-type aldehyde starting from the natural and cheap (L)-serine. The Marcantoni’s research group has yet studied this type of aldehyde and its related protecting group identifying which are the best in terms of stereoselectivity, racemization and yield. The result of this study is that the best protecting group for amine is the hindered di-tert-butyl dicarbonate (Boc), and for the formation of oxazolidine ring it used cyclohexanone thai is more bulky than the usually used acetone. [70] The scheme 11 shows the synthetic pathway to Garner-type aldehyde 91 .

OH O TBSCl TBSO O NaBH4 TBSO imidazolo EtOH OCH3 OCH3 OH CH2Cl2 NH2 NH2 NH2 84 85 86 O 87 PTSA benzene

HO TBSO TBSO

O TBAF O Boc2O 1eq O N THF N HN Boc Boc I2, Solvent free

90 89 88 NEt3 DMSO . Py SO3 CH2Cl2 O

H O N Boc

91

Scheme11. Synthesis of Garner-type aldehyde

- 76 -

The synthesis starts from the methyl-ester of serine 84 , a commercially available starting material (scheme 12). The first step of the synthesis is the protection of hydroxyl group as silyl ether, by tert-butyldimethylsilyl chloride (TBS), which has been chosen for it easy introduction and for the nonchelating property of the resulting silyl ether.

OH O TBSCl TBSO O imidazolo OCH3 OCH3 CH2Cl2 NH2 NH2 84 85

Scheme 12. Protection of the hydroxyl group of L-methyl ester serine.

The second step is the reduction of compund 85 by NaBH 4 in order to generate the alcoholic group (scheme 13). During this reaction we noticed a byproduct, boronate ester, that was converted into 86 by hydrolysis with phosphate buffer at pH=3, increasing the yield until 94%. [71]

TBSO O NaBH4 TBSO O EtOH HO B OCH3 OH N H NH2 NH2 OSBT 85 86 92

pH=3

Scheme 13. Reduction of the ester and conversion of boronate ester into 86 .

- 77 -

The third step is the formation of oxazolidine ring (as protecting group) using cyclohexanone 87 and p-Toluenesulfonic Acid (PTSA) in catalytic amount in benzene for 24 h at room temperature (scheme 14). The reaction gives a good yield.

O TBSO TBSO O 87 PTSA OH HN NH2 benzene 86 88

Scheme 14. Protection of amino and hydroxyl groups by ciclohexanone.

The fourth step is the protection of amino group by di-tert -butyldicarbonate (Boc) (scheme 15). This reaction was performed in different condition in order to reach the higher yield.

In the first test the compound 88 was treated with Boc 2O in ratio 1:2 and catalytic DMAP in Et 3N-CH 3CN 3:1, but the reaction didn’t work and only the starting materials was recovered. The second test was performed using Boc 2O in toluene and a saturated solution of Na 2CO 3; the reaction works, but the yield was only 45%. The third test was done with

88 e Boc 2O in ratio 1:2,5 in Et 3N (10 eq) and CH 2Cl 2 but the reaction time was quite long in fact, after 12 hours it wasn’t complete. The best conditions were founded with the fourth test, in which only one eq. of Boc 2O and I 2 10% mol, are necessary under solvent-free condition. [72] The reaction is fast (after 1 h the conversion is 63% and after 2:30 h is complete) and the yield is 90%.

TBSO TBSO

O Boc2O 1eq O HN N I2, Boc Solvent free

88 89

Scheme 15. Protection of amino group by Boc.

- 78 -

The fifth step of the our synthesis is the removal of the TBS protecting group by tetrabutylamonium fluoride (TBAF), in order to restore the hydroxyl functionality (scheme 16). TBAF is highly selective for unmasking the hydroxyl functionality and this great efficiency comes from the great affinity between the atoms of fluorine and silicon that establish a strong bond. This reaction involves 89 and a slight excess of TBAF (ratio 1:1,2) in THF. After 2h the reaction is finished with a 93% of yield.

TBSO HO

O TBAF O N Boc N THF Boc

89 90

Scheme 16. Deprotection of hydroxyl group.

The last step of this scheme is the oxidation of the hydroxyl group of 90 in order to obtain our Garner-type aldehyde (scheme 17). This reaction is carried out using dimethyl sulfoxide, as the oxidant, activated by the sulfur trioxide pyridine complex in the presence of triethylamine as a base. This condition are know as Parikh-Doering oxidation. [73] The reaction gives as Garner-type aldehyde in 83% of yield.

HO O NEt3, DMSO, H O . O N Py SO3 N Boc Boc

CH2Cl2

90 91

Scheme 17. Oxidation of alcoholic functionality to obtain aldehyde.

