Thesis for Printing
Chemistry of Hydroxycinnamate Esters and their Role as Precursors to Dekkera Produced Off-flavour in Wine
A thesis presented in fulfilment of the requirements for the degree of
Doctor of Philosophy
Josh L. Hixson BTech (Forens&AnalytChem), BSc (Hons)
School of Agriculture, Food and Wine
March 2012
Table of Contents
Abstract ...... iv
Declaration ...... vii
Acknowledgements ...... viii
Publications and Symposia ...... xi
Abbreviations ...... xiii
Figures, Schemes and Tables ...... xvi
Chapter 1: Introduction ...... 1
1.1 General Introduction ...... 1
1.2 Dekkera/Brettanomyces bruxellensis ...... 1
1.3 Volatile Phenols ...... 5
1.3 Introduction to Tartrate Esters ...... 11
1.4 Introduction to Glucose Esters ...... 16
1.5 Introduction to Ethyl Esters ...... 18
1.5 Research Aims ...... 20
Chapter 2: Synthesis of Hydroxycinnamoyl Esters ...... 22
2.1 Synthesis of Hydroxycinnamic Acids and Derivatives ...... 22
2.2 Synthesis of Hydroxycinnamoyl Tartrate Esters ...... 24
2.2.1 Introduction to Tartrate Ester Synthesis ...... 24 2.2.2 Synthesis of Hydroxycinnamoyl Tartrate Esters ...... 26
2.3 Synthesis of Hydroxycinnamoyl Glucose Esters ...... 34
2.3.1 Introduction to Glucose Ester Synthesis ...... 34 2.3.2 Synthesis of Hydroxycinnamoyl Glucose Esters ...... 37
2.4 Conclusions ...... 47 i
Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters ...... 50
3.1 Introduction...... 50
3.2 Research Aims ...... 54
3.3 Theoretical Studies into Acyl Migration of Hydroxycinnamoyl Glucoses ...... 55
3.3.1 Thermodynamics of Migration ...... 55 3.3.2 Kinetics of Migration ...... 60
3.4 Liquid Chromatography of Wine...... 67
3.5 Conclusions...... 76
Chapter 4: Photoisomerisation of Hydroxycinnamic Acids ...... 79
4.1 Introduction...... 79
4.1.1 Hydroxycinnamate Photoisomerisation ...... 79 4.1.2 cis -Hydroxycinnamate content in grapes and wine ...... 81 4.1.3 Enzymatic Specificity ...... 83
4.2 Research Aims ...... 86
4.3 Synthesis of cis -Hydroxycinnamic Acids...... 87
4.4 Theoretical Studies into the Isomerisation of Hydroxycinnamic Acids ...... 91
4.5 Conclusions ...... 105
Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis ...... 107
5.1 Bioconversion of trans -Hydroxycinnamate Esters ...... 107
5.1.1 Ethyl Esters ...... 107 5.1.2 Ethyl Esterase Substrate Selectivity ...... 110 5.1.3 Tartrate Esters ...... 111 5.1.4 Glucose Esters ...... 113 5.1.5 Conclusions for Chapter 5.1 ...... 114
5.2 Stereoselectivity of D. bruxellensis Enzyme Activities ...... 115
5.2.1 Decarboxylase Stereoselectivity ...... 115
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5.2.2 Ethyl Esterase Stereoselectivity ...... 121 5.2.3 Conclusions for Chapter 5.2 ...... 125
5.3 Thesis Conclusions and Future Directions ...... 126
Chapter 6: Experimental ...... 130
6.1 General Experimental ...... 130
6.2 Experimental Procedures for Chapter 2 ...... 133
6.2.1 Hydroxycinnamoyl Derivatives ...... 133 6.2.2 Synthesis of Hydroxycinnamoyl Tartrate Esters ...... 142 6.2.3 Synthesis of Hydroxycinnamoyl Glucose Esters ...... 157
6.3 Experimental Procedures for Chapter 3 ...... 172
6.4 Experimental Procedures for Chapter 4 ...... 175
6.5 Experimental Procedures for Chapter 5 ...... 180
6.5.1 General Procedures for Chapter 5 ...... 180 6.5.2 Fermentation of trans -Hydroxycinnamate Esters ...... 184 6.5.3 Stereoselectivity of D. bruxellensis Enzyme Activities ...... 184
Appendix 1: Data for Migration Thermodynamics ...... 186
Appendix 2: Data for Migration Kinetics ...... 188
Appendix 3: Data for Energy Profiles ...... 190
Appendix 4: Data for Vertical Excitations and HOMO-LUMO Gaps ...... 192
Appendix 5: Data from Ethylphenol Analyses ...... 196
References ...... 198
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Abstract
The potential for malodour in wine caused by the accumulation of ethylphenols has been widely studied with respect to the breakdown of the hydroxycinnamic acids, p-coumaric and ferulic acid, by D. bruxellensis . The presence of esterified hydroxycinnamate conjugates in grapes and wine is well established and they account for a large proportion of the hydroxycinnamate content. There exists the possibility that these conjugates could also provide the potential for spoilage, though they have never been linked to the direct formation of ethylphenols. The research highlighted within this thesis examines the potential role of a number of esterified conjugates in the production of ethylphenols by D. bruxellensis . Two classes of berry derived esters, the tartaric acid and glucose bound hydroxycinnamates, as well as the vinification formed ethyl esters, were synthesised and used for model fermentation experiments.
Chapter 2 describes the preparation of a number of protected hydroxycinnamic acid derivatives that were used in the synthesis of the hydroxycinnamoyl tartrate esters ( 7 and 8) for the first time. Coupling 1-O-chloroacetyl protected p-coumaric and ferulic acids ( 21 and 22 ) with di-tert -butyl-L-tartrate ( 34) followed by selective hydrolysis of the tert -butyl esters yielded p-coumaroyl tartrate ( 7) and feruloyl tartrate ( 8). Hydroxycinnamoyl glucose esters ( 9 and 10 ) were prepared using the same hydroxycinnamates ( 21 and 22 ), esterifying with a prepared trichloroacetimidate glucosyl donor sequence, though purification of the glucose esters resulted in undesired chemical transformations. It was found that photoisomerisation of the glucose esters could be prevented via synthesis under red light, which gave trans -9 and 10 , however migration of the hydroxycinnamoyl moiety around the glucose ring, which yielded mainly the 2-O-α- and 6-O-α-esters, was a product of submitting the esters to non-aqueous solvents and could not be avoided.
The acyl migration of the glucose esters that was observed in Chapter 2 has been researched at a DFT B3LYP 6-31G* theoretical level in Chapter 3 with respect to both the thermodynamics and kinetics of the transformations. The desired 1-O-β-esters were thermodynamically favoured only in water, while in any other solvent studied the 2-O-α- and 6-O-α-esters would prevail. Kinetically, migration to the 3-O-position involved lower energy barriers which can be equated to a more rapid process, although the ring-flipped
iv conformation needed to achieve the migration would promote subsequent migration to the 6-O-position. Step-wise migration, from the 1-O- to the 2-O-position, was found to be thermodynamically less favoured than other migrations investigated. This effect can be rationalised by the formation of a 5-membered cyclic intermediate in comparison to the 6- membered intermediate produced during 1-O- to 3-O-migration. However, the energy barriers involved in 1-O-β- to 2-O-β-migration better explain the comparative extent of migration observed between the p-coumaroyl and feruloyl glucose esters. The possibility of multiple glucose esters existing in wine was the focus of a brief study, finding two separate p-coumaroyl glucose esters in red and white wine, while a lesser extent of migration in feruloyl glucose limited observation to concentrated wine alone. However, due to co-elution of feruloyl glucose ( 10 ) with suspected p-coumaroyl anthocyanin derivatives in red wine, HPLC-MRM was required to detect it, which is the first report of this compound in red wine.
Theoretical studies into observed photoisomerisations and the synthesis of cis - hydroxycinnamates are described in Chapter 4. The cis -ethyl hydroxycinnamates were isolated and hydrolysed to give a mixture of cis/trans -hydroxycinnamic acids ( 3 and 4), which could be separated by flash chromatography, though the pure cis -isomers isomerised rapidly under ambient conditions and slowly under red light back to the trans -isomers. Stable isomeric mixtures were achieved by irradiation with ultra-violet light giving mixtures of 40-50% of the cis -isomer which could be used further in fermentation studies. Computational evidence suggested that isomerisation of the hydroxycinnamic acids was favoured with greater resonance throughout the molecule. Those with deprotonated phenolic moieties possessed the most intramolecular electron movement, decreasing the HOMO-LUMO gap and promoting photoisomerisation. Smaller solvent and substrate effects were also noted, though the nature of the phenol and carboxyl clearly played the most important role in determining stability of each isomer.
Fermentation in the presence of the synthesised trans -hydroxycinnamoyl esters ( 7-12 ) and investigation into the stereospecificity of D. bruxellensis enzyme activities was performed as detailed in Chapter 5. In Australia, three genetic groups of D. bruxellensis account for 98% of isolates, with the largest of these groups making up 85%. AWRI 1499 is a representative of the largest genetic group, with AWRI 1608 and AWRI 1613 belonging to the two remaining significant genetic groups. In the presence of AWRI 1499, the trans - v ethyl esters ( 11 and 12 ) were metabolised to varying extents with the preference for breakdown of ethyl coumarate ( 11 ) over ethyl ferulate ( 12 ). This selectivity was investigated further and found to be common for both AWRI 1499 and AWRI 1608, while AWRI 1613 was unable to breakdown either ester. The preference for formation of 4- ethylphenol ( 1) over 4-ethylguaiacol ( 2) from the ethyl esters could accentuate the ratio of these compounds as seen in wine, initially thought to be brought about by the relative concentration of the precursor acids.
Of the berry derived esters, the tartrate esters ( 7 and 8) were not metabolised by AWRI 1499, and subsequent fermentations with AWRI 1608 and 1613 yielded the same result. This confirmed that the tartrate esters cannot contribute directly to the formation of ethylphenols during exposure to D. bruxellensis . The glucose esters were metabolised by AWRI 1499 to a moderate extent (35% conversion), providing information that these can contribute to the accumulation of ethylphenols during barrel ageing. Furthermore, the isomerisation of the glucose esters lead to studies into the stereoselectivity of D. bruxellensis enzyme activities, whereby the decarboxylase as well as the ethyl esterase showed selectivity for the trans -isomers and that the cis -hydroxycinnamate content of grapes and wine are not important in the accumulation of ethylphenols. The experimental procedures employed throughout Chapters 2-5 are outlined in Chapter 6.
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Declaration
This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.
I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying, subject to the provisions of the Copyright Act 1968.
I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library catalogue, the Australian Digital Thesis Program (ADTP) and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.
………………………………….. Josh L. Hixson
…………………………………..
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Acknowledgements
In no particular order, other than chronologically, I would like to thank my supervisors for their commitment to my learning. Dr. Gordon Elsey…… Gordy, you have been an absolute inspiration since I met you in 2006 and you are the reason I started this particular journey. You have pushed me to know more and work harder and have been a constant source for knowledge outside of the field of chemistry as well as within it. It saddens me that we didn’t get to finish this journey together, and it saddens me even more that it was easier for me to dismiss you and carry on without you, rather than help you through some tough times and for that I am sorry. I will always consider you a friend, regardless of the past, and I truly believe that you have contributed as much by leaving me to research and become independent as you have to actively increasing my knowledge.
Dr. Chris Curtin for taking me on during my Ph.D. after seeing my complete lack of microbiology skills during my honours year and still wanting to get the best out of me and instill into me as much microbiology knowledge as possible. Also for being a fantastic outlet when synthetic chemistry became too much and we could discuss fermentation experiments or I could listen to you get excited about potential enzyme activities that could be expressed.
When the notion of leaving Flinders University in 2008 arose, the choice to relocate to The University of Adelaide was made so much easier by being ‘adopted’ by Prof. Dennis Taylor. Den, thank you for the opportunity of working under you. You have given me such an insight into the workings of a university always open to discuss which grants you were applying for and what the outcomes were, when you really didn’t have to. From finding me a scholarship at very short notice, to offering me roles in the lab to keep my mind off of what was making me unhappy. You have shown me that there is nothing wrong with breaking the mold, because it’s not held together that well to start off with.
The final member of the supervision team, who picked up the slack when it was needed, Dr. Mark Sefton. It has been a pleasure to work with such a fantastic and knowledgeable flavour and aroma chemist and I sometimes forget how lucky I am. I have honestly been approached with ‘you work with Dr Sefton? He is a legend, I have read so many of his
viii papers’ and that was on the other side of the world. Thank you for giving me advice when I felt like there was nobody else who wanted to give me any.
The members of the original Elsey/AWRI group who moved to Adelaide with me, Natoiya and Jo, and those who I found when I arrived, Pete and Nicole. You have been good friends, and have learnt when to leave me alone and when to make me laugh. I have probably spent as much time complaining to you about all sorts of things than I have talking to you about science, but by letting me vent, you have definitely helped me get through.
People that haven’t been on the whole journey, but those that have helped along the way, Dr. Simon Mathew for advice about theoretical calculations, Dr. Eric Dennis for advice on anything I needed or just a random message to keep the spirits up, Dr. Dave Jeffery for being the only person to come and visit me in my red lab in the basement and discuss deprotection strategies. Ms. Dimi Capone for running ethylphenol analyses and keeping the instruments running so very well and Dr. Edward Tiekink at the University of Malaysia for performing X-ray crystallography.
To my family, especially my parents, thank you for understanding why I am doing this and not out getting a job, and also for pushing me into university when I would have been just as happy lying on the couch…at the time that is. I am sorry it took me until I started honours to actually apply myself to do anything, I am hoping you have largely forgotten my first 21 years of complete laziness and contentment with achieving the minimum amount to survive.
The biggest thanks of all has to go to my wife and best friend Suey. When you met me I was a lay about undergraduate student who was about to dropout of uni and find something more exciting to do, but you have stuck with me, and supported me throughout my extended university stay, financially, emotionally and physically. Thank you for being there when I have needed you, and for knowing when I have needed support, even if I was too tired or grumpy or hungry to figure it out. Thank you for putting up with long days, long nights, long sleeps and long ramblings about my project, without you I wouldn’t have made it through this. Also for giving me the ultimate inspiration to get this done and start
ix my working life, I can’t wait to meet Googy and finally take my place as the provider for my new family.
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Publications and Symposia
Publications: Hixson, J. L.; Sleep, N. R.; Capone, D. L.; Elsey, G. M.; Curtin, C. D.; Sefton, M. A.; Taylor, D.K. Hydroxycinnamic acid ethyl esters as precursors to ethylphenols in wine. J. Agric. Food Chem. Accepted 12/02/2012.
Hixson, J. L.; Curtin, C. D.; Sefton, M. A.; Taylor, D. K. Stereospecificity of D. bruxellensis in the production of ethylphenol off-flavour in wine. Proceedings of the 13 th Weurman Flavour Research Symposium. In press.
Hixson, J. L.; Taylor, D. K.; Ng, S. W.; Tiekink, E. R. T. Di-tert -butyl (2 R,3 R)-2-({(2 E)-3- [4-(acetyloxy)-3-methoxyphenyl]prop-2-enoyl}oxy)-3-hydroxybutanedioate. Acta Crystallographica, Section E 2012 , 68 (3), o509-o510.
Hixson, J. L.; Taylor, D. K.; Ng, S. W.; Tiekink, E. R. T. Di-tert -butyl (2 R,3 R)-2-({( 2E )-3- [4-(acetyloxy)phenyl]prop-2-enoyloxy)-3-hydroxybutanedioate. Acta Crystallographica, Section E 2012 , 68 (2), o568-o569.
Hixson, J. L.; Elsey, G. M.; Curtin, C. D.; Sefton, M. A.; Taylor, D.K. Hydroxycinnamoyl glucose and tartrate esters and their role in the formation of ethylphenols in wine. J. Agric. Food Chem. In draft.
Symposia: Hixson, J. L.; Curtin, C. D.; Taylor, D. K.; Elsey, G. M. Mapping the Metabolic Inputs of ‘Brett’ Taint. Poster presented at the 2009 YPD conference (Meeting of the Australasian Yeast Group).
Hixson, J. L.; Curtin, C. D.; Taylor, D. K. Stereospecificity of the Decarboxylase Enzyme of D. bruxellensis. Poster presented at the 14 th Wine Industry Technical Conference, 2010.
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Hixson, J. L.; Elsey, G. M.; Curtin, C. D.; Taylor, D. K. Isomerisation of the Hydroxycinnamic Acids and their Role in the Production of Wine Off-aroma. Seminar presented at the 2010 Adelaide Synthetic Chemistry Symposium.
Hixson, J. L.; Curtin, C. D.; Sefton, M. A.; Taylor, D. K. Determination of Alternative Precursors to Brettanomyces/Dekkera Produced Off-flavour. Seminar presented at the 13 th Weurman Flavour Research Symposium, 2011.
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Abbreviations
4-EG 4-Ethylguaiacol 4-EP 4-Ethylphenol Å Angstroms Ac Acetyl AcCl Chloroacetyl AcCN Acetonitrile app. d Apparent doublet Ar Aromatic AWRI Australian Wine Research Institute Bn Benzyl br Broad COSY Correlation spectroscopy d Doublet DAD Diode array detector DCM Dichloromethane dd Doublet of doublets ddd Doublet of doublet of doublets DFT Density functional theory EIC Extracted ion chromatogram ESI Electrospray ionization Et Ethyl
Et 2O Diethyl ether EtOAc Ethyl acetate g Grams GC Gas chromatography Glc Glucose HCA Hydroxycinnamic acid HMBC Heteronuclear multiple bond correlation HMQC Heteronuclear multiple quantum coherence HOMO Highest occupied molecular orbital HPLC High-performance liquid chromatography HRMS High resolution mass spectroscopy xiii
Hz Hertz hν Light J Coupling constant kJ Kilojoules L Litre LC Liquid chromatography Lit. Literature LUMO Lowest unoccupied molecular orbital m Multiplet M Molar (moles/litre) m/z Mass to charge ratio mg Milligrams
MgSO 4 Magnesium sulphate MHz Megahertz ML Megalitre mL Millilitre MMFF Merck Molecular Force Field mmol Millimoles mol Moles m.p. Melting point MRM Multiple reaction monitoring MS Mass spectrometry MYPG Malt, yeast extract, peptone, glucose nm Nanometres NMR Nuclear magnetic resonance p Para Ph Phenyl ppb Parts per billion ppm Parts per million q Quartet
Rf Retension factor rpm Revolutions per minute s Singlet
S0 Singlet ground state xiv
S1 Singlet first excited state t Triplet
T1 Triplet first excited state tert Tertiary THF Tetrahydrofuran TIC Total ion chromatogram TLC Thin layer chromatography TMS Tetramethyl silane UV Ultra-violet Vis Visible VNBC Viable but non-culturable X4 Hexane fraction YNB Yeast extract, nitrogen, base YPD Yeast extract, peptone, dextrose δ Chemical shift µ Micro
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Figures, Schemes and Tables
List of Figures: Figure 1.1: Ethylphenols produced by D. bruxellensis in red wine...... 5 Figure 1.2: Enzymatic conversion of hydroxycinnamic acids to volatile phenols...... 6 Figure 1.3: L-Tartaric acid esters of p-coumaric acid ( 7) and ferulic acid ( 8)...... 11 Figure 1.4: 1-O-β-D-Glucose esters of p-coumaric acid ( 9) and ferulic acid ( 10 )...... 16 Figure 1.5: Ethyl hydroxycinnamates...... 18 Figure 1.6: Evolution of ethyl coumarate in Shiraz wine...... 19 Figure 1.7: Hydroxycinnamoyl tartrate ( 7 and 8), glucose ( 9 and 10 ) and ethyl esters ( 11 and 12 ) to be synthesised and used in these studies...... 20 Figure 2.1: Molecular structure and crystallographic numbering scheme for 35 ...... 31 Figure 2.2: Molecular structure and crystallographic numbering scheme for 36 ...... 32 Figure 2.3: 1H proton NMR spectrum of the chloroacetyl protons in 2,3,4,6-O- tetrachloroacetyl-β-D-glucopyranosyl cinnamate ( 48 )...... 40 Figure 2.4: NMR spectrum of isomerised glucose esters. a) cis/trans -Feruloyl glucose ( 10 ). b) cis/trans -Cinnamoyl glucose ( 53 )...... 42 Figure 2.5: Hydroxycinnamate esters to be used in fermentation experiments...... 48 Figure 2.6: Dominant equilibria in hydroxycinnamoyl glucose ester mixtures to be used in fermentation experiments...... 48 Figure 3.1: Acyl migration in p-coumaroyl glucose...... 50 Figure 3.2: Initial silica catalysed 3-S- to 6-O-migration observed by Whistler et al...... 51 Figure 3.3: Migration intermediates. a) Base-catalysed 1-O-β- to 2-O-β-migration intermediate proposed by Iddon et al. b) Acid-catalysed 4-O-α- to 6-O-α-migration intermediate proposed by Horrobin et al...... 51 Figure 3.4: Proposed migration of mono-O-chloroacetyl derivatives to the 6-O-position. . 53 Figure 3.5: Twenty possible esters of p-coumaroyl glucose ( 9) and feruloyl glucose ( 10 ). 55 Figure 3.6: Energy of p-coumaroyl and feruloyl glucose esters in water, relative to the 1-O- β-esters...... 56 Figure 3.7: Energy of p-coumaroyl and feruloyl glucose esters in dichloromethane, relative to the 1-O-β-esters...... 57 Figure 3.8: Energy of p-coumaroyl and feruloyl glucose esters in ethanol, relative to the 1- O-β-esters...... 58
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Figure 3.9: Energy of p-coumaroyl and feruloyl glucose esters in toluene, relative to the 1- O-β-esters...... 58 Figure 3.10: p-Coumaroyl glucose ( 9) ester energies calculated in changing solvents, relative to the 1-O-α-esters...... 59 Figure 3.11: Key intermediates (Int. 1-4) for the acid-catalysed 1-O-β- to 2-O-β- acyl migration of p-coumaroyl glucose ( 9)...... 62 Figure 3.12: Energy of the intermediates in 1-O-β- to 2-O-β-p-coumaroyl glucose migration, relative to intermediate 1...... 63 Figure 3.13: Energy of the intermediates in 1-O-β- to 2-O-β-feruloyl glucose migration, relative to intermediate 1...... 63 Figure 3.14: Energy of the intermediates in 1-O-β- to 6-O-β-p-coumaroyl glucose migration, relative to intermediate 1...... 65 Figure 3.15: Energy of the intermediates in 1-O-β- to 6-O-β-feruloyl glucose migration, relative to intermediate 1...... 65 Figure 3.16: Energy of the intermediates in 1-O-β- to 3-O-β-p-coumaroyl glucose migration, relative to intermediate 1...... 66 Figure 3.17: Energy of the intermediates in 1-O-β- to 3-O-β-feruloyl glucose migration, relative to intermediate 1...... 66 Figure 3.18: Glucose ring-flip to facilitate 1-O- to 3-O-migration and 1-O- to 6-O- migration...... 67 Figure 3.19: p-Coumaroyl glucose. a) Extracted ion chromatogram of m/z 325. b) Mass spectrum at 29.6 to 29.8 minutes...... 69 Figure 3.20: Feruloyl glucose. a) Extracted ion chromatogram of m/z 355. b) Mass spectrum at 36.5 to 36.6 minutes...... 69 Figure 3.21: Concentrated white wine, extracted ion chromatogram of m/z 325...... 70 Figure 3.22: Mass spectra of compounds identified in extracted ion chromatogram of m/z 325...... 71 Figure 3.23: Concentrated white wine, extracted ion chromatogram of m/z 355...... 72 Figure 3.24: Mass spectra of compounds identified in extracted ion chromatogram of m/z 355...... 72 Figure 3.25: Red wine chromatogram (DAD)...... 73 Figure 3.26: Concentrated red wine, extracted ion chromatogram of m/z 325...... 73 Figure 3.27: Concentrated red wine, extracted ion chromatogram of m/z 355...... 74
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Figure 3.28: HPLC-MRM traces (aglycone - blue, aglycone minus water - red) of hydroxycinnamoyl glucose esters. a) Pure glucose esters. b) Neat white wine. c) Concentrated white wine. d) Concentrated red wine...... 75 Figure 4.1: Photoisomerisation of the hydroxycinnamoyl glucose esters...... 79 Figure 4.2: Electron configuration of π bonding and anti-bonding molecular orbitals in ground and excited states...... 80 Figure 4.3: Compounds investigated in decarboxylation studies...... 85 Figure 4.4: Proposed resonance assisted conversion of cis -p-coumaric acid to trans -p- coumaric acid...... 88 Figure 4.5: Intended effect of metal coordination on cis -hydroxycinnamates...... 91 Figure 4.6: Frontier molecular orbital diagrams of trans-p-coumaric acid ( 3). a) HOMO of
S0 trans -p-coumaric acid. b) LUMO of S 0 trans -p-coumaric acid. c) HOMO of T 1 trans -p- coumaric acid...... 92
Figure 4.7: Electron spin density in T 1 trans -p-coumaric acid...... 93 Figure 4.8: Energy profile of p-coumaric acid ( 3)...... 94 Figure 4.9: Energy profile produced from forward and reverse dynamic, and manual constraint of ethyl coumarate ( 11 )...... 95 Figure 4.10: Pyramidilised alkene resulting from rotation of the dihedral angle from 180 o to 0 o in ethyl coumarate ( 11 )...... 95 Figure 4.11: cis -Ethyl coumarate conformers produced by: a) drawing trans -ethyl coumarate and rotating the dihedral; and b) drawing cis -ethyl coumarate...... 96
Figure 4.12: Energy profile for p-coumaroyl glucose ( 9), relative to S 0 trans -isomer...... 96
Figure 4.13: a) T 1 energy profile for p-coumaric acid ( 3) in water, relative to the S 0 trans - acid. b) T 1 energy profile for p-coumaroyl glucose ( 9) water, relative to the S 0 trans - isomer...... 97
Figure 4.14: S 0-T1 vertical excitation energy for trans -p-coumaric acid ( 3) and trans -p- coumaroyl glucose ( 9)...... 98 Figure 4.15: HOMO-LUMO gap for trans -p-coumaric acid and trans -p-coumaroyl glucose...... 99
Figure 4.16: a) Vertical excitation energies (S 0-T1) of trans -p-coumaroyl glucose phenolate in solvents of differing polarity. b) HOMO-LUMO gap...... 99 Figure 4.17: a) HOMO-LUMO gap of trans -hydroxycinnamates. b) HOMO-LUMO gap of cis -hydroxycinnamates...... 101
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Figure 4.18: HOMO-LUMO gaps of cis -hydroxycinnamates during base-catalysed ester hydrolysis...... 102 Figure 4.19: Numbering of oxygen atoms in hydroxycinnamate skeleton...... 103 Figure 4.20: HOMO-LUMO gap of p-coumaric acid carboxylate...... 103 Figure 4.21: HOMO-LUMO gaps of hydroxycinnamate derivatives against ratio of charge between oxygen 1 and oxygen 3...... 104 Figure 4.22: Relationship between HOMO-LUMO gap and double bond length in hydroxycinnamate derivatives...... 105 Figure 5.1: Ethyl coumarate ( 11 ) and ethyl ferulate ( 12 )...... 107 Figure 5.2: Percentage of the theoretical maximum conversion of ethyl esters ( 11 and 12 ) to ethylphenols...... 108 Figure 5.3: Percentage recovery of coumarates in fermentations...... 109 Figure 5.4: Percentage recovery of ferulates in fermentations...... 109 Figure 5.5: Percentage of the theoretical maximum conversion from ethyl coumarate ( 11 ) and ethyl ferulate ( 12 ) to ethylphenols by different strains of D. bruxellensis ...... 110 Figure 5.6: p-Coumaroyl L-tartrate ( 7) and feruloyl L-tartrate ( 8)...... 111 Figure 5.7: p-Coumaroyl glucose ( 9) and feruloyl glucose ( 10 )...... 113 Figure 5.8: Percentage of the theoretical maximum conversion of hydroxycinnamoyl glucose esters ( 9 and 10 ) to ethylphenols...... 113 Figure 5.9: Percentage of the theoretical maximum conversion to 4-ethylguaiacol for the trans - and cis /trans - fermentations...... 116 Figure 5.10: Evolution of 4-ethylguaiacol in cis/trans -fermentations as a percentage of maximum conversion observed in trans -fermentations...... 117 Figure 5.11: Compounds by percentage in end-point fermentation samples...... 119 Figure 5.12: Percentage of the theoretical maximum conversion to 4-ethylphenol in trans - and cis /trans - fermentations...... 120 Figure 5.13: Evolution of 4-ethyphenol in cis/trans -fermentations as a percentage of maximum conversion observed in trans -fermentations...... 120 Figure 5.14: Percentage of the theoretical maximum ethylphenol conversion from cis -ethyl esters...... 122 Figure 5.15: Total coumarate recovery from cis -fermentations...... 124 Figure 5.16: Total ferulate recovery from cis -fermentations...... 124 Figure 5.17: Breakdown of ethyl ferulate in a single fermentation...... 125
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List of Schemes: Scheme 2.1: Synthesis of hydroxycinnamic acid derivatives...... 22 Scheme 2.2: Literature syntheses of mono-esters of tartaric acid...... 25 Scheme 2.3: Literature syntheses of chicoric acid...... 26 Scheme 2.4: Synthesis of benzylated hydroxycinnamoyl tartrate esters...... 27 Scheme 2.5: Attempted debenzylation procedures...... 28 Scheme 2.6: Synthesis of di-tert -butyl tartrate...... 29 Scheme 2.7: Esterification of hydroycinnamic acids and di-tert -butyl tartrate...... 30 Scheme 2.8: Synthesis of hydroxycinnamoyl tartrate esters...... 34 Scheme 2.9: Modified Koenigs-Knorr reaction conditions employed within this research group...... 35 Scheme 2.10: Glycosylation reactions of Ziegler...... 36 Scheme 2.11: Glycosylation method described by Galland...... 37 Scheme 2.12: Synthesis of 1-O-benzyl hydroxycinnamoyl glucopyranoses...... 39 Scheme 2.13: Synthesis of glucose esters with free hydroxycinnamic acids...... 41 Scheme 2.14: Glycosylation with 1-O-acetyl hydroxycinnamic acids and partial deacetylation using XAD-8 resin...... 44 Scheme 2.15: Glycosylation of 1-O-chloroacetyl hydroxycinnamates, and migration of the free glucose esters...... 46 Scheme 3.1: Mechanism for acid catalysed 1-O-β- to 2-O-β- acyl migration of p- coumaroyl glucose ( 9)...... 61 Scheme 4.1: Attempted synthesis of cis -hydroxycinnamic acids...... 88
List of Tables: Table 1.1: Hydroxycinnamoyl tartrate ester concentrations in different grape varieties. .... 12 Table 1.2: Changes in p-coumaroyl tartrate concentration during malolactic fermentation...... 13 Table 1.3: Changes in p-coumaroyl tartrate concentration during wine storage...... 14 Table 2.1: 1H NMR shifts for migrated hydroxycinnamoyl glucose esters...... 47 Table 4.1: Content of cis - and trans-p-coumaroyl tartrate in the skin and juice of red and white grapes...... 82 Table 4.2: Isomeric ratio of p-coumaric acid ( 3) under different storage conditions...... 90
Table 4.3: Solvent polarities and ET 30 values...... 98 xx
Table 5.1: Ethylphenol content in tartrate ester fermentation experiments...... 112 Table 5.2: Concentration of cis - and trans-ferulic acid in end-point fermentation samples...... 119 Table 5.3: Final trans -ethyl ester content in cis -ethyl ester fermentations...... 123
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“Success is the ability to go from one failure to another with no loss of enthusiasm”
Sir Winston Churchill
“I’m a great believer in luck, and I find the harder I work the more I have of it”
Thomas Jefferson
Chapter 1: Introduction
Chapter 1: Introduction.
