Development of a Protecting Group for Sulfate Esters.
Andrew D. Proud.
FIIJ The University of Edinburgh. II,9J C) Abstract:
This work describes the development of a protecting group for sulfate monoesters in carbohydrates. After initial trial studies, the trifluoroethyl ester was selected as a suitable protecting group. The ester was formed from the sulfate by treatment with trifluorodiazoethane. This protection method is shown to be compatible with other common protecting groups used in carbohydrate chemistry. The protecting group is proven to be stable to the manipulations of many other protecting groups. The utility of monosaccharides incorporating protected sulfate esters in synthesis has been examined. It has been proven that they are useful and versatile building blocks in the synthesis of more complex structures. Selective cleavage of the trifluoroethyl ester was achieved with potassium tert-butoxide. The mechanism for this deprotection reaction has been studied and this investigation has led to the advancement of alternative deprotection methods.
I declare that, this Thesis has been composed by m,yself and that the work is my own.
Andrew David Proud. Acknowledgements:
I would like to thank my supervisor Prof. Sabine Flitsch for her help and support throughout this project. I also wish to thank Dr. Jeremy Prodger, of GlaxoWelicome and Prof. Nick Turner of The University of Edinburgh for their many suggestions and useful advice.
I wish to acknowledge the EPSRC and GlaxoWelicome for financial support.
I wish to thank all the people that run the NMR and X-ray crystallography services at Edinburgh University but especially John Miller and Simon Parsons.
I wish to thank all those that have worked in the research groups of Prof. Flitsch, Prof. Turner and Dr. Hume, who over the last three years have become great friends and from whom I have learned so much.
I thank my family for encouraging me in this continuation of my studies and for helping me realise that there are bigger things in life than this project.
And finally I wish to thank my wife Louise for her love and care and support. This PhD project has been part of our journey together where will we go next? "It is not always realised how widespread is the occurrence of carbohydrate sulphates in Nature."
E. G. V. Percival, Quart. Rev. (1949).
"Twa birds sat on a bara, one was a spug, the other a spara."
By Wullie Shakespeare. Table of Contents.
Section One: Introduction . 8 1. Biological Background to Carbohydrates ...... 9 2. Sulfated Carbohydrates ...... 14 2.l. Heparin ...... 14 2.2. Nod Factors...... 17 2.3. Sialyl Lewis Antigens...... 20 3. Non Carbohydrate Sulfates ...... 23 3.1. Sulfated Proteins ...... 23 3.2. Sulfated Marine Natural Products...... 24 4. Synthesis of Sulfated Carbohydrates ...... 26 4.1. Sulfotransferase Enzymes ...... 26 4.2. Chemical Methods ...... 28 4.3. Selective Sulfation ...... 32 4.4. The Problem with Synthesis ...... 35 5. Sulfate Protecting Groups ...... 37 5.1. Requirements of a Sulfate Protecting Group ...... 37 5.2. Development of a Sulfate Protecting Group ...... 38 5.3. History of Sulfate Protecting Groups...... 41 5.4. Conclusion . ...... 48 Section Two: Results and Discussion ...... 49
6 . Preliminary Work...... 50 6.1. Trihaloethyl Sulfonates ...... 50 7. Development of a Synthesis of Sulfate Diesters...... 53 7.1. Chlorosulfates . ...... 54 7.2. Trifluoroethyl Chlorosulfate ...... 58 7.3. Alkylation of Sulfates ...... 59 7.4. Tnfluoroethylation of Sulfates...... 66 8. Synthesis of Protected Sugar Sulfates...... 68 8.1. Protection of Sulfated Monosaccharides...... 68 8.2. Disaccharides . 74 8.3. Conclusions...... 74 Stability of Sulfate Protecting Group ...... 75 9.1. Acid Stability ...... 75 9.2. Stability to Bases...... 76 9.3. Stability to Hydrogenation ...... 76 9.4. Fluoride Stability...... 77 9.5. Conclusions...... 77 Utility: Use in synthesis ...... 78 10.1. Glycosyl Donors ...... 79 10.2. Glycosyl Acceptors ...... 90 10.3. Conclusions...... 95 Deprotection of Protected Sulfate Diesters...... 96 11.1. Deprotection using Sodium Methoxide ...... 97 11.2. Other Bases for the Deprotection of Trifluoroethyl Esters ...... 99 11.3. Potassium tert-Butoxide for the Deprotection of Trifluoroethyl Esters.. 101 11.4. Other Attempts at Deprotection Conditions...... 103 11.5. Oxidative Cleavage ...... 104 11.6. Conclusions...... 107 Mechanistic Studies ...... 108 12.1. Simple Nucleophilic Displacements ...... 108 12.2. Mechanisms Involving Multiple Steps ...... 112 12.3. Conclusions...... 125 Conclusions...... 126 Section Three: Experimental...... 128 General Experimental...... 129 Experimental ...... 130 SectionFour: Bibliography...... 176 Appendix1: Publications ...... 189 Appendix2: X-Ray Data...... 194 Abbreviations:
The use of abbreviations has been kept to a minimum throughout this thesis however the following have been used within the text:-
DCC Dicyclohexylcarbodiimide. DCM Dichioromethane. DMAP NN-Dimethylaminopyridine. DMF Dimethyl fomiamide. JR Infra Red Spectroscopy. LCMS Liquid Chromatography Mass Spectrometry. MS Mass Spectrometry. NBS N-Bromosuccinimide. NIS N-Iodosuccinimide. NMR Nuclear Magnetic Resonance Spectroscopy. TLC Thin Layer Chromatography. THF Tetrahydrofuran. UV Ultra Violet. Section One: Introduction.