- 79 -

4.5 Part two: synthesis of sphingosine

The second part of the synthesis is dedicated to the formation of organocerium reagent and consequent addition to the Garner-type aldehyde in order to obtain sphingosine. As we saw earlier, sphingosine is characterized by a long aliphatic chain with 18 atoms of carbon and a double bond at C4 position. In order to generate the sphingosine we thought to a 15 atom of carbon linear chain containing a double bond to be added to Garner-type aldehyde. To do this, we choose economic and commercially available 1-tetradecanol as starting material. It was oxidized and converted into 1-iodopentadecene by the Takay’s reaction,[74] and then used as substrate to generate organolithium. At this point the organolithium reagent was reacted with the CeCl 3 and once obtained the organocerium species, it was added to our aldehyde, thus obtaining the protected sphingosine. The scheme 18 shows the synthetic pathway that we selected among the different trials performed, and we will discuss in detail every step of the synthesis.

- 80 -

O OH PCC H CH2Cl2 93 94

CrCl2, CHI3 THF, 0° C

n-BuLi Li Ether, -78°C I 96 95

O H O N Boc

CeCl3 THF, -78°C 91

OH OH + O O N N Boc Boc 97 98

Scheme 18. Synthesis of protected sphingosine.

In this scheme the first step is the oxidation of 1-tetradecanol 93 to tetradecanal 94 , using pyridinium chlorocromate (PCC) as oxidizing agent in dichloromethane. [75] The reaction is fast easily was performed at room temperature with a yield of 88%.

O OH PCC H CH2Cl2 93 94

Scheme 19. Oxidation of 1-tetradecanol.

- 81 -

At this point the aldehyde undergoes Takai reaction (or Takai olefination). [76] It works at

0°C with 6 equivalents of cromium(II) chloride (CrCl 2) and 2 equivalents of iodoform

(CHI 3) with respect to the aldehyde, using THF as solvent. This reaction allowed us to convert the aldehydic function into an alkenyl iodide with the needed E conformation of the double bond with a 93% yield (scheme 20). The only one particular precaution achieve the best results is to carry out the procedure in dry environment .

O CrCl2, CHI3 H I 94 THF, 0° C 95

Scheme 20. The Takai reaction.

We have also testing the variant of Takai’s reaction which use a catalytic amount of CrCl 3, reduced by metallic Zn into CrCl 2 and subsequently transformed into CrCl 3 by Me 3SiCl, [77] thus closing the redox cycle. The presence of Zn and Me 3SiCl is essential for the reaction but is also very important the presence of NaI that avoid the halogen exchange. The ratio among the different reagent are in the order aldehyde 1 eq., cromium(III) chloride 0.2 eq., iodoform 2 eq., zinc 6 eq., trimethylsylilchloride 6 eq. and sodium iodide 1 eq.. We tried this type of reaction, the yield was only 45% (scheme 21).

O CrCl3, CHI3, Zn, Me3SiCl, NaI H I 94 dioxane, 25° C 95 45%

Scheme 21. Alternative Takai reaction.

The third step of the synthesis is the conversion of iodotetradecene into the organolithium reagent. Initially the idea thought to a Grignard reagent to generate the organocerium species. We tried several time to perform the reaction in the scheme 22 but in every test we

- 82 - had negative out comes as the only product an allene deriving from the reaction between two iodotetradecenes. The presence of this compound was detected by GC and NMR analysis; the most plausible explanation for this result is the increased reactivity of the Grignard reagent due to the presence of the double bond and Iodine.

OH

O C13H27 O Desired N product H O Boc Boc N 97 Mg 91 C13H27 C H I 13 27 MgI THF 95 99 CeCl3 THF, -78°C

C13H27 Byproduct obtained C13H27 100

Scheme 22. Test with Grignard reagent.

So we tried the preparation of organolithium compound following the concept seen in chapter three about the reactivity of organolithium and Grignard reagent. In particular is highlighted the better nuclophilicity and low basicity of RLi/CeCl 3 complexes with respect to the RMgX/CeCl 3 complexes. In the scheme 23 is shown the reaction that we performed and the best condition; the product obtained was immediately used in the following step. [78]

n-BuLi I Li Ether, -78°C 95 96

Scheme 23. Formation of organolithium compound.

The fourth step is the rapid reaction of the organolithium compound with anhydrous CeCl 3 in THF at -78°C to generate organocerium reagent. The subsequent addition of Garner-

- 83 - type aldehyde to the organocerium species allowed us to obtain the all-protected sphingosine. [70] In order to reach the best condition we tried with different dilutions and different amount of organolithium and CeCl 3. The results showed that: (i) if the solution of organolithium is slightly diluted, the reaction lead to byproducts but is no trace of the desired one; (ii) the same situation appears if we perform the reaction with too diluted solution of RLi; (iii) an intermediate dilution and 3 equivalents of organolithium and CeCl 3 for each equivalent of aldehyde, leads to the formation of the desired product. As we can see in the scheme 24, the reaction leads to a diastereoisomeric mixture of product; we in fact obtein anti and syn adducts. Through the study of 1H and 13 C NMR spectra and using the comparative method, we determined that the diastereoselectivity is 4:1 in favor of the anti product. We were also able to separate the diastereoisomeric mixture by flash column chromatography. This is also due to the presence of bulky protecting groups on the Garner- type aldehyde, which as we saw in chapter 3 provide a selective attack on the Si face and leads to anti diasteroisomer.