1.1 General Introduction.
The global history of wine production spans back many thousands of years, supported by the discovery of wine vessels that have been dated to as early as circa 5400-5000 B.C., as well as there being numerous biblical references to wine. 1 The history of Australian wine, however, begins with European settlement in 1788 when the Lady Penrhyn, 2 one of the eleven ships in the First Fleet, arrived carrying on it vine cuttings and seeds from the species Vitis vinifera obtained en route from Brazil and the Cape of Good Hope. 3 Within days of landing, the vines had been planted in the governor’s garden in Sydney Cove but failed to thrive due to the humid coastal climate. Late in 1788 the first inland farming settlement was established at Rose Hill (now Parramatta), some 24 km further inland. This region had a much drier climate allowing for cultivation of a vineyard and by 1791 boasted 3 acres consisting of 8000 vines and the following year produced approximately 150 kg of table grapes. 3 In 1795 Philip Schaffer became the colony’s first vigneron when he produced some 90 gallons, or approximately 400 litres of wine. 2
From humble beginnings, the Australian wine industry has developed extensively, 4 now consisting of 2477 wineries, crushing 1,603,000 tonnes of grapes and producing 1,533 ML of wine, 5 of which, 777 ML is exported worldwide, with a value of A$2,167,200,000. 6 South Australia contributes 48% of the total volume of Australian wine production, which is a product of crushing 689,000 tonnes of grapes. 7
Even though the world of wine has largely moved past the fortuitous fermentation of grape juice caused by indigenous yeast, and into an industry of more controlled and predictable fermentations with cultured or purchased yeasts, 8-9 there still remains uncertainties that can be brought about through the presence of unwanted microrganisms. 10
1.2 Dekkera/Brettanomyces bruxellensis.
A constant issue encountered in wine making throughout the world, 11 and of interest in Australia since the first reported occurrence in 1986, is that of contamination caused by 1 Chapter 1: Introduction yeast of the Brettanomyces and Dekkera genera, 12 through their potential to cause a prolific economic impact on the wine industry. 13-14
The name Brettanomyces was originally introduced in 1904 as the characteristics produced by this yeast were similar to the English beers of the time, with the prefix ‘Brettano’ a reference to the British brewing industry. The first report of this yeast in wine came much later, when in 1930 Mycotorula intermedia was isolated from a French must, later to be reclassified as Brettanomyces . The early 1950’s saw the first report of this genus in bottled wine, which was closely followed by the discovery of a sporulating form of Brettanomyces ,15 which was categorised into the new genus, Dekkera .16
Since then species within Brettanomyces and Dekkera have been classified and re- classified numerous times, 17 with the work of Smith and Poot 18 and Boekhout 19 being largely responsible for the current taxonomy of these genera. There consists five species of Brettanomyces (bruxellensis , anomolus , naardenensis , custersianus , nanus ), of which bruxellensis and anomolus have teleomorphs in the genus Dekkera .17 However, due to the difficulties associated with characterising yeasts into either the Brettanomyces or Dekkera genera on the basis of sporulation, 20 these two names are often used interchangeably. 11 The current preference is for Dekkera on the basis of molecular identification. 21
While there have been numerous wine-related studies focusing on different strains and species of Brettanomyces and Dekkera , recent attempts to characterise grape, wine and winery isolates have failed to identify a Dekkera species other than D. bruxellensis .22-27 Also, early research may have used outdated yeast classifications describing species that have since been re-classified. As such, reference to previous literature will reflect the author’s original classifications, but wine related instances of these yeasts will be referred to as D. bruxellensis .
Many studies into Dekkera and Brettanomyces yeasts have shown great variety between strains within the same species for easily observable characteristics including growth, nutritional requirements and metabolic output, 23-24, 28-36 but the development of genetic characterisation has allowed for a more in depth study of these yeasts. Conterno et al. obtained 47 wine strains of B. bruxellensis from around the world, of which 35 isolates were studied in great detail. While no two strains displayed the same characteristics in
2 Chapter 1: Introduction terms of growth, temperature dependence, ethanol tolerance, sulfite tolerance and metabolic output, the genetic characteristics of these strains could link them with their geographic origin, vintage year and wine variety. 23
With respect to Australian winemaking, Curtin et al. characterised 244 D. bruxellensis isolates from wine making regions across the country and showed that all isolates could be placed into closely related genetic groups. One group dominated, accounting for 85% of all the isolates, with another two genetic groups contributing 6% and 7% respectively. 22 The second two groups, while less represented in Australia, were shown to be closely related to reference isolates from France and California, which indicates that these secondary D. bruxellensis groups in Australia may be representative of internationally isolated strains. While Dekkera strains can be classified into distinct genetic clusters, it is the more generic characteristics that lead to yeast being classified into the Brettanomyces or Dekkera genera.
Prior to the work of Renouf and Lovaud-Funel, 37 the origin of these yeasts in the winery was not completely understood, but through the development of an enrichment media specifically for growth of B. bruxellensis , detection on grape berries and the vineyard origin was inferred. These wild yeasts encounter the grapes by becoming airborne and settling on them in the vineyard, or can be spread by fruit fly and bees with traces of these yeasts having being found in the feeding and breeding areas of both insects, as well as on their legs, bodies and wings.15 Once present in the winery, D. bruxellensis can become established in any area with which the affected wine comes in contact.11, 15
Even though D. bruxellensis can be found throughout the winery, and has been isolated as early in the winemaking process as the completion of alcoholic fermentation, 33 malolactic fermentation is an important period in the development of D. bruxellensis due to the low sulphur dioxide levels needed for growth of lactic acid bacteria, as well as the residual sugar still remaining in the wine. 38 The ability to grow and thrive with few nutrients,39-40 along with the increased tolerance for high ethanol concentrations make the later stages of winemaking ideal for D. bruxellensis , with little competition from other wine microorganisms. 20 This allows for survival throughout vinification until conditions are more encouraging for growth, 40 which is why the most common place of Dekkera infection is in the barrel, during wine ageing. 15, 41-42 D. bruxellensis can live solely off of cellobiose, a product of cellulose degradation, allowing it to remain in empty barrels and contaminate
3 Chapter 1: Introduction subsequent wine additions. 11, 15, 23 Once a barrel has become contaminated, sterilisation can be attempted through shaving, toasting, steaming or hot water treatments, 15, 43 though there is no guaranteed method by which to eradicate Dekkera , with prevention being the recommended course of action. 43
While re-use of barrels can increase the chance of further infection by D. bruxellensis ,44-45 the use of new barrels can provide the yeast with additional sugar and oxygen in which to establish themselves, 20 and techniques such as micro-oxygenation which is used to accelerate wine ageing can provide a more favourable environment for growth. 44-45 This is not to say that lack of oxygen is a great inhibitor, Dekkera can adapt to conditions of low oxygen, with anaerobiosis only restricting growth and not preventing it. 44, 46
In the suspended volume of wine, sulphur dioxide has proven relatively successful in controlling D. bruxellensis growth. 47 Along with the use of barrels during red wine production, 15, 42 the increased sulphur dioxide efficacy at the lower pH of white wine is probably the main reason why this yeast is more commonly found in red wines. 20 However, due to the ability of wine to penetrate deep into the barrel, Dekkera can be carried deep into cracks, between staves, and around the bung, proliferating away from the dissolved sulphur dioxide. 14 Furthermore, the use of sulphur dioxide may induce a viable but non-culturable state (VBNC) whereby the yeast is no longer active but can become viable again given favourable conditions. In a VBNC state Dekkera cells can shrink from an average 5-8 x 3-4 µm to be small enough to pass through a 0.45 µm filter and then proliferate in the ‘filtered’ wine, 48-50 with the potential to become the major organism in bottled wine. 51 Alternatively trialled methods for controlling Dekkera include dimethyldicarbonate, 52-53 sorbic acid, 54 increased temperatures, 55-56 low-voltage electric current, 57 ozone, 38 and high-power ultra sonic radiation. 58
Furthermore, detection of D. bruxellensis in wine has proven to be difficult due to its comparatively slow growing nature and limited carbon dioxide production.11 Agar plate smears can be used for the detection of yeasts in winemaking, but D. bruxellensis is often overgrown by other yeasts that are present and missed, or can even develop long after the agar plate has been disposed of. 15 Current techniques for detection revolve around additions of compounds inhibitory to the growth of other organisms, allowing Dekkera to
4 Chapter 1: Introduction be the sole occupant and more easily identified, 59 in a similar manner to that previously described for detection on grapes. 37
If able to grow in wine, D. bruxellensis is associated with formation of several “spoilage” compounds. It has been connected with the production of acetic acid in wines, 60 and though this effect is lessened with decreasing amounts of oxygen, it can still be produced under full anaerobiosis. 44-46, 60 D. bruxellensis has also been directly linked with the production of ‘mousy’ aromas in wine from tetrahydropyridines, 12, 29 the formation of isovaleric acid,15 which has been described as rancid or cheesy, 43 and the production of volatile phenols. 61-62
1.3 Volatile Phenols.
Of the spoilage compounds produced by D. bruxellensis , those of greatest interest, especially in red wine, are 4-ethylphenol (1) and 4-ethylguaiacol (2). These compounds, their presence in wine, and their link to D. bruxellensis has been extensively researched over the past 25 years, 15, 41-43, 61-67 and they are produced almost exclusively by Dekkera under oenological conditions.61-62 Only trace amounts of 4-ethylphenol and 4-ethylguaiacol have been identified in grape musts, with very little present at the conclusion of malolactic fermentation. The resulting wines can have much higher concentrations of ethylphenols, with the greatest increase usually occurring during barrel ageing, 41 where Dekkera proliferates.
OCH3
OH OH 1 2 Figure 1.1: Ethylphenols produced by D. bruxellensis in red wine.
Phenols, 1 and 2, are formed via the activity of two enzymes that are active towards the hydroxycinnamic acids, p-coumaric acid (3) and ferulic acid (4). The first of these activities, a decarboxylase, converts compounds 3 or 4 into 4-vinylphenol or 4-
5 Chapter 1: Introduction vinylguaiacol (5 or 6), respectively, by removing the carboxylic acid moiety and releasing carbon dioxide. 68 The second activity is a vinyl reductase which acts by reducing the C-C double bond, generating ethylphenols ( 1 or 2).68-69
COOH Decarboxylase Vinyl Reductase
R R R
OH OH OH 3 R = H 5 R = H 1 R = H 4 R = OCH3 6 R = OCH3 2 R = OCH3 Figure 1.2: Enzymatic conversion of hydroxycinnamic acids to volatile phenols.
In studies of B. bruxellensis and B. anamolus , decarboxylase activity towards caffeic acid as well as p-coumaric and ferulic acids ( 3 and 4) has been shown. 68, 70 However, it has not been until recently that the metabolite of caffeic acid, 4-ethylcatechol, has been quantified in wine. 71 While caffeic acid concentrations in wine can exceed that of p-coumaric and ferulic acid, 28, 72-73 4-ethylcatechol concentrations are much lower than 4-ethylphenol and are closer to that of 4-ethylguaiacol. 71 The sensory threshold of 4-ethylcatechol is yet to be adequately determined in wine. Initial reports suggest a detection threshold around 50 µg/L, 74 though unpublished investigations by the Australian Wine Research Institute (AWRI) suggest that it is much higher. A recent study into the detection thresholds in cider also support a significantly increased threshold when compared to that of 4-ethylphenol, 75 implying that 4-ethylcatechol is of little importance in the production of volatile phenol off-flavour in wine.
Other microbes present during winemaking possess the necessary enzymatic abilities to breakdown p-coumaric and ferulic acids and can produce varying amounts of volatile phenols. However, unlike Dekkera , the activity of the Saccharomyces cerevisiae decarboxylase is inhibited by the presence of polyphenols in red wines. 41, 76 As such, S. cerevisiae is able to perform decarboxylation and contribute to the accumulation of vinylphenols in white wine alone, 76 although does not possess the ability to subsequently reduce the vinylphenols to the ethyl analogues. 77-78
6 Chapter 1: Introduction
This is also the case for lactic acid bacteria. For many that possess the ability to decarboxylate hydroxycinnamic acids and produce vinylphenols, the activity is inhibited by the presence of polyphenols, though in situations where decarboxylation can be performed, subsequent formation of ethylphenols is hindered by limited vinyl reductase activity. 61-62 Specifically, Oenococcus oeni , the organism largely responsible for malolactic fermentation, displays limited decarboxylase activity even when uninhibited. 61, 79 For those that do possess decarboxylase activity it has been found that it needs to be induced, whereby the bacteria need to grow in the presence of the hydroxycinnamic acids for it to be activated. 80
Chatonnet et al. investigated numerous bacteria from the genera Leuconostoc , Lactobacillus , Pediococcus and Acetobacter , and yeasts Candida , Hanseniaspora , Metchnikovia , Pichia , Hansenula , Kluyveromyces , Torulaspora and Zigosaccharomyces with respect to production of volatile phenols. 41 Other non-wine related microorganisms that have been studied include Lactobacillus plantarum , Lactobacillus hilgardii , Lactobacillus brevis , Pediococcus pentosaceus , Pediococcus damnosus ,61 Klebsiella oxytoca ,81-82 Erwinia uredovora ,82 Aerobacter aerogens ,83 Cladosporium phlei ,84 Polyporus circinata ,85 Bacillus subtilis 86 and Pichia guilliermondii .87-88
Of these Lactobacillus plantarum can produce ethylphenols in synthetic media (2.55% conversion, compared with 68.6% by D. bruxellensis ), but like most other organisms is inhibited by the presence of polyphenolic compounds. 61 P. guilliermondii can also produce ethylphenols but only in grape juice, and as such can only contribute to volatile phenol accumulation prior to alcoholic fermentation. 88 Furthermore, this organism has only been associated with wine prior to alcoholic fermentation, having been isolated from grapes, grape juice and winery equipment, but not from wine itself. 87 Other than L. plantarum and P. guilliermondii , the remaining organisms were either studied with respect to decarboxylase activity and the ability to produce vinylphenols, or did not possess the necessary enzymatic abilities to produce ethylphenols.
Thus, while winemaking micro-organisms other than D. bruxellensis can possess the enzymatic abilities to breakdown the hydroxycinnamic acids, they do not produce ethylphenols in the quantities seen by D. bruxellensis in wine, 61-62 due to either inhibition of the necessary activities by polyphenolic compounds present in red wine or poor survival
7 Chapter 1: Introduction under oenological conditions. Organisms that do possess the ability to produce volatile phenols are not present during barrel ageing where the majority of the spoilage occurs. 41
Studies into the sensory impact of the volatile phenols found that 4-ethylphenol has a detection threshold of 605 µg/L and a rejection threshold of 620 µg/L in a red wine, while 4-ethylguaiacol was detected at 110 µg/L with the wine being rejected at 140 µg/L. 41 However, the amounts of these compounds found in wine differs greatly depending on wine variety, with ratios of 4-ethylphenol:4-ethylguaiacol in Australian red wine varying from 10:1 in Cabernet Sauvignon to 3.5:1 in Pinot Noir and with an average ratio of approximately 8:1, 63 which is said to be a result of the relative amounts of precursors present in the grape. 41 Also, differences in yeast nutrients, winemaking practices, D. bruxellensis strains, temperature and use of oak can contribute to altering the ratios and concentration of ethylphenols in finished wine. 43
At an 4-ethylphenol:4-ethylguaiacol ratio of 10:1, which has become known as the Bordeaux ratio, due to first being determined in Bordeaux red wine by Chatonnet and Pons (1988, cited in Romano et al. 2009), 89 a combined 4-ethylphenol and 4-ethylguaiacol detection threshold of 369 µg/L and a rejection threshold of 426 µg/L was determined. 41 A study of the ethylphenol concentration of Australian red wines reported a combined concentration of ethylphenols in excess of 2500 µg/L in three wines, and across the entire survey an average concentration of 795 µg/L for 4-ethylphenol and 99 µg/L for 4- ethylguaiacol, with approximately 60% of the wines possessing combined concentrations in excess of 426 µg/L. 63 The threshold of these compounds relies heavily on the wine variety in which they are found, with a lighter wine being spoiled at a much lower threshold than a full-bodied wine. 43 The descriptors used for each compound are spicy, phenolic, medicinal, wet horse, woody and smoky for 4-ethylphenol, and smoky or clove- like for 4-ethylguaiacol. 62, 66, 90
A more recent study into the sensory properties of the ethylphenols has linked both isovaleric acid and isobutyric acid with masking effects. 89 This study indicates that the presence of these two acids can increase the detection thresholds of the ethylphenols by as much as four times. This would have a similar effect as the wine style, whereby a wine with higher isovaleric and isobutyric acid concentrations could contain slightly more
8 Chapter 1: Introduction ethylphenols than a wine with lower concentrations of these acids and exhibit the same sensory properties.
While the ethylphenols are the main contributors to Dekkera related spoilage in red wines, 31 the vinylphenols, while less thoroughly researched, are of greater importance in white wines. 76 Their relative scarcity in red wine is brought about by the efficacy of the vinyl reductase that D. bruxellensis possesses, converting most to give the ethyl analogues, 41 as well as the potential incorporation of vinylphenols into pyranoanthocyanins. 91 As mentioned previously, the vinylphenols in white wines are due to the decarboxylase ability of S. cerevisiae when not impeded by polyphenolics, and when these vinylphenols are present in wine, a 1:1 mixture (4-vinylphenol:4-vinylguaiacol) imparts pharmaceutical or phenolic nuances at concentrations above 770 µg/L. 76
The production of volatile phenols in wine is proportional to the Dekkera population, 41 with 4-ethylphenol able to be used as a marker for growth, 11 while yeast growth is proportional to the sugar concentration. Sugar at a level of 300 mg/L allows for proliferation of 1 x 10 3 cells/mL, enough to yield 600 µg/L of 4-ethylphenol. In wine that has completed malolactic fermentation, up to 1 g/L or more of residual sugar can be found. 61
Research towards the removal of ethylphenols has shown promise in lowering concentrations, with reverse-osmosis found to reduce the concentrations of 4-ethyphenol and 4-ethylguaiacol in wine, but the reduction in the volatile phenols was matched with losses to other desirable aroma compounds. 92 Experimentation with lyophilised yeast as an adsorbent for 4-ethylphenol also resulted in reductions of desirable compounds, in this case the loss of anthocyanins produced a reduction in wine colour. 93
With current efforts at ethylphenol removal resulting in concomitant reductions in wine quality, one effective way of avoiding spoilage, apart from limiting Dekkera growth, is to minimise the initial concentration of precursors in the wine. 64 As such, the role of hydroxycinnamic acids in the production of volatile phenols should come under further scrutiny.
9 Chapter 1: Introduction
There have been conflicting reports as to the presence of p-coumaric and ferulic acids in grapes with some studies identifying these compounds in grapes, juice or must, 73, 94-97 while others have failed to do so. 72, 98-99 This could in part be due to insufficient extraction from the berry, as those that could identify them, found large concentrations in the skin. 94- 96 Reported concentrations in juice or must generally range from not present, or not detected, to around 0.2 mg/L 73, 97 which is seen to increase throughout vinification with changes observed due to skin contact, alcoholic fermentation, malolactic fermentation and ageing or storage. 97, 99-105 A comprehensive study of 547 red wines from multiple countries and wine regions found p-coumaric acid in concentrations ranging from not detected through to 6.7 mg/L, 106 and although this study did not quantify ferulic acid, it is often present in lower concentrations than p-coumaric acid, 72-73, 101, 103 hence lower 4- ethylguaiacol concentrations following breakdown by D. bruxellensis . The increase of free hydroxycinnamic acids during vinification is largely attributed to the release from conjugated forms, 99-100 with the hydroxycinnamic acids having been found as tartaric acid esters, glucose esters, glucosides, ethyl esters, bound to anthocyanins, or in combinations of these. 107-108 The hydroxycinnamic acids are known to possess antimicrobial properties 109-110 and can be stored in the grape in an inert form until needed to fight off unwanted organisms, also they may be conjugated to assist in both solubility and transport. 111 Therefore, before p-coumaric and ferulic acids can be decarboxylated and reduced, yielding ethylphenols, they must be freed from the conjugated forms in which they are found in grapes.
A common oenological technique is to employ commercial enzyme preparations during maceration to aid in the release of phenolic compounds from the grape berries. Those containing undesired cinnamoyl esterase capabilities can be effective at hydrolysing bound forms, which then leaves the free hydroxycinnamic acids available for conversion to the associated volatile phenols. As such, enzyme preparations that do possess cinnamoyl esterase activity are not recommended for use in winemaking as they can increase the chance of spoilage. 64, 112 Furthermore, Dekkera has been shown to be active in the formation and degradation of ethyl esters, 113 and in the release of aglycones from glycosidically bound forms. 114 However, the study of the direct volatile phenol production from bound hydroxycinnamates by Dekkera has not been studied.
10 Chapter 1: Introduction
1.3 Introduction to Tartrate Esters.
The first report of L-tartaric acid conjugates of the hydroxycinnamic acids in grapes was by Ribereau-Gayon in 1965, from paper chromatography of black grape skin extracts and of red wine, identifying the caffeoyl tartaric acid ester along with the p-coumaroyl tartaric acid ester (7) and feruloyl tartaric acid ester (8). This study failed to identify the quinic acid esters of the hydroxycinnamic acids, which is the form in which they exist in many other plants, and were believed to exist in grapes.115 Since this discovery, much research has been done on the tartaric acid esters of hydroxycinnamic acids, including the discovery of them in the whole grape berry and not just the skin, 116 and showing that they are largely found in the juice of the grape. 117 These esters are also the main phenolic constituent of fresh grape juice and it has been confirmed that they exist mainly in their trans -form, with the cis -isomer being present at lower levels. 118 The hydroxycinnamoyl tartrates have no odour, but can add to the taste and astringency of wine, possessing a bitter taste above a concentration of 10 ppm.119
COOH COOH
HOOC HOOC OH OH
O O O O
OCH3
OH OH 7 8 Figure 1.3: L-Tartaric acid esters of p-coumaric acid ( 7) and ferulic acid ( 8).
The highest concentration of hydroxycinnamoyl tartrates are found in immature berries and they decrease as the berry ripens, 94-96, 120-122 which is accentuated by an increase in berry volume, though in most cases the weight of compound per berry also shows a decline as the berry matures. 95, 121 One study observed an initial drop during ripening followed by a slow re-accumulation to the original amounts as the berries matured. 122
The identification and quantification of the tartrate esters in grapes, 72, 94-96, 122-125 skin, 98, 126- 127 juice 73, 120, 127 and must 97, 128-129 has been performed across both red and white grape
11 Chapter 1: Introduction varieties. The literature data has been collected from different grape varieties from around the world, and the concentrations determined using a number of analytical techniques which quantify varied parts of the grape berry or during different stages of winemaking. As such, a summary of the tartrate ester content in grapes and wine would, at best, only be an average of many different analyses which possess significant variation. Also, feruloyl tartrate can be present at low enough concentrations that some analytical methods, if it can be detected, may give unreliable results.128 The table below shows the results achieved by Ong and Nagel from analysing different grape varieties for the presence of p-coumaroyl and feruloyl tartrate in the berry. 122
Table 1.1: Hydroxycinnamoyl tartrate ester concentrations in different grape varieties.122
NOTE: This table is included on page 12 of the print copy of the thesis held in the University of Adelaide Library.
The tartrate ester content continues to change during winemaking due to the effects of skin contact,97, 102, 129 alcoholic fermentation,97, 99, 101-102, 104, 129 malolactic fermentation,97, 101-103 and storage or ageing.99-100, 102, 104, 129-130 Some studies have focused simply on the difference between either grape berry, juice or must concentration, and that of finished wine, with significant reductions in concentration noted.72-73, 128
In separate studies Gil-Munoz et al.,102 Somers et al.,99 and Nagel and Wulf129 monitored the changes in p-coumaroyl tartrate throughout fermentations of Monastrell, Chardonnay, and Cabernet Sauvignon and Merlot wines, respectively. All authors reported an initial increase in concentration, peaking during alcoholic fermentation, followed by a large decrease throughout either malolactic fermentation and storage for the red varieties or just storage for Chardonnay (3-5 fold reductions). Any reductions witnessed in the early stages 12 Chapter 1: Introduction of winemaking have been associated with enzymatic cleavage of the tartrate esters resulting in liberation of the free hydroxycinnamic acids. 99
Observing malolactic fermentation alone (Table 1.2), Hernandez et al. employed different lactic acid bacteria as well as studying spontaneous malolactic fermentation reporting increases and decreases in p-coumaroyl tartrate concentration, depending on the conditions. 103 From grapes that were crushed at ambient temperature, Gil-Munoz et al. witnessed close to a 3-fold reduction of p-coumaroyl tartrate concentration during malolactic fermentation of Monastrell wine, compared with an increase in wine made with grapes crushed at 10 oC. 102
Table 1.2: Changes in p-coumaroyl tartrate concentration during malolactic fermentation. 102-103 Initial (mg/L) Final (mg/L) Variety Details Reference 8.68 Spontaneous 17.98 O. oeni -18 13.75 13.84 Tempranillo O. oeni -159 Hernandez et al. (2007) 8.98 L. plantarum -51 12.77 L. plantarum -39
397 148 Normal temp. Monastrell Gil-Munoz et al. (1999) 163 255 Low temp.
Ageing or storage of wines, both red and white, results in a reduction of the tartrate ester concentration over different lengths of storage and under a number of conditions (Table 1.3). The bottle ageing of red wine has been studied from 8 months to 26 months, with large reductions seen in Monastrell wine over shorter storage times, 130 small reductions observed in Cabernet Sauvignon and Merlot over longer periods, 129 and differences between wine varieties with Monagas reporting a dramatic loss of p-coumaroyl tartrate in Tempranillo, but only slight fluctuations in Graciano and Cabernet Sauvignon. 100 In the study of Gil-Munoz mentioned above, a 2-fold reduction through 210 days of ageing is described.
13 Chapter 1: Introduction
Table 1.3: Changes in p-coumaroyl tartrate concentration during wine storage. 99-100, 102, 104, 129-130 Concentration (mg/L) Initial Final Variety Storage Length Reference 1.9 0.11 1.9 0.13 Monastrell 8 months Bautista-Ortin et al. (2007) 2.1 0.15 1.9 0.17
15 11.8 Cab. Sav. 192 days Nagel and Wulf (1979) 10.9 6.6 Merlot
0.77 0.14 Tempranillo 0.9 0.8 Graciano 18.5 months Monagas et al. (2005) 1.12 0.9 Cab. Sav.
148 88 Monastrell 210 days Gil-Munoz et al. (1999)
0.8 Steel - 94 days 2.6 Chardonnay Somers et al. (1987) 1 Oak - 94 days
4.5 3.4 3.8 3.3 Pinot Blanc 11 months Vrhovsek and Wendelin (1998) 3.2 3.2 1.7 1.5
These results were mimicked in white wine with different vinification treatments of Pinot Blanc showing only slight reductions of tartrate esters over 11 months of ageing, 104 while Chardonnay after 125 days contained around one-third of the p-coumaroyl tartrate seen before storage and experienced no loss in feruloyl tartrate. 99
Though there are variations between studies regarding the concentration changes, with different varieties and techniques utilised, the overall observation is for a reduction in the tartrate ester concentration from grape through to wine, which is mirrored in simpler studies comparing grape content with wine content alone. 72-73, 128 However, with respect to D. bruxellensis , the interest lies in the hydroxycinnamoyl tartrate concentration in red wines from the completion of alcoholic fermentation, throughout storage when this yeast would be in contact with the wine.
Very few studies have quantified feruloyl tartrate in wine as well as p-coumaroyl tartrate. A study by Nagel et al. monitored the changes in concentration of both compounds from must to wine in 3 white and 3 red varieties, observing feruloyl tartrate at concentrations of
14 Chapter 1: Introduction
1.4, 1.9 and 1.2 ppm in Cabernet Sauvignon, Merlot and Pinot Noir, respectively, compared with p-coumaroyl tartrate at 5.2, 3.1 and 4.7 ppm. 128
In other studies, p-coumaroyl tartrate has been found in wine that has undergone malolactic fermentation in concentrations ranging from less than 1 mg/L 101 up to greater than 20 mg/L, 103 and in wines with different lengths of ageing approximately 1 mg/L 100, 131 through to around 10 mg/L. 97, 129 With typical p-coumaric concentrations in red wines ranging from trace to 6.7 mg/L, as reported by Goldberg et al., 106 the role of the hydroxycinnamoyl tartaric acid esters in the formation of ethylphenols has the potential to exceed that of the free acids. However, due to the relative molecular weights of the tartrate esters ( 7 and 8) compared with the acids ( 3 and 4), breakdown of an equal concentration of each substrate will result in a greater amount of ethylphenols in the case of the acids.
While the decrease in hydroxycinnamoyl tartrate ester concentration during winemaking has been linked to typical winemaking practices such as fermentation and the yeast Saccharomyces cerevisiae ,97, 99, 101-102, 104, 129, 132 malolactic fermentation and the bacteria Oenococcus oeni ,97, 101-104, 129 as well as wine ageing,99-100, 102, 104-105, 129-130 the use of commercial enzyme preparations has also been studied.64, 112 The breakdown of the tartrate esters, followed by fermentation with either S. cerevisiae or B. bruxellensis resulted in an increase in the volatile phenol concentration, linking the use of enzyme preparations to the liberation of free hydroxycinnamic acids, which can be further modified by yeast.
There has also been a single study linking the p-coumaroyl tartrate to D. bruxellensis ,28 but this study failed to link any observed losses of the tartrate ester with an increase in volatile phenol production. Instead, only p-coumaroyl tartrate and p-coumaric acid were quantified in wine having undergone fermentation with multiple strains of D. bruxellensis , with both compounds exhibiting inconsistent changes in concentration. In some cases the concentration of p-coumaric acid in the samples inoculated with D. bruxellensis were higher than in the uninoculated control, and did not exhibit a corresponding loss in the tartrate ester, leaving it unclear as to whether p-coumaroyl tartrate could be enzymatically hydrolysed by D. bruxellensis and subsequently broken down to yield volatile phenols.
15 Chapter 1: Introduction
1.4 Introduction to Glucose Esters.
The presence of glucose esters of hydroxycinnamic acids was first postulated in 1978 by Ong and Nagel who tentatively identified them during a study on the constituents of grapes using high pressure liquid chromatography. 120 Soon after this discovery, Herrmann and Reschke analysed a number of different fruits to isolate and determine their constituents,133 including white grapes, from which the authors confirmed the presence of p-coumaroyl glucose ( 9) and feruloyl glucose (10 ) (Figure 1.4). More recently Baderschneider and Winterhalter isolated both the p-coumaroyl and feruloyl glucose esters from a German Riesling, 108 which was the first reported occurrence of these two compounds in wine. The first report of the isolation of the p-coumaroyl glucose ester from red wine was in 2004 by Monagas, 107 though no feruloyl glucose was detected.
OH OH
O O HO HO HO O O HO O O
OH OH
H3CO
OH OH 9 10 Figure 1.4: 1-O-β-D-Glucose esters of p-coumaric acid ( 9) and ferulic acid ( 10 ).
Very little research has been performed with regard to grape and wine concentrations of 9 and 10 . Through extraction of Riesling wine and purification, Baderschneider was able to isolate 24.2 mg of p-coumaroyl glucose and 23.9 mg of feruloyl glucose, although in this publication there is no mention of the scale of the extraction. 108 In a later report the co- author, Winterhalter, states that the earlier study was performed on 100 litres of wine, 134 which would indicate an approximate glucose ester concentration of approximately 0.24 mg/L, without taking into account losses during extraction and purification.
The identification of p-coumaroyl glucose in four monovarietal red wines via liquid-liquid extraction was described by Monagas et al. 131 Two compounds with identical fragmentation patterns were observed, denoted ‘Hexose ester of trans -p-coumaric acid (1)’
16 Chapter 1: Introduction and ‘Hexose ester of trans -p-coumaric acid (2)’, which were rationalised by attachment to glucose via different glucose hydroxyls. Both esters were found in the four wines at similar concentrations with combined amounts ranging from 0.46 mg/L in Merlot to 0.71 mg/L in Tempranillo, which were determined after 1.5 months of bottle ageing. An extended study by the same author investigated the concentration changes in the two p-coumaroyl glucose ester conjugates through 26 months of ageing in the bottle for three of the four varieties used above. 100 From the figures in their publication, which utilise two x-axes without specification of which axis the data corresponds with, it is initially unclear as to the concentration of the glucose esters, although the initial data points correspond with those described in their previous publication, 131 and can therefore be established. In all three varieties studied, the glucose ester concentrations only fluctuate mildly over 26 months.