Section One: Introduction. Section One: Introduction. 9
This thesis project forms part of the ongoing research of Professor Sabine Flitsch at The University of Edinburgh into the development of methods for the synthesis of complex oligosaccharides. This work examines chemical and enzymatic routes to naturally occurring oligosaccharides which have uncommon constituent monosaccharides, unusual linkages or interesting additional functional groups.
This project is aimed at the development of methods for the synthesis of sulfated oligosaccharides and has been specifically focused on the invention of a protecting group for sulfate monoesters. Such an invention will greatly aid further synthetic projects.
The objective of this section is to introduce a number of important and interesting sulfated oligosaccharides and through a discussion of their biology, to make some justification for the interest in such compounds. This brief biological introduction is covered in Chapters 1, 2 and 3. Chapter 4 discusses the various methods used to synthesis sulfated carbohydrates and why these methods are often inadequate. The section then discusses the advantages a sulfate protecting group would bring to a synthesis of sulfated compounds in Chapter 5, followed by a short explanation of the properties that such a group would need. The introductory section finishes with a short discussion of the strategy adopted in this thesis project and the problems that were foreseen.
1. Biological Background to Carbohydrates.
This section discusses the importance of carbohydrates in biology and explains the background into which the work on sulfated carbohydrates fit.
The history of carbohydrate chemistry stretches back almost to the beginnings of organic chemistry. Studies of saccharides are partly responsible for modern understanding of chirality and stereochemistry and historical figures in carbohydrate Section One: Introduction. 10 chemistry have had a pronounced influence on our current concept of molecular structure in 3-dimensional space. However the area of carbohydrate chemistry has undergone a renaissance in the last two decades due in part to the advances in glycobiology.
It was not until the 1970's that the role of carbohydrates in nature was found to extend beyond the structural role that polysaccharides, such as cellulose and starch, play in plants. Up until this time it was known that carbohydrates played an essential part in energy storage and transport but they were not thought to have any real role in biological processes.
"Polysaccharides seem at the moment to be less spectacular substances than proteins and nucleic acids. They are, however, of universal importance in living matter and significant developments throwing light on structure-function relations will surely emerge in the near future." Gerald 0. Aspinall (1970)'
However in the early 1980's technology became available that greatly simplified the determination of oligosaccharide sequences 2. This technological advance allowed the rapid characterisation of the carbohydrate portions of complex glycoproteins. Subsequent to this experiments were conducted to identify the importance of the carbohydrate to the function of a glycoprotein. A number of experiments then illustrated that the carbohydrate was central to the function of the glycoprotein3' 4. Glycoconjugates have now been identified as being of primary importance to several significant biological processes 5 such as mammalian fertilisation6' 7, viral replication810, parasitic infection11' 12, cell growth 13, cell-cell adhesion 14 and immune response 15. It is due to this biological significance that the study of carbohydrate chemistry is once again at the forefront of chemical research. Section One: Introduction. 11
Carbohydrates are commonly implicated in biological recognition processes. At their simplest level such processes involve the transfer of information through the recognition of one molecule by another. This molecular recognition is dependant on the 3-dimensional shape of both molecules. Therefore nature requires large repertoire of molecular shapes to differentiate between biological functions. Carbohydrates are important in nature because their versatility of structure and 3-dimensional shape means that very different shapes can be constructed from a small number of building blocks. The potential for diversity in oligosaccharides is greater than other biological polymers such as proteins and DNA/RNA because;
• There are a larger number of potential monomer building blocks (although only about ten monosaccharide units that are commonly found in mammalian glycoproteins and glycolipids 16 (see Figure 1:3). • The pattern of monomer connection has greater scope for variation (i.e. monomers may be connected between the 1 and the 4 positions or between the 1 and the 6 positions etc.). • Branching can occur at any monomer unit. • Each linkage between two monomers can occur in one of two possible configurations (either the (x or P anomer (see Figure 1:1)).