OH O O H O N N 97 Boc Boc

Li 91 + CeCl 96 3 OH THF, -78°C O N Boc 98

d.r.= 80:20

Scheme 24 . Synthesis of protected sphingosines.

- 84 -

4.6 Part three: synthesis of C6 (L)-erythro-ceramide

This part of the total synthesis consists in the deprotection of sphingosine and its subsequent N-acylation using hexanoyl chloride (scheme 25)

OH OH HCl 1 M O THF, 70°C OH N NH 97 Boc 10h 14 2

O MgO Cl THF/H2O 101

OH (E) (R) (S) OH NH

O 77 L-erythro-ceramide C6

Scheme 25. Synthesis of L-erythro -ceramide.

The first step of this sequence is the deprotection of the amino and hydroxyl group of molecule 97 in order to generate the L-erythro -sphingosine. This reaction employs hydrochloridric acid 1 M in THF at 70°C. The strong acid accompanied with the high temperature provides to remove both protecting group avoiding an additive step for the deprotection one by one of each protecting group. We also tried the deprotonation under acis condition using HCl 2 M I MeOH. Unfortunately we could deprotected only the more labile N,O-acetal.

OH OH HCl 1 M O THF, 70°C OH N NH 97 Boc 10h 14 2

Scheme 26. Deprotection of sphingosine.

- 85 -

The last step of the our total synthesis is the N-acylation of the amino group which lead to the formation of L-erythro -ceramide C6. The reaction is conducted in THF/H 2O 3:1 with addition of 5 eq. MgO and 2,2 eq. of hexanoyl chloride. [80] In this reaction is really important to control the temperature, which must be maintained under that is better not exceed 20°C in order to avoid the O-acylation. [ 81]

O OH OH (E) Cl (R) 101 (S) OH OH NH 14 NH2 MgO THF/H O O 2 77

L-erithro-ceramide C6

Scheme 27. Acylation of amino group.

- 86 -

4.7 Total synthesis of L-erythro-ceramide C6

The result of all the tests and the speculations reported in the previous chapters, we was the first total synthesis of L-erythro -ceramide C6, starting from the natural aminoacid (L)- serine. The utility of retrosynthetic analysis helped us to find the right intermediate and the best reactions conditions to reach our goal. Our convergent total synthesis could be also an attractive process from the industrial point of view due to the choise of inexpensive starting material and the efficiency and reproducibility of all the steps reported in the scheme 28. It report the synthesis which is composed by 9 steps plus 3 additional steps for the formation of the linear alkyl chain. The overall yield of the synthesis is 40%.

O TBSO OH O TBSO O TBSO TBSO HO TBSCl NaBH 4 87 Boc O 1eq imidazolo EtOH O 2 TBAF OCH3 OCH3 OH O O HN I2, NH CH2Cl2 PTSA N THF N 2 NH2 NH2 Solvent free Boc Boc benzene 84 85 86 88 89 90

NEt3 DMSO SO3pyr CH2Cl2 O O PCC CrCl2, CHI3 n-BuLi C13H27 OH C H C H + H O C H H 13 27 13 27 Li 13 27 I Ether, -78°C N 93 CH2Cl2 THF, 0° C Boc 94 95 96

91

CeCl3 THF, -78°C

OH OH O (E) OH C13H27 O C H (S) Cl HCl 1 M 13 27 (R) OH 101 N NH C13H27 OH Boc MgO THF, 70°C NH2 10h 97 O THF/H2O 14 77

+

OH

C13H27 O N Boc 98

Scheme 28. Total synthesis of L-erythro -ceramide C6.

- 87 -

4.8 Conclusion and future project

In conclusion, in this thesis work performed in the research group of Prof. Marcantoni, we reached successfully a brilliant first total synthesis of L-erythro -ceramide C6. This could be a very useful method to synthesize this important small molecule with high biological activity. Our purpose is to employ the L-erythro -ceramide C6 in biological tests, in order to discover other potentialities in addition to those yet reported in the literature. Moreover from the previous analysis it has emerged the notable activity of natural ceramide C6 (see paragraph 4.2) and the test that we want to perform will be critical to find out if the enantiomer interacts in the same way of the natural one and with the same receptors. Another project for the future is a structural modification moving the double bond on C3- C4 and the hydroxyl group on the C5 atom, in order to study the relationship between double bond position and interaction with the receptors. This modification is relevant from the synthetic point of view, because it will be employed the Wittig reaction, one on the most powerful reaction in organic synthesis, that will allow the effective formation of a new carbon-carbon double bond. The scheme 29 shows the synthetic pathway for this future project. Starting from Garner-type aldehyde 91 , it will react with triphenylphosphoranylidene acetaldehyde phosphonium salt, to give the product 103 .

