Chiral Retinoid Derivatives: Synthesis and Structural Elucidation of a New Vitamin A Metabolite Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Madalina Andreea Stefan aus Ploiesti (Rumänien) 1. Referent : Prof. Dr. Henning Hopf 2. Referentin: Prof. Dr. Monika Mazik eingereicht am: 08.05.2006 mündliche Prüfung (Disputation) am: 14.07.2006 Druckjahr 2006 Vorveröffentlichungen: Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Tagungsbeiträge: M. Stefan, H. Hopf, J. P. Schuchardt, G. Eichele, H. Nau: Synthesis and Characterization of a new vitamin A metabolite (Poster), Chemical Society of Japan, Annual Meeting, Frühjahrsymposium, Yokohama, Japan 2005 M. Stefan, H. Hopf, N. Giese, H. Nau, A New Vitamin A Metabolite: Synthesis and Characterization (Poster), ORCHEM, Bad Nauheim 2004 M. Stefan, H. Hopf, N. Giese, H. Nau, Synthesis and Characterization of a New Vitamin A Metabolite (Poster), GDCh Frühjahrsymposium, Heidelberg 2004 M. Stefan, H. Hopf, N. Giese, H. Nau, Total Synthesis of New Vitamin A Metabolite (Poster), GDCh Jahrestagung, München 2003 Die vorliegende Arbeit wurde in der Zeit von August 2002 bis September 2005 am Institut für Organische Chemie der Technischen Universität Braunschweig unter der Leitung von Prof. Dr. H. Hopf angefertigt. I wish to express my sincere gratitude to Prof. Dr. Henning Hopf for his supervision, support and encouragement throughout this research. Without his advice and endless interest, this study could not have been carried out and completed. Personally and scientifically I have developed myself learning from his advices and experience. I am very grateful to Prof. Y. Okamoto for the research opportunity he gave me during my visit in Nagoya, Japan. I also wish to thank him for his advice, ideas, valuable discussions and remarks. I thank Prof. H. Nau and his co-workers J.-P. Schuchardt and N. Giese for their cooperation and valuable discussions during this research project. I offer my sincere thanks to Ms. P. Holba-Schulz for high resolution and 2D NMR spectra; Ms. K. Kadhim for UV and IR spectra; Dr. U. Papke and Ms. D. Döring for MS spectra, Dr. T. Beuerle for GC/MS analyses; Dr. J. Grunenberg for his supervision on the molecular modeling studies. I am very grateful to Prof. Dr. L. Ernst for his discussions and support during assignment of the isomers of 11 by NMR spectra . I am thankful to Prof. M. Mazik for agreeing to be the co-referee of my thesis. I thank Dr. U. Jahn for his advices and to his group and all the group members of Prof. Dr. H. Hopf for the great moments spend together at the Institute of Organic Chemistry. My special thanks is going to Dr. Heino Hinrichs, Emanuela Dinca, Susanne Kritsch, Dr. Jens Reichwagen and Harald Berger. I also thank all the members of the Okamoto group who made my time in Nagoya to be a great experience. Dedicated to my parents, my brother and to Heino Table of Contents 1 Introduction .............................................................................................................1 1.1 Biological Importance of Vitamin A Derivatives .............................................3 1.2 Aim of this Work...............................................................................................6 2 Retrosynthesis........................................................................................................11 3 Syntheses ................................................................................................................14 3.1 β-Ionone ( 22 ) and 4-Oxo-β-ionone ( 23 ) as Starting Materials .......................14 3.2 Synthetic Approach to 13,14-Dihydro-retinoids by Horner-Wadsworth- Emmons Reaction. Retrosynthetic Path a ...................................................................14 3.3 Synthesis of Retinoic Acid Derivatives by Wittig Reaction with C 15 +C 5 Backbones. Retrosynthetic Path b...............................................................................17 3.4 Consecutive Double Bond Formation of the Retinoid Skeleton. Retrosynthetic Path c .........................................................................................................................20 3.4.1 Synthesis of β-Ionylidene-acetaldehyde ( 47 ) and 4-Oxo-β-ionylidene- acetaldehyde ( 53 ) ....................................................................................................20 3.4.2 Racemic and Enantioselective Synthesis of Phosphonium Salt 66 and its Derivatives ..............................................................................................................24 3.4.3 Synthesis of 13,14-Dihydro-retinoic Acid ( 10 ) and 4-Oxo-13,14- dihydro-retinoic Acid ( 11 ) ......................................................................................28 3.4.4 Synthesis of Modified 13,14-Dehydro-retinoids.....................................30 3.4.5 Synthesis of Labeled 13,14-Dihydro-retinoic Acids...............................31 3.4.6 Synthesis of 4-Hydroxy-retinoids ...........................................................33 3.4.7 Horner-Wadsworth-Emmons Approach .................................................35 4 Analysis of Retinoids by High Performance Liquid Chromatography............39 4.1 Separation of Retinoids by Reversed Phase HPLC.........................................39 4.2 Chiral Separation.............................................................................................45 4.3 Chiral Separation of 4-Oxo-13,14-dihydro-retinoic acid ( 11 ) and its Ester ...48 4.4 Chiral Separation of 13,14-Dihydro-retinoic acid ( 10 ) and its Ester.............. 51 4.5 Chiral Separation of 4-Oxo-13,14-dihydro-13-phenyl-retinoic acid ( 83 ) and its Ester ....................................................................................................................... 51 4.6 Chiral Separation of 13,14-dihydro-13-phenyl-retinoic acid ( 82 ) and its Ester. ........................................................................................................................ 53 4.7 Chiral Separation Mechanism......................................................................... 54 5 Spectroscopic Analysis ......................................................................................... 57 5.1 Infrared Spectroscopy..................................................................................... 57 5.2 UV/Vis-Spectroscopy ..................................................................................... 62 5.3 Mass Spectrometry ......................................................................................... 68 5.4 NMR Spectroscopy......................................................................................... 74 5.4.1 4-Oxo-13,14-dh-RA ( 11 )........................................................................ 74 5.4.2 13,14-Dihydro-RA ( 10 ) .......................................................................... 81 6 Circular Dichroism............................................................................................... 83 6.1 Introduction..................................................................................................... 83 6.2 Experimental Determination of Circular Dichroism ...................................... 86 6.3 Theoretical Calculations of Circular Dichroism Spectra................................ 88 6.4 Circular Dichroism of Substituted Retinoids................................................ 100 6.4.1 CD Spectra of 75 ................................................................................... 100 6.4.2 CD Spectra of Phenyl-substituted Retinoids ........................................ 102 7 Receptor-Ligand Interactions of Vitamin A Metabolites ............................... 106 7.1 Possible Metabolization Pathway of ROH to the New Vitamin A Metabolite .. ...................................................................................................................... 106 7.2 Ligand-Protein Interactions in Retinoid Receptors ...................................... 107 8 Molecular Modeling - Proton Affinity Calculations........................................ 114 9 The New Vitamin A Metabolite: Structure and Conformation Assignment 117 10 Summary and Conclusion ..............................................................................121 11 Experimental Section ......................................................................................124 11.1 Instrumentation and general experimental considerations ............................124 11.2 General experimental procedures..................................................................125 11.3 Experimental procedures...............................................................................127 12 Abbreviations...................................................................................................183 13 References ........................................................................................................184 1 Introduction The importance of vitamin A in health and disease has been recognized relatively late in human history, if we consider that humans have been confronted with night blindness, a typical disease resulting from vitamin A deficiency, for thousands of years. The curative role of the juice of cooked liver extracts