Using a similar extraction method, Hernandez et al. studied polyphenolic compounds in red wine during malolactic fermentation, and again, two p-coumaroyl hexose esters were observed. 103 Initial concentrations in wine were 1.23 and 1.51 mg/L, with increases seen throughout both spontaneous and inoculated malolactic fermentation with different species and strains. Final concentrations of p-coumaroyl glucose (1) were between 1.39 and 7.16 mg/L, and 1.95 to 2.63 mg/L for p-coumaroyl glucose (2). While there appears little reason for such a disproportionate increase, this study infers the potential for either chemical or enzymatic formation of the glucose esters during vinification.
More recently, results of Boido et al. mirror those detailed above with a solid-phase extraction resulting in identification of two p-coumaroyl glucose esters in Tannat red wine, both in near identical concentrations. 98 Along with quantification in wine, the content of the two esters in skin was monitored during grape ripening with concentrations of 1.8 and 1.2 mg/kg 20 days after veraison, rising to 2.4 and 1.4 mg/kg 10 days before harvest and finally 2.6 and 1.7 mg/kg at harvest.
All of the studies mentioned above used p-coumaric acid to quantify the p-coumaroyl glucose esters, and employ extraction techniques that can result in loss of analyte with little means of determining the extraction efficiency due to a lack of a pure standard. However, it can be concluded that the glucose esters which accumulate during grape ripening, continue to increase during malolactic fermentation, and are stable throughout wine ageing.
17 Chapter 1: Introduction
Though there exists very little information on the breakdown of the glucose esters, potential pathways to degradation during the vinification process include the use of enzyme preparations containing the appropriate esterases, 64, 112 or by the activity of microbiological esterases, either by those of intended wine microflora or by those of unwanted microorganisms.135-136 Finally, the acidic environment of the wine could promote acid-catalysed hydrolysis,135 though the results of Monagas imply that the glucose esters are stable, or that any hydrolysis is in equilibrium with re-formation. 100
To date, no research has been performed on this class of compounds with respect to the potential for breakdown by D. bruxellensis into volatile phenols.
1.5 Introduction to Ethyl Esters.
While numerous grape derived hydroxycinnamoyl derivatives have been identified, there also exist those that are a product of the winemaking process. This is the case for ethyl coumarate ( 11 ) which was identified by Somers et al. and assumed to be the product of the free hydroxycinnamic acid and ethanol during alcoholic fermentation. 137
O O O O
OCH3
OH OH 11 12 Figure 1.5: Ethyl hydroxycinnamates.
The quantities observed in wine are presumed to be dependent on the original concentration of hydroxycinnamic acid, and the ester synthesising abilities of the yeast employed for alcoholic fermentation. Somers et al. reported that ethyl coumarate was not present in the must of commercial Chardonnay wine, but increased throughout the course of alcoholic fermentation to finally be observed at 2.7 mg/L at day 31. 99 This increase in
18 Chapter 1: Introduction ethyl coumarate was preceded by an initial increase in p-coumaric acid concentration, followed by a steady decline as ethyl coumarate was formed.
Compared with ethyl coumarate ( 11 ), ethyl ferulate ( 12 ) has been less frequently observed in wine, which is most likely to be a product of the relative quantities present, though it has previously been identified using both HPLC and GC in red wine, 138 and also in Riesling wine. 139-140
Both ethyl coumarate and ethyl ferulate were quantified in 3 red wines and 3 white wines in 2003 when Sleep developed a method for analysis using the deuterated analogues. 141 Both esters were identified in all 6 wines analysed, with ethyl coumarate found at 0.35 to 1.02 mg/L and ethyl ferulate at 0.01 to 0.14 mg/L. Furthermore, the ethyl esters were not observed in standard ratios, and wines with the highest ethyl ferulate did not necessarily correspond to those with the highest ethyl coumarate concentrations.
Using the above SIDA method, the evolution of ethyl coumarate was monitored in a Shiraz wine throughout vinification and ageing (AWRI unpublished results) with a sigmoidal increase during alcoholic fermentation, which was followed by a slow accumulation during ageing, peaking at 3600 ppb after 300 days (Figure 1.6).
Figure 1.6: Evolution of ethyl coumarate in Shiraz wine.
Previously described evidence indicated that the activity of alcoholic fermentation has the ability to break down the tartrate esters to give the free acids, which, as shown above, can then be esterified again to give the ethyl hydroxycinnamates. However, there has been no
19 Chapter 1: Introduction evidence as to the ability of D. bruxellensis to then breakdown the ethyl esters to give volatile phenols.
1.5 Research Aims.
With D. bruxellensis reported to express esterase activites, 113 it is surprising that to date no studies have actively measured the volatile phenol output when fermentation is conducted in the presence of common and known esters of hydroxycinnamic acids. The hydroxycinnamoyl tartrate and glucose esters, which are present in the grape berry, survive throughout the winemaking process. Additionally, the evolution of free acids through cleavage of esterified forms, and subsequent esterification to yield the ethyl esters results in a number of hydroxycinnamoyl derivatives that are present during barrel ageing. These esterified forms could contribute to the accumulation of ethylphenols, but are yet to be examined.
As such, this study aims to synthesise the aforementioned hydroxycinnamoyl esters (Figure 1.7) and examine the ability of D. bruxellensis to metabolise these compounds and determine whether they can act as direct precursors to the volatile phenols, or if enzyme preparations containing cinnamoyl esterase are indeed required for the release of the free hydroxycinnamic acids before this conversion can take place.
OH HO
COOH OH
HOOC HO O OH
O O O O O O
R R R
OH OH OH 7 R = H 9 R = H 11 R = H 8 R = OCH3 10 R = OCH3 12 R = OCH3 Figure 1.7: Hydroxycinnamoyl tartrate ( 7 and 8), glucose ( 9 and 10 ) and ethyl esters ( 11 and 12 ) to be synthesised and used in these studies.
20 Chapter 1: Introduction
As previously mentioned, there exist three significant genetic groups of D. bruxellensis in Australia, with one of these accounting for 85% of isolates. By including the hydroxycinnmamoyl esters in fermentation studies with a representative strain (AWRI 1499) from the most common group, the results will be largely representative of what could be expected from Australian isolates of D. bruxellensis .22 As such, small scale D. bruxellensis fermentations will be conducted in the presence of each of the esters with the result determined by analysis for the production of 4-ethylphenol and 4-ethylguaiacol as previously described by Pollnitz et al. 63
21 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
Chapter 2: Synthesis of Hydroxycinnamoyl Esters.
2.1 Synthesis of Hydroxycinnamic Acids and Derivatives.
The synthesis of hydroxycinnamic acids and protected derivatives is a matter of simple organic transformation. More importantly, the hydroxycinnamate derivative must be compatible with the conditions required for esterification with glucose or tartaric acid and subsequent deprotection so that the desired product can be achieved. As such, for use in the synthesis of glucose and tartrate esters, a variety of protected hydroxycinnamic acid derivatives were synthesised, as outlined below (Scheme 2.1).
O
R R i O O
HO HO
13 R = H 11 R = H (93%) 14 R = OCH3 12 R = OCH3 (72%)
ii iii
O O
R R OH O
HO BnO
3 R = H (97%) v 17 R = H (74%) iv ii 4 R = OCH3 (96%) 18 R = OCH3 (69%)
O O O
R R R OH OH OH
AcO ClAcO BnO
19 R = H (83%) 21 R = H (35%) 15 R = H (93%) 20 R = OCH3 (79%) 22 R = OCH3 (65%) 16 R = OCH3 (85%) i) (carbethoxymethylene)triphenylphosphorane. ii) potassium hydroxide. iii) anhydrous potassium carbonate, benzyl bromide. iv) pyridine, acetic anhydride. v) sodium hydroxide, chloroacetyl chloride. Scheme 2.1: Synthesis of hydroxycinnamic acid derivatives.
22 Chapter 2: Synthesis of Hydroxycinnamoyl Esters p-Hydroxybenzaldehyde ( 13 ) and vanillin ( 14 ) underwent Wittig olefination to afford the ethyl esters (11 and 12 ) in good yields, initially being achieved at room temperature in the presence of 2-3 equivalences of the appropriate stabilised ylide over a number of weeks. Alternatively, microwave assisted synthesis proved more facile as a method of synthesis and also afforded 12 with only a small excess of ylide required, but could only be performed on small scales with our system due to the energetic nature of the process, with reaction volumes limited to 15 mL. An attempted scale-up, though minor, for the synthesis of 11 proved unsuccessful given the small volume of the vessel and much of the reaction mixture was lost. As such, the original method at ambient temperature over a number of weeks was the method of choice.
The synthesis of the 1-O-benzyl hydroxycinmamic acids ( 15 and 16 ) was achieved by benzylation of 11 or 12 to give 17 or 18 in 74 and 69% yield respectively, followed by base-catalysed hydrolysis to furnish 15 and 16 in 93 and 85% yield. Previously, the benzylation of caffeic acid was described by Galland et al. and was originally achieved via benzylation of the free acid to give the benzylic ester as well as the ether, followed by hydrolysis of the ester to yield the di-phenolic protected analogue. 142 However, in this case, the production of 11 and 12 allowed for benzylation of the phenol alone.
Unlike in the preparation of 15 and 16 , the phenolic protecting groups with ester functionality were not installed directly onto 11 and 12 due to the potential for removal of the protecting group during hydrolysis of the ethyl ester. Hence hydrolysis of 11 and 12 , in an analogous method to that used for production of 15 and 16 , afforded the free hydroxycinnamic acids (3 and 4) which were subsequently protected.
1-O-Acetyl protection to furnish 19 and 20 in good yields (83 and 79%) was achieved by allowing 3 or 4 to react with acetic anhydride in pyridine.143 The successful incorporation of the acetyl group was confirmed by the inclusion of a 3-proton singlet in each of the 1H NMR spectra corresponding to the acetyl methyl, and the downfield shift of the ring proton signals caused by the electron-withdrawing nature of the protecting group. However, when an analogous process was attempted for preparation of the 1-O-chloroacetyl derivatives (21 and 22 ), it proved unsuccessful. After 16 hours of reaction the mixture had solidified and by dissolving in methanol, analysis by TLC showed no desired product. Instead, these derivatives were prepared using 2M sodium hydroxide solution and chloroacetyl
23 Chapter 2: Synthesis of Hydroxycinnamoyl Esters chloride. 144 This process had to be optimised as the original procedure employed a large volume of sodium hydroxide solution which was found to promote degradation of the chloroacetyl chloride. Therefore it was determined that the hydroxycinnamic acid should be dissolved in a minimal amount of sodium hydroxide solution, aided with sonication, followed by addition of chloroacetyl chloride which afforded 21 in 35% yield and 22 in 65% yield. The yields were diminished by the inability to successfully separate 21 or 22 from 3 or 4, as only the initial recrystallisation yielded pure product with subsequent recrystallisation attempts yielding mixtures of the product and the free acid ( 3 or 4). Characterisation of 21 and 22 by 1H NMR showed the inclusion of a 2-proton singlet at approximately 4.6 ppm in accordance with the literature data. 144-145
These aforementioned processes gave four different derivatives for each hydroxycinnamic acid (free acids 3 and 4, 1-O-benzyl protected 15 and 16 , 1-O-acetyl protected 19 and 20 and 1-O-chloroacetyl protected 21 and 22 ) which would be used in the synthesis of the target glucose and tartrate esters, as well as 11 and 12 which could be used directly in later fermentation experiments.
2.2 Synthesis of Hydroxycinnamoyl Tartrate Esters.
2.2.1 Introduction to Tartrate Ester Synthesis While the existence of tartrate esters in grapes and wines has been widely researched, the synthesis of tartrate conjugates has received less attention. Synthetically, they are mostly built as di-conjugates but because of the prevalence of mono-esterified tartrates in grapes it has become desirable to synthesise mono-esters. 146-147 The synthesis of mono-esterified tartaric acid conjugates has been reported on only a few occasions, but all follow a similar path. The esterification of dibenzyl tartrate with either the acid in the presence of trifluoroacetic anhydride, 148-149 or DMAP and DCC, 150-151 or with an acid chloride, 152 was followed by removal of the benzyl groups via hydrogenolysis (Scheme 2.2).
24 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
OMe OR
HO COOBn COCl COOH HO COOBn
HO COOBn OMe OR HO COOBn
R = H, Me, Pri
DMAP, DCC Et N, DMAP or 3 TFAA
HO COOBn HO COOH OR O OR O
H2, Pd/C O COOBn O COOH
OR OR Scheme 2.2: Literature syntheses of mono-esters of tartaric acid.
The only reported syntheses of hydroxycinnamoyl tartrates are that of chicoric acid, or dicaffeoyl tartrate (Scheme 2.3). Scarpati and Oriente managed the preparation of chicoric acid through reaction of tartaric acid and the acid chloride of caffeic acid protected as a cyclic carbonate (Pathway A), 153 and many other methods for esterification directly from tartaric acid have been utilised. 147 However, the polarity of the products would provide limited methods by which the mono- and di-esters could be separated. In the preparation of di-esters the issue of purification can be simplified by reacting with an excess of the hydroxycinnamate and avoiding the formation of the mono-ester, this would not be the case when attempting to produce mono-esters alone. As such, protection of the tartaric acid moiety is required to simplify the handling of the esterified products.
Zhao and Burke described the preparation of chicoric acid by reaction of diacetyl caffeoyl acid chloride and di-tert -butyl tartrate, followed by treatment with trifluoroacetic acid to remove the tert -butyl groups, and mild acid hydrolysis of the acetyl groups to yield the desired product (Pathway B). 143 However, the synthesis described by Lamidey et al. employs a synthetically simpler starting tartrate derivative and a single protecting group, removing the need for a two-step deprotection. 154
Esterification of dibenzyl caffeic acid with dibenzyl tartrate in the presence of 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and DMAP yielded the hexabenzyl-protected chicoric acid (Pathway C). Removal of the benzyl ethers and esters was achieved in a single step via hydrogenolysis in the presence of triethylsilane and 25 Chapter 2: Synthesis of Hydroxycinnamoyl Esters triethylamine. While these examples describe the preparation of a di-ester, and the exact procedures may not be useful in synthesising mono-esters, at the very least they provide evidence as to the conditions under which the hydroxycinnamoyl esters are, and will be, stable.
O HO COOH R''O R'Cl Pathway A) O
HO COOH R''O 80% Acetic acid O
t t 1) TFAA HO COOBu R'''O COOBu RO COOH R''Cl 2) HCl Pathway B)
t t HO COOBu R'''O COOBu RO COOH
Chicoric acid
HO COOBn R'O COOBn R'''OH Et3SiH, Pd(OAc)2, Et3N Pathway C)
HO COOBn R'O COOBn
O O
R = OH R'' = OAc
OH OAc
O O
OBn R' = O R''' = O O OBn Scheme 2.3: Literature syntheses of chicoric acid.
2.2.2 Synthesis of Hydroxycinnamoyl Tartrate Esters The method by which all of the mono-esters have been created, utilising dibenzyl tartrate, has also been employed in the production of chicoric acid, and was utilised here in the synthesis of requisite hydroxycinnamic acid esters ( 7 and 8). Protection of L-tartaric acid (23 ) with benzyl alcohol in the presence of p-toluenesulphonic acid proceeded smoothly in excellent yields (94%). Esterification of dibenzyl L-tartrate ( 24 ) with acids 15 or 16 catalysed by trifluoroacetic anhydride yielded the tri-benzyl protected L-tartrate esters ( 25 and 26 ). Furthermore, flash chromatography gave two fractions, one of the pure mono- 26 Chapter 2: Synthesis of Hydroxycinnamoyl Esters esters and a second consisting largely of the di-esters ( 27 and 28 ) with only minor co- elution of the mono-esters.
Formation of the mono-esters was confirmed by the unsymmetrical nature of the tartrate proton shifts and the downfield shift of H 2’ , while the undesired di-esters were identified by a symmetrical tartrate as outlined in the literature, 143, 154 a downfield shift of the tartrate
H2’ and H 3’ shifts from 4.63 ppm observed in 24 to approximately 5.9 ppm corresponding to formation of the desired ester functionality, and a 1:1 ratio between hydroxycinnamoyl and tartrate shifts. The di-esters were not fully purified, with 1H NMR data extracted from the crude mixture and simply compared with that for benzyl protected chicoric acid as reported by Lamidey, 154 to explain the reduced formation of the mono-esters.
HO COOH
HO COOH
23
BnOH/p-TsOH
O
HO COOBn R OH +
HO COOBn BnO
24 (94%) 15 R = H 16 R = OCH3
TFAA
O COOBn
R COOBn BnOOC COOBn O + OH R'O OR' BnO
25 R = H (47%) 27 R' = 15 28 R' = 16 26 R = OCH3 (50%) Scheme 2.4: Synthesis of benzylated hydroxycinnamoyl tartrate esters.
Removal of the benzyl groups by hydrogenolysis was first attempted as described by Galland et al. for 1-O-benzyl-caffeoyl glucose, 142 using 1,4-cyclohexadiene as the proton donor, but when attempted on 26 this method resulted in reduction of the double bond in 27 Chapter 2: Synthesis of Hydroxycinnamoyl Esters preference to debenzylation to give 29 , as determined by the loss of the alkene proton shifts in the proton spectrum and the appearance of saturated alkane shifts between 2.84 and 2.48 ppm. The characterisation of multiple reduced hydroxycinnamate derivatives show all signals for the alkane protons observed in the region of 2.5 to 3.1 ppm. 155-158 In addition, minor shifts due to this second tartaric acid derivative were observed, moving upfield from those corresponding to 26 . Furthermore, limited debenzylation was suspected as the entire reaction mixture was soluble in chloroform-d, which is not expected for the desired products ( 7 and 8).
A subsequent attempt using triethylsilane as a more mild hydrogen donor,154 again resulted in reduction of the double bond, though concomitant debenzylation also occurred affording 30, which was characterised by a further downfield shift of the tartrate proton shifts and the loss of the signals corresponding to the benzyl moieties. Altering the stoichiometry of the reactants showed evidence of the desired product, but only in very small quantities, and not consistently. In conclusion, the use of benzyl protection was an insufficient method for consistent and reproducible production of the desired hydroxycinnamoyl tartrates, thus an alternative approach was investigated.
O COOBn
H3CO COOBn O
OH BnO 26
1) 1,4-cyclohexadiene, Pd/C 2) Et3SiH, Pd/C
O COOBn O COOH
H3CO COOBn H3CO COOH O O + OH OH BnO HO 29 30 Scheme 2.5: Attempted debenzylation procedures.
In a similar fashion to Zhao or Lamidey et al. the synthesis of the tartrate esters was attempted using di-tert -butyl tartrate ( 34 ). However, due to the cost of the starting material it was synthesised rather than purchased.
28 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
The use of 34 was described by Uray and Lindner including details of the conversion of di- O-acetyl tartaric acid ( 32 ) to 34 using an undesirable reagent, isobutylene. 159 The use of gaseous isobutylene was avoided by Wright et al., who reported a method for the in-situ formation of isobutylene from the acid-catalysed dehydration of tert -butanol. 160 A review of tartrate synthesis by Syndoradzki et al. outlined the procedure for synthesis of 32 , which proceeded through di-O-acetyl tartaric anhydride ( 31 ). 146
O HO COOH AcO AcO COOH AcO COOBut AcCl H2O SOCl2, t-BuOH O
t HO COOH AcO AcO COOH AcO COOBu O 23 31 (82%) 32 (99%) 33 (49%)
KOH
HO COOBut
HO COOBut
34 (29%) Scheme 2.6: Synthesis of di-tert -butyl tartrate.
Procedures for the preparation of 31 are well known, 161-163 though under these conditions, one study noted the formation of 32 directly. Barros et al. reported the formation of 32 directly, without first yielding 31 , and furthermore was characterised in deuterated chloroform, in which 32 was found to be insoluble, unlike the anhydride. 162 Here, 31 was achieved in 82% yield, but the simplicity of the proton NMR led to ambiguity. The 1H
NMR spectra of 31 and 32 (run in acetone-d6) both display only a shift for the CH protons at either 6.17 and 5.72 ppm and a shift for the acetyl protons at 2.19 or 2.11 ppm, respectively, and differ slightly between solvents.161-163 As such, the formation of 31 was confirmed via determination of the melting point, with the literature value differing by around 20 oC to that of 32. Preparation of 32 was achieved through stirring with 2 equivalences of water in acetone, though recommendations are for the use of water alone to ring-open the anhydride, 146 it was discovered however, that reacting in an organic solvent with minimal water lead to a more simple work-up. Concentration followed by trituration with hexane gave 32 as a white solid in 99% yield after removing excess water in vacuo .
29 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
Installation of the tert -butyl ester functionality was successful on small scales using the method of Wright, but yields diminished as the scale of the reaction was increased. Nonetheless, this proved superior to another method which involved first producing the di- acid chloride and reacting with tert -butanol, 161-162 which tended to cause dehydration of 32 with concomitant formation of 31. In the preparation of 33 , mono-O-acetyl di-tert -butyl tartrate was produced as a minor product, which could also be used in the subsequent deacetylation. Initially, the two products were separated for characterisation, though a subsequent synthesis used a mixture of the two for the following reaction.
Uray describes the use of potassium hydroxide to deacetylate 33 , although the amount of potassium hydroxide required proved less than reported.159 The literature procedure involves a larger amount of potassium hydroxide and a very quick reaction time, though the reaction was much more controllable with smaller amounts of potassium hydroxide for a longer period. Minor amounts of tert -butyl methyl tartrate was isolated during purification and characterised by 1H NMR, with the data corresponding to that previously reported. 159 Presumably upon addition of potassium hydroxide to the reaction mixture a portion of the methanol was deprotonated and the resulting methoxide was responsible for the replacement of a tert -butyl group for a methyl ester.
Synthesis of 35 or 36 was achieved by converting the hydroxycinnamate to the acid chloride, and then reacting with 34, but this could only be achieved for hydroxycinnamates with phenolic protection (19 -22 ).
O O COOBut
R R COOBut OH 1) SOCl2 O
2) Pyridine, 34 OH AcO AcO
19 R = H 35 R = H (31%) 20 R = OCH3 36 R = OCH3 (48%) Scheme 2.7: Esterification of hydroycinnamic acids and di-tert -butyl tartrate.
Preparation of 35 and 36 was achieved in relatively good yields, with minor formation of the di-ester, which could be lessened by using a greater excess of 34 . The unreacted 34 was easily recovered during flash chromatography of the reaction mixture, eluting with 10%
30 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
EtOAc/X4 while 35 and 36 , with similar Rf values, eluted with 20-30% EtOAc/X4.
Interestingly, the di-esters displayed higher Rf values by TLC, but eluted after the mono- esters from flash chromatography, which is presumably an effect caused by the differences in the silica gel used during flash chromatography and that on the TLC plates. However, purification with flash chromatography provided adequate separation of 35 and 36 from the di-esters to obtain pure samples, with the mixtures analysed by 1H NMR to confirm the presence the di-esters. While the data is not shown, the di-esters exhibited the same hydroxycinnamoyl 1H shifts as 35 and 36 , a symmetrical tartrate lacking a free hydroxyl and integration of all signals confirmed two hydroxycinnamoyl moieties for every tartrate, as described by Zhao. 143
Recrystallisation of 35 and 36 from ethyl acetate/X4 yielded hygroscopic white crystals which were submitted to X-ray crystallography (Figures 2.1 and 2.2), which confirm retention of the ( R,R )-stereochemistry during preparation of 34 and esterification to afford 35 and 36 .
Figure 2.1: Molecular structure and crystallographic numbering scheme for 35.
31 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
Figure 2.2: Molecular structure and crystallographic numbering scheme for 36 .
The 1H and 13 C shifts of the tert -butyl groups within 35 and 36 were assigned using the shifts of the previously synthesised tert -butyl derivatives. The 1H tert -butyl shifts of 33 appear at approximately 1.44 ppm, whereas they appear at 1.52 ppm in 34 . It was assumed that the tert -butyl group adjacent to the esterified hydroxyl would possess the upfield shift, which was confirmed with the tert -butyl shifts for the di-esters appearing at 1.44 ppm.
Attempted deprotection of 35 using a previously described method which employed trifluoroacetic acid to remove the tert -butyl groups followed directly by acid promoted deacetylation, 143 resulted in the desired product ( 7), which by 1H NMR analysis was shown to exist in a mixture with other p-coumaroyl tartrate derivatives. Four components were observed all possessing overlapping signals corresponding to a p-coumaroyl moiety bound to tartaric acid, which was rationalised by the formation of tartaric acid sodium salts during work-up. Purification using reverse-phase chromatography was unsuccessful as all components co-eluted, and in an attempt to protonate the sodium salts the mixture was taken up in methanol, the pH adjusted to 1 (2M HCl solution) and stirred at room temperature for 16 hours, which was unsuccessful as shown by 1H NMR analysis. Furthermore, the use of acetyl protection in the synthesis of hydroxycinnamoyl tartrates was discontinued when it was discovered that removal of these groups within the glucose esters ( 54 and 55 , described in a later section), which was performed concurrently, was not possible while retaining the hydroxycinnamoyl ester linkage.
Utilising a more labile phenolic protection strategy the 1-O-chloroacetyl derivatives, 21 and 22 , were esterified with 34 , yielding the chloroacetylated products 37 and 38 . During 32 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
purification of 38 with flash chromatography, a second compound (of lower R f) was isolated and identified as 40 , having undergone phenolic deprotection under the reaction conditions. To test the efficacy of the dechloroacetylation, the p-coumaroyl analogue ( 37 ) was dissolved in 1:1 pyridine/benzene and stirred at room temperature for 24 hours. Concentration and separation by flash chromatography, gave an approximate 60:40 ratio of 39 :37 .
For the following esterification attempts reaction times were increased, which gave 39 and 40 as the major products (19 and 28% yields), with some 37 and 38 remaining (11 and 6%). This reaction was not further optimised, as it gave the desired products in acceptable yields, and further deprotection of the chloroacetylated compounds under the same conditions could be achieved.
Recrystallisation of 39 and 40 in an analogous manner to 35 and 36 yielded white solids, which were not suitable for X-ray crystallography, and though the microanalysis of 40 was within acceptable limits, the hygroscopic nature of the compounds resulted in incorrect microanalytical data for 39 . Nonetheless, the crystallographic data and optical rotations of 35 and 36 and the microanalysis of 40 achieved under the same conditions, along with the similar 1H and 13 C spectra, optical rotations and expected high resolution accurate mass determination strongly support the structure of 39 and 40.
Deprotection of 39 and 40 was achieved using trifluoroacetic acid, concentrated and purified with reversed-phase chromatography. 143 The crude product was dissolved in methanol and loaded onto a pre-packed 4 g C18 cartridge which was washed with water/formic acid (99:1) and 7 and 8 eluted with water/acetonitrile/formic acid (69:30:1). Concentration of the fractions containing only pure product yielded 7 and 8 which could be used directly in fermentation experiments, described in Chapter 5.
33 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
O
R OH
ClAcO
21 R = H 22 R = OCH3
1) SOCl2 2) Pyridine, 34
O COOBut O COOBut
R COOBut R COOBut O + O OH OH ClAcO HO
37 R = H (30%) Reacted for 16 hours 38 R = OCH3 (34%) 40 R = OCH3 (24%)
37 R = H (11%) 39 R = H (19%) Reacted for 45 hours 38 R = OCH3 (6%) 40 R = OCH3 (28%)
TFA, C18-RP chromatography
O COOH
R COOH O
OH HO
7 R = H (82%) 8 R = OCH3 (41%) Scheme 2.8: Synthesis of hydroxycinnamoyl tartrate esters.
2.3 Synthesis of Hydroxycinnamoyl Glucose Esters.
2.3.1 Introduction to Glucose Ester Synthesis Glycosylation methods have been extensively studied, and also widely reviewed in books and journal articles, 164-170 including specific articles discussing effects that control glycosylation such as the anomeric effect. 171
34 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
Previous attempts in this research group to synthesise glucose conjugates, have taken advantage of a modified Koenigs-Knorr reaction. Again, since the original publication which employed glucopyranosyl bromide, and silver carbonate in the presence of the alcohol to achieve glycosylation, 172 this reaction has been modified and improved, and these advancements have also been reviewed numerous times. 173-175
Glucosylation has been achieved via pivaloyl or acetyl protection of glucose followed by activation of the glucosyl donor via installation of anomeric bromine (Scheme 2.9). 175-177 The glucose ether linkage was created using silver triflate as a catalyst, followed by removal of glucosyl protecting groups under basic conditions. However, the synthesis of a glucose ester differs slightly to that of a glucoside due to the ease of hydrolysis of the ester linkage under the basic conditions required to remove the glucosyl protection.
OR1 OR1
R OH, 2,6-lutidine O 2 O R1O R1O OR R1O AgOTf, CH2Cl2 R1O 2
R1O R1O Br R1 = Ac, Piv Scheme 2.9: Modified Koenigs-Knorr reaction conditions employed within this research group.
This procedure was employed for glycosylation of hydroxycinnamic acids by the current author using acetyl glucose protection, which could not be removed with the glucose ester linkage remaining intact, 178 even though Birkofer et al. has reported the use of acetyl protection on both the glucosyl hydroxyls and on the phenol to achieve hydroxycinnamoyl glucose esters. 179 To ensure consistent and adequate yields for the target esters, a different protecting group was needed for the glycosyl protection in order to achieve synthesis of hydroxycinnamoyl glucose esters.
The use of the chloroacetyl group in carbohydrate synthesis was first described by Bertolini and Glaudemans which provides an alternative to the acetyl group in that the former can be removed under more mildly nucleophilic conditions due to the decreased electron density of the carbonyl carbon caused by the electron withdrawing nature of the chlorine. 180 Chloroacetyl protection was utilised by Ziegler and Pantkowski to prepare cinnamic and hydroxycinnamic acid esters via numerous modified Koenigs-Knorr
35 Chapter 2: Synthesis of Hydroxycinnamoyl Esters glycosylations (Scheme 2.10). 144 The glycosylation attempts, with either cinnamic acid ( i) or 3,4,5-trimethoxybenzioc acid ( ii ), gave 51-58% of a mixture of α- and β-glucose ester (77-81% α-ester) using a glucosyl fluoride in the presence of boron trifluoride etherate (Method 1). Purification of the crude mixtures afforded 39% of the α-anomer and 27% of the α-anomer for reactions i and ii , respectively. Using chloroacetyl protected galactopyranosyl bromide and the silver carboxylate yielded 70% of the β-galactose ester (Method 2). However, improving on either selectivity for the β-anomer (over method 1) or simplicity of reaction (eliminating light sensitive reagents in method 2), the use of a glucopyranosyl trichloroacetimidate in the presence of cinnamic acid and trimethylsilyl trifluoromethanesulfonate (TMSOTf) resulted in a 75% yield with a ratio of 92:8 β:α- glucose ester (Method 3).
Method 1) OAcCl OAcCl OAcCl
RCO2H ClAcO O ClAcO O ClAcO O ClAcO ClAcO ClAcO OC(O)R ClAcO ClAcO ClAcO F OC(O)R i) R = cinnamic acid, 58% crude, 81:19 (α:β) ii) R = 3,4,5-trimethoxybenzoic acid, 51% crude, 77:23 (α:β) Method 2) OAcCl OAcCl OAcCl ClAcO ClAcO ClAcO RCO Ag O 2 O O ClAcO ClAcO ClAcO OC(O)R
ClAcO ClAcO ClAcO Br OC(O)R 70% 0:100 (α:β) Method 3) OAcCl OAcCl OAcCl
RCO H 2 O O ClAcO O ClAcO ClAcO ClAcO ClAcO ClAcO OC(O)R
ClAcO O CCl3 ClAcO ClAcO OC(O)R 75% 8:92 (α:β) NH Scheme 2.10: Glycosylation reactions of Ziegler.