Again it react with an organolithium or the Grignard reagent in the presence of CeCl 3 in order to produce a compound 104 . After the deprotection will get 105 a potential biologically active analogue of L-erythro -sphingosine.

O O OH C13H27MgBr Ph3P O or H H C H O 102 O C13H27Li 13 27 O N N N Boc Boc Boc CeCl3 THF, -78°C 91 103 deprotection 104

OH

C13H27 OH NH2 105

Scheme 29. Future project.

- 88 -

5 EXPERIMENTAL SECTION

5.1 Instrumentation

All the reaction were monitored by thin layer chromatography silica gel Merck Kieselgel 60 F254 and gas chromatography. The instrument employed is a gas chromatograph 6850 Agilent Technologies, with a capillary column (0.32mm x 30m) and stationary phase HP-1 Agilent of 0.40-0.45 m thickness.

Separation and purification of compounds were realized by column flash chromatography on silica gel Merck (0.040-0.063mm).

Characterization of the products was carried out by mass spectra, IR spectra and Nuclear Magnetic Resonance spectra (NMR) of 1H and 13 C.

Mass spectra were obtained through the serial work of a gas chromatograph and mass spectrometer: Hewlett-Packard GC/MS 6890N. The mass spectrometer uses the EI ionization mode with an electronic beam of 70eV.

IR spectra were obtained by using a Perkin-Elmer 1310 spectrometer operating in the range 4000-600 cm -1 with NaCl as the transmitting material and solid state IR spectra were carried out by a Perkin-Elmer 100 solid state FT-IR spectrometer.

NMR spectra were acquired by using a magnetic resonance spectrometer Varian Mercury Plus 400, operating at 400 MHz for 1H and 100 MHz for 13 C. Chemical shifts are expressed in δ (ppm) compared to the signal of the residual solvent.

- 89 -

5.2 Synthetic Procedures and Characterization of Products

5.2.1 Protection of hydroxyl group

OH O TBSCl TBSO O imidazolo OCH3 OCH3 CH2Cl2 NH2 NH2 84 85

In a three-necked round-bottomed flask equipped with condenser, thermometer, magnetic stirrer and under N 2 flow, were placed 5.27g (0034 mol) of the methyl ester of (L)-serine

(84 ) and 10.69g (0.075 mol) of TBSCl, and dissolved in 40 ml of dry CH 2Cl 2. Through the aid of an ice bath the temperature was lowered to 0°C; when the mixture inside the flask had reached the desired temperature were added 7.98 g of imidazole (0.115 mol) in several portions. Finished the addition the reaction proceeds at room temperature for 48h. After disappearance of the starting material a solution of 2N HCl was added to the mixture until the pH = 5.

The product was extracted with CH 2Cl 2 (6 × 50 ml), the organic fractions collected in a flask and washed with brine (2 × 20 ml) by means of a separating funnel. The organic phase was then dried over Na 2SO 4, filtered and pulled in a rotavapor. The product 85 was a yellow oil used without further purification.

- 90 -

Characterization of product 85

TBSO O

OCH3 NH2 85

(S)-methyl 2-amino-3-(tert -butyldimethylsilyloxy)propanoate m.w. 233 g/mol

GC analysis: 10.8 min. MS (EI, 70eV) m/z: 218, 176, 158, 144, 116, 102, 89, 73, 59.

- 91 -

5.2.2 Reduction of the ester

TBSO O NaBH4 TBSO EtOH OCH3 OH NH2 NH2 85 86

In a three-necked round-bottomed flask equipped with condenser, thermometer, magnetic stirrer and under a stream of N2 were dissolved 7.92 g (0.039 mol) of the methyl ester 85 in

150 ml of absolute EtOH and 5.9 g of NaBH 4 were added. Through an oil bath, the mixture was warmed to 35°C. The reaction was left under stirring for about 5h and, once the starting material finished, the mixture was treated with approximately 40 ml of solution of a phosphate buffer (citric acid /phosphate) at pH = 3 to allow the conversion of the ester boronate 92 (see section 4.3). The product was extracted with CHCl 3 (6 × 50 mL) and dried over Na 2SO 4. The purification was carried out by flash chromatography using as eluent cyclohexane-ethyl acetate-ethanol in the ratio 6:3:1. 6.55 g of product 86 were obtained as colorless oil, with a yield of 94%.

