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