The trichloroacetimidate glycosylation method was developed by Schmidt and involved preparation from trichloroacetonitrile under basic conditions. 181 This method has been explored in great detail appearing as book chapters authored or co-authored by Schmidt. 168- 169 The trichloroacetimidate method, along with chloroacetyl protection was implemented by Galland to synthesise caffeoyl glucose esters which were used for co-pigmentation studies in wines (Scheme 2.11). 142 36 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
HO O
OAcCl OAcCl OBn
O ClAcO O Sieves, AgOTf ClAcO ClAcO ClAcO O OBn ClAcO ClAcO O CCl3 OBn O
NH OBn
1) Pyridine, water
2) 1,4-cyclohexadiene, Pd/C
OH OH
HO O HO O OH OH O
Scheme 2.11: Glycosylation method described by Galland.
While Galland employed silver triflate to catalyse the glycosylation, Ziegler utilised trimethylsilyl triflate, as is outlined by numerous examples in Preparative Carbohydrate Chemistry, 169 which removes the need for the reaction to be carried out in the dark. This minor change aside, preparation on the glucosyl donor via chloroacetylation, anomeric deprotection and activation through preparation of trichloroacetimidate via previously described methods, 142, 144, 180-181 along with the phenolic protection strategies of Galland to give the protected glucose esters, deprotection was expected to yield p-coumaroyl and feruloyl glucose esters ( 9 and 10 ).
2.3.2 Synthesis of Hydroxycinnamoyl Glucose Esters Formation of 41 was achieved by addition of chloroacetylchloride (46 ) to D-glucose yielding an approximate 55:45 mixture of α-:β-41 after purification and in excellent yields (91%). Subsequent preparation of 42 gave some residual unreacted 41 solely as the α- anomer, from which it could be seen by 1H NMR that the shifts for the chloroacetyl groups are not singlets, as previously reported, 142, 182 but distorted AB quartets. This effect is displayed as a large central signal representing the overlapping inner lines, with very minor outer lines which when obscured leaves only the single signal corresponding to the inner lines.
37 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
One chloroacetyl group in α-41 showed separated inner lines, appearing as two singlets, as the satellite peaks were unresolved from other shifts and the coupling in the distorted doublets cannot be determined. This effect is difficult to observe in a spectrum of both anomers, possibly why the previous reports give a two proton singlet for each chloroacetyl group.142, 182 In the experimental section (Chapter 6) the chloroacetyl shifts are referred to as apparent singlets (app. s) as they are technically highly distorted pairs of doublets where the coupling cannot be sufficiently determined.
In the purification of 41 or 42 both anomers largely co-eluted on column chromatography, and appeared as one spot by TLC. However, the α-anomer did elute slightly before the β- anomer, meaning that discarded fractions containing one anomer and an impurity could alter the anomeric ratio which, if important to the synthesis, should be determined prior to the final purification.
Preparation of 42 gave a 55% yield with an anomeric ratio of 70:30 (α:β) as expected, 182 but subsequent synthesis of 43 did not give the pure α-anomer as was reported by Galland. 142 Instead 43 was isolated in a 2:1 ratio ( α:β), supporting the findings of Ziegler, 144 having isolated a 2.6:1 mixture of α:β-anomer. The anomers of 43 possess different R f values by both TLC and flash chromatography, and as such the β-anomer, eluting second, could potentially be mistaken for a by-product and discarded. Furthermore, 43 undergoes hydrolysis, as shown by Skouroumounis, 174 and it is was found that the β-anomer was hydrolysed preferentially to the α-anomer, with the hydrolysis product eluting between the α- and β-43 . From column chromatography the order of elution is α-43 , the hydrolysis product, then β-43 .
38 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
OAcCl OAcCl OH
O O ClAcCl, pyridine ClAcO Hydrazine acetate ClAcO HO O ClAcO ClAcO HO ClAcO OH ClAcO OAcCl HO OH
42 (55%, 70:30 α:β) 41 (91%, 55:45 α:β)
Cl3CCN, DBU
O OH
OAcCl OAcCl
R1
ClAcO O Sieves, TMSOTf ClAcO O ClAcO ClAcO O R2 + ClAcO O CCl3 ClAcO O R2 43 (75%, 67:33 α:β) NH R1
15 R1 = OBn, R2 = H 44 R1 = OBn, R2 = H (52%) 16 R1 = OBn, R2 = OCH3 45 R1 = OBn, R2 = OCH3 (54%) 47 R = H, R = H 1 2 48 R1 = H, R2 = H (54%) Scheme 2.12: Synthesis of 1-O-benzyl hydroxycinnamoyl glucopyranoses.
The glycosylation procedure was initially trialled with cinnamic acid ( 47 ) with the desired product, 48 , isolated in 54% yield and possessing a distinct 1H NMR shift for the anomeric proton, with a coupling constant of 8.2 Hz supporting production of the β-ester and matching the literature data. 144 While synthesis of 48 proved useful in confirming the efficacy of the glycosylation technique and determining the stoichiometric conditions, the lack of phenol functionality meant that it was not useful in confirming the entire synthetic pathway due to the inability to investigate phenolic deprotection strategies. However, synthesis of a single anomer of 48 allowed for confirmation of the AB quartet nature of some chloroacetyl protons (Figure 2.3). The inner lines at 4.03 and 4.04 ppm corresponded with satellite peaks at 3.99 and 4.08 ppm, giving 4.05 and 4.02 ppm doublets with 14.5 Hz coupling.
39 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
Figure 2.3: 1H proton NMR spectrum of the chloroacetyl protons in 2,3,4,6-O- tetrachloroacetyl-β-D-glucopyranosyl cinnamate ( 48 ).
Synthesis of 44 and 45 gave very similar yields to the cinnamate ( 48 ), and the formation of the β-esters was confirmed by the shift of the anomeric proton signal from 6.60 ppm (3.7 Hz) and 5.94 ppm (7.6 Hz) observed for 43 , to a single signal at 5.91 ppm possessing an 8.2 Hz coupling constant corresponding to the 1,2-trans conformation of the sugar. However, debenzylation in the presence of the α,β-double bond was investigated concurrently during the synthesis of the hydroxycinnamoyl tartrate esters ( 7 and 8, described above) and due to those findings 44 and 45 were not used further in the production of the glucose esters.
Glucosylation using 3 and 4 gave inadequate yields of 49 and 50 which co-eluted with a sugar impurity from column chromatography. In one instance 50 was prepared as a pure compound, though the outcome of the reactions were inconsistent with 49 only being achieved as a mixture of compounds identified based on the 1H NMR spectra, of which the data for 50 matched that later reported by Zhu and Ralph. 183
Regardless, impure 49 , as well as 48 and 50 , were deprotected followed by attempted purification on XAD-2, which proved insufficient. Although multiple purification attempts
40 Chapter 2: Synthesis of Hydroxycinnamoyl Esters using XAD-8 resin provided separation of the desired products and the 6-O-chloroacetyl- β-D-glucopyranose derivatives ( 51 and 52 ) which were identified by the 1H NMR characterisation of 51 alone.
O OH
OAcCl OH
43, Sieves, TMSOTf ClAcO O ClAcO O R ClAcO R O 49 R = H (29% crude) OH 50 R = OCH3 (39%) 3 R = H 4 R = OCH3
Pyridine, water
OH OAcCl OH OH
HO O + HO O HO O HO O R R OH OH O O 9 R = H 51 R = H (25%) 10 R = OCH3 52 R = OCH3
XAD-8
OH OH OH
O HO + HO O HO O HO O R OH OH O O
trans-9 R = H trans-10 R = OCH3 R
OH cis-9 R = H (33% both isomers) cis-10 R = OCH3 (20% both isomers) Scheme 2.13: Synthesis of glucose esters with free hydroxycinnamic acids.
Compounds 9, 10 and 53 were isolated with minor impurities where the excess signals were attributed to only a single compound as indicated by 1H NMR integration. By identifying the distinct ring coupling present in 10 , the presence of a β-anomeric proton signal consistent with a glucose ester, and coupling constants of 13.0 Hz for the α,β- doublets, led to identification of the impurity as cis -10 . Upon investigation of the literature,
41 Chapter 2: Synthesis of Hydroxycinnamoyl Esters the changes in 1H signals between trans -10 and the impurity were consistent with that observed between cis - and trans -ferulic acid ( 4)184 and between cis - and trans -ethyl ferulate ( 12 )185 supporting the presence of the cis -isomer, which was also found to be the case for 9 and 53 (Figure 2.4).
From the work of Kahnt, 186-187 isomerisation was determined to be a result of exposure to ultra-violet radiation during purification. However, the nature of ambient light conditions that 9, 10 and 53 were exposed to indicated that photoisomerisation was not limited to ultra-violet light and exposure to laboratory lighting would also induce this effect. Isomeric ratios differed slightly between glucose esters, but were in the vicinity of 4:1 ( trans :cis ).
Figure 2.4: NMR spectrum of isomerised glucose esters. a) cis/trans -Feruloyl glucose ( 10 ). b) cis/trans -Cinnamoyl glucose ( 53 ).
Furthermore, photoisomerisation was limited to 9, 10 and 53 , and not experienced during the synthesis of any other hydroxycinnamate derivative. As a precaution further synthetic attempts towards 9 and 10 were conducted under red light while further investigation into the photoisomerisation is outlined in Chapter 4. While 9 and 10 could be synthesised directly from 3 and 4 without employing phenolic protection, the yields were lower than desired and unpredictable enough to warrant further investigation into the use of phenolic protection to develop a reproducible and reliable method of synthesis.
42 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
The use of 19 and 20 in glucosylation gave good yields of 54 and 55 , but removal of the acetyl group could not be achieved with retention of glucose ester functionality. Previously, attempted removal of glucosyl acetates resulted in hydrolysis of the hydroxycinnamoyl glucose linkage, 178 though the use of phenolic acetyl protection was attempted based on the documented successful removal in the presence of a hydroxycinnamoyl ester. 143, 179
Removal of the glycosyl chloroacetyl groups from 54 in 1:1 pyridine/water failed to remove the phenolic acetyl group, yielding 56 as evidenced by crude 1H NMR analysis. Attempted purification on XAD-8 resulted in the appearance of another compound with a lower R f value than 56. Partial separation by flash chromatography gave a small amount of 1 the lower R f compound which was determined to be 9 by H NMR, as well as a mixture of 9 and the original 56 . While 56 could only be isolated as a mixture, with subsequent purification attempts removing some of the residual 9 but also giving slight deacetylation producing additional 9, the 1H NMR could be assigned by ignoring those signals known to belong to 9. Removal of all chloroacetyl groups was evident by the glucosyl proton signals all appearing between 3.86 and 3.38 ppm, indicating the deprotected species, but also possessing a 3-proton signal at 2.29 ppm and ring proton signals slightly downfield from those observed in 9, which is consistent with the presence of a phenolic acetyl group.
43 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
O OH
OAcCl OAc
43, Sieves, TMSOTf ClAcO O ClAcO O R ClAcO R O 54 R = H (40%) OAc 55 R = OCH3 (44%) 19 R = H 20 R = OCH3
OAcCl OAc
ClAcO O ClAcO O ClAcO O 54
1:1 Pyridine/water XAD-8
OH OH OAc OH
HO O HO O + HO O HO O OH OH O O 56 (20%) 9 (4%) Scheme 2.14: Glycosylation with 1-O-acetyl hydroxycinnamic acids and partial deacetylation using XAD-8 resin.
While XAD-8 gave minor deprotection of the acetyl group, it was not enough to be an effective method in which to synthesise 9 and 10 , with only trace amounts produced. Alternative methods for deacetylation included stirring with Amberlite IR-120 (H + form), 188 Amberlite IR-4B ( -OH form) and catalytic amounts of triethylamine, which failed to yield 9, with increasing hydrolysis of the glucose ester bond as the methods became less mild. As such, preparation of 54 (and 55 ) was not a viable pathway to 9 (and 10 ), giving very small yields through accidental deprotection on XAD-8.
The use of 21 and 22 for glycosylation has been attempted by Ziegler using a glucosyl fluoride in the presence of boron trifluoride etherate, which gave a moderate yield (51%) and largely the α-glucose ester (Scheme 2.10). Purification of the mixture gave 27% of the α-glucose ester, though the use of similar glycosyl acceptors and varying glucosyl donors
44 Chapter 2: Synthesis of Hydroxycinnamoyl Esters suggests that the preference for production of the α-anomer is a factor of the glucosyl donor, and the use of 21 with the trichloroacetimidate method might therefore yield largely the β-anomer, as was seen when used with cinnamic acid. Though the deprotection of this compound is not reported, 144 the phenolic chloroacetyl group was not labile under glycosylation conditions and was expected to be labile under the same deprotection conditions used to remove the glucosyl chloroacetates.
Compounds 57 and 58 were prepared in 48 and 64% yield respectively, and were characterised fully, following which the data reported for the synthesis of 58 by Zhu and Ralph matches that obtained here. 183 Deprotection of 57 and 58 required reaction times that were 50% longer (6 hours) to remove all five chloroacetyl groups than previously experienced for the removal of four groups (4 hours, 48 -50, 54 ). Again, a large proportion of the incomplete reaction mixture existed as the 6-O-chloroacetyl protected species.
Given the large proportion of mono-protected species, and co-elution from XAD-8, alternative purification attempts included flash chromatography using 1% formic acid/ethyl acetate, 5% methanol/ethyl acetate and 10% methanol/dichloromethane. The two former solvent systems resulted in co-elution, the later afforded pure fractions of each, from which re-reaction of the mono-protected analogues yielded the desired products, 9 and 10 . Given full conversion to 9 and 10 , purification by flash chromatography using 5% methanol in ethyl acetate is a convenient and quick method, but fails to remove the mono-protected species.
Furthermore, TLC of 9 and 10 in aqueous solvent systems utilised heat to evaporate excess solvent between applications, which resulted in three spots, which were found to correspond to the desired product ( 9 or 10 ), D-glucose and the aglycone (based on corresponding R f values of TLC standards) in varying ratios depending on the amount of heat applied. The decomposition of the compound on silica by heat could have resulted in previously purified samples being deemed impure and further purifications attempted. As such, TLC of the glucose esters should be performed without the use of a heat gun, with the excess solvent allowed to evaporate at room temperature.
Following an adequate method of purification, a previously pure sample of glucose ester contained impurities that did not show up by TLC, but as seen by 1H NMR possessed the
45 Chapter 2: Synthesis of Hydroxycinnamoyl Esters correct shifts to be a hydroxycinnamoyl glucose derivative with ester functionality. The ratio of the hydroxycinnamate to glucose shifts remained 1:1, but various shifts that were consistent with anomeric protons in different environments. After investigation of the literature, the appearance of the extra proton shifts were determined to be caused by acyl migration, or movement of the hydroxycinnamate onto different glucose hydroxyls, which was observed to occur to a greater extent in p-coumaroyl glucose than in feruloyl glucose. In labelling the migrated structures, the attachment to glucose is designated by the hydroxyl number (1, 2, 3, 4 or 6) and the orientation of the anomeric hydroxyl designated either α or β.
O OH
OAcCl OAcCl
43, Sieves, TMSOTf ClAcO O ClAcO O R ClAcO R O 57 R = H (48%) OAcCl 58 R = OCH3 (64%) 21 R = H 22 R = OCH3
Pyridine, water OH OH
HO O HO O R OH O
9 R = H (43% all esters) 10 R = OCH3 (20% all esters)
Migration
HCA = O
OR2
HO O HO
R1O R OH
OH α R1 = HCA, R2 = H, 2-O- -ester α R1 = H, R2 = HCA, 6-O- -ester Scheme 2.15: Glycosylation of 1-O-chloroacetyl hydroxycinnamates, and migration of the free glucose esters.
46 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
The shifts for the migrated esters (Table 2.1) were assigned using data from Brecker who studied the migration of glucosyl acetate and formate esters, 189 and the main esters produced, apart from the 1-O-β-esters, are the 2-O-α-esters and the 6-O-α-esters. However, shifts could be assigned for the 1-O-α-ester and the 3-O-α-ester, as well as minor production of the corresponding 2/3/6-O-β-esters. In addition to altered positions and coupling constants for the anomeric protons, the shifts for the proton at the point of attachment appeared further downfield. Coupling constants of 3.7 Hz corresponded to α- anomeric hydroxyls, with 7.8-8 Hz for β-anomeric hydroxyls, with the nature of the aglycone ( p-coumaroyl or feruloyl) having no impact on the shifts of the glucose protons.
1 Table 2.1: H NMR shifts for migrated hydroxycinnamoyl glucose esters in CD 3OD.
Ester H1' (ppm) H2' (ppm) H3' (ppm) H6a' (ppm) H6b' (ppm) 1-O- ααα 6.21 (d, 3.7 Hz) 1-O- βββ 5.6 (d, 7.8 Hz) 2-O- ααα 5.33 (d, 3.7 Hz) 4.67 (dd, 10.0 and 3.7 Hz) 2-O- βββ 4.69 (d, 8.0 Hz) 4.77 (dd, 9.6 and 8.0 Hz) 3-O- ααα 5.14 (d, 3.7 Hz) 4.97 (dd) 3-O- βββ 4.6 (d, 7.8 Hz) 5.04 (dd) 6-O- ααα 5.08 (d, 3.7 Hz) 4.5 (dd, 11.8 and 2.1 Hz) 4.31 (dd, 11.8 and 5.4 Hz) 6-O- βββ 4.48 (d, 8.0 Hz) 4.45 (dd, 11.8 and 2.2 Hz) 4.29 (dd, 11.8 and 6.0 Hz)
When the migrated mixtures were submitted to wine-like conditions (10% ethanol, pH of 3.5) the 1-O-β-esters predominated, but attempts at isolation of these gave a mixture of esters, as determined by 1H NMR. The migration of these esters was studied further (Chapter 3), although a mixture of esters was used in fermentation experiments (Chapter 5). While the desired 1-O-β-esters should proliferate in the fermentation media, the evolution of 4-ethylphenol and 4-ethylguaiacol would require metabolism of a hydroxycinnamoyl glucose ester, regardless of its position of attachment to glucose.
2.4 Conclusions.
Along with ethyl esters ( 11 and 12) , which could be used directly in fermentation experiments, the synthesised hydroxycinnamoyl derivatives ( 3, 4, 15 , 16 , 19 -22 ) were used to explore a new method for successful synthesis of 7 and 8 for the first time, which can
47 Chapter 2: Synthesis of Hydroxycinnamoyl Esters now be investigated directly with respect to ethylphenol formation in the presence of D. bruxellensis .
O COOH O
R COOH R O O
OH HO HO
7 R = H 11 R = H 8 R = OCH3 12 R = OCH3 Figure 2.5: Hydroxycinnamate esters to be used in fermentation experiments.
The synthesis of 9 and 10 was achieved, following which the synthetic methodology was further confirmed by Zhu and Ralph, 183 who published the synthesis of feruloyl glucose with only slight differences to that detailed above. However, the transformations that were observed for 9 and 10 have not been reported by any other author, and will be investigated in greater detail. The speed of photoisomerisation of 9 and 10 during synthesis was not observed for any other hydroxycinnamoyl derivative, and as such should be investigated to determine what factors contributed to this phenomenon, with the ultimate aim of achieving synthesis of pure trans -glucose esters without having to handle them exclusively under red light. Additionally, if the ratio of cis:trans -9 and 10 that was observed in the laboratory is seen in the grape berry, the contribution of the cis -isomers to the production of ethylphenols, or otherwise, could have a large effect on the organoleptic properties of wine.
OH OR OH
HO O HO O HO O HO OR HO HO OH HO RO OH OH
R = trans-p-coumaroyl or trans-feruloyl Figure 2.6: Dominant equilibria in hydroxycinnamoyl glucose ester mixtures to be used in fermentation experiments.
The migration of the glucose esters could not be controlled under experimental conditions, though the 1-O-β-esters were observed to predominate in a wine-like environment. The potential for migration in wine should be investigated to determine if esters other than the
48 Chapter 2: Synthesis of Hydroxycinnamoyl Esters
1-O-β-esters isolated by Baderschneider 108 could be present, and if the two p-coumaroyl hexose esters observed by Monagas 100, 131 and Hernandez 103 are indeed present in wine and not products of the analysis, or if additional esters are present that were not observed. Regardless, 9 and 10 can be submitted to D. bruxellensis to determine their role in the production of ethylphenols, given that under fermentation conditions they should exist mainly as the 1-O-β-esters.
49 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters.
3.1 Introduction.
Previously (Chapter 2) it was observed that the prepared 1-O-β-hydroxycinnamoyl glucose esters ( 9 and 10 ) underwent acyl migration under acidic conditions to give a range of undesired mono-hydroxycinnamoyl esters which exhibited alternative glycosyl attachment, affording largely the 2-O-α- and 6-O-α-esters, in addition to the initial 1-O-β-esters. While the extent of migration and the ratio of the resulting esters differed between synthetic attempts, a common factor for production was flash chromatography on silica gel, with no migration observed during alternative purification attempts.
OH OR2 6 OH 6 4 4 H+ O HO O HO 1 HO O HO 1 2 3 2 3 1 OH R O OH O
R1 = HCA, R2 = H, 2-O-α-ester R1 = H, R2 = HCA, 6-O-α-ester
HCA = O
OH Figure 3.1: Acyl migration in p-coumaroyl glucose.
A similar phenomenon was observed for an S- to O-acetyl group migration occurring for a furanosyl derivative in the presence of silica (Figure 3.2), which the authors rationalised by the formation of a pseudo hydrogen bond between the silica and the carbonyl oxygen, which decreased the electron density around the carbonyl carbon, promoting nucleophilic attack and allowed for easy migration to the neighbouring oxygen. 190 The extent of
50 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters migration was lessened with decreasing activities of silica, which was achieved by hydrating the silica, reducing the electron accepting abilities. While it was found not to occur for an O- to O-acetyl migration, this report confirms that silica can promote acyl migration, which appeared to be the case for 9 and 10 .
OH OH HO AcO
O Silica O
SAc O SH O O O
Figure 3.2: Initial silica catalysed 3-S- to 6-O-migration observed by Whistler et al.
Iddon et al. studied the migration of phenyl acetic acid glucosides under basic conditions, describing cyclic transition states, and suggested that the speed of migration was largely related to the stability of the intermediates. 191 Migration from the 4-O- to that 6-O-position proceeded through a 6-membered cyclic intermediate and was found to be much favoured over those created by nucleophilic attack of the neighbouring hydroxyl groups, which proceed through a 5-membered intermediate. Similar work by Horrobin et al. investigated 4-O- to 6-O-migrations under acid-catalysed conditions which resulted in the proposal of a mechanism that proceeded through a cationic cyclic intermediate (Figure 3.3). Again, the speed of migration was governed by the stability of the intermediate, but the relative ratios of the products were purely under thermodynamic control. 192 Furthermore, the speed of the 4-O- to 6-O-migrations shown by both Horrobin and Iddon rationalise the lack of 4-O- glucose ester observed in the migrated mixtures of 9 and 10 , which can quickly migrate to the 6-O-position.
a) b)
OH OH H
O O HO O O HO O AcO O O AcO OAc R Figure 3.3: Migration intermediates. a) Base-catalysed 1-O-β- to 2-O-β-migration intermediate proposed by Iddon et al. b) Acid-catalysed 4-O-α- to 6-O-α-migration intermediate proposed by Horrobin et al.
51 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
Yoshimoto and Tsuda monitored migrations to and from multiple positions in glucose and discovered that a direct 1-O- to 6-O-migration was not possible, but occurred in a step- wise fashion via first a 1-O- to 3-O-, then a 3-O- to 6-O-migration. 193 By protecting the 3- O-position, the previously observed 1-O- to 6-O-migration was inhibited, indicating the involvement of the 3-O-position in the process. They also documented that the 4-O- to 6- O-migration occurred rapidly, as did a 1-O-α- to 2-O-transformation, while 2-O- to 3-O-, 3-O- to 4-O- and 4-O- to 2-O-migrations occurred at much slower rates. 194 These findings support the assigned composition of the mixtures for the hydroxycinnamoyl glucose esters (9 and 10 ), with rapid migration away from the 4-O-position limiting its presence, while the ease of 3-O- to 6-O-migration can explain both the prevalence of the 6-O-esters and limited production of the 3-O-esters. However, Yoshimoto and Tsuda failed to induce a 1- O-β- to 2-O-migration and claimed that the 1-O-β-ester was stable to migration, in contrast to the hydroxycinnamoyl glucose esters. 194 Furthermore, most of the studies into glucose acyl migrations have found that migrations to yield the preferred 6-O-esters are largely irreversible, 194-195 which again, is not the case for hydroxycinnamoyl esters, with a migrated mixture returning to the 1-O-β-ester under wine-like conditions.
As studied in sucrose migrations, Mollinier et al. discovered that basic conditions favoured the formation of the 6-O-ester, though in acidic conditions the 6-O- as well as the 3-O- and 2-O-esters were produced, 196 mimicking that seen for the hydroxycinnamoyl glucoses (9 and 10 ) which migrated to give the 6-O- and 2-O-esters, with traces of the 3-O-esters detected. Furthermore, the reported preference for the 6-O-ester under basic conditions may explain the occurrence of 51 and 52, the 6-O-chloroacetyl derivatives, which were formed during the deprotection of 49 and 50 to produce 9 and 10 , as explained in Chapter 2. It may not be that the 6-O-chloroacetyl is the last group removed, but instead, when a single chloroacetyl group remains, regardless of the position, migration to the 6-O-position is promoted under the influence of basic conditions (Figure 3.4).
52 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
OH OAcCl OH
ClAcO O B- HO O B- HO O HO OR HO OR ClAcO OR 4-O- to 6-O- 3-O- to 6-O- OH OH OH
B- 2-O- to 6-O-
OH
HO O HO OR OAcCl Figure 3.4: Proposed migration of mono-O-chloroacetyl derivatives to the 6-O-position.
The above studies confirm that acyl migrations within glucose derivatives are common, and can be achieved under acidic or basic conditions, with the conditions playing an important role in the final products. Largely, the ratios of the products formed are under thermodynamic control, with the stabilities of the intermediates only determining the speed at which the final products are formed. If these processes are controlled completely by thermodynamics, then by mapping the relative stabilities of each possible ester one should be able to gain an indication as to the potential for the formation of each ester under given conditions.
A density functional study of acyl migration in formyl nucleosides determined that mapping a step-wise mechanism was a more valid pathway than a concerted migration, and that the geometry of the products should be optimised in the desired solvent rather than a geometry optimisation in a vacuum, followed by a single-point energy calculation in solvent. 197 The use of a step-wise mechanism to study migration supports those proposed by both Horrobin and Iddon, using a number of cyclic intermediates. 191-192
In addition, it was discovered that the energy of migration was lowered by increasing the polarity of the solvent, 197 most likely by stabilising the charge of the intermediates. However, for the glucose esters ( 9 and 10 ) a greater extent of migration away from the 1- O-β-ester was observed in less polar solvents. Whether this was for kinetic or thermodynamic reason was initially unclear.
53 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
3.2 Research Aims.
Acyl migrations can occur under acidic, basic and neutral conditions 198 which can lead to the formation of different products. 196 The hydroxycinnamoyl glucose esters ( 9 and 10 ) were seen to migrate away from the 1-O-β-esters under acidic non-aqueous conditions and the resulting mixtures migrated back to the 1-O-β-esters in an acidic aqueous environment (Chapter 2). The role of thermodynamics has been identified in the literature as the main factor in determining both if migration will occur and to what extent it will occur under given conditions, 192, 196 while the kinetics of each migration relied on the stability of the intermediates. 191-192
Initially the thermodynamics of migration were studied here in an attempt to determine why migration occurred, and to justify the ratios of esters observed, including the propensity for increased migration for the p-coumaroyl glucose system compared with the feruloyl derivative. Following the findings of Rangelov et al., the equilibrium geometry of each ester was optimised in the desired solvents, rather than optimising the geometry in a vacuum and then performing single-point energy calculations in each solvent using the vacuum optimised geometry common to all. 197
Additionally, by applying an analogous mechanism to that described by Horrobin 192 the energy of key intermediates was expected to provide insight into how quickly each transformation can occur. This should indicate the likely pathway of migration and whether the ratios obtained experimentally are purely under thermodynamic control, or whether the kinetics do contribute.
Once the nature of migration is determined, protocols for avoiding, minimising, controlling or even predicting migration can be developed so that synthesis can be achieved more simply and without having to characterise and utilise mixtures.
Finally, several analyses have identified two separate hexose esters of p-coumaric acid without designation of which esters were present. 100, 103, 131 If the formation of multiple esters is found to be possible in wine, then the nature of the esters identified could be determined, also providing information as to whether multiple esters will need to be considered during quantification in grapes and wine.
54 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
3.3 Theoretical Studies into Acyl Migration of Hydroxycinnamoyl Glucoses.
3.3.1 Thermodynamics of Migration Experimentally the 1-O-β-, 2-O-α- and 6-O-α-esters were produced preferentially, though each of the twenty possible esters ( α- and β-anomers for 1-O-, 2-O-, 3-O-, 4-O- and 6-O- esters, Figure 3.5) need to be considered, not only explain the presence of those that were seen, but also why others were absent from the “migrated” mixtures.
βββ-D-glucopyranosyl ααα-D-glucopyranosyl OH OH OH O HO OH HO 1-O- HO O HO O HO R O OH R O O OH OH
HO O HO O HO OH HO
2-O- O O OH R R O O
HO HO
OH OH HO HO
3-O- HO O HO O O OH O R R OH HO OH O O
OH OH O O
R R O 4-O- O O O HO OH HO
OH HO HO HO OH
O O
R R O O 6-O- HO HO O HO O HO HO OH HO HO OH OH
9 R = H 10 R=OCH3 Figure 3.5: Twenty possible esters of p-coumaroyl glucose ( 9) and feruloyl glucose ( 10 ).
55 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
The equilibrium geometries of each of the ten p-coumaroyl and ten feruloyl esters were determined in water using the DFT B3LYP level of theory and a 6-31G* basis set, and the energy of the optimised structures compared to the energy of the desired 1-O-β-ester (Figure 3.6).
80 p-Coumaroyl glucose Feruloyl glucose 60
40
20 Energy Energy (kJ/mol)
0
rs rs rs rs rs rs rs rs rs te te te te te te te te te s s s s s s s s s e e e e e e e e e ααα- βββ- ααα- βββ- ααα- βββ- ααα- βββ- ααα------O -O O -O O -O O -O O - 2 - 3 - 4 - 6 - 1 2 3 4 6 Figure 3.6: Energy of p-coumaroyl and feruloyl glucose esters in water, relative to the 1-O- β-esters. See Appendix 1, Table A1.1 for ground state energies and relative differences.
The relative energy of each of the glucose esters as calculated in water indicate precisely what was observed during the synthesis of the glucose esters; that the 1-O-β-esters are thermodynamically favoured, and when exposed to conditions conducive to migration in an aqueous environment, the 1-O-β-esters should prevail. As such, when migrated mixtures of 9 and 10 were subjected to storage in acidic aqueous conditions, the 1-O-β- esters could be recovered as a result of thermodynamic influences. Notably, the relative energies of the 2-O-α- and 6-O-α-esters are lower than the remaining seven esters suggesting that given migration away from the 1-O-β-ester in water, the formation of these two species would be favoured.
Experimentally, migration was observed during flash chromatography on silica employing solvent systems consisting of a small fraction of methanol in dichloromethane. To examine the effect of the solvent system the energy of each ester was calculated in dichloromethane with the results indicating that thermodynamically, the 6-O-α- and the 2-O-α-esters are more favoured than the 1-O-β-ester (Figure 3.7). Already it can be seen why migrated
56 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters mixtures of the glucose esters possessed the ratios that they did, with the preferred product in water, migrating in dichloromethane to give a mixture heavily favouring the formation of the 2-O-α- and 6-O-α-esters. While very little regarding the relative extent of migration observed between p-coumaroyl glucose and feruloyl glucose can be explained by the thermodynamic influences, Figures 3.6 and 3.7 explain the occurrence of different esters under changing solvent conditions. However, these results suggest that in dichloromethane complete migration to the 6-O-position would eventuate, and also that the ratios observed experimentally were under kinetic as well as thermodynamic control. The length of exposure to dichloromethane, or the kinetics of migration determined the extent of migration from initially being exposed to dichloromethane to the time of characterisation, as equilibrium has not yet been established.