Characterization of product 86

TBSO

OH

NH2 86 (R)-2-amino-3-(tert -butyldimethylsilyloxy)propan-1-ol m.w. 205 g/mol

GC analysis: 10.45 min. IR: 3449, 3352, 1648, 1389 1361 cm -1. MS (EI, 70eV) m/z: 174, 158, 148, 131, 119, 101, 75, 60, 30. 1 H-NMR (400 MHz, CDCl 3): δ=0.02 (s, 6H), 0.90 (s, 9H), 2.27 (br s, 3H), 2.99 (t, J=4.9 Hz, 1H), 3.53 (dd, J=10.7, 6.0 Hz, 1H), 3.58-3.68 (m, 3H).

- 92 -

5.2.3 Protection of amino and hydroxyl group with cyclohexanone

O TBSO TBSO O 87 PTSA OH HN NH2 benzene 86 88

In a three-necked round-bottomed flask equipped with condenser, thermometer and magnetic stirrer were placed 0.150 g of amino-alcohol 86 , dissolved in about 20 ml of dry benzene and the system was equipped with Dean-Stark trap, keeping the environment under N 2 flow. Then 1.2 equivalents of cyclohexanone and PTSA in catalytic amounts (0.002 g) were added. The reaction was left under stirring for about 4h and the disappearance of the starting material was monitored by GC. 25 ml of H2O were added approximately to the reaction mixture and it was extracted with CH 2Cl 2 (4 × 25 mL). The organic phases were combined and washed with NaHCO 3 (2 × 25 ml) and brine (2 × 20 mL). The organic phase was dried over Na 2SO 4 and the solvent was removed by rotavapor. Because of the decomposition of product on silica gel, the purification of 88 was carried out via Kugelrohr distillation, removing the excess of cyclohexanone. The product as a cololess oil was obtained with a yield of 83%.

- 93 -

Characterization of product 88

TBSO

O HN

88 (R)-3-[( tert -butyldimethylsiloxyl)methyl]-1-oxa-4-azaspiro[4.5]decane m.w. 285 g/mol

GC Analysis: 14.5 min IR: 3296, 2932, 2360, 1448, 1362, 1254, 1063 cm -1 MS (EI, 70eV) m/z: 285 [M +], 256, 242, 228, 172, 140, 123, 73. 1 H-NMR (400 MHz, CDCl 3): δ=0.05 (s, 6H), 0.88 (s, 9H), 1.380-1.649 (m, 10H), 2.33 (br s, 1H), 3.41-3.47 (m, 1H), 3.55 (t, J=7.69 Hz, 1H), 3.66-3.69 (m, 1H), 3.76-3.79 (m, 1H), 3.83 (t, J=6.9 Hz, 1H).

- 94 -

5.2.4 Protection of amino group with Boc

TBSO TBSO

O Boc2O 1eq O HN N I2, Boc Solvent free

88 89

In a two-necked round-bottomed flask equipped with condenser and magnetic stirrer were added 0.200 g (0.702 mmol) of 88 , 0.153 g (0.702 mmol) of Boc 2O and a catalytic amount 0.018g of iodine (10 mol%)under solvent-free conditions at room temperature. After stirring the reaction mixture for 2.5h 10 ml of ether were added. The reaction mixture was washed with 5 ml of solution at 5% of Na 2S2O3, 10 ml of saturated NaHCO 3 and dried over

Na 2SO 4. After the filtration, the solvent was evaporated by rotavapor and the residue was purified by silica gel column with eluent hexane-EtOAc in ratio 9:1 obtaining the product 89 with a yield of 90%.

Characterization of product 89

TBSO

O N Boc

89 tert -butyl-(3S)-3-(tert -butyldimethylsiloxylmethyl)-1-oxa-4-azaspiro[4,5]decane-4- carboxylate m.w. 385,61 g/mol

GC Analysis: 14.83 min

- 95 -

5.2.5 Deprotection of the alcoholic group

TBSO HO

O O N TBAF 1M Boc N THF Boc

89 90

In a two-necked round-bottomed flask equipped with condenser and magnetic stirrer were added 3,45 g (8,96 mmol) of 89 , 150 ml of dry THF and 13 ml (10,75 mmol) of solution 1

M of TBAF in THF. After stirring for 2 h, 50 ml of H 2O were added, and the reaction mixture was extracted with EtOAc (4 x 30 ml) and the organic phase was dried with

Na 2SO 4. The solvent was evaporated by rotavapor and the crude of reaction was purified by silica gel column chromatography with eluent cyclohexane-EtOAC in ratio 6:4 , to give the alcohol 90 in a 93% of yield.

