Synthesis of Chlorogenic Acids & Chlorogenic Acid Lactones by Birgul Surucu A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry Approved Dissertation Committee Prof. Nikolai Kuhnert Prof. Dr. Thomas Nugent Dr. Ian Cunningham Defense Date: 08 March 2011 School of Engineering and Science Department of Chemistry Campus Ring 1 28759 Bremen Germany Abstract The chlorogenic acids are a family of esters formed between certain trans -cinnamic acids and quinic acid. Chlorogenic acids are among the most abundant polyphenols, which are naturally occurring in plants, with various biological activities beneficial to human health. Chlorogenic acids are secondary metabolites that are found in plants. As a group they generally contain several subgroups and usually several isomers within each subgroup. The development of methods for the synthesis of existing chlorogenic acids and new series of chlorogenic acids were developed in this thesis in order to have the compounds available as analytical standards, for confirmation of previous and future LC/MS work, and most importantly for individual biological testing in various assays. The target compounds were obtained with two strategies 1.selective synthesis using appropriate protecting groups strategies, 2.nonnselective synthesis producing mixtures of isomers. The general strategy, which was applied through out the thesis in order to synthesise the target compounds was, as follows; • Preperation of acid chlorides of cinnamic acids • Preparation of protected quinic acid derivatives • Acylation • Deprotection A series of mono-acyl chlorogenic acids and esters, in particular of those substituted in the 1-, 3-, 4- and 5- positions of the quinic acid with coumaroyl, feruloyl, caffeoyl and dimethoxy cinnamoyl and cinnamoyl substituents, have been successfully synthesised. As intermediates in these syntheses a series of quinide esters, potential metabolites and degradation products of naturally occurring chlorogenic acids have also been obtained. The first examples of di-acyl chlorogenic acids, have been successfully synthesised. A general protection and deprotection strategy for the synthesis of chlorogenic acids has been established. Poly-acyl chlorogenic acids starting from qunic acid and quinic acid lactones also have been established. The right conditions for the synthesis of certain type of chlorogenic acids and esters have been established in this work. i “Imagination is more important than knowledge” Albert Einstein (1879-1955) ii Acknowledgements I would like to thank Prof. Nikolai Kuhnert for giving me the opportunity to work in this project and for everything he has done during my seven years study. I would like acknowledge the TOBB University and Jacobs University Bremen for funding my studies. I also greatly appreciated the feedback I received during my PhD Viva. Both of my examiners, Dr. Ian Cunningham and Prof. Thomas Nugent, provided helpful and thoughtful suggestions which improved the quality of this work’s presentation. My special thank to Prof. U. Kortz for allowing me to use his lab and instruments during my final analysis, and his students for being in the lab whenever I needed. In addition I would also like to thank Dr. M. Dickman for his time and contribution in solving the X-ray structures presented in this thesis. I would also like to thank Prof. M. Clifford for his cooperation, my co-supervisor Dr. Ian Cunningham for his help and support, Mr. Jim Bloxsidge for his excellent NMR technical support throughout my time at the University of Surrey, Ms. Judith Peters for processing my elemental analysis, Mr. Richard Chaundy at the University of Surrey and Ms. Anja Mueller at Jacobs University for running my mass spectral determinations. I would like to thank all my family for their unlimited love and support which has been only the impulse to achieve my goals, especially for believing in me always and forever. I would also like to thank “my little brother” Cihangir for being the most cheerful part of my life. Finally, I have to say 'thank-you' to all my friends wherever they are. iii Abbreviations Ac Acetyl Ac 2O Acetic anhydride AcOH Acetic acid MeCN Acetonitrile CGA Chlorogenic acids CHCl 3 Chloroform (COCl) 2 Oxalyl chloride CoQA p-coumaroylquinic CQA Caffeoylquinic acid CinCl Cinnamoyl chloride DCM Dichloromethane DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMP Dimethoxypropane DMSO Dimethyl sulfoxide Et 3N Triethylamine EtOAc Etyl acetate EtOH Ethanol FT Fourier transform MeOH Methanol IR infrared I Iodine THF Tetrahydrofuran TLC Thin-layer chromatography TMS-Cl Trimethylsilyl chloride TMSOTf trimethylsilyl trifluoromethanesulfonate TrocCl 2,2,2-trichloroethyl chloroformate PE Petroleum ether PTSA p-toluenesulfonic acid iv Abbreviations for NMR NMR Nuclear magnetic resonance COSY 1H-1H Correlation spectroscopy HMQC Heteronuclear Multiple Quantum Coherence (heteronuclear correlation spectroscopy) HMBC Heteronuclear Multiple Bond correlation NOESY Nuclear Overhauser Effect Spectroscopy bs broad signal s singlet d doublet dd doublet of doublet ddd doublet of doublet of doublet t triplet m multiplet q quartet J coupling constant Hz hertz Abbreviations for MS MS Mass Spectrometry CI chemical ionization EI electron ionization m/z mass-to-charge ratio M+ parent molecular ion HPLC High Performance Liquid Chromatography LC-MS Liquid Chromatography Mass Spectroscopy v CONTENT Chapter 1 vi Content Chapter 1 TABLE OF CONTENTS Abstract……………………………………………………………………………..... i Acknowledgements…………………………………………………………………..iii Abbrevations………………………………………………………………………....iv Contents……………………………………………………………………………....vi CONTENT ................................................................................................................... vi BACKGROUND ........................................................................................................ 11 Phenolic Phytochemicals ............................................................................................. 1 Types of polyphenols ................................................................................................... 3 Phenolic acids ............................................................................................................... 6 Hydroxybenzoic acids ................................................................................................ 6 Hydroxycinnamic acids ............................................................................................. 6 Biosynthesis of phenolic compounds in plants .......................................................... 7 Biological activity of Polyphenols ............................................................................... 9 Functions of phenolic acids in plants ....................................................................... 10 Polyphenol content in food and dietary intake ....................................................... 11 THE CHLOROGENIC ACIDS ................................................................................ 14 Natural occurrence of chlorogenic acids and dietary intake .................................... 16 Other cinnamate conjugates ..................................................................................... 18 Cinnamate transformation products ......................................................................... 21 Analytical methods for determination of chlorogenic acids .................................. 21 Functions of hydroxycinnamic acids in plants ........................................................ 24 Biosynthesis of chlorogenic acids in plants .............................................................. 25 Metabolism of chlorogenic acids and health ........................................................... 27 Mechanisms of absorption ........................................................................................ 28 Chemical synthesis of chlorogenic acids .................................................................. 30 Protection of Quinic acid ......................................................................................... 31 Carboxy protection ................................................................................................... 31 Protection of hydroxyl groups ................................................................................. 31 Reported Synthesis of chlorogenic acids in literature ............................................ 33 Synthesis of 5-caffeoylquinic acid ........................................................................... 33 1-O-p-coumaroylquinic acid .................................................................................... 33 1-O-caffeoylquinic acid ........................................................................................... 34 1-O-galloylquinic acid ............................................................................................. 34 Synthesis of 3-O-cinnamoyl and 3-O-p-coumaroylquinic ....................................... 34 1-O-Cinnamoylquinic acid ....................................................................................... 35 3-O-Cinnamoylquinic acid ....................................................................................... 35 4- and 5-O-Cinnamoylquinic acid ........................................................................... 36 Synthesis of 1-, 4- and 5-O-p-coumaroylquinic acid ............................................... 37 3-O-o-Coumaroyl, 3-O-Feruloyl and 3-O-Sinapyl