60 p-Coumaroyl glucose Feruloyl glucose 40
20
0 Energy Energy (kJ/mol)
-20
rs rs rs rs rs rs rs rs rs te te te te te te te te te s s s s s s s s s e e e e e e e e e ααα- βββ- ααα- βββ- ααα- βββ- ααα- βββ- ααα------O -O O -O O -O O -O O - 2 - 3 - 4 - 6 - 1 2 3 4 6 Figure 3.7: Energy of p-coumaroyl and feruloyl glucose esters in dichloromethane, relative to the 1-O-β-esters. See Appendix 1, Table A1.2 for ground state energies and relative differences.
To further investigate the role of solvents in determining the thermodynamics of migration for both 9 and 10 , the energy of each of the esters was determined in ethanol (Figure 3.8) and toluene (Figure 3.9), representing polar non-aqueous and non-polar solvents respectively, to investigate whether a trend in ester energy with respect to solvent properties could be established.
57 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
40 p-Coumaroyl glucose Feruloyl glucose 20
0 Energy Energy (kJ/mol)
-20
rs rs rs rs rs rs rs rs rs te te te te te te te te te s s s s s s s s s e e e e e e e e e ααα- βββ- ααα- βββ- ααα- βββ- ααα- βββ- ααα------O -O O -O O -O O -O O - 2 - 3 - 4 - 6 - 1 2 3 4 6 Figure 3.8: Energy of p-coumaroyl and feruloyl glucose esters in ethanol, relative to the 1- O-β-esters. See Appendix 1, Table A1.3 for ground state energies and relative differences.
60 p-Coumaroyl glucose Feruloyl glucose 40
20
0 Energy Energy (kJ/mol)
-20
rs rs rs rs rs rs rs rs rs te te te te te te te te te s s s s s s s s s e e e e e e e e e ααα- βββ- ααα- βββ- ααα- βββ- ααα- βββ- ααα------O -O O -O O -O O -O O - 2 - 3 - 4 - 6 - 1 2 3 4 6 Figure 3.9: Energy of p-coumaroyl and feruloyl glucose esters in toluene, relative to the 1- O-β-esters. See Appendix 1, Table A1.4 for ground state energies and relative differences.
The thermodynamic preference for the 1-O-β-esters only occurred for water, while the other three solvents studied theoretically show a preference for formation of the 6-O-α- esters with the 2-O-α-esters closely following. The relative energies in ethanol show not only a strong preference for the 2-O-α- and 6-O-α-esters, but also suggest that the 3 -O-α- and β-esters are as likely to occur as the 1-O-β-esters. NMR characterisation of the glucose esters was performed in d 4-methanol, and while the effect of methanol on the energies of the esters could not be studied due to the restraints of the program, it is expected that a similar trend would be experienced to that calculated for ethanol. As such, any migration that resulted from exposure to silica in dichloromethane could be compounded by exposure
58 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters to methanol during characterisation. As such, the limited exposure to methanol could have increased the thermodynamic preference for the 3-O-esters, as well as the 6-O-and 2-O- esters, and allowed for minor formation of these esters which could be detected in minor quantities by 1H NMR analysis.
The relative energies shown in Figures 3.6-3.9 show large changes in the energy of every ester with changing solvent, though this is only a product of plotting the data relative to the 1-O-β-esters. Extracting the data for the p-coumaroyl glucose esters in all four solvents and plotting the energies relative to the less important 1-O-α-esters furnishes Figure 3.10. The largest changes in energy between solvents is present for the 1-O-β-esters, indicating that in different environments, the energies of the other esters does not change to a great extent, but really only relative to the 1-O-β-esters. The four energies obtained for the 1-O-β-esters (relative to the 1-O-α-ester) show the greatest variation across the four solvents with energies of -59.44, -45.53, -34.66 and -51.77 kJ/mol producing a standard deviation of 10.5 kJ/mol. By performing the same analysis for the remaining esters, the 2-O-α-esters produce a standard deviation of 4.5 kJ/mol, 4.2 kJ/mol for the 2-O-β-esters, 3.9 kJ/mol for the 4-O-β-esters and the deviations of the remaining esters between 2.2 and 2.9 kJ/mol. A similar trend can be observed when the data is plotted relative to any ester other than the 1- O-β, and shows that as the environment moves away from aqueous, the preference for the 1-O-β-esters decreases as opposed to preference for the 2-O-α- or 6-O-α-esters increasing.
0 Dichloromethane Water -20 Ethanol Toluene
-40 Energy (kJ/mol) Energy
-60
βββ βββ ααα βββ ααα βββ ααα βββ ααα ------O -O -O -O -O -O -O -O -O 1 2 2 3 3 4 4 6 6 Figure 3.10: p-Coumaroyl glucose ( 9) ester energies calculated in changing solvents, relative to the 1-O-α-esters. See Appendix 1, Table A1.5 for relative energies and standard deviations.
59 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
By studying ethanol and toluene in addition to water and dichloromethane, it can be seen that preference for the 1-O-β-esters is not a product of solvent polarity and these appear to only be favoured in aqueous environments, with migration to the 2-O-α- and 6 -O-α-esters likely to occur in any solvent other than water. Though formation of other esters may be favoured, the occurrence of mixtures with differing ratios indicates that equilibrium has not yet been achieved, and the thermodynamic products will prevail once equilibrium has been reached. However, instantaneous determination of which esters are present will rely on both the thermodynamics to determine which esters are being formed and the kinetics to describe to what extent it has occurred at the time of measurement.
3.3.2 Kinetics of Migration While base-catalysed migration under some reaction conditions might be expected to a certain extent, the observed migrations as well as any expected to occur in wine, must be acid-catalysed, and the lack of free acid seen experimentally in the migrated mixtures indicate that this is not a case of ester hydrolysis followed by re-attachment, but that the process is one of intramoleular transesterification. By combining the observations proposed by Horrobin et al., 192 and that known for an acid-catalysed transesterification, the mechanism of 1-O-β- to 2-O-β-migration for the p-coumaroyl glucose analogue ( 9) can be hypothesised (Scheme 3.1).
60 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
O O O H O O O O O H OH HO HO O H
OH OH OH
H O O O OH OH O OH O O O O O H
OH OH OH Scheme 3.1: Mechanism for acid catalysed 1-O-β- to 2-O-β- acyl migration of p- coumaroyl glucose ( 9).
In an acidic environment a pH dependant equilibrium will exist between the protonated and unprotonated carboxyl oxygen, with the energy difference between the two forms being of little consequence to the kinetics of the migration. As such, the four cationic intermediates of interest in mapping the kinetics of this particular migration are given below (Figure 3.11). While migration has been mapped for the 1-O-β- to 2-O-migration in p-coumaroyl glucose, a similar mechanism is expected for all those investigated.
61 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
OH OH
HO O HO O HO O O HO O H OH OH O H
OH OH
Int. 1 Int. 2
OH OH
H HO O HO O HO O HO OH OH O O O H
OH OH Int. 3 Int. 4 Figure 3.11: Key intermediates (Int. 1-4) for the acid-catalysed 1-O-β- to 2-O-β- acyl migration of p-coumaroyl glucose ( 9).
The transformation shown (Scheme 3.1) involves formation of the 2-O-β-ester, though experimentally the 2-O-α-ester was favoured with very little of the 2-O-β-ester able to be detected. Ignoring any conversion between α- and β-glucose that could possibly occur during migration (in an attempt not to complicate the study with concurrent reactions), migration from the 1-O-β-ester to form the 2-O-α-ester must occur either via mutarotation before or after migration. Due to mechanistic constraints in mutarotation between the 1-O- β- and the 1-O-α-ester it is assumed that the migration occurs first, from the 1-O- to the 2- O-position, followed by mutarotation to convert the 2-O-β- to the 2-O-α-ester. Each four intermediates (Int. 1-4) were optimised at the B3LYP 6-31G* level and the calculated energy compared with that of intermediate 1.
62 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
150 Water Dichloromethane 100 Vacuum
50
0 Energy Energy (kJ/mol) Int. 1 Int. 2 Int. 3 Int. 4 -50 Figure 3.12: Energy of the intermediates in 1-O-β- to 2-O-β-p-coumaroyl glucose migration, relative to intermediate 1. See Appendix 2, Table A2.1 for ground state energies and relative differences.
Unlike the glucose ester energies calculated in the previous section, the energies of the migration intermediates were calculated for water and dichloromethane alone, rather than in all four solvents as were employed above (removing ethanol and toluene). Experimentally, water and dichloromethane were the most common solvents that the glucose esters ( 9 and 10 ) experienced, having been allowed to react in an aqueous system and purified in either water/methanol or dichloromethane/methanol. Analogous intermediates to those used for p-coumaroyl glucose (as shown in Figure 3.11), were used in calculation of the 1-O-β- to 2-O-β-feruloyl glucose migration (Figure 3.13).
200 Water Dichloromethane 150 Vacuum
100
50 Energy Energy (kJ/mol)
0 Int. 1 Int. 2 Int. 3 Int. 4
Figure 3.13: Energy of the intermediates in 1-O-β- to 2-O-β-feruloyl glucose migration, relative to intermediate 1. See Appendix 2, Table A2.2 for ground state energies and relative differences.
The relative energies of the intermediates for the p-coumaroyl and feruloyl glucose esters show similar patterns with intermediates 2 and 3 being highest in energy, which is accentuated in water. Therefore more energy is required for migration in water than dichloromethane, which is the opposite effect to that seen by Rangelov, 197 whereby more
63 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters polar solvents reduced the energy barriers to migration. However, in this case all four intermediates investigated are cationic, with intermediate 1 expected to exist in equilibrium with the neutral species under acidic conditions. Therefore, the energy involved in the formation of the cationic intermediates is not considered and there is no expected effect between the intermediates based on charge stabilisation.
Not only is the formation of the 2-O-α-esters more favoured in solvents other than water, but the energy of migration is lessened in dichloromethane also. When the glucose esters were synthesised and purified by column chromatography in a dichloromethane based solvent system, migration away from the 1-O-β-esters under acidic conditions was thermodynamically favoured and kinetically more favoured also.
Furthermore, the energy to migration in feruloyl glucose ( 10 ) compared with p-coumaroyl glucose ( 9) in dichloromethane is almost twice as much. For feruloyl glucose the migration is limited by intermediate 2, lying some 60 kJ/mol higher in energy than intermediate 1, whereas the same transition for p-coumaroyl glucose requires only 35 kJ/mol. Experimentally, it was seen that migration occurred faster, or to a greater extent for p- coumaroyl glucose than for feruloyl glucose, which can be explained by the kinetics of migration. With a faster process occurring for p-coumaroyl glucose, migration had occurred to a greater extent at the time of characterisation than for feruloyl glucose.
As mentioned previously, it is expected that by performing the NMR characterisation of the glucose esters in methanol, migration would continue beyond that caused simply by purification. As a result, the equilibrium of the mixtures may not have been achieved over the course of 2-3 hours needed for purification and characterisation, with the kinetically favoured process (migration in p-coumaroyl glucose) having occurred to a greater extent. While it would have been relatively simple to allow the mixture to equilibrate over hours, or even days, the key outcome of this study was to obtain pure 1-O-β-esters and as such, migration was not encouraged.
Additionally, intermediate 4 was lower in energy than intermediate 1 for p-coumaroyl glucose under all the conditions investigated, showing a preference for formation of the cationic 2-O-β-ester over the cationic 1-O-β-ester, which is not the case for feruloyl
64 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters glucose, indicating that kinetically, migration to the 2-O-position within p-coumaroyl glucose is comparatively favoured.
While step-wise migration around the glucose ring has been proposed, 189 migration to the 2-O-position is not the only path of migration. Yoshimoto dismissed direct 1-O- to 6-O- migration, though their observations differ from those seen in these studies enough for it not to be completely discounted. 193 Calculating the energy barriers for migration to the 6- O-position should support or eliminate direct migration, as such analogous intermediates to the 1-O- to 2-O-migration were optimised and the energies calculated, the same mechanism was assumed, with the attacking nucleophile changed to the hydroxyl of the final ester.
150 Water Dichloromethane 100 Vacuum
50
0 Energy Energy (kJ/mol) Int. 1 Int. 2 Int. 3 Int. 4 -50 Figure 3.14: Energy of the intermediates in 1-O-β- to 6-O-β-p-coumaroyl glucose migration, relative to intermediate 1. See Appendix 2, Table A2.3 for ground state energies and relative differences.
150 Water Dichloromethane 100 Vacuum
50
0 Energy Energy (kJ/mol) Int. 1 Int. 2 Int. 3 Int. 4 -50 Figure 3.15: Energy of the intermediates in 1-O-β- to 6-O-β-feruloyl glucose migration, relative to intermediate 1. See Appendix 2, Table A2.4 for ground state energies and relative differences.
65 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
Figures 3.14 and 3.15 show that direct migration to the 6-O-β-esters is as likely as migration to the 2-O-β-esters, with very similar energy barriers to overcome. In a similar fashion to the 2-O-β-p-coumaroyl glucose migration in dichloromethane (Figure 3.12), the protonated 6-O-β-esters are more favoured than the protonated 1-O-β-esters under all conditions for both 9 and 10 , suggesting that in an aqueous acidic environment direct migration to the 6-O-position would be favoured over migration to the 2-O-position.
Both transitions involve intermediates of unfavourable conformations, with the 1-O- to 2- O-migration involving a 5-membered cyclic intermediate, and the 1-O- to 6-O-migration a 7-membered transition state. Given the evidence of Iddon, 191 the migration to the 3-O- position involving a 6-membered cyclic intermediate should be more favourable than both of the previously calculated transformations.
60 Water 40 Dichloromethane Vacuum 20
0 Int. 1 Int. 2 Int. 3 Int. 4
Energy Energy (kJ/mol) -20
-40 Figure 3.16: Energy of the intermediates in 1-O-β- to 3-O-β-p-coumaroyl glucose migration, relative to intermediate 1. See Appendix 2, Table A2.5 for ground state energies and relative differences.
40 Water Dichloromethane 20 Vacuum
0 Int. 1 Int. 2 Int. 3 Int. 4 -20 Energy Energy (kJ/mol)
-40 Figure 3.17: Energy of the intermediates in 1-O-β- to 3-O-β-feruloyl glucose migration, relative to intermediate 1. See Appendix 2, Table A2.6 for ground state energies and relative differences.
66 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
As expected, the migration to the 3-O-position involves smaller energy barriers, and can be expected to occur more quickly than migration to the 2-O- and 6-O-positions. However, thermodynamics suggests a preference for the formation of the 2-O- and 6-O-esters, which would be a case of subsequent migration.
The optimised intermediates 2 and 3 produced in studying both the 1-O- to 3-O- and 1-O- to 6-O-migrations involve a glucose ring-flip, with the result being that for the 1-O-β-ester the 1-OH, the 3-OH and the 6-OH are all axial and on the same face above the ring, leading to a less hindered migration.
OH OH OR OH Ring-flip O O HO OR HO
OH OH OH Figure 3.18: Glucose ring-flip to facilitate 1-O- to 3-O-migration and 1-O- to 6-O- migration.
This effect is seen more greatly for the 1-O- to 3-O-migration where very small energy barriers are seen. The unfavoured ring-flipped conformation is stabilised by the formation of the bicyclic intermediates, or more accurately that the formation of the cyclic intermediate in the ring-flipped conformer is more favourable than in the original conformation. A glucose ring-flip would explain the findings of Yoshimoto, that 1-O- to 3- O- to 6-O-migrations were occurring rapidly, and potentially why they did not experience 1-O-β- to 2-O-migration, with these groups being too far removed for the transformation to take place. 193
3.4 Liquid Chromatography of Wine.
The ease of migration in non-aqueous solvents provides an explanation as to why two p- coumaroyl hexose esters were seen by both Monagas and Hernendez, 100, 103, 131 and brings into question whether one or both esters observed are an artifact of the extraction being performed in non-aqueous solvents, rather than a grape or wine product. A study by Perez-
67 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
Magarino looked at the ability of different resins to absorb and retain phenolic compounds by loading, then washing with water, ether and finally ethyl acetate. 199 From a single resin one p-coumaroyl glucose ester was eluted with water, but when non-aqueous solvents were used, two p-coumaroyl glucose esters were observed. It is unlikely that the difference in chemical properties of the two p-coumaroyl glucose esters would result in a single ester eluting while the other is retained and further supports the theory that the 1-O-β-esters are solely found in wine, and any other esters are produced upon exposure to non-aqueous solvents.
To investigate the presence of multiple hydroxycinnamoyl glucose esters in wine, the extraction method of Monagas was employed to extract red and white wine spiked with both p-coumaroyl and feruloyl glucose ester ( 9 and 10 ). By comparing the amount remaining in the “spiked” extraction to that of a pure sample, the liquid-liquid extraction efficiency was approximately 20% for both compounds, and thus considered inadequate for detecting small quantities in wine.
Solid-phase extraction by loading spiked wine onto XAD-8, elution with 25%, 50% and 75% methanol in water, followed by HPLC analysis indicated that the majority of the glucose ester content was found in the 50 and 75% methanol fractions and that the extraction efficiency was approximately 60%. When analysed by LC-MS, the pure glucose ester standards, which were not subjected to extraction, consisted largely of single esters, while the extracted wines contained two glucose esters, the most predominant being the 1- O-β-ester, with the minor peak expected to be either the 2-O-α- and 6-O-α-esters, indicating that the spiked glucose esters were migrating under the extraction conditions.
With liquid-liquid extraction giving poor extraction efficiencies, the nature of the solvents likely to yield migration, and the solid-phase extraction also resulting in migration, neat red and white wine along with concentrated samples (5 times concentrated under reduced pressure) were submitted to analysis by LC-MS. The extracted ion chromatograms of the pure esters again showed largely single esters (Figures 3.19a and 3.20a) which suggests that migration is not an effect of the HPLC method. The fragmentation pattern of p- coumaroyl glucose matches literature data, 131 and feruloyl glucose fragmented in an analogous manner, as reported 200 (Figures 3.19b and 3.20b).
68 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
Figure 3.19: p-Coumaroyl glucose. a) Extracted ion chromatogram of m/z 325. b) Mass spectrum at 29.6 to 29.8 minutes.
Figure 3.20: Feruloyl glucose. a) Extracted ion chromatogram of m/z 355. b) Mass spectrum at 36.5 to 36.6 minutes.
69 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
Injection of neat white wine, as well as concentrated white wine gave the same results, with 5 main peaks appearing in the area of interest in the extracted ion chromatogram for m/z 325 (Figure 3.21). Based on the fragmentations, the first peak observed in the EIC for m/z 325 corresponds to the p-coumaroyl glucoside (A), lacking the fragmentation which corresponds to the loss of water from the aglycone seen in the glucose ester fragmentation, the second two (B and C), with aglycone peaks matching ferulic acid are likely to be feruloyl tartrate derivatives, 131 and the fourth and fifth peaks (D and E) are the two p- coumaroyl glucose esters (Figure 3.22).
C
A B D E
Figure 3.21: Concentrated white wine, extracted ion chromatogram of m/z 325.
70 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
A)
B)
C)
D)
E)
Figure 3.22: Mass spectra of compounds identified in extracted ion chromatogram of m/z 325.
The fragmentations of the two peaks identified in the extracted ion chromatogram of m/z 355 imply that the first peak (A) is the feruloyl glucoside and the second (B) is the feruloyl glucose ester (Figure 3.24).
The observation that the white wine, when analysed neat, or concentrated appears to possess two p-coumaroyl glucose esters, but only single feruloyl glucose ester, is consistent with the reluctance of feruloyl glucose ester to migrate which was observed during synthesis and supported by theoretical studies.
71 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
B
A
Figure 3.23: Concentrated white wine, extracted ion chromatogram of m/z 355.
A)
B)
Figure 3.24: Mass spectra of compounds identified in extracted ion chromatogram of m/z 355.
With little difference in the results from neat and concentrated white wine, the analysis of red wine was only repeated with concentrated red wine (Figure 3.25), with the extracted ion chromatogram for m/z 325 possessing additional peaks, which are tentatively identified
72 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters as p-coumaroyl anthocyanin derivatives (Figure 3.26). However, the presence of the extra peaks (at 32.6, 34.3, 36.7 and 40.0 minutes) in the same region of the chromatogram as feruloyl glucose (approximately 36.6 minutes) led to decreased resolution of the m/z 355 extracted ion chromatogram (Figure 3.27), with no fragmentations able to be found matching that of the reference sample.
Figure 3.25: Red wine chromatogram (DAD).
Figure 3.26: Concentrated red wine, extracted ion chromatogram of m/z 325.
73 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
Figure 3.27: Concentrated red wine, extracted ion chromatogram of m/z 355.
Due to decreased sensitivity for feruloyl glucose resulting from the presence of additional peaks in red wine, the samples were submitted to HPLC-MRM, with the fragmentation from the parent ions to the aglycone and the aglycone-water being monitored. For p- coumaroyl glucose, p-coumaric acid ( m/z 163, red line) and p-coumaric acid minus water (m/z 145, blue line) fragmentations are shown on the left of Figure 3.28, and for feruloyl glucose, ferulic acid ( m/z 193, red line) and ferulic acid minus water ( m/z 175, blue line) are shown on the right of Figure 3.28.
74 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
p-Coumaroyl glucose Feruloyl glucose
a)
b)
c)
d)
Figure 3.28: HPLC-MRM traces (aglycone - blue, aglycone minus water - red) of hydroxycinnamoyl glucose esters. a) Pure glucose esters. b) Neat white wine. c) Concentrated white wine. d) Concentrated red wine.
From the chromatograms in Figure 3.28, the presence of multiple p-coumaroyl glucose esters in white and red wine can be observed, but the presence of a second glucose ester of ferulic acid is not immediately obvious in white wine. Although the concentration of feruloyl glucose in concentrated red wine is somewhat lower than in white wine, evidence
75 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters of a second peak in the concentrated red wine sample can be seen. Even though this data confirms the finding of Monangas, Hernandez and Perez-Magarino that there are multiple glucose esters in wine, it has also shown that the extraction method can contribute to the extent of migration, with pure glucose esters undergoing migration during solid-phase extraction. These results also indicate why the feruloyl glucose ester was not observed in previous studies, as the presence of what are likely to be p-coumaroyl anthocyanin derivatives in red wine co-elute, and prevent identification and quantification. Feruloyl glucose could only be seen in this case by determining the retention time and comparing fragmentations with the pure reference compound.
3.5 Conclusions.
Previous studies have evaluated the migrations to and from multiple positions of glucose, and the reverse processes. However, in this study, only the migrations away from the 1-O- β-esters were examined as the destruction and formation of these esters were specifically of interest. Migrations involving 5-, 6- and 7-membered cyclic intermediates have been studied with kinetic preference for migration through the 6-membered intermediate, to an ester that is thermodynamically unfavourable. 191-192 Migration between neighbouring hydroxyls, in this case 1-O- to 2-O-migration, occurs through a 5-membered intermediate, while migration to the 3-O-position, which was found to be kinetically more favoured, involves an extra carbon atom, resulting in a 6-membered intermediate. Whereas migration from the 1-O- to the 6-O-position requires formation of a 7-membered intermediate, and was found to be kinetically as favoured as formation through a 5-membered intermediate.
Studying the kinetics of migration away from the 1-O-β-esters is valuable in determining the likelihood of migration, and it can now be rationalised why previous authors 193 have seen migration through the 3-O-position to get to the 6-O- and 2-O-position. However, the fact that the 3-O-esters do not accumulate in the migrated glucose ester mixtures strengthens the argument that these migrations are ultimately under thermodynamic control. Thus, while in water the energy barriers for migration to the 3-O-position are quite favourable, the energies of the end products suggest that this wouldn’t be the case, indicating that the 3-O-esters are intermediates and the secondary migration to the 6-O- position is extremely facile.
76 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters
However, these results do indicate that in wine like conditions (largely aqueous) that the 1- O-β-esters will be thermodynamically favoured, and that most migrations are suppressed due to higher energy barriers to migration, with the only exception being the migration to the 3-O-position which requires a ring flip of the glucose ring before the migration can take place.
It can be seen that the migrations observed in the hydroxycinnamoyl glucose esters are largely under thermodynamic control and that under conditions that favour products other than the 1-O-β-ester, migration will most likely occur rapidly. This study did not investigate the base-catalysed migration, as it would be of little importance in acidic wine medium and the 1-O-β-esters appeared stable under mildly basic conditions. There is the potential that synthetically, basic conditions should also be avoided, not the least because of the greater likelihood of hydrolysis.
While the kinetics would suggest that migration away from the 1-O-β-esters should proceed through the 3-O-position, the relative ratios of the different glucose esters observed during synthesis showed the p-coumaroyl moiety migrating to a greater extent than the feruloyl, which suggests that migration of p-coumaroyl glucose should be kinetically more favoured. This effect is only predicted for a 1-O- to 2-O-migration (Figures 3.12 and 3.13) with the energy required for migration much higher in the feruloyl derivative than in the p-coumaroyl. Furthermore, if migration to the 3-O-position is experienced, the ring-flip required for this transformation to occur would most likely promote further migration to the 6-O-position, resulting in formation of the thermodynamically more stable esters.
In future synthetic attempts, the use of solvents other than water should be limited, especially under conditions conducive to migration. If organic solvents are employed, they should be done so under neutral conditions, or preferably in the presence of a buffer. In the event of migration away from the desired 1-O-β-esters, it has been shown that storage under aqueous acidic conditions will again yield the desired esters.
Furthermore, it can be expected that in wine, and wine-like environments such as model fermentations, that the 1-O-β-esters will predominate and other esters would be a product
77 Chapter 3: Acyl Migration of Hydroxycinnamoyl Glucose Esters of the conditions that the compounds are exposed to. An analytical method using a liquid- liquid or solid-phase extraction could promote formation of other esters and render the quantification inaccurate. As such, to quantify these compounds in wine, care must be taken to ensure that migration hasn’t occurred, and that the compounds seen are products of the grape or wine conditions, rather than artifacts of the methodology. If extraction and concentration is required, analysis of a neat wine sample might assist in determining if any migration has occurred as a result of the processing. The differences in the ratio of esters observed between model wine, where the 1-O-β-ester is predominant, and red wine, where multiple esters were observed, are most likely a product of the matrices and effected by such factors as pH and dielectric constant.
In addition, this study also describes the identification of feruloyl glucose ( 10 ) for the first time in red wine, which, along with p-coumaroyl glucose can exist as multiple esters in both red and white wine.
78 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
Chapter 4: Photoisomerisation of Hydroxycinnamic Acids.
4.1 Introduction.
4.1.1 Hydroxycinnamate Photoisomerisation In addition to the acyl migrations that were observed (Chapter 2) and studied (Chapter 3), trans -p-coumaroyl glucose ( 9) and trans -feruloyl glucose ( 10 ) were found to undergo photoisomerisation resulting in formation of cis -analogues (Figure 4.1). Interconversion between trans - and cis -hydroxycinnamic acids has been known since Kahnt reported and investigated the nature of the conversion and found the equilibrium to be effected by the solvent in which the transition occurred, the concentration of the hydroxycinnamic acids, and also the nature of the compounds, with caffeic acid existing in different isomeric ratios to an esterified analogue.187
OH OH OH hν O O HO HO HO O HO O R OH OH O O
9 R = H cis-9 R = H 10 R = OCH3 cis-10 R = OCH3 R OH Figure 4.1: Photoisomerisation of the hydroxycinnamoyl glucose esters.
A further investigation by Kahnt measured the pH dependence of the photoisomerisation and observed that maximum conversion to the cis -acids was achieved within a pH range of 5-7. 186 Subsequent studies have shown there to be changes in equilibria due to: additional substituents, with TMS ethers producing different isomeric ratios than the free hydroxycinnamic acids, although the direction of the equilibrium change was not consistent; 201 subtle differences in conversion to the cis -analogue in differing solvents; 202 as well as due to the wavelength of incident light.203 Longer wavelengths of ultra-violet light induce a slower isomerisation, while shorter wavelengths induce a quicker and more complete isomerisation but can also result in degradation after prolonged exposure. 203
79 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
Under irradiation, the majority of the hydroxycinnamic acids exist at isomeric ratios favouring the trans -isomer, with the cis -acid contributing between 40 and 49%. 204 While many studies have shown the effect of ultra-violet light on isomerisation, other reports suggest that it can be promoted under more innocuous light conditions, providing the recommendation that the hydroxycinnamic acids should be handled in the dark to avoid any potential isomerisation. 205-207 p-Coumaric acid ( 3) plays an important role in bacteria, being the basis for the chromophore for the photoactive yellow protein or PYP, and as such the isomerisation of p-coumaric acid, and derivatives, have been extensively studied at various theoretical levels. 208-213 Kort et al. first investigated the photoisomerisation in the PYP and found that the thioester of p-coumaric acid is in equilibrium between the cis - and trans -forms, but also concluded that not only can the cis -isomer be photochemically converted to the trans - isomer, but that the cis - to trans -isomerisation could be facilitated thermally, which is not the case for the reverse process.214 In the case of the true PYP, the photoisomerisation can be induced by light up to 430 nm, although many investigations into the PYP however, have begun with p-coumaric acid as a model system.
Common to these studies is the concept that the isomerisation proceeds via an excited electronic state through promotion of an electron from the alkene π-bond into an anti- bonding orbital, allowing for free bond rotation with the product determined by the nature of the preferred conformation in the excited state. There still remains conjecture as to whether the photoisomerisation of p-coumaric acid proceeds through a singlet excited state
(S 1) with paired electron spin, or a triplet excited state (T 1) with unpaired electron spin (Figure 4.2).
π∗
π
S 0 S1 T1 Figure 4.2: Electron configuration of π bonding and anti-bonding molecular orbitals in ground and excited states.
80 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
Li and Fang studied the excited states of trans-p-coumaric acid with respect to the nature of the excitation, finding that the lowest singlet transition (S 0-S1) possessed more n-π* character which resulted from excitation of the unpaired electrons of the carbonyl oxygen, but that the S 0-T1 transition was dominated by a π - π * electron promotion. The S0-T1 transition resulted in T1 p-coumaric acid possessing the lowest energy conformation with o o the alkene p-orbitals at a 90 dihedral angle, compared with 180 seen in the S 0 state. o Relaxation from T 1 at a 90 conformation to S 0 accounted for the formation of cis - and trans -isomers with either able to be formed. 208
Furthermore, Sergi et al. compared the excitation energies of p-coumaric acid against the phenolate anion (the form in which it is found in the PYP) and found that the vertical excitation energy of the anion was 20% lower than that of the protonated form, 211 though in acidic wine-like conditions (pH 3.5) it is extremely unlikely that the phenolate anion would be found.
4.1.2 cis -Hydroxycinnamate content in grapes and wine There are few examples of grape or wine quantifications that include both the cis - and trans -hydroxycinnamates, with the majority of these studies focusing on p-coumaroyl tartrate, 72-73, 96, 101, 103, 118, 123-127, 131 although feruloyl tartrate 72 and p-coumaric acid have also been considered. 101, 103 Of the studies that do consider hydroxycinnamate stereochemistry, it is sometimes only specified for some compounds and not others, 28, 72, 98, 101, 104 or only the trans -isomer is (or can be) quantified, 73, 99-100, 103, 123-124, 126, 131 while other studies do not consider the stereochemistry at all.97, 102, 105-106, 119, 121-122, 128-130, 215-216 For some studies it is unclear whether the quantification techniques fail to distinguish the two forms, or if the cis -isomers are present in concentrations lower than the detection threshold. However, it is understandable that quantifying both cis - and trans -isomers has been achieved most regularly for the most prevalent form, p-coumaroyl tartrate.