Characterization of product 90

HO

O N Boc

90 tert -butyl-(3S)-3-(hydroxymethy)-1-oxa-4-azaspiro[4,5]decane-4-carboxylate m.w. 271,35 g/mol

GC analysis: 12.705 min m.p. 104°C IR (neat): 3477, 1667, 1295, 1276 cm -1 1 H-NMR (400 MHz, CDCl 3): δ = 1.00-1.80 (m,17H), 1.85-2.45 (m, 2H), 2.74 (br s, OH, 1H), 3.45-4.15 (m, 5H). 13 C-NMR (100 MHz, CDCl 3): δ = 22.6, 23.4, 25.7, 28.6, 31.8, 35.8, 45.0, 63.3, 65.8, 95.7, 98.6, 155.0.

- 96 -

5.2.6 Oxidation of the alcoholic group to aldehyde

HO O NEt3, DMSO, H O SO pyr O N 3 N Boc Boc CH2Cl2

90 91

In a three-necked round-bottomed flask equipped with condenser, thermometer, magnetic stirrer and under N 2 flow, were placed 1g (3.7 mmol) of alcohol 90 , 25 ml of CH 2Cl 2 and freshly distilled 3,61 ml (25,9 mmol) of Et 3N and 3.67 ml (51.8 mmol) of DMSO and the solution was cooled until 0°C. SO 3py complex was added portionwise and the reaction mixture was stirred at the same temperature for 15 min. Then the solution was allowed to warm to room temperature, stirred for 2,5 h, diluted with CH 2Cl 2 (4 x 60 ml). The combined organic phase extracts were treated with H2O (2 x 35) and brine (2 x 20 ml), dried over Na 2SO 4 and concentrated. The crude was purified by column chromatographic with eluent cyclohexane-EtOAc 9:1 and the aldehyde 91 , was obtained with a yield of 83%.

- 97 -

Characterization of product 91

O

H O N Boc

91 tert -butyl-(3S)-3-formyl-1-oxa-4-azaspiro[4,5]decane-4-carboxylate m.w. 269,34 g/mol

GC analysis: 11.911 min. 25 [α]D +65.6 ( c 1.57, CHCl 3). IR (neat): 2863, 1738, 1709, 1452, 1365 cm -1 1 H-NMR (400 MHz, CDCl 3): δ = 1.01-1.78 (m,17H), 1.95-2.55 (m, 2H), 3.92-4.35 (m, 3H), 9.53 (s, 1H), 9.58 (s, 10 H). 13 C-NMR (100 MHz, CDCl 3): δ = 23.4, 24.7, 25.1, 28.5, 31.6, 32.4, 34.3, 35.3, 63.6, 64.1, 64.9, 81.3, 96.3, 151.3, 200.2. MS (EI, 70eV): m/z = 269 [M +], 240, 213, 196, 184, 140,126, 96, 57, 41, 29.

- 98 -

5.2.7 Oxidation of tetradecanol

O OH PCC H CH2Cl2 93 94

In a two-necked round-bottomed flask equipped with condenser and magnetic stirrer were placed 1.5 g (7 mmol, 1.5 eq.) of PCC and 25 ml of CH 2Cl 2 in order to have a stirred suspension. 1 g (4.67 mmol) of alcohol was added in one portion at rt. After 2 h the mixture was diluted with 40 ml of ether, and filtered through a pad of Florisil, filtrate was concentrated, dissolved in ether (50 ml), and filtered again through a pad of silica. The filtrate was concentrated to afford the compound 94 as a colourless oil with 88% of yield which was used without further purification.

Characterization of product 94

O

H 94 Tetradecanal m.w. 212 g/mol

GC analysis: 10.995 min. 1 H-NMR (400 MHz, CDCl 3): δ = 0.873 (t, J= 6.4 Hz, 3H), 1.27 (d, J= 16.67 Hz, 20 H), 1.639 (t, J= 7.3 Hz, 2 H), 2.391-2.433 (m, 2 H), 9.758 (s, 1 H) 13 C-NMR (100 MHz, CDCl 3): δ = 14.346, 22.307, 22.916, 29.229, 29.389, 29.581, 29.653, 29.803, 29.862, 29.892, 32.143, 44.153, 203.198. MS (EI, 70eV): m/z = 212 [M +], 194, 168, 152, 138, 124, 110, 96, 82, 57, 41, 29.

- 99 -

5.2.8 Takai olefination

O CrCl2, CHI3 H I 94 THF, 0° C 95

In a two-necked round-bottomed flask equipped with condenser and magnetic stirrer were placed 0.348 g (2.83 mmol, 6 eq.) of CrCl 2 and gently flame-dried under high vacuum.

