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 - 2 - 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. - 3 - 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. - 4 - PALAVRAS CHAVE Ácidos de Lewis, Aldeído de tipo Garner, Ceramida, Esfingolípidos, Esfingosina, Moléculas biologicamente activas, Moléculas pequenas, Síntese total. - 5 - 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. - 7 - 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 - 8 - 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 - 9 - 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 - 10 - 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.