Both isomers of feruloyl tartrate were quantified and expressed as molar percentages in Cencibel grapes and the resulting wine, with an initial cis -content of 26.3%, dropping to 9.4% in wine. 72 The effect of malolactic fermentation on cis - and trans-p-coumaric acid was studied, with an initial isomeric ratio of 54.8% cis-p-coumaric acid observed; resulting in 25.2% of the cis -isomer after malolactic had been conducted in steel, against 32.2% in a
81 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids barrel. 101 In the same study, red wine was aged for 14 months resulting in p-coumaric acid content consisting of 6.5% of the cis -isomer. The same author performed malolactic fermentation with several lactic acid bacteria and found that the p-coumaric acid content changed from 48.1% of the cis -isomer to between 3.1 and 38.1% after malolactic fermentation. 103
The effect of ultra-violet light on cis/trans -ratios in grapes can be observed by compiling the cis - and trans-p-coumaroyl tartrate content in red and white skins of Montealegra et al., 126 and the content in red and white juices as determined by Singleton et al. (Table 4.1). 127
Table 4.1: Content of cis - and trans-p-coumaroyl tartrate in the skin and juice of red and white grapes. 126-127
Average Concentration trans -p -coumaroyl tartrate cis-p -coumaroyl tartrate % cis -isomer Skin content (mg/kg) White (n = 6) 7.63 2.92 27.65 Red (n = 4) 6.28 1.79 22.15
Juice content (mg/L) White (n = 19) 15.32 3.11 16.87 Red (n = 21) 18.38 3.43 15.73
The highest cis -content is in the skin of white grapes (27.7%), where increased exposure to ultra-violet light is expected, followed by the skin of red grapes (22.2%) where pigmentation can provide some relief from ultra-violet radiation. The juice content of cis - p-coumaroyl tartrate is lower than observed in the skins likely caused by the absorbance of radiation by compounds in the skins protecting the “juice” hydroxycinnamates, again with white grapes (16.9%) having a higher cis -content than red grapes (15.7%). While this data has come from two separate sources, the approximate ratio of cis - to trans-p-coumaroyl tartrate in the entire berry decreases with decreasing exposure to ultra-violet radiation. In red or white grapes the approximate cis :trans ratio agrees with those observed during synthesis of the glucose esters, which were observed to exist as approximately 20-25% of the cis -isomer.
The data obtained by Lee and Jaworski in Pinot Blanc grapes supports that shown in Table 4.1. They observed ratios of 16.6 and 21.8% of cis -p-coumaroyl tartrate at harvest across two separate vintages, 96 as well as Gomez-Alonso reporting 21% of the cis -isomer in Cencibel grapes. 72 Meanwhile, other studies have described much different ratios. Betes- Saura et al. analysed free run juice of 3 white grape varieties and found an average cis -
82 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids content of 47.6 %, 73 while a further study by Lee and Jaworski found cis -contents ranging from 10 – 67% across 21 white cultivars. 124
In wines, the isomeric ratio of p-coumaroyl tartrate also varies, with one study by Monagas et al. observing ratios between 10.9 to 55.3% of the cis -isomer in four different red wine varieties, 131 while other studies reported contents anywhere between 6.5 and 47.4% of the cis -isomer. 72-73, 101, 103
Of the hydroxycinnamates of interest in this study, p-coumaroyl tartrate is the analogue to have been most thoroughly quantified, although the presence of other cis - hydroxycinnamates in wine have been documented. Baderschnieder 108 investigated the phenolic content of a Riesling wine identifying a cis -isomer for most trans - hydroxycinnamate species identified (excluding the glucose esters of p-coumaric and ferulic acid), including p-coumaric acid, p-coumaric and ferulic-4-O-glucosides, and p- coumaroyl and feruloyl tartrate esters. With the extent of isomerisation observed for the glucose esters in the laboratory (described in Chapter 2), it is possible that a more sensitive technique might have detected cis -glucose esters also.
The hydroxycinnamates, a class of compound that are widely researched because of the possibility that they contribute to spoilage during winemaking, have not been considered in microbial breakdown with respect to stereochemistry. The cis -hydroxycinnamates which, as shown above, can contribute to around 20% of the hydroxycinnamate content of the berry, have not been specifically evaluated with respect to metabolism by D. bruxellensis and whether these can contribute to the accumulation of ethylphenols in wine during barrel ageing.
4.1.3 Enzymatic Specificity The breakdown of hydroxycinnamate esters to yield ethylphenols involves two potentially stereospecific enzymes, an esterase and a decarboxylase. Submitting cis -esters to D. bruxellensis would simultaneously test both of these enzymes in the same experiment, potentially leading to ambiguous results. Furthermore, once Dekkera has expressed decarboxylase activity, the product of the cis - and trans -acids, the vinylphenols ( 5 and 6), do not possess differing alkene stereochemistry and as such the subsequent vinyl reductase
83 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids will not be a factor in stereospecific metabolism. Formation of ethylphenols from acids as well as esters must both proceed via decarboxylation, and as such, this stage of hydroxycinnamate metabolism is a key step at which the stereospecificity should be scrutinised.
A purified p-coumarate decarboxylase from B. bruxellensis has been tested for substrate specificity with p-coumaric acid, caffeic acid, ferulic acid and m-coumaric acid. 68 The decarboxylase was active towards caffeic, p-coumaric and ferulic acids in that order of preference, and inactive towards m-coumaric acid. Although, it showed that a para - hydroxyl group was required, and by shifting it to the meta position, decarboxylation was retarded, the specificity within this study towards cis -acids was not tested.
Similar results were seen from a purified hydroxycinnamate decarboxylase enzyme from B. anomalus when tested towards a number of similar acids. 70 Again, caffeic acid was preferentially decarboxylated before p-coumaric and ferulic acids with relative activities of 37.5 and 31.3%, but the decarboxylase was inactive towards cinnamic acid, sinapic acid, hydrocaffeic acid, o-coumaric acid, m-coumaric acid, p-methoxycinnamic acid, p- hydroxybenzoic acid, iso-ferulic acid, 5-hydroxyferulic acid, 3,4-methylenedioxycinnamic acid, phenylalanine and pyruvic acid. This provides further information that a para - methoxy group is insufficient to facilitate decarboxylation, but again, no cis -acids were tested.
Gramatica et al. showed the ability of Saccharomyces cerevisiae to decarboxylate p- coumaric acid, p-methoxycinnamic acid, ferulic acid and 3,4-dimethoxycinnamic acid, but found that it was not active towards cinnamic acid, caffeic acid and methylenedioxycinnamic acid. 217 cis -3,4-Dimethoxycinnamic acid was then tested and the decarboxylase was not active towards it, indicating an inability to decarboxylate this particular cis -acid. Unlike the previous reports, this study shows the ability for a decarboxylation to occur for a compound possessing a para -methoxy group, though possessing a para -hydroxyl group doesn’t appear to guarantee decarboxylation with caffeic acid not being affected.
84 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
COOH H3CO COOH HO COOH
HO HO HO p-Coumaric acid Ferulic acid Caffeic acid
HO COOH COOH COOH
OH H3CO m-Coumaric acid o-Coumaric acid p-Methoxycinnamic acid
H3CO COOH COOH HO COOH
HO HO H3CO OH p-Hydroxybenzoic acid iso-Ferulic acid 5-Hydroxyferulic acid
O COOH COOH COOH COOH HO
NH O 2 O O 3,4-methylenedioxycinnamic acid Phenyl alanine Pyruvic acid Hydroxypyruvic acid
OH COOH COOH COOH
HO
Mandelic acid Hydrocinnamic acid 4-Hydroxyphenyl acetic acid
COOH HO COOH COOH
HO HO 4-Phenyl but-3-enoic acid Phloretic acid Dihydrocaffeic acid
H3CO COOH H CO COOH 3 COOH COOH
HO O O HO OCH3 Dihydroferulic acid Sinapic acid Acrylic acid Crotonic acid Figure 4.3: Compounds investigated in decarboxylation studies.
Goodey and Tubb studied S. cerevisiae in relation to the gene then designated as POF1 (now known as PAD1 218 ), which is responsible for the decarboxylation of hydroxycinnamic acids and resulting production of phenolic off-flavour as observed in beer.219 Those strains possessing the ability to decarboxylate were designated Pof +. S. cerevisiae strains possessing the Pof + phenotype showed the ability to decarboxylate ferulic acid, cinnamic acid and p-coumaric acid, in that order of preference, while caffeic acid, 4-hydroxypyruvic acid, mandelic acid, hydrocinnamic acid, 4-hydroxyphenylacetic
85 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids acid, 4-hydroxybenzoate and 4-phenylbut-3-enoate were unaffected by the S. cerevisiae decarboxylase. Again, compounds possessing a para -hydroxyl group weren’t necessarily decarboxylated, and additionally, cinnamic acid that has no aromatic ring substitution was able to be decarboxylated.
Harada and Mino studied the substrate specificity of the decarboxylase activity of Cladosporium phlei .84 It was active towards cis-p-coumaric acid, trans-p-coumaric acid, caffeic acid and ferulic acid, in that order of preference, and was inactive towards cinnamic acid, m-coumaric acid, o-coumaric acid, phloretic acid, p-methoxycinnamic acid, dihydrocaffeic acid, dihydroferulic acid, sinapic acid, acrylic acid and crotonic acid.
With these examples of decarboxylases, one fungus which showed otherwise similar substrate specificity to Dekkera did possess the ability to metabolise cis -acids, while brewing yeasts, which showed very different substrate specificity lacked the ability to metabolise cis -acids. However, there are no reports as to the stereospecificity of the decarboxylase of D. bruxellensis and whether cis -hydroxycinnamates, in addition to the trans -isomers, can be metabolised and contribute to the accumulation of ethylphenols in wine.
4.2 Research Aims.
Following the photoisomerisation of the hydroxycinnamoyl glucose esters ( 9 and 10 ) which resulted in partial conversion to furnish cis -analogues, and the ease by which it occurred, the role of the cis -hydroxycinnamates in the production of ethylphenols has become of importance to this research. Investigation into the known cis -hydroxycinnamate content of grapes has indicated that for most trans -hydroxycinnamates there exists a corresponding cis -isomer, and of those quantified, the cis -content is in the vicinity of 20% of the total hydroxycinnamates. The ability of D. bruxellensis to metabolise the cis - isomers, or otherwise, could have an impact on the production of ethylphenols by as much as 20% within wine.
The decarboxylase of D. bruxellensis is active in the bioconversion of the free acids and esters to ethylphenols and will be tested for stereospecificity by conducting fermentation in
86 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids the presence of both the cis - and trans -hydroxycinnamic acids. The trans -hydroxycinnamic acids have already been synthesised, being isolated during preparation of the esters (Chapter 2), with the cis -hydroxycinnamic acids now requiring synthesis.
In addition to synthesis of cis-p-coumaric and cis -ferulic acids, the photoisomerisation of the hydroxycinnamic acids will be investigated at a theoretical level beyond that of the free acids to explain the rapid isomerisation experienced for the glucose esters, which was not observed for any other hydroxycinnamate derivative. Using the existing theoretical studies of p-coumaric acid, the procedures will be extended to the hydroxycinnamate esters in an effort to determine the energy barriers associated with photoisomerisation, and develop protocols to increase the ease of synthesis of the glucose esters (9 and 10 ) while maintaining stereoisomeric purity.
4.3 Synthesis of cis-Hydroxycinnamic Acids.
By isolating the minor products from the Wittig reaction (as described in Chapter 2) via column chromatography, pure cis -11 and cis -12 could be achieved, though these were only produced in limited quantities as the trans -products are thermodynamically favoured. The formation of ethyl coumarate (11 ) affords approximately 90% trans -11 and 10% cis -11 , while the same reaction for the production of ethyl ferulate (12 ) yields around 70% trans- 12 and 30% cis -12 , which can largely be separated by flash chromatography with only minor co-elution of isomers. While cis -11 and cis -12 could be isolated and characterised under ambient light conditions, attempted base-catalysed ester hydrolysis to yield the cis - acids ( cis -3 and cis -4) proved unsuccessful, resulting in isomeric mixtures. Hydrolysis of cis -11 gave a mixture of cis - and trans -3 in a ratio of 20:80, while hydrolysis of cis -12 afforded a 35:65 mixture of cis - and trans -4.
87 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
O OEt
O H O
Wittig OEt + R R R OH OH OH 13 R = H cis/trans-11 R = H 14 R = OCH3 cis/trans-12 R = OCH3
KOH
O
OH
R
OH
cis-3 R = H cis-4 R = OCH3 Scheme 4.1: Attempted synthesis of cis -hydroxycinnamic acids.
Initial conclusions for the production of the isomeric mixtures of 3 and 4 were that the basic reaction conditions caused deprotonation of the phenolic hydroxyl, generating a phenolate resonance structure that interrupted the α,β-unsaturated double bond and causing a conversion back to the thermodynamically more favoured trans -isomer (Figure 4.4).
- O OEt O OEt
- O O
OEt OEt H+
O- O O OH Figure 4.4: Proposed resonance assisted conversion of cis -p-coumaric acid to trans -p- coumaric acid.
Separation of cis- and trans -4 was achieved through flash column chromatography using 10% methanol in dichloromethane, yielding the pure cis -4 acid as indicated by TLC. After standing for 16 hours in solution, analysis by NMR showed a 2:1 mixture of cis - to trans -4,
88 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids contradicting the previous hypothesis that isomerisation to the trans -acid was facilitated by formation of the phenolate.
Though the trans -acids proved stable under ambient light conditions during synthesis, this was not the case for the cis -acids, undergoing isomerisation under laboratory lighting. As such, isolation of the pure cis -acids by chromatography was then performed under red light in an attempt to minimise photoisomerisation, although under these conditions the cis -acids still underwent a slow conversion back to the trans -acids. This indicated that spiking a fermentation with pure cis -acids in the dark would result in some conversion back to the trans -isomer which D. bruxellensis could metabolise and yield ethylphenols. With the cis - acids ( cis -3 and cis -4) found to isomerise to give trans -acids even under conditions of low light exposure, attempted synthesis and isolation of the pure cis -3 and cis -4 was discontinued.
As the decarboxylase of D. bruxellensis is active towards only a few hydroxycinnamic acids, a more stable alternative cis -substrate could not be utilised in the investigation of stereoselective metabolism. Furthermore, an alternative substrate would not provide adequate information as to the ability of D. bruxellensis to produce the ethylphenols of interest to this study. Therefore it was required that fermentation experiments be performed on isomeric mixtures of cis - and trans -hydroxycinnamic acids. These could be produced through ultra-violet irradiation with literature observations indicating that the hydroxycinnamates, upon irradiation, exist in approximately 40-49% in the cis -form. 204
Photoisomerisation of p-coumaric acid ( trans-3 to cis-3) was initially performed under 254 nm ultra-violet light (a readily available lamp in organic laboratories used to view TLC plates) on small scale in NMR tubes for simple analysis requiring no sample preparation. After 66 hours of irradiation a stable 52:48 ratio of trans - to cis -3 was produced as indicated by 1H NMR analysis. Concurrently trans -ethyl coumarate ( 11 ) was irradiated to investigate the thermodynamic effects of the isomerisation, assuming that cis -11 would be produced to a greater extent given the stability comparative to cis -3 during synthesis. After 66 hours a 60:40 mixture of trans - and cis -11 was observed suggesting that the stability of cis -11 during synthesis and chromatography is not due to thermodynamic stabilities of the cis -isomers, but more likely a result of the higher energy required to facilitate the conversion.
89 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
One study, of o-coumaroyl glucoside isomerisation, reported a quicker trans - to cis - conversion with a shorter wavelength of ultra-violet light, but noted that prolonged exposure of the glucosides to 254 nm light gave significant degradation of the product, with only 40% remaining after 1 hour of exposure. The isomerisation induced by using a 365 nm lamp was found to be slower, but produced the cis -isomer to greater extents and resulted in very little degradation of the substrate. 203
Isomerisation using 365 nm light was performed for both hydroxycinnamic acids (trans -3 and trans -4), with the percentage conversion to the cis -isomer higher in 4. The extent of isomerisation for 3 was less than experienced under 254 nm irradiation, however 365 nm light was used in preference to avoid any potential and unnecessary degradation of the acids. The isomeric ratios of the cis:trans -mixtures were determined by integrating the signal for H 8 of each isomer as these signals not only show a large change in chemical shift between isomers, but are removed from other signals thus avoiding overlapping shifts.
The stability of the cis/trans -mixtures were tested by storing a small amount of cis/trans-p- coumaric acid (3) under different conditions. A 61:39 mixture of trans :cis -3 was stored in acetone or as a solid under the conditions outlined in Table 4.2, with the final ratios determined after two weeks of storage.
Table 4.2: Isomeric ratio of p-coumaric acid ( 3) under different storage conditions.
% of isomer Condition trans cis Room temperature, Solid 63 37 Dark In acetone 62 38 Room temperature, Solid 63 37 Light In acetone 63 37 Solid 60 40 4 oC In acetone 61 39 Solid 62 38 -20 oC In acetone 63 37
Slight deviations in the observed isomeric ratios are most likely a product of the variability of the analytical method (NMR), otherwise the mixtures of cis/trans-3 proved stable, indicating that the mixtures produced by irradiation would remain constant over the course of the planned fermentation studies.
90 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
Additionally, in an attempt to promote formation of cis -ethyl coumarate (cis -11 ) by binding transition metals between the aromatic ring and carbonyl, and effectively “holding” the molecule in the cis -configuration (Figure 4.5), palladium and platinum were stirred in the presence of 11 . Both metals were found to retard photoisomerisation in an undesired manner, and had the effect of yielding trans -11 from the cis -isomer.
O
O
M
OH Figure 4.5: Intended effect of metal coordination on cis -hydroxycinnamates.
Submitting trans -ethyl coumarate ( 11 ) to ultra-violet radiation in the presence of either palladium or platinum on activated carbon, isomerisation to cis -11 was inhibited by approximately the same amount as the molar ratio in which the metal was present. Additionally, by submitting cis -ethyl coumarate ( cis -11 ) to palladium acetate, which was utilised instead of 10% palladium on carbon to increase the number of moles of metal per mass of reagent, had a large effect of converting cis -11 to the trans -isomer. With an excess of palladium, 100% conversion from cis -11 to trans -11 was achieved under ambient light conditions. So, not only did complexation of metals fail in promoting formation of cis -11 , but assisted in achieving the opposite, production of trans -11 , which is consistent with literature reports. 220 However, future use of transition metals to maintain isomeric purity, or convert unwanted cis -analogues to the trans -isomer may be of use. In the case of undesired formation of cis -glucose esters ( cis-9 and cis -10 ), future synthetic attempts may involve producing isomeric mixtures under ambient light conditions, followed by recovery of the trans -isomers by exposure to palladium or platinum.
4.4 Theoretical Studies into the Isomerisation of Hydroxycinnamic Acids.
Ab initio studies into the photoisomerisation of p-coumaric acid have been well documented, 208-213 but this study aimed to explain the differences in isomerisation observed
91 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids between hydroxycinnamate derivatives in the laboratory, namely: the ease of trans - to cis - isomerisation for the glucose esters ( 9 and 10 ) that has not been observed previously; the instability of the cis -hydroxycinnamic acids (cis -3 and cis -4) relative to the cis -ethyl hydroxycinnamates (cis -11 and cis -12 ); and the rapid formation of the trans -acids seen during the base-catalysed ester hydrolysis of the cis -ethyl esters ( cis -11 and cis -12 ).
Using a DFT B3LYP 6-31G* level of theory, trans-p-coumaric acid underwent an equilibrium geometry optimisation at the ground state (S 0) and then again at the first excited state. While there remains some discrepancy in the literature regarding whether the 208 isomerisation proceeds via excitation to the S 1 or T 1 state, optimisation of the ‘first excited state’ of trans -p-coumaric acid identifies this as the T 1 state. As the S 1 and T 1 state occupy the same molecular orbital, with the HOMO of each theoretically corresponding with the LUMO of the S 0 state, the only difference in energy should arise from the opposing spin of the promoted electron, which should be constant across all the geometries and of little consequence to this study.
The HOMO and LUMO of S 0 trans -p-coumaric acid ( 3), along with the HOMO of T 1 trans -p-coumaric acid were calculated for the optimised structures and are shown below
(Figure 4.6). These support that promotion of an electron from S 0 trans -p-coumaric acid will result in a molecular orbital corresponding to T 1 trans -p-coumaric acid.
Figure 4.6: Frontier molecular orbital diagrams of trans-p-coumaric acid ( 3). a) HOMO of
S0 trans -p-coumaric acid. b) LUMO of S 0 trans -p-coumaric acid. c) HOMO of T 1 trans -p- coumaric acid.
92 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
Also, the spin density of T 1 trans -p-coumaric acid (Figure 4.7) is largely concentrated around the α,β-unsaturated double bond, suggesting that the alkene is highly affected by the excitation into the T 1 state.
Figure 4.7: Electron spin density in T1 trans -p-coumaric acid.
The energy profile of p-coumaric acid ( 3) resulted from constraining the dihedral angle of trans -p-coumaric acid around the α,β-unsaturated double bond to 180 o, optimising the geometry at a DFT B3LYP 6-31G* level and calculating the energy of the conformation. Using a dynamic constraint, the dihedral angle was rotated from 180 o to 0 o through 19 possibilities, optimising the geometry and calculating the energy every 10 degrees. This process was applied to the singlet ground state (S 0), then repeating for the triplet state (T 1) representing the photoisomerisation of p-coumaric acid in a vacuum (Figure 4.8).
93 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
300 T1 S0 200
100 Energy Energy (kJ/mol)
0 0 30 60 90 120 150 180 Dihedral Angle
Figure 4.8: Energy profile of p-coumaric acid (3). See Appendix 3, Table A3.1 for calculated energies and relative differences.
The potential for isomerisation of the hydroxycinnamic acids, or compounds possessing a similar molecular backbone, can be observed in the energy profile of p-coumaric acid.
Excitation of S 0 trans -p-coumaric acid to the T 1 state results in the preferred conformation at a 90 o dihedral angle, which corresponds to the two p-orbitals sitting orthogonal.
Relaxation back to S 0 state can give either trans - or the cis -p-coumaric acid, which corresponds to literature evidence. 213
Repeating the analysis for ethyl coumarate ( 11 ) the S 1 profile was generated with questionable results (Figure 4.9). Rotation of the dihedral from 180 o results in geometries of increasing energy until the 70 o conformation whereby calculations for the 60 o and 50 o conformations fail to converge, and expected values are achieved for the remaining conformations (0-40 o). This effect also observed for calculations using a dynamic dihedral constraint and for calculation using individually drawn structures (manual constraint), though the manually drawn structures are a product of the preceding conformation (170 o drawn from the 180 o, and so on). As the dihedral angle of the carbon skeleton is altered, the alkene protons remain at 180 o to each other producing structures with increasing degrees of pyramidilisation and energy (50 and 60 o conformations) until the calculations fail to converge, whereby the protons are forced to a 0 o dihedral angle (0-40 o conformations) (Figure 4.10). As such, the S 0 profile was achieved by dynamically rotating the dihedral from 180 o to 90 o (forwards) and from 0 o towards 90 o (backwards), producing improved results.
94 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
300 Manual S 0 Forwards Dynamic S 0 Backwards 200 Dynamic S 0 Fowards T1
100 Energy Energy (kJ/mol)
0 0 30 60 90 120 150 180 Dihedral Angle
Figure 4.9: Energy profile produced from forward and reverse dynamic, and manual constraint of ethyl coumarate (11 ). See Appendix 3, Table A3.2 for calculated energies and relative differences.
RH RH H H HH R R H RH RH R H R R R
o o o o o 180 140 90 60 30 Figure 4.10: Pyramidilised alkene resulting from rotation of the dihedral angle from 180 o to 0 o in ethyl coumarate ( 11 ).
o The dynamic S 0 profiles (forwards and backwards) differ for the 0-40 conformations by approximately 17 kJ/mol, a product of optimising different ethyl coumarate conformers. Beginning with trans -ethyl coumarate and rotating the alkene to give cis -ethyl coumarate produces a different conformer to simply drawing cis -ethyl coumarate, which differ by rotation around the C 8-C9 bond (Figure 4.11). Both configurations, when optimised, produce geometries that are in their own right in potential energy wells of the surface. As such, the optimisation of the higher energy conformer determines a local energy minimum which does not correspond to the global energy minimum. Calculations started from an initial MMFF geometry, gave higher energy structures than those started from the MMFF conformer, which identifies the global energy minimum.
95 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
a) b) EtO O
C8 EtO O
C9 O OEt OR
OH OH OH Figure 4.11: cis -Ethyl coumarate conformers produced by: a) drawing trans -ethyl coumarate and rotating the dihedral; and b) drawing cis -ethyl coumarate.
The energy profile of p-coumaroyl glucose (9) (Figure 4.12) incorporates first identifying the MMFF conformer before calculating the S 0 profile from both the trans - and cis -isomers towards the 90 o dihedral. The results suggest that different p-coumarate substrates do not appear to have a large enough effect on the excitation energy to heavily effect the extent or speed of isomerisation. The energy profiles of p-coumaric acid (3), ethyl coumarate (11 ) and p-coumaroyl glucose (9) display similar characteristics and energy barriers.
300 T1 S0 Backwards 200 S0 Forwards
100 Energy Energy (kJ/mol)
0 0 30 60 90 120 150 180 Dihedral Angle
Figure 4.12: Energy profile for p-coumaroyl glucose (9), relative to S 0 trans -isomer. See Appendix 3, Table A3.3 for calculated energies and relative differences.
The potential for the different solvent conditions that the glucose esters encountered during synthesis (aqueous) compared with the other hydroxycinnamates (organic) to have an effect on the isomerisation exists, as noted by Kahnt. 187 Repeating the theoretical analysis for p-coumaric acid ( 3) and p-coumaroyl glucose ( 9) in water (Figure 4.13) yields near identical energy profiles as shown by the T 1 energy profiles which mimic that observed respectively within Figures 4.8 and 4.12, which were calculated in a vacuum.
96 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
a) b) 260 280
240 260
220 240
200 220 Energy Energy (kJ/mol) Energy Energy (kJ/mol)
180 200 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Dihedral Angle Dihedral Angle
Figure 4.13: a) T 1 energy profile for p-coumaric acid (3) in water, relative to the S0 trans -
acid. b) T 1 energy profile for p-coumaroyl glucose (9) water, relative to the S0 trans - isomer. See Appendix 3, Table A3.4 for calculated energies and relative differences.
The solvated and vacuum calculated T1 profiles appear to have similar energies, and all o display diabatic profiles, whereby the lowest energy conformation in the T1 state is at a 90 dihedral angle, which will relax to the S0 state at the highest energy point and potentially produce either isomer. The shape of the T 1 and S 0 profiles, and the energy differences between the S0 and T 1 states (the vertical excitation energies) do not change excessively as a result of either solvation or substrate. Furthermore, optimising the geometry of the T 1 state may lead to incorrect vertical excitation energies as the S 0 geometry of a molecule is excited directly to the T 1 state, rather than excited to a different, optimised T1 geometry. Therefore, the vertical excitation energies should be a result of an optimised geometry for the S 0 state, followed by a single point energy calculation for that geometry at the T 1 state.
In addition to the S 0-T1 vertical excitation energy, the HOMO-LUMO gap was determined for trans -p-coumaric acid (3) and trans -p-coumaroyl glucose (9) under the effect of solvation (Figure 4.14). The vertical excitation energy, along with the HOMO-LUMO gap should decrease with increasing solvent polarity for a π- π * transition due to the comparative stabilising effect on the π* orbital compared with the π orbital.221
Determination of solvation effects was investigated using solvent ET 30 values as shown in
Table 4.3, with a solvent of higher polarity possessing a greater ET 30 value.
97 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
Table 4.3: Solvent polarities and ET 30 values.
Solvent Dielectric Constant ET 30 Value Water 80 63.1 Ethanol 24.6 51.9 Acetone 21 42.2 DCM 9.1 40.7 THF 7.5 37.4 Ether 4.3 34.5
265 trans -p-Coumaric Acid trans -p-Coumaroyl 260 Glucose
255
250 Energy Energy (kJ/mol)
245 30 35 40 45 50 55 60 65
ET 30
Figure 4.14: S 0-T1 vertical excitation energy for trans -p-coumaric acid (3) and trans -p- coumaroyl glucose (9). See Appendix 4, Table A4.1 for calculated vertical excitation energies.
The vertical excitation energies of trans -p-coumaric acid and trans -p-coumaroyl glucose in numerous solvents (Figure 4.14) differ by approximately 7 kJ/mol throughout the solvents tested, and it is unlikely that such a small difference in vertical excitation energy between the substrates would explain the vast difference in the ease of isomerisation that was observed during synthesis. However, the HOMO-LUMO gaps of the S 0 compounds in each of the solvents (Figure 4.15) indicate a similar change in energy as displayed for the vertical excitation energies, without having to calculate the energy of the T 1 state, providing a more rapid indication of the vertical excitation energies.
98 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
420 trans -p-Coumaric Acid trans -p-Coumaroyl 410 Glucose
400
390 Energy Energy (kJ/mol)
380 30 35 40 45 50 55 60 65
ET 30
Figure 4.15: HOMO-LUMO gap for trans -p-coumaric acid and trans -p-coumaroyl glucose. See Appendix 4, Table A4.1 for calculated HOMO-LUMO gaps.
The effects of solvent and substrate on isomerisation as described by Kahnt 187 have not yielded justification to the changes in isomerisation observed during synthesis, although Kahnt also identified pH as another determining factor in the isomeric equilibrium.186 In addition, Sergi et al. found a decrease in excitation energy of p-coumaric acid upon formation of the phenolate, 211 and during synthesis, the glucose esters were believed to have been deprotonated on XAD-2 resin. Slight deprotonation of the glucose esters on XAD-8 resin could facilitate a more rapid isomerisation which was experienced for these compounds alone as only they were submitted to XAD resins. Repeating the vertical excitation energy and HOMO-LUMO gap calculations for the trans -p-coumaroyl glucose phenolate afforded Figure 4.16.
a) b) 200 320
195 318
316 190 314 185 Energy Energy (kJ/mol)
Energy Energy (kJ/mol) 312
180 310 30 35 40 45 50 55 60 65 30 40 50 60
ET 30 ET 30
Figure 4.16: a) Vertical excitation energies (S 0-T1) of trans -p-coumaroyl glucose phenolate in solvents of differing polarity. b) HOMO-LUMO gap. See Appendix 4, Table A4.1 for calculated vertical excitation energies and HOMO-LUMO gaps.
99 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
As seen by Sergi, a significant reduction of the vertical excitation energy is observed for the trans-p-coumaroyl glucose phenolate to between 185 and 195 kJ/mol from 250-255 kJ/mol calculated for the protonated form. Again, evidence of this trend is observed in the reduction of the HOMO-LUMO gap, indicating that phenolic deprotonation facilitates the isomerisation away from the trans -isomer. Furthermore, if deprotonation of p-coumaroyl glucose significantly effects the energy barrier to isomerisation, then the earlier hypothesis regarding the base-catalysed ester hydrolysis inducing isomerisation from the cis - hydroxycinnamates back to the trans -isomers, may in part be justified.
By comparing the HOMO-LUMO gaps of the cis - and trans -hydroxycinnamates examined throughout this study (p-coumaric acid, ethyl coumarate and p-coumaroyl glucose), similar conclusions can be made, that the trans -glucose esters are slightly more prone to isomerisation away from the trans -isomer, and the energy barrier to isomerisation is lowered with increasing solvent polarity (Figure 4.17). Additionally, the HOMO-LUMO gaps of the cis -isomers gave the same trend with regard to solvent polarity, again with only minor differences observed between substrates.
100 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
a) 420 trans -Ethyl Coumarate trans -p-Coumaric Acid 410 trans -p-Coumaroyl Glucose 400
390 Energy (kJ/mol) Energy
380 30 40 50 60
ET 30
b) 410 cis -Ethyl Coumarate cis -p-Coumaric Acid 405 cis -p-Coumaroyl Glucose
400
395 Energy (kJ/mol) Energy
390 30 40 50 60
ET 30
Figure 4.17: a) HOMO-LUMO gap of trans -hydroxycinnamates. b) HOMO-LUMO gap of cis -hydroxycinnamates. See Appendix 4, Table A4.2 and A4.3 for orbital energies HOMO- LUMO gaps.
The relative stability of the cis -ethyl esters (cis -11 and cis -12 ) compared with the cis -acids (cis -3 and cis -4) can only be attributed to the different solvents that the compounds experienced, with the ethyl esters being synthesised and purified with less polar solvents (i.e. dichloromethane), while the acids were prepared in ethanol and water, with this study providing no other explanation as to the ease with which the acids isomerise.
In order to assess the hypothesis that during the attempted cis -hydroxycinnamic acid synthesis, base-catalysed ester hydrolysis was the main contributing factor to isomerisation, the nature of the compounds that existed under the reaction conditions have to be determined. Literature pK a values for p-coumaric acid are 4.35 and 8.80 which 222 correspond to the carboxyl group and the phenol group, respectively. With a pK a of 4.35, the carboxylate would be heavily deprotonated at the pH needed to form the phenolate, as such, the phenolate needs only be considered if the carboxylate anion is also taken into account. At the pH of reaction, determined to be 13 as shown by pH strips, cis -
101 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids ethyl coumarate (cis -11 ) would exist as the phenolate. Ester hydrolysis yields cis -p- coumaric acid dianion, which is protonated fully during acidic work-up (to pH 3) yielding cis -p-coumaric acid (cis -3). The HOMO-LUMO gaps of each of the two anionic structures present during reaction have been calculated under different solvent conditions, and compared with cis -ethyl coumarate and c is -p-coumaric acid (Figure 4.18).
420 cis -Ethyl Coumarate 400 cis -Ethyl Coumarate anion 380 cis -p-Coumaric Acid dianion 360 cis -p-Coumaric Acid
Energy (kJ/mol) Energy 340
320 30 35 40 45 50 55 60 65
ET 30
Figure 4.18: HOMO-LUMO gaps of cis -hydroxycinnamates during base-catalysed ester hydrolysis. See Appendix 4, Table A4.4 for orbital energies and HOMO-LUMO gaps.
The HOMO-LUMO gap of the cis -ethyl coumarate phenolate is considerably lower than for the protonated form, implying that the greatest potential for cis - to trans -isomerisation during this reaction is observed for the phenolate. While the cis -p-coumaric dianion has a reduced HOMO-LUMO gap compared with the protonated forms, and would be more likely to isomerise back to the trans -isomer. Thus, the cis -ethyl coumarate phenolate must be largely responsible.
Additionally, those species existing as phenolic anions would be expected to have a much greater electron donating character, leading to increased resonance forms and hence reducing the double bond character of the alkene. It can only be assumed that this effect is not as great for the p-coumaric dianion, as the formation of the carboxylate anion reduces the electron withdrawing character of the carboxyl and retarding electron movement throughout the molecule. If this is indeed the case, then calculation of the p-coumaric carboxylate (with a protonated phenol) HOMO-LUMO would result in an increase in HOMO-LUMO gap due to a similar electron donating character of the phenol as observed for p-coumaric acid, but with reduced electron withdrawing character at the top of the molecule, as determined by the charge on oxygen 3 (Figure 4.19).
102 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
R 2 O O 3
O 1 R' Figure 4.19: Numbering of oxygen atoms in hydroxycinnamate skeleton.
trans -p-Coumaric 440 carboxylate cis -p-Coumaric carboxylate 420
400 Energy (kJ/mol) Energy 380
30 40 50 60
ET 30
Figure 4.20: HOMO-LUMO gap of p-coumaric acid carboxylate. See Appendix 4, Table A4.5 for HOMO-LUMO gaps.
The HOMO-LUMO gap of the p-coumaric acid carboxylate in less polar solvents is similar to those observed for p-coumaric acid, but in more polar solvents, there is a great increase in the HOMO-LUMO gap of the carboxylate. This supports the theory that the energies of isomerisation within the hydroxycinnamates are largely dependent on the nature of intramolecular electronics, and the ability to reduce the double bond character of the alkene.
The natural charge on the phenolic oxygen (Oxygen 1) and on the single-bonded carboxylic oxygen (Oxygen 3) was determined for all of the hydroxycinnamates investigated throughout this study (p-coumaric acid, p-coumaric acid carboxylate, p- coumaric acid dianion, ethyl coumarate, ethyl coumarate phenolate, p-coumaroyl glucose and p-coumaroyl glucose phenolate). The charge ratio (O1/O3) was then calculated in order to provide an indication of the nature of the electronics of each group and what effect they will have directly on the HOMO-LUMO gap (Figure 4.21). With good linear
103 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids correlation observed between oxygen charge ratio and HOMO-LUMO gap, additional substrates were investigated including 1-O-acetyl p-coumaric acid (19 ) and 1-O- chloroacetyl p-coumaric acid (21 ), with an expected reduction in electron donating character of the ring giving a higher HOMO-LUMO gap, and the carboxylate anions of 19 and 21 , in which the effect should be intensified.
2.5
2.0
1.5
1.0
0.5 Oxygen Charge Ratio Oxygen 0.0 300 350 400 450 HOMO-LUMO gap
Figure 4.21: HOMO-LUMO gaps of hydroxycinnamate derivatives against ratio of charge between oxygen 1 and oxygen 3. See Appendix 4, Table A4.6 and A4.7 for charges on the oxygen atoms and calculated ratios.
The HOMO-LUMO gap shows a rough linear relationship with the charge ratio of the compounds investigated. Those with a large charge on oxygen 3 compared with oxygen 1 (lower ratio) are expected to have a lesser effect on the double bond and display an increased HOMO-LUMO gap, where as those with large negative phenolic oxygen charges and less negative charge on oxygen 3 are expected to have a much larger effect on the double bond. Increased electron movement within the molecule decreases the HOMO- LUMO gap and suggest that they will be more prone to isomerisation, which is also seen by mapping the HOMO-LUMO gap against double bond length (Figure 4.22). The linear correlation between the bond length or oxygen charge ratio and the HOMO-LUMO gap indicates that increased resonance of the substrates increases the chance of photoisomerisation.
104 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
1.38
Å) 1.37
1.36
1.35 Bond Length ( Length Bond
1.34 300 350 400 450 HOMO-LUMO gap
Figure 4.22: Relationship between HOMO-LUMO gap and double bond length in hydroxycinnamate derivatives. See Appendix 4, Table A4.6 and A4.7 for alkene bond length.
4.5 Conclusions.
The synthesis and attempted isolation of the cis -hydroxycinnamic acids ( cis -3 and cis -4) resulted in isomeric mixtures, which proved stable and could be maintained under fermentation conditions. The cis -ethyl esters ( cis -11 and cis -12 ) could be isolated and handled under ambient light conditions without noticeable isomerisation back to the trans - isomers. As such, fermentation studies into the stereospecificity of the D. bruxellensis decarboxylase activity will need to be limited to spikes of isomeric mixtures of cis/trans - hydroxycinnamic acids, and be fermented against pure trans -acids with the difference in ethylphenol formation between them examined (Chapter 5).
Base-catalysed ester hydrolysis of the cis -ethyl esters involved formation of cis -ethyl ester phenolates which were found to have a much lower HOMO-LUMO gap that could be largely implemented in the conversion back to the trans -isomers. The relative stabilities of the ethyl esters compared with the hydroxycinnamic acids could only be attributed to a lowering of the HOMO-LUMO gap in solvents of increasing polarity, with the environments needed to handle (synthesise or analyse) each compound contributing to isomerisation. Additionally, the use of group 10 metals in the presence of cis - or trans - ethyl coumarate was found to encourage formation of the trans -isomer. The use of transition metals may have future applications in stereochemical control of the hydroxycinnamic acids in synthetic attempts.
105 Chapter 4: Photoisomerisation of Hydroxycinnamic Acids
The energy required to excite p-coumaroyl glucose (9) to the T 1 state and facilitate cis/trans -isomerisation was lower than for p-coumaric acid (3), but was further lowered by formation of the phenolate, of which there was evidence of occurring during contact with XAD resins. As such, the use of XAD resins with the hydroxycinnamic acids should be performed under strictly acidic conditions, preventing phenolate formation, or it should be performed under light conditions of lower energy. In this study, red light proved useful in preventing isomerisation.
Further studies into the isomerisation of hydroxycinnamates observed during synthesis showed a relationship between the electronic make-up of the molecule and the energy needed to facilitate photoisomerisation, with compounds allowing increased electron movement, having reduced HOMO-LUMO gaps. This result indicates that even if the glucose esters had not been completely deprotonated on the XAD resin, any extent of hydrogen bonding to the phenol that would increase the electron donating character of the phenolic oxygen would also decrease the energy required for photoisomerisation to occur.
106 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis .
5.1 Bioconversion of trans-Hydroxycinnamate Esters.
5.1.1 Ethyl Esters
O O O O
OCH3
OH OH 11 12 Figure 5.1: Ethyl coumarate ( 11 ) and ethyl ferulate ( 12 ).
Bioconversion of the ethyl hydroxycinnamates ( 11 and 12 ) by D. bruxellensis strain AWRI 1499, a representative of the predominant strain grouping in Australian winemaking, 22 was studied and the outcome determined by the production of 4-ethylphenol and 4- ethylguaiacol. The self-anaerobic fermentations were conducted to maximise the conversion from precursors, through vinylphenols to ethylphenols. 21 Yeast biomass peaked at day 6 and the fermentations were conducted for a further 3 days, concluding shortly after the yeast entered stationary phase to maximise the potential metabolism of the ethyl esters.
107 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
60 4-Ethylphenol 4-Ethylguaiacol 40
20 Percentage Conversion Percentage 0 2 4 6 8 10 Fermentation Progress (Days)
Figure 5.2: Percentage of the theoretical maximum conversion of ethyl esters ( 11 and 12 ) to ethylphenols.
AWRI 1499 displayed a much greater affinity for metabolism of ethyl coumarate (51.4% conversion to 4-ethylphenol), over ethyl ferulate (4.0% conversion to 4-ethylguaiacol). This appears to be a product of the esterase activity as such a preference is not observed for the decarboxylase during the metabolism of the free hydroxycinnamic acids, as seen in a later experiment and in literature reports.
Furthermore, Godoy et al. studied a purified p-coumarate decarboxylase enzyme from B. bruxellensis and tested for substrate specificity with p-coumaric acid, caffeic acid and ferulic acid. The decarboxylase was effective in metabolism of all three substrates with an activity of 120 and 80% for caffeic and ferulic acids relative to that of p-coumaric acid. 68 Similar results were observed by Edlin et al. for a hydroxycinnamate decarboxylase from B. anomalus , although preferential breakdown was witnessed for caffeic acid, followed by p-coumaric and ferulic with relative activities of 37.5 and 31.3%, respectively.70
The preferential breakdown of p-coumaric acid compared with that for ferulic acid, as detailed in these two studies, occurred with relative activities of 1.25 and 1.20, rendering unlikely the possibility that in this instance the decarboxylase could account for a favoured formation of 4-ethylphenol over 4-ethylguaiacol by a factor of 12.85.
Therefore the substrate selectivity is presumably a product of the esterase activity of AWRI 1499 which could be substantiated by the recovery of the remaining ethyl esters in the fermentation samples using the GC-MS SIDA method as described by Sleep. 141 This method has been validated for quantification up to 10 mg/L in wine, and as such could be
108 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis employed without dilution of the fermentation samples. Combining the recovery of the initially spiked ethyl esters with the evolution of ethylphenols provides a total recovery (Figures 5.3 and 5.4).
100 4-Ethylphenol Ethyl Coumarate 80 Total Recovery
60
40
20 Percent Percent Recovered
0 2 4 6 8 10 Fermentation Progress (Days)
Figure 5.3: Percentage recovery of coumarates in fermentations.
100 4-Ethylguaiacol Ethyl Ferulate 80 Total Recovery
60
40
20 Percent Percent Recovered
0 2 4 6 8 10 Fermentation Progress (Days)
Figure 5.4: Percentage recovery of ferulates in fermentations.
The total recovery at the conclusion of fermentation was approximately 80% for both the ferulate series and the coumarate series. In addition to minor contributions by the acids and vinylphenols, slight losses are expected through adsorption onto the yeast, 93, 223 as well as through loss of the volatile ethylphenols through the gas-lock. Although, with the significant amount of remaining ethyl ferulate in the ferments, it can be concluded that, for AWRI 1499, uptake and metabolism of ethyl coumarate is preferential over ethyl ferulate. This suggests that the ethyl esterase activity of AWRI 1499 exhibits a substrate selectivity.
109 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
5.1.2 Ethyl Esterase Substrate Selectivity As well as establishing the potential for spoilage caused by the breakdown of ethyl coumarate ( 11 ), the ethyl ester substrate selectivity shown for AWRI 1499 has the potential to accentuate the ratio of 4-ethylphenol:4-ethylguaiacol in wine. To determine whether this selectivity is strain dependant or is common throughout D. bruxellensis , fermentation experiments with strains representing the two remaining significant genetic groups (AWRI 1608 and AWRI 1613), in the presence of ethyl coumarate ( 11) and ethyl ferulate ( 12 ) were conducted, along with a repeat fermentation with AWRI 1499. By using D. bruxellensis strains AWRI 1499, 1608 and 1613, representatives of the three genetic groups that contribute to 98% of Australian wine isolates were studied. End-point analyses of 4-ethylphenol and 4-ethylguaiacol were conducted for all fermentations (Figure 5.5).
80 4-Ethylphenol 4-Ethylguaiacol 60
40
20 Percentage Conversion Percentage 0 3 99 1 I 14 I 1608 I 16 R WR WR A AW A Figure 5.5: Percentage of the theoretical maximum conversion from ethyl coumarate ( 11 ) and ethyl ferulate ( 12 ) to ethylphenols by different strains of D. bruxellensis .
The preference shown in the previous experiment by AWRI 1499 was observed again, with ethyl coumarate metabolised over ethyl ferulate by a factor of 8.75. In this instance the fermentations were conducted over a longer period as the evolution of ethylphenols was still showing an upward trend after 9 days in the previous experiment (Figure 5.2). AWRI 1608 also displayed a preferential metabolism for ethyl coumarate, however in this case by a factor of 18.75, due largely to lesser production of 4-ethylguaiacol.
D. bruxellensis AWRI 1613 did not convert either ethyl ester to the respective ethylphenol. Subsequent analysis of the end-point fermentation samples by HPLC using ethyl coumarate and ethyl ferulate as external standards indicated that both esters remained at
110 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis the same concentration at which they were spiked. The experiment with AWRI 1613 was repeated to confirm these findings, and again did not metabolise the ethyl esters, indicating an inability to express the necessary esterase activity to facilitate this breakdown.
Greater concentrations of 4-ethylphenol over 4-ethylguaiacol in wine is well documented, 41, 63 and has been attributed to the relative amounts of precursors present in the grapes. However, these results show that not only will this ratio be defined by the relative amounts of free hydroxycinnamic acids present in the berry, but also by the relative amounts of ethyl esters produced during vinification. A 10:1 ratio of 4- ethylphenol:4-ethylguaiacol could imply an initial 10:1 ratio of p-coumaric acid:ferulic acid, but it could also imply a 1:1 ratio of ethyl coumarate:ethyl ferulate and a 10:1 selectivity in the metabolism of the esters, or a combination of these two effects.
As the ethyl esters are able to be metabolised by some strains of D. bruxellesis and contribute to the accumulation of ethylphenols, the esterification of the free acids during alcoholic fermentation is not a means to avoiding spoilage caused by D. bruxellensis . Instead, this confirms that additional factors need to be considered when assessing a wine for the potential production of ethylphenols, such as the extent of formation of esters as well as the strain of D. bruxellensis that proliferates, given that in the presence of AWRI 1613 the ethyl esters are benign in the accumulation of ethylphenols.
5.1.3 Tartrate Esters
COOH COOH
HOOC HOOC OH OH
O O O O
OCH3
OH OH 7 8 Figure 5.6: p-Coumaroyl L-tartrate ( 7) and feruloyl L-tartrate ( 8).
111 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
Fermentation experiments with AWRI 1499 in the presence of p-coumaroyl and feruloyl tartrate ( 7 and 8) yielded no conversion to 4-ethylphenol (4-EP) or 4-ethylguaiacol (4-EG) after 10 days of fermentation, and to ensure the legitimacy of the result the experiment was repeated, and identical results observed. As such, the ability of different strains of D. bruxellensis to metabolise the tartrate esters were tested in fermentation experiments with AWRI 1608 and AWRI 1613. Analysis for ethylphenol content in the end-point fermentation samples again failed to detect either 4-ethylphenol (4-EP) or 4-ethylguaiacol (4-EG) for both strains examined (Table 5.1).
Given, the dilutions used prior to analysis, and the limit of detection for the methodology at 10 µg/L, the minimum detectable concentration of ethylphenols in the AWRI 1499 fermentations corresponds to 1.25% conversion from the tartrate esters. Detection in the AWRI 1608 and AWRI 1613 fermentations was limited at around 0.7% conversion, having employed a smaller dilution factor in preparation of the samples for analysis, due to the lack of ethylphenol production observed in the initial experiments.
Table 5.1: Ethylphenol content in tartrate ester fermentation experiments. 4-EP 4-EG AWRI 1499 rep. 1 N.D. N.D. AWRI 1499 rep. 2 N.D. N.D. AWRI 1608 N.D. N.D. AWRI 1613 N.D. N.D.
As outlined in Chapter 1, with the abundance of the tartrate esters, they are often the major hydroxycinnamates found in grape juice, although the potential for them to contribute to ethylphenol spoilage is now understood to be somewhat limited and unlike the ethyl esters appears not to be determined by the yeast strain.
While D. bruxellensis appears to lack the capability to hydrolyse the tartrate esters, and hence they are unlikely to contribute to the accumulation of the ethylphenols during barrel ageing, the tartrate ester content of the berries still remains a source of the free hydroxycinnamic acids ( 3 and 4). The previously mentioned studies of Dugelay and Gerbeaux showed that the tartrates could be hydrolysed using commercial enzyme preparations, 64, 112 and there is a loss observed during vinification, 97, 99-105, 129-130, 132 as such the hydroxycinnamoyl tartrate esters ( 7 and 8) cannot be completely ignored during the entire vinification process. 112 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
5.1.4 Glucose Esters
OH OH
O O HO HO HO O O HO O O
OH OH
H3CO
OH OH 9 10 Figure 5.7: p-Coumaroyl glucose ( 9) and feruloyl glucose ( 10 ).
The synthetic samples of the glucose esters ( 9 and 10 ) were initially spiked as mixtures of multiple esters, with the majority existing as the 1-O-β-, 2-O-α- and 6-O-α-esters, though the 1-O-β-esters were found to be thermodynamically favoured in wine-like environments, as shown by theoretical studies (Chapter 3) and by their prevalence during storage in fermentation-like conditions (Chapter 2). Regardless of the ratio of esters present at the time of fermentation, the release of the free hydroxycinnamic acids must be achieved by hydrolysis of a hydroxycinnamoyl glucose ester. Fermentation studies with AWRI 1499 were concluded after 16 days, and all samples that were taken throughout the experiment were analysed for content of the ethylphenols (Figure 5.8).
4-Ethylphenol 40 4-Ethylguaiacol 30
20
10 Percentage Conversion Percentage 0 4 8 12 16 Fermentation Progress (Days)
Figure 5.8: Percentage of the theoretical maximum conversion of hydroxycinnamoyl glucose esters ( 9 and 10 ) to ethylphenols.
113 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
At the conclusion of fermentation, 4-ethylphenol and 4-ethylguaiacol were detected at levels corresponding to 35% conversion from the glucose esters ( 9 and 10 ), and remained relatively constant from day 10 onwards. Unlike the ethyl esters, p-coumaroyl glucose and feruloyl glucose were metabolised to a similar extent (Figure 5.8).
Due to the previously described stability of the glucose esters in fermentation-like conditions, both experimentally observed in Chapter 2 and reported in the literature, 100 it can be concluded that the evolution of the ethylphenols is not a product of chemical hydrolysis of the glucose esters followed by the expected metabolism of the acids. The moderate conversion of the glucose esters to ethylphenols, along with the observed glucose ester concentrations in wine (outlined in Chapter 1), indicate that while they can contribute to the accumulation of ethylphenols during barrel ageing, the metabolism of the glucose esters alone would not have major effects on the organoleptic properties of the resulting wine.
5.1.5 Conclusions for Chapter 5.1 For the first time, this work has shown the ability of D. bruxellensis to metabolise esterified hydroxycinnamic acids directly to ethylphenols. Furthermore, the differences in breakdown observed between different classes of esters implies the presence of multiple pathways, or enzyme activities, involved in the release of free hydroxycinnamic acids from an esterified form. The formation of 4-ethylphenol and 4-ethylguaiacol from the ethyl esters ( 11 and 12 ) shows an overall preference for the breakdown of ethyl coumarate, though the reasons for stereoselective bioconversion remain to be identified. The high proportions of ethyl ferulate in the fermentation samples could be a result of a decrease in transport into the yeast cell for an intracellular esterase, equivalent transport into the cell but decreased conversion due to the nature of the enzyme, or selective activity of an extracellular esterase.
The inability of D. bruxellensis to metabolise the tartrate esters ( 7 and 8), and the moderate conversion observed for the glucose esters, mean that apart from the metabolism already known for p-coumaric and ferulic acids, ethyl coumarate has the largest potential to contribute to wine spoilage, having been found at concentrations high enough to generate sufficient ethylphenols to affect wine aroma and flavour. However, the formation and
114 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis metabolism of ethyl coumarate depends on many variables which would need to be considered. Initially, the concentration of p-coumaric acid in the wine will be determined both by that present in the berry, as well as the release from p-coumaroyl tartrate through enzymatic or chemical hydrolyses. Following this, the formation of ethyl coumarate from p-coumaric acid via esterification with ethanol could be effected by the enzymatic abilities of the wine microflora (transferases), or by conditions affecting the equilibrium of a chemical esterification, or trans -esterification, such as pH and ethanol concentration. Finally, as shown above (Figure 5.5), the strain of D. bruxellensis can determine the conversion of ethyl coumarate to 4-ethylphenol, if any.
The glucose esters could contribute to the accumulation of ethylphenols in barrels, though the concentrations in which they are present they are unlikely to be able to produce sufficient ethylphenols to cause wine spoilage on their own. However, in addition to the contribution from the ethyl esters and the free acids, the glucose esters could be the determining factor in whether the ethylphenol content is above or below the perception threshold.
It can now be seen that not only the free acids contribute to the accumulation of ethylphenols, but that certain hydroxycinnamate esters are also potential sources of spoilage, although this cannot be assumed for all hydroxycinnamates. With the extent of bioconversion differing between substrate, class of ester and strain of D. bruxellensis , the breakdown of each esterified hydroxycinnamate must be tested individually and strain dependencies must be considered.
5.2 Stereoselectivity of D. bruxellensis Enzyme Activities.
5.2.1 Decarboxylase Stereoselectivity Following the photoisomerisation of the hydroxycinnamoyl glucose esters during synthesis, the ability of D. bruxellensis to produce ethylphenols through the metabolism of cis -hydroxycinnamates was of interest. As the breakdown of all hydroxycinnamates to form ethylphenols must proceed via decarboxylation of the acid, synthesis of the cis -acids was attempted (Chapter 4). The inability to synthesise a pure sample of cis -ferulic acid
115 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis resulted in the use of an isomeric mixture of known stable ratio was chosen. In the case of ferulic acid (4) a thermodynamically stable 50:50 mixture of the cis - and trans -isomers was achieved, whereas p-coumaric acid ( 3) existed as a 39:61 mixture of the cis- and trans - isomers. The analytical technique most readily available for real-time determination of the isomeric ratio during fermentation was NMR. Due to overlap of crucial proton shifts between p-coumaric and ferulic, the decarboxylase stereoselectivity could not be examined for both acids concurrently. As ferulic acid was obtained with an equal cis:trans -ratio, it was tested prior to p-coumaric acid, and the isomeric mixture was submitted to fermentation studies with AWRI 1499 ( cis /trans -fermentations) and compared with fermentations spiked with pure trans -ferulic acid ( trans -fermentations).
The isomeric ratio during the fermentation experiments was monitored by extracting uninoculated controls and analysing by NMR. Enough of each acid needed to be present in the uninoculated controls to ensure that adequate spectra could be obtained to determine the isomeric ratio. Thus, allowing for losses during extraction, 10 mg of the mixture was required in each control (200 mL), which equated to initial spiked concentrations of 50 mg/L. As Kahnt described the changes in isomerisation equilibrium with changing substrate concentrations, 187 to avoid any effects of concentration on the isomeric ratio the inoculated fermentations were also spiked at 50 mg/L, five times higher than previously used for the trans -hydroxycinnamate esters. Throughout fermentation the ratio of cis:trans -ferulic acid in the uninoculated controls remained stable, and the results for 4- ethylguaiacol analysis of samples taken throughout fermentation are given below (Figure 5.9).
80 trans -Fermentations cis/trans -Fermentations 60
40
20 Percentage Conversion Percentage 0 2 4 6 8 10 Fermentation Progress (Days)
Figure 5.9: Percentage of the theoretical maximum conversion to 4-ethylguaiacol for the trans - and cis /trans - fermentations.
116 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
The fermentations supplemented with a mixture of 50:50 cis:trans -ferulic acid produced approximately half as much 4-ethylguaiacol as the fermentations containing only trans- ferulic acid. The evolution of 4-ethylguaiacol in the cis /trans -fermentations as a percentage of the maximum conversion observed in the trans -fermentations is displayed in Figure 5.10, and shows more clearly the 50% reduction of 4-ethylguaiacol produced during the cis /trans -fermentations.
50
40 -Ferment
30 trans
20
10
0 Percentage of Percentage 2 4 6 8 10 Fermentation Progress (Days)
Figure 5.10: Evolution of 4-ethylguaiacol in cis/trans -fermentations as a percentage of maximum conversion observed in trans -fermentations.
While these results strongly suggest that cis -ferulic acid is not at all metabolised by D. bruxellensis , quantification of the remaining ferulic acid in the fermentation samples would confirm the inability to metabolise cis -ferulic acid.
Ferulic acid quantification techniques as outlined in a review by Barberousse et al. largely employ reverse-phase HPLC, methanol-water-acid ternary solvent systems and run times in excess of 20 minutes. 224 However, a method previously developed by the AWRI describes quantification of p-coumaric and ferulic acid using ion-exchange HPLC, resulting in retention times of around 3 minutes for trans -ferulic acid (unpublished method). Although analysis times are reduced using ion-exchange HPLC, the previous analysis did not consider both isomers of ferulic acid and required optimisation to achieve resolution of the cis - and trans -ferulic acid.
Using formic acid in water (0.1:99.9, solvent A) and formic acid in acetonitrile (0.1:99.9, solvent B), the AWRI method (45% B, isocratic elution) gave very little separation of the
117 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis isomers. This method was further refined, requiring less of the organic solvent (A), with an isocratic profile of 30% B resulting in similar separation to less organic systems, but with significantly shorter run times.
To adequately quantify both trans - and cis -ferulic acid, a calibration curve for each had to be produced. Preparing trans -ferulic acid samples in the concentration range of 1-75 mg/L gave calibration curves with correlation coefficients of 0.9998 and 0.9973 for absorbance at 280 and 320 nm, respectively. To overcome the instability of pure cis -ferulic acid, cis /trans -ferulic acid mixtures of differing, yet known, concentrations and ratios were produced and analysed. A maximum concentration of 75 mg/L was used with a 50:50 isomeric ratio equating to a maximum cis -ferulic acid content of 37.5 mg/L. Using the trans -ferulic acid calibration curves the trans -isomer could be quantified in the mixture, and the remaining cis -ferulic acid in the sample determined, giving calibration curves with correlation coefficients of 0.9968 and 0.9937 for 280 and 320 nm, respectively. While a number of different wavelengths were originally used for detection (280, 320, 353, 370 and 520 nm), only 4 of these gave reliable calibration curves for trans -ferulic acid (280, 320, 353 and 370 nm), and only two of those gave reliable calibration curves for cis -ferulic acid, 280 and 320 nm, the most common wavelengths used to quantify ferulic acid. 224
With initially spiked concentrations of 50 mg/L for trans -ferulic acid and 25 mg/L for cis - ferulic acid (50% of a 50 mg/L spike), preparation of the calibration curves from 1-75 mg/L and 0.5-37.5 mg/L, respectively allowed for the fermentation samples to be analysed without dilution. After the first 6 samples, overlap of the cis - and trans -ferulic acid peaks occurred, caused by the drift in retention time of cis -ferulic acid. After refreshing the column, the analysis could be resumed, though after analysis of the calibration samples and only a few additional fermentation samples, peak overlap was experienced again. Ion- exchange HPLC was not an adequate method of analysis for multiple samples due to the limited number of samples that could be run before the column required refreshing. Fortuitously, the original 6 samples analysed were the final (triplicate) samples of each of the trans - and cis /trans -fermentations, as shown in Table 5.2.
118 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
Table 5.2: Concentration of cis - and trans -ferulic acid in end-point fermentation samples. Concentration (mg/L) Initial spike (mg/L) trans -Ferulic acid cis -Ferulic acid trans -Ferulic acid cis -Ferulic acid trans -Ferments 1.02 ± 0.05 2.23 ± 0.11 50.0 0.0 cis/trans -Ferments 0.44 ± 0.07 12.43 ± 0.07 25.0 25.0
The final ferulic acid concentrations (expressed as the percentage remaining), along with the initial 4-ethylguaiacol quantifications (expressed as percentage conversion) are combined to give total recoveries of approximately 70% (Figure 5.11). For the trans - fermentations, most of the spiked acid is converted to 4-ethylguaiacol (70%) with around 3% remaining as ferulic acid, existing as 2% trans - and 1% cis -ferulic acid. As expected, metabolism of trans -ferulic acid had occurred to a great extent leaving very little acid, though some had isomerised to give a small cis -content. The cis/trans -fermentations contain 4-ethylguaiacol concentrations corresponding to 40% conversion from the spiked acid, with around 30% remaining as ferulic acid (25% cis -isomer and 5% trans -isomer).
80 4-Ethylguaiacol trans -Ferulic Acid 60 cis -Ferulic Acid
40
20 Percentage Recovery Percentage 0
ts nts en e
s-Ferm n a rans-Ferm tr /t is c
Figure 5.11: Compounds by percentage in end-point fermentation samples.
Recovery of cis -ferulic acid was approximately 50% of the spiked 25 mg/L indicating a potential for uptake by the yeast, but an inability to decarboxylate it. However, as incomplete recovery of cis -ferulic acid was observed, the inability of AWRI 1499 to metabolise cis -ferulic acid cannot be definitively confirmed.
The above fermentation experiment was repeated under the same conditions with cis - and trans -p-coumaric acid ( 3) to determine whether the same effect was observed, fermenting
119 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
D. bruxellensis in the presence of a 39:61 cis :trans -mixture alongside trans -p-coumaric acid. The evolution of 4-ethylphenol in both sets of fermentations was monitored throughout the experiment (Figure 5.12), also the isomeric ratio in the blanks were observed to be stable by NMR.
80 trans -Ferments cis/trans -Ferments 60
40
20
0
Percentage Conversion to 4-EP Conversion Percentage 2 4 6 8 10 12 Fermentation Progress (Days)
Figure 5.12: Percentage of the theoretical maximum conversion to 4-ethylphenol in trans - and cis /trans - fermentations.
In an analogous fashion to the ferulic acid fermentations, conversion to 4-ethylphenol in the cis /trans -p-coumaric acid fermentations corresponded to the trans -content, giving final 4-ethylphenol concentrations 39% lower than observed in the trans -fermentations (Figure 5.13).
60 -Ferment 40 trans
20
0 Percentage of Percentage 4 8 12 Fermentation Progress (Days)
Figure 5.13: Evolution of 4-ethyphenol in cis/trans -fermentations as a percentage of maximum conversion observed in trans -fermentations.
120 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
Slight deviations in 4-ethylphenol concentration may arise from a) partial isomerisation of cis -p-coumaric acid back to the trans -isomer, b) from slight loss of 4-ethylphenol in the trans -ferments due to the high concentrations produced, or c) from slight variations in the amount of p-coumaric acid spiked into, or remaining in the fermentations produced from solubility issues, caused by performing these fermentation experiments at higher concentrations than employed previously.
The quantification of ferulic acid in the previous experiments did not provide additional evidence as to the role of the cis -acid, with the final conclusions based purely on the isomeric ratio of the spikes compared with the ratio of produced 4-ethylguaiacol. As such, quantification of the cis - and trans -p-coumaric acids was not performed for these fermentations. However, these results confirm that the metabolism of p-coumaric acid occurs to a much greater extent for the trans -isomer and the role of the cis -acids in the production of ethylphenols is not significant.
5.2.2 Ethyl Esterase Stereoselectivity During the synthesis of the cis -hydroxycinnamic acids ( 3 and 4) detailed in Chapter 4, the cis -ethyl esters were found to be stable under ambient light conditions. As the ability of D. bruxellensis to metabolise the ethyl esters had been shown, if the cis -ethyl esters could also be enzymatically hydrolysed, they were a potential source of pure cis -hydroxycinnamic acids. Thus, cis -ethyl coumarate ( 11 ) and cis -ethyl ferulate ( 12 ) were simultaneously submitted to fermentation experiments with AWRI 1499 and the evolution of ethylphenols monitored (Figure 5.14).
121 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
1.0 4-Ethylphenol 0.8 4-Ethylguaiacol
0.6
0.4
0.2 L.o.Q. Percentage Conversion Percentage 0.0 2 4 6 8 10 Fermentation Progress (Days)
Figure 5.14: Percentage of the theoretical maximum ethylphenol conversion from cis -ethyl esters.
The amounts of 4-ethylphenol and 4-ethylguaiacol produced from metabolism of the cis - ethyl esters were so low that some data points were below the limit of quantification (L.o.Q.) for the analytical method (10 µg/L). The values that lay under the L.o.Q. are approximated only but do indicate an upward trend that continues to above the limit of detection.
Conversion to the ethylphenols under the same conditions (9 days of fermentation, Figure 5.2) was observed at 51 and 4% from trans -ethyl coumarate and trans -ethyl ferulate, respectively. The production of ethylphenols observed during the cis -ethyl ester fermentations, though minimal, could be explained by either the esterase being active towards the cis -ethyl esters, or by partial isomerisation to yield trans -ethyl esters. For production of ethylphenols via metabolism of the cis -ethyl esters, the resulting cis -acids must isomerise and the trans -acids then metabolised. Otherwise the cis -ethyl esters partially isomerised during the experiment to give a small amount of the trans -ethyl esters, which were then broken down, or the samples of cis -ethyl esters used to spike the fermentations contained trace impurities of the trans -isomers. The difference in conversion observed between the cis -ethyl coumarate and cis -ethyl ferulate would suggest the latter, brought about by the esterase substrate selectivity established earlier. Using the quantification method of Sleep, the trans -ethyl esters in the fermentation samples could be determined 141 and the method was also applied in an attempt to quantify the cis -ethyl esters.
122 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
Table 5.3: Final trans -ethyl ester content in cis -ethyl ester fermentations. trans -Ester concentration (mg/L) Initial cis -ester Inoculated Uninoculated spike (mg/L) Ethyl coumarate 1.1 ± 0.1 1.5 ± 0.1 10.0 Ethyl ferulate 1.8 ± 0.1 1.8 ± 0.1 10.0
The uninoculated samples contain a minor but significant proportion of the trans -ethyl esters which is not expected to occur purely over the course of the fermentation experiments, and is most likely a result of the length of storage (due to instrument availability the fermentation samples had to be stored for 9 months prior to analysis of the ethyl esters content). If conversion from cis -ethyl coumarate to trans -ethyl coumarate had occurred during fermentation a much larger concentration of 4-ethylphenol would be expected, based on the conversions observed in previous experiments.
However, the minor differences in the amounts of trans -ethyl coumarate between the inoculated and uninoculated fermentations suggest that minor conversion could have occured during fermentation, followed by metabolism of the resulting trans -ethyl coumarate to yield 4-ethylphenol.
The cis -ethyl esters could not be accurately quantified using the same method as the trans - esters due to differences in both the extraction efficiencies and the mass spectral responses. Mixtures of known cis - and trans -ethyl ester ratios were analysed by GCMS to determine the differences in ionisation potentials, then mixtures were extracted and analysed to determine the differences in responses, which were then factored into the quantification to afford a more accurate indication of the cis -ethyl ester concentration. When combined with the trans -ethyl ester and the ethylphenol quantifications, the total content at the end of fermentation could be determined (Figures 5.15 and 5.16).
123 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
100 4-Ethylphenol trans -Ethyl coumarate 80 cis -Ethyl coumarate 60
40
20 Percentage Recovery Percentage 0
lated
Inocu noculated ni U Figure 5.15: Total coumarate recovery from cis -fermentations.
60 4-Ethylguaiacol trans -Ethyl ferulate 40 cis -Ethyl ferulate
20 Percentage Recovery Percentage 0 d
oculated In Uninoculate Figure 5.16: Total ferulate recovery from cis -fermentations.
While the recovery for the coumarates were good, this was not the case for the ferulates. Rough GCMS quantifications of ethyl ferulate (not specific to either isomer) from a single fermentation throughout the experiment were performed using an abbreviated SIDA method (Figure 5.17).
124 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
10
8
6
4
2 Ethyl Ferulate (mg/L) Ferulate Ethyl 0 0 2 4 6 8 10 Fermentation Progress (Days)
Figure 5.17: Breakdown of ethyl ferulate in a single fermentation.
The spiked cis -ethyl ferulate within the inoculated fermentation samples was slowly degraded, and as the final concentration of cis -ethyl ferulate was reduced in both inoculated and uninoculated ferments it can be assumed a chemical breakdown rather than a microbiological breakdown took place. With many potential causes, it was not further investigated. In any case the role of cis -ethyl ferulate in the production of 4-ethylguaiacol is negligible. Even allowing for a 50% reduction in cis -ethyl ferulate in the fermentation, the amount of 4-ethylguaiacol produced would correspond to a conversion of no more than 0.5%.
5.2.3 Conclusions for Chapter 5.2 The results of the first part of this study, limited by the reduced recovery of the spiked cis - acids, indicate that the decarboxylase of D. bruxellensis is specific to metabolism of the trans -hydroxycinnamic acids ( 3 and 4). However, from the isomeric ratio of the spiked acids and the subsequent production of ethylphenols, the decarboxylase of D. bruxellensis has little or no activity towards the cis -acids. For the cis -acids to be completely disregarded as precursors to the ethylphenols one of two things must be achieved: either the experimental conditions must be improved to allow for a pure cis -acid spike and it remains isomerically pure; or the full recovery of the cis -acids from isomeric mixtures needs to be achieved. The nature of the photoisomerisation does not allow for use of a pure cis -acid spike, and an attempt to use one would most likely result in slow conversion back to the trans -acid, giving a dynamic mixture that would be difficult to characterise over the course of these experiments. The incomplete recovery of the cis -acids is most likely a result of adsorption onto the yeast cell wall or by uptake, which could be overcome by examining
125 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis the enzyme directly, through isolation and purification, as opposed to examining the indirect products of the activity of the decarboxylase enzyme which has been done in this study.
Even though the cis -hydroxycinnamic acids cannot be completely eliminated as precursors to ethylphenols, the conversions observed from the isomeric mixtures strongly suggest that they are not metabolised by D. bruxellensis , and implies that the decarboxylase possesses stereospecificity.
In reviewing the content of the cis -hydroxycinnamates in grapes and wine (Chapter 4) it was noted that the tartrate esters exist in both isomeric forms, with the cis -esters contributing around 20%. While it has been shown that the tartrate esters are not metabolised by D. bruxellensis , rendering them of little relevance to the direct stereoselective breakdown, the tartrate esters are hydrolysed either enzymatically and/or chemically prior to barrel ageing (described in Chapter 1). Assuming that the cis -tartrate esters also undergo the same hydrolyses, then the production of cis -acids would not lead to further metabolism by D. bruxellensis .
By displaying stereoselectivity in decarboxylase activity, the cis -hydroxycinnamate content of wine, esterified or otherwise, can be ignored in relation to the build-up of ethylphenols during barrel ageing, as cis -hydroxycinnamate esters could either be enzymatically hydrolysed, which may be limited, or chemically hydrolysed yielding cis - acids which will not contribute to the production of ethylphenols (unless enough time is allowed between hydrolysis and D. bruxellensis growth for some trans -acid to form).
5.3 Thesis Conclusions and Future Directions.
This study has investigated the synthesis and chemical transformations of hydroxycinnamate esters, their role in the production of ethylphenols in wine by D. bruxellensis and the stereochemical factors contributing to the enzymatic breakdown of hydroxycinnamates in wine.
126 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
In Chapter 2, the synthesis of p-coumaroyl and feruloyl tartrate ( 7 and 8) has been described for the first time, via the coupling of the 1-O-chloroacetyl protected acids and di- tert -butyl tartrate. This has allowed them to be dismissed in the accumulation of ethylphenols by D. bruxellensis unless first hydrolysed via an alternative pathway. Moreover, the synthetic methodology developed can now be applied for the synthesis of other hydroxycinnamoyl tartrate derivatives such as caffeoyl tartrate ester or the grape reaction product. 225 Furthermore, incorporating isotopically labelled hydroxycinnamate moieties into the synthesis would allow for accurate and convenient quantification of these compounds in grapes and wine, an addition that could also be applied to the glucose esters.
Synthesis of the glucose esters from the 1-O-chloroacetyl protected acids and a trichloroacetimidate glucosyl donor and deprotection in pyridine/water resulted in both photoisomerisation and acyl migration, which have not previously been detailed for the hydroxycinnamoyl glucose esters. During the course of this study the synthesis of feruloyl glucose ( 10 ) using a similar synthetic pathway was described by Zhu, 183 although no migration or photoisomerisation was described. Theoretical studies into the migration of the glucose esters identified the role of non-aqueous conditions in altering the thermodynamic preference for different esters, and in changing kinetic aspects of the migration away from the desired 1-O-β-esters (Chapter 3). Investigation into the migration during previously reported analytical procedures showed that the extraction process can have an effect on the ratios of esters present, but that wine naturally contains multiple esters for both p-coumaroyl glucose and feruloyl glucose. During these studies, feruloyl glucose was identified for the first time in red wine, which is rationalised to have gone unnoticed by other authors due to coelution with p-coumaroyl derivatives tentatively identified as anthocyanin derivatives.
As outlined in Chapter 4, photoisomerisation of the glucose esters under ambient light conditions was found to be accelerated in comparison to other synthesised hydroxycinnamate derivatives, resulting in production of cis -glucose esters in roughly 1:4 ratio with the trans -isomers. For the duration of synthesis this was subdued by working under red light. Theoretical investigations into the photoisomerisation found that while solvent and substrate do play a minor role in the ease of isomerisation, the nature of the phenol and carboxyl contribute to a much larger extent. By increasing the electron donating character of the phenol or increasing the electron withdrawing character of the
127 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis carboxyl, electron movement within the molecule was increased resulting in a decrease in the α,β-double bond length. This reduced the HOMO-LUMO gap of the molecule, affecting the S 0-T1 vertical excitation energy and altering the energy required for photoisomerisation. To avoid photoisomerisation, production of the phenolate should be prevented either by using phenolic protection as far into a synthetic route as possible, or by preventing exposure to basic conditions.
The ease by which the glucose esters isomerised encouraged the investigation into the ability of D. bruxellensis to metabolise cis -hydroxycinnamates in addition to the trans - isomers. Attempts at synthesising pure cis -p-coumaric and ferulic acids were unsuccessful, with pure cis -ethyl esters undergoing isomerisation during base-catalysed ester hydrolysis, assisted by formation of the phenolate of the ethyl esters. Instead stable isomeric mixtures were produced with ferulic acid existing in a 50:50 ratio of cis :trans -ferulic acid and p- coumaric as a 39:61 ratio, which could be used in fermentation experiments, along with the pure cis -ethyl esters that were synthesised and remained pure under ambient light conditions.
Fermentation of the ethyl esters with multiple strains of D. bruxellensis showed that a substrate selectivity exists in some strains for the preferential breakdown of ethyl coumarate over ethyl ferulate, which was observed in two strains representing nearly 92% of Australian D. bruxellensis isolates, while a third strain was unable to metabolise either. This work showed the ability of D. bruxellensis to form ethylphenols directly from esterified hydroxycinnamates for the first time, also identifying a substrate preference that could contribute to the 4-ethylphenol:4-ethylguaiacol ratio seen in red wines. The bioconversion of ethyl coumarate and subsequent production of 4-ethylphenol has also been established.
The tartrate esters, while abundant enough to have a large impact on the ethylphenol content of wine, were unable to be metabolised by D. bruxellensis , showing that for this compound to be of significance it must first be hydrolysed by alternative enzymatic methods, or chemically. The inability of D. bruxellensis to breakdown the tartrates was common to all three strains tested.
128 Chapter 5: Bioconversion of Hydroxycinnamates by D. bruxellensis
Metabolism of migrated mixtures of p-coumaroyl glucose and feruloyl glucose by D. bruxellensis showed a moderate 35% conversion to the ethylphenols. Given the previous quantifications in wine, the glucose esters have the ability to contribute to the production of ethylphenols, but are not abundant enough to solely spoil wine.
In fermentation experiments to examine the stereoselectivity of the decarboxylase activity, metabolism of cis -ferulic and cis -p-coumaric acids to yield 4-ethylguaiacol and 4- ethylphenol, was at most limited. The inability to recover all of the spiked cis -acid in conjunction with the nature of the isomerisation did not allow determination of whether the decarboxylase activity towards the cis -acids was non-existent or just very small. A similar result was achieved in testing the ethyl esterase for stereoselectivity, indicating that the cis - ethyl esters were not broken down, with minor production of ethylphenols attributed to isomerisation or the presence of trace impurities. Regardless, both decarboxylase and ethyl esterase were more active towards the trans -isomers, rendering the cis -hydroxycinnamates unimportant in the accumulation of ethylphenols in wine.
Following the findings that hydroxycinnamates other than the free acids can contribute to ethylphenol accumulation, further conjugates should be tested in order to determine all metabolic inputs in the production of 4-ethylphenol and 4-ethylguaiacol. In addition to those tested in this study, other derivatives have been identified which have the potential to contribute to a greater extent than those shown here. During the LCMS studies of red wine, a peak attributed to the glucoside of p-coumaric acid was identified, a compound which has been identified in red wine and isolated from white wine previously, 108, 131 and appeared to more abundant than the glucose esters. Furthermore, p-coumaroyl anthocyanin derivatives have been identified in red wine, 107 which could also be examined as potential precursors to ethylphenols.
Regardless of the hydroxycinnamate under investigation, the work here has shown that metabolism of each substrate by D. bruxellensis must be tested individually, as the result can be dependent on the substrate, the stereochemistry, and the strain used. While much has been reported regarding the breakdown of trans -p-coumaric and trans -ferulic acid, and this thesis has detailed the role of esterified precursors to ethylphenols and the stereoselective metabolism by D. bruxellensis , the potential for spoilage of this species in wine still requires further investigation.
129 Chapter 6: Experimental
Chapter 6: Experimental.
6.1 General Experimental.
Solvents and reagents Dry organic solvents were purchased and dispensed using a Puresolv™ solvent purification system. Pyridine was dried by storage on 4 Å molecular sieves and tert -butanol was distilled from CaH 2 onto 4 Å molecular sieves. General organic solvents were obtained and distilled where needed. Reagents other than those synthesised were purchased from Sigma- Aldrich Chemical Company Ltd. and used without further purification.
Naming of synthesised compounds Compounds are named using common nomenclature as they would appear in literature, followed by the IUPAC name as generated using ACD/Labs 12.0 software. Where alkene stereochemistry is not denoted the trans -isomer was produced. For the assignment of NMR shifts, the numbering systems are shown below.
O OH 9
8 7 O OH 4 O 2' 6' 5 3 1' OH 4' 5' O HO HO O 6 2 4' OH 3' 3' 2' 1' 1 HO OH OH O
Chromatography Column chromatography was performed using Davisil 40-63 µm silica gel. Thin layer chromatography was performed using Merck silica gel 60 F 254 alumina sheets (20 x 20 cm) and viewed under UV light.
130
Chapter 6: Experimental
Melting points Melting points were obtained using a Buchi B-540 melting point apparatus and are uncorrected.
Optical Rotation Rotation was measured with a polAAr 21 polarimeter and referenced to the sodium D line (589 nm) at 20 oC in a cell with 1 dm path length. The concentration is specified in g/100 mL and in the solvent as reported.
Infrared spectroscopy Spectra were acquired with a Perkin Elmer Spectrum One FT-IR spectrometer using neat samples.
High-resolution mass spectrometry Accurate mass determination was performed by The Organic Mass Spectrometry Facility, University of Tasmania. Where an appropriate spectrum was obtained prior to accurate mass determination, other significant fragmentations are quoted.
Mass spectra of compounds 9 and 10 Spectra were obtained during LCMS studies of 9 and 10 in wine, as detailed in Chapter 6.3 (Experimental Procedures for Chapter 3).
Elemental analysis Analysis was performed at the University of Otago, New Zealand.
X-ray crystallography Crystallographic data was performed by Dr Edward R. T. Tiekink at the Department of Chemistry, University of Malaysia.
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Chapter 6: Experimental
NMR spectroscopy The 1H and 13 C spectra were acquired with either a Bruker Ultrashield Plus 400 MHz Spectrometer or a Bruker Ultrashield Plus 600 MHz Spectrometer, where indicated. Spectra were recorded in the specified solvent, and referenced as described by Gottlieb et al. 226 In cases of overlapping solvent and compound shifts, the spectra were referenced to the TMS peak at 0.00 ppm. Chemical shifts ( δ ) are reported in ppm and coupling constants (J) in Hz. 13 C assignments were made using 2D correlation experiments HMQC and HMBC.
Photoisomerisation
Was carried out using either a 365 nm or 254 nm ultra-violet lamp, as specified.
Computational Chemistry Was performed using Spartan ’08 software package, with final calculations employing density functional theory (DFT) and the supplied B3LYP 6-31G* basis set.
132
Chapter 6: Experimental
6.2 Experimental Procedures for Chapter 2.
6.2.1 Hydroxycinnamoyl Derivatives (Carbethoxymethylene)triphenylphosphorane
Ethyl bromoacetate (17 mL, 0.15 mmol) and triphenylphosphine (40.31 g, 0.15 mmol) were heated under reflux in toluene (150 mL) for 15 hours. The resulting precipitate was filtered and washed with toluene (3 x 50 mL). The prepared salt was then stirred with sodium hydroxide (11.88 g, 0.30 mmol) in water (500 mL) for 1 hour. The product was extracted with ethyl acetate, dried (Na 2SO 4) and concentrated to give (carbethoxymethylene)triphenylphosphorane as a beige solid (44.50 g, 91%); m.p. 117-122 oC (lit. m.p. 126-127 oC). 227 1 H NMR: (400 MHz, CDCl 3) δ: 7.69-7.64 (m, 6H, ArH), 7.57-7.52 (m, 3H, ArH), 7.48-
7.44 (m, 6H, ArH), 3.96 (q, 2H, J = 7.1 Hz, OC H2CH 3), 2.80 (br. s, 1H, CH), 1.03 (t, 3H, J 227 = 7.1 Hz, OCH 2CH3). Physical and spectral properties were as previously reported.
Ethyl coumarate (11) Ethyl 3-(4-hydroxyphenyl)prop-2-enoate
(Carbethoxymethylene)triphenylphosphorane (10.07 g, 28.91 mmol) and p- hydroxybenzaldehyde (3.34 g, 27.37 mmol) were stirred in dry dichloromethane (70 mL) under nitrogen at ambient temperature. After 10 days a further portion of (carbethoxymethylene)triphenylphosphorane (8.01 g, 23.01 mmol) was added and the mixture stirred for a further 12 days. The reaction mixture was concentrated and purified using column chromatography (30% EtOAc/X4) which gave partial separation of isomers, yielding 4.87 g (93%) of trans -ethyl coumarate as a white solid and 0.20 g (4%) of a mixture of cis /trans -ethyl coumarate (9:1) as a colourless oil. trans-Ethyl coumarate 133
Chapter 6: Experimental
Ethyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate m.p. 72.1-73.0 oC (lit. m.p. 73 oC). 185
Rf (50% EtOAc/X4): 0.47 1 H NMR: (400 MHz, CDCl 3) δ: 7.63 (d, 1H, J = 15.9 Hz, H 7), 7.42 (app. d, 2H, J = 8.6 Hz,
H3,5 ), 6.85 (app. d, 2H, J = 8.6 Hz, H 2,6 ), 6.29 (d, 1H, J = 15.9 Hz, H 8), 4.27 (q, 2H, J = 7.1
Hz, O CH 2CH 3), 1.33 (t, 3H, J = 7.1 Hz, OCH 2CH 3). cis-Ethyl coumarate Ethyl (2Z)-3-(4-hydroxyphenyl)prop-2-enoate
Rf (50% EtOAc/X4): 0.50 1 H NMR: (400 MHz, CDCl 3) δ: 7.63 (app. d, 2H, J = 8.6 Hz, H 3,5 ), 6.85 (d, 1H, J = 12.7
Hz, H 7), 6.80 (app. d, 2H, J = 8.6 Hz, H 2,6 ), 5.83 (d, 1H, J = 12.7 Hz, H 8), 4.21 (q, 2H, J =
7.1 Hz, O CH 2CH 3), 1.29 (t, 3H, J = 7.1 Hz, OCH 2CH 3). Data was extracted from the mixture of isomers. For both isomers, all physical and chemical properties were as previously reported. 185, 228
Ethyl ferulate (12) Ethyl 3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate
Reaction of vanillin (3.56 g, 23.41 mmol) using the same procedure as described for 11 (above), yielded 3.73 g (72%) of trans -ethyl ferulate as a pale yellow solid and 0.89 g (17%) of a mixture of cis /trans -ethyl ferulate (8:2) as a yellow oil. trans-Ethyl ferulate Ethyl (2E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate m.p. 39.6-41.2 oC (lit. m.p. 39 oC). 185
Rf (50% EtOAc/X4): 0.45 1 H NMR: (400 MHz, CDCl 3) δ: 7.61 (d, 1H, J = 15.9 Hz, H 7), 7.07 (dd, 1H, J = 8.2 and
1.9 Hz, H 5), 7.03 (d, 1H, J = 1.9 Hz, H 3), 6.91 (d, 1H, J = 8.2 Hz, H 6), 6.28 (d, 1H, J = 15.9
Hz, H 8), 4.27 (q, 2H, J = 7.1 Hz, O CH 2CH 3), 3.92 (s, 3H, OCH 3), 1.33 (t, 3H, J = 7.1 Hz,
OCH 2CH 3). cis-Ethyl ferulate 134
Chapter 6: Experimental
Ethyl (2Z)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate
Rf (50% EtOAc/X4): 0.53 1 H NMR: (400 MHz, CDCl 3) δ: 7.77 (d, 1H, J = 1.9 Hz, H 3), 7.11 (dd, 1H, J = 8.5 and 1.9
Hz, H 5), 6.88 (d, 1H, J = 8.5 Hz, H 6), 6.79 (d, 1H, J = 12.9 Hz, H 7), 5.81 (d, 1H, J = 12.9
Hz, H 8), 4.21 (q, 2H, J = 7.1 Hz, O CH 2CH 3), 3.92 (s, 3H, OCH 3), 1.29 (t, 3H, J = 7.1 Hz,
OCH 2CH 3). Data was extracted from the mixture of isomers. For both isomers, all physical and chemical properties were as previously reported. 185, 229
Microwave synthesis of ethyl ferulate (12)
Vanillin (0.52 g, 3.44 mmol) and (carbethoxymethylene)triphenylphosphorane (1.17 g, 3.36 mmol) were added to dry dichloromethane (15 mL) in a 30 mL reaction vessel and heated at 50 oC under microwave radiation (CEM discover microwave reactor) for 5 minute intervals. After 20 minutes the reaction mixture showed no further change by TLC and was concentrated and purified using column chromatography (20% EtOAc/X4) which yielded 0.46 g (60%) of trans -ethyl ferulate and 0.13 g (17%) of a mixture of trans - and cis -ethyl ferulate (15:85).
Attempted microwave synthesis of ethyl coumarate (11)
(Carbethoxymethylene)triphenylphosphorane (2.00 g, 5.73 mmol) and p- hydroxybenzaldehyde (0.59 g, 4.79 mmol) were added to dry dichloromethane (15 mL) in a 30 mL reaction vessel and heated at 50 oC under microwave radiation for 5 minutes. After 5 minutes most of the reaction mixture had leaked into the microwave reactor, and a repeat procedure of this scale provided the same result.
135
Chapter 6: Experimental
1-O-Benzyl p-coumaroyl ethyl ester (17) Ethyl (2E)-3-[4-(benzyloxy)phenyl]prop-2-enoate
Anhydrous potassium carbonate (0.38 g, 2.71 mmol) and benzyl bromide (0.47 mL, 3.95 mmol) were added to a mixture of ethyl coumarate ( 11 ) (0.50 g, 2.60 mmol) in dry dichloromethane (10 mL) and the reaction mixture heated under reflux. After 20 hours, further benzyl bromide (0.47 mL, 3.95 mmol) was added. After 4 days, the reaction was quenched with water, the organics extracted with ethyl acetate (3 x 10 mL), dried (MgSO 4) and concentrated. Purification by column chromatography (10% EtOAc/X4) gave 0.54 g (74%) of 17 ester as a white solid. m.p. 64.8-65.7 oC (lit. m.p. 65-67 oC). 230
Rf (50% EtOAc/X4): 0.61 1 H NMR: (400 MHz, CDCl 3) δ: 7.64 (d, 1H, J = 16.0 Hz, H 7) 7.47 (app. d, 2H, J = 8.8 Hz,
H3,5 ), 7.44-7.34 (m, 5H, ArH), 6.98 (app. d, 2H, J = 8.8 Hz, H 2,6 ), 6.31 (d, 1H, J = 16.0 Hz,
H8), 5.10 (s, 2H, OCH2Ph), 4.28 (q, 2H, J = 7.1 Hz, O CH 2CH 3), 1.34 (t, 3H, J = 7.1 Hz, 230 OCH 2CH 3). All physical and chemical properties were as previously reported.
1-O-Benzyl feruloyl ethyl ester (18) Ethyl (2E)-3-[4-(benzyloxy)-3-methoxyphenyl]prop-2-enoate
Using the same reaction conditions as described for 17 (above), ethyl ferulate ( 12 ) (1.00 g, 4.50 mmol) gave 0.96 g (69%) of 18 as a white solid. m.p. 67.7-68.4 oC (lit. m.p. 64 oC). 231
Rf (50% EtOAc/X4): 0.69 1 H NMR: (600 MHz, CDCl 3) δ: 7.61 (d, 1H, J = 15.9 Hz, H 7), 7.44-7.42 (m, 2H, ArH),
7.38-7.36 (m, 2H, ArH), 7.33-7.30 (m, 1H, ArH), 7.07 (d, 1H, J = 2.0 Hz, H 3), 7.03 (dd,
1H, J = 8.4 and 2.0 Hz, H 5), 6.87 (d, 1H, J = 8.4 Hz, H 6), 6.30 (d, 1H, J = 15.9 Hz, H 8),
5.19 (s, 2H, O CH 2Ph), 4.26 (q, 2H, J = 7.1 Hz, O CH 2CH 3), 3.92 (s, 3H, OCH 3), 1.33 (t,
3H, J = 7.1 Hz, OCH 2CH 3). Physical and spectral properties were as previously reported. 231-232
136
Chapter 6: Experimental
1-O-Benzyl p-coumaric acid (15) (2E)-3-[4-(Benzyloxy)phenyl]prop-2-enoic acid
Potassium hydroxide (0.10 g, 1.71 mmol) was dissolved in water (5 mL) and added to a mixture of 1-O-benzyl coumaroyl ethyl ester ( 17 ) (0.21 g, 0.73 mmol) in ethanol (5 mL). The reaction mixture was stirred at room temperature for 20 hours then concentrated under reduced pressure. The residue was taken up in water (10 mL), acidifed to pH 3 with 10% HCl solution and caused precipitation of a white solid, which was extracted into ethyl acetate (3 x 10 mL), washed with saturated brine solution (2 x 10 mL), dried (MgSO 4) and concentrated. This gave 0.17 g (93%) of 15 as a white solid. m.p. 200.2-201.8 oC (lit. m.p. 198-201 oC). 233
Rf (50% EtOAc/X4): 0.27 1 H NMR: (400 MHz, CDCl 3) δ: 7.74 (d, 1H, J = 15.9 Hz, H 7), 7.50 (app. d, 2H, J = 8.7 Hz,
H3,5 ), 7.44-7.30 (m, 5H, ArH), 6.99 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.32 (d, 1H, J = 15.9 Hz,
H8), 5.11 (s, 2H, OCH2Ph). 1 H NMR: (600 MHz, Acetone-d6) δ: 7.65-7.62 (m, 3H, H 3,5,7 ), 7.50-7.49 (m, 2H, ArH),
7.42-7.39 (m, 2H, ArH), 7.36-7.34 (m, 1H, ArH), 7.08 (app. d, 2H, J = 8.8 Hz, H 2,6 ), 6.39
(d, 1H, J = 15.9 Hz, H 8), 5.20 (s, 2H, OC H2Ph). Physical properties and spectral properties in acetone were as previously reported. 233
1-O-Benzyl ferulic acid (16) (2E)-3-[4-(Benzyloxy)-3-methoxyphenyl]prop-2-enoic acid
1-O-Benzyl feruloyl ethyl ester ( 18 ) (0.31 g, 1.00 mmol) was submitted to the same procedure as described for 15 (above). This gave 0.24 g (85%) of 16 as a pale yellow solid. m.p. 189.6-190.3 oC (lit. m.p. 191 oC). 231
Rf (50% EtOAc/X4): 0.14
137
Chapter 6: Experimental
1 H NMR: (400 MHz, CDCl 3) δ: 7.70 (d, 1H, J = 15.9 Hz, H 7), 7.43 (app. d, 2H, J = 7.1 Hz, ArH), 7.38 (app. t, 2H, J = 7.3 Hz, ArH), 7.31 (app. t, 1H, J = 7.2 Hz, ArH), 7.09 (d, 1H, J
= 2.0 Hz, H 3), 7.06 (dd, 1H, J = 8.3 and 2.0 Hz, H 5), 6.89 (d, 1H, J = 8.3 Hz, H 6), 6.31 (d,
1H, J = 15.9 Hz, H 8), 5.20 (s, 2H, O CH 2Ph), 3.93 (s, 3H, OCH 3). 1 H NMR: (600 MHz, Acetone-d6) δ: 7.61 (d, 1H, J = 15.9 Hz, H 7), 7.51-7.49 (m, 2H,
ArH), 7.41-7.39 (m, 2H, ArH), 7.36 (d, 1H, J = 2.0 Hz, H 3), 7.35-7.32 (m, 1H, ArH), 7.19
(dd, 1H, J = 8.3 and 2.0 Hz, H 5), 7.08 (d, 1H, J = 8.3 Hz, H 6), 6.42 (d, 1H, J = 15.9 Hz,
H8), 5.19 (s, 2H, C H2Ph), 3.91 (s, 3H, OCH 3). Physical properties and spectral properties in chloroform were as previously reported. 231, 234
p-Coumaric acid (3) (2E)-3-(4-Hydroxyphenyl)prop-2-enoic acid
trans -Ethyl coumarate ( 11 ) (1.00 g, 5.20 mmol) was dissolved in 1:1 aqueous ethanol (v/v, 20 mL) followed by the addition of potassium hydroxide (0.87 g, 15.52 mmol), then the reaction mixture was stirred at room temperature for 3 days. The mixture was then diluted with water (10 mL), unwanted organics extracted with diethyl ether (2 x 20 mL), the aqueous layer acidified to pH 3 with 2 M hydrochloric acid solution and extracted with ethyl acetate (2 x 20 mL). Concentration at reduced pressure gave 0