Upon cooling, the vacuum was released under N 2 atmosphere and the flask was charged with 5 ml of dry THF. The slurry was stirred for 15 min to prevent the aggregation of the

CrCl 2. In another flask containing 0.100g ( 0.47 mmol) of 94 were added 10 ml of THF dry followed by 0.372 g (0.94 mmol, 2 eq.) of CHI 3. This resulting mixture was added dropwise by syringe to the CrCl 2 slurry at 0°C. The reaction was stirred at the same temperature for 3h and the color change from dark green to dark red. Once the reaction finished 25 ml of H 2O were added and the mixture was extracted with ether. The organic layer was collected and washed with Na 2S2O3, brine and dried over Na 2SO 4. The solvent was removed under vacuum by rotavopor and the crude product was purified by silica gel column chromatography using hexane as eluent. The product 95 was obtained as colorless oil in 93% of yield.

- 100 -

Characterization of product 95

I 95 (E)-1-iodopentadec-1-ene m.w. 336 g/mol

GC analysis: 13.567 min. 1 H-NMR (400 MHz, CDCl 3): δ = 0.880 (t, J= 6.4 Hz,3 H), 1.254 (br s, 22 H), 2.013-2.137 (m, 2 H), 5.965 (d, J=14.478 Hz, 1 H), 6.165 - 6.544 (m, 1 H). 13 C-NMR (100 MHz, CDCl 3): δ = 14.355, 22.922, 28.584, 29.158, 29.589, 29.770, 29,880, 32.151, 33.430, 34.058, 36.279, 74.463, 147.155. MS (EI, 70eV): m/z = 336 [M +], 295, 280, 266, 252, 238, 223, 209, 195, 182, 166, 125, 111, 97, 83, 69, 55, 41, 29.

- 101 -

5.2.9 Formation of organolithium

n-BuLi I Li Ether, -78°C 95 96

In a three-necked round-bottomed flask equipped with condenser, thermometer, magnetic stirrer and under N 2 flow were introduced 0.749 g (2.23 mmol, 1 eq.) of 95 and 40 ml of ether and the mixture was cooled to -78°C. Reached this temperature were added 1.5 ml of solution 1.6 M of n-BuLi and the mixture was stirred for 40 min. The product was immediately used in the following reaction without any type of characterization or purification.

5.2.10 Addition of the organocerium to aldehyde

OH O O H O N N 97 Boc Boc

Li 91 + CeCl 96 3 OH THF, -78°C O N Boc 98

In a three-necked round-bottomed flask equipped with condenser, thermometer, magnetic stirrer and under N 2 flow were introduced, previously quickly and finely ground to a powder in a mortar, 0.830 g (2.23 mmol, 3 eq.) of CeCl 3. The flask was immersed in an oil bath and heated gradually to 135-140°C under vacuum. The powder was heated without stirring for 1 h, and then it was stirred at the same temperature for an additional hour to be dried completely. While the flask was still hot, N 2 was introduced, and the powder was

- 102 - cooled in an ice bath. 50 ml of freshly distilled THF were added and after the removal of the ice bath, the suspension was allowed to stir overnight under N 2 at rt. The flask was cooled at -78°C and 0.749 g (2.23 mmol, 3 eq.) of 96 were added via canula and stirred for 1 h. It was added dropwise the solution of 0.100 g (0.74 mmol) of aldehyde 91 in 4 ml of THF dry, and the mixture was strirred for 3 h at -78°C. 15 ml of saturated solution on

NH 4Cl were added to the mixture, and the aqueous phase was extracted with ether. The collected organic phase was washed with 20 ml of NaHCO 3, 20 ml of brine and dried over

MgSO 4. The crude diastereomers were separated and purified by column chromatography with cyclohexane-EtOAc in ratio 95:5 as a eluent. The two diastereomers 97 and 98 obtained were both yellow oils and the stereoselectivity was 4:1 anti-syn with a total yield of 79%.

Characterization of product 97

OH

O N Boc 97

m.w. 479 g/mol

1 H-NMR (400 MHz, CDCl 3, -50 °C): δ = 0.83 (t, J= 6.8 Hz, 3 H), 1.19 (br s 20 H), 1.31 – 1.71 (m, 17 H), 1.99 (br s, 2 H), 2.11 – 2.24 (m, 1 H), 2.33 – 2.46 (m, 1 H), 3.74 – 3.98 (m, 3 H), 4.73 – 4.74 (m, 1 H), 5.64- 5.73 (m, 1 H), 5.38 – 5.43 (m, 1 H). 13 C-NMR (100 MHz, CDCl 3, -50 °C): δ = 14.43, 22.77, 22.92, 23.52, 23.66, 24.74, 25.23, 25.87, 27.14, 27.24, 27.38, 28.54, 28.63, 29.18, 29.36, 29.45, 29.59, 29.74, 29.83, 29.91, 32.15, 32.63, 34.29, 34.89, 35.31, 37.71, 42.21, 62.35, 63.59, 64.15, 64.90, 73.74, 74.48, 75.17, 95.98, 98.94, 128.52, 133.60, 200.15 excess of carbon atom due to the coexistence of rotamers of the carbamate.

- 103 -

Characterization of product 98

OH

O N Boc 98

m.w. 479 g/mol

1 H-NMR (400 MHz, CDCl 3, -50 °C): δ = 0.83 (t, J = 6.8 Hz, 3H), 1.18 (br s m 20 H), 1.30- 1.63 (m, 17 H), 1.92 – 2.05 (m, 2 H), 2.16-2.33 (m, 2 H), 3.79 – 4.05 (m, 3 H), 5,06 – 5.07 (m, 1 H), 5.29 – 5.35 (m, 1 H), 5.68 – 5.75 (m, 1 H). 13 C-NMR (100 MHz, CDCl 3, -50 °C): δ = 14.29, 22.88, 23.15, 23.56, 23.66, 25.09, 28.62, 29.21, 29.41, 29.55, 29.68, 29.87, 30.23, 30.61, 31.12, 32.11, 32.53, 34.19, 35.76, 37.29, 37.63, 38.94, 62.03, 64.67, 66.99, 81.45, 95.99, 125.71, 127.13, 129.83, 135.478, 155.711, 173.714 excess of carbon atom due to the coexistence of rotamers of the carbamate.

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5.2.11 Acid hydrolysis for the deprotection of sphingosine

OH OH HCl 1 M O THF, 70°C OH N NH 97 Boc 10h 14 2

In a two-necked round-bottomed flask equipped with condenser and magnetic stirrer were placed 0.042 g (0.088 mmol) of 97 , 2 ml of 1 M HCl and 2 mL of THF. The mixture was heated at 70 °C for 10 h under nitrogen. The reaction mixture was cooled to rt and neutralized with 2 ml of 1M NaOH. The product was extracted with EtOAc (3 x 10 ml), and the combined organic layers were washed with brine and dried over Na 2SO 4. Removal of the solvent provided crude sphingosine analogue 14 as a white solid, which was used in the next reaction without further purification.

Characterization of product 14

OH

OH NH 14 2

(E,2R,3S)-2-amino octa dec-4-ene-1,3-diol L-erythr o-sphingosine m.w. 299

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5.2.12 N-acylation of sphingosine

O OH OH (E) Cl (R) 101 (S) OH OH NH 14 NH2 MgO THF/H O O 2 77

In a two-necked round-bottomed flask equipped with condenser and magnetic stirrer were added 0.026 g ( 0.088 mmol) of 14,1 ml of THF, 250 l H 2O and (THF/H 2O 3:1) and 0.017 g (0.44 mmol, 5 eq.) of MgO. The mixture was stirred vigorously for 30 minutes at 20°C, then was added dropwise in 30 min 1 ml of solution containing 0.027 ml of hexanoyl chloride 101 . It was stirred for an additional hour, and once the reaction was complete, it was filtered through a bed of Celite. The filtrate was washed with 30 ml

EtOAc and with H 2O. The organic phase was dried over MgSO 4 and the solvent was evaporated under vacuum. The crude product was purified by silica gel column chromatography with eluent CHCl 3-MeOH 99:1, to give 77 as a white solid.

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Characterization of product 77

OH (E) (R) (S) OH NH

O 77

N-((E,2R,3S)-1,3-dihydroxy octadec-4-en-2-yl)hexanamide L-erythro -ceramide C6 m.w. 397 g/mol

IR: 3604, 3416, 2927, 2854,1725, 1531, 1457, 1409,1371,1340 cm -1 1 H-NMR (400 MHz, CDCl 3): δ = 0.87 (t, J= 6.57 Hz, 6 H), 1.25 (br s, 28 H), 1.62-1.65 (m, 2 H), 2.04-2.09 (m, 2 H), 2.32-2.36 (m, 1 H), 3.72-3.76 (dd, J= 3.1 Hz,1 H), 3.87 (br s, 1 H) 3.92-3.94 (m, 1 H ) 4.11 (d, J= 11.531 Hz, 1 H), 4.39 (t, J= 5.317 Hz, 1 H), 4.50 (br s, 1 H), 5.51-5.56 (dd, J=6.4 Hz, 1 H), 5.80-5.87 (m, 1 H). 13 C-NMR (100 MHz, CDCl 3): δ = 14.35, 22.92, 29.21, 29.40, 29.59, 29.68, 29.81, 29.90, 32.15, 32.45, 54.26, 61.37, 63.15, 63.24, 64.50, 69.25, 74.40, 133.92, 134.35, 135.57, 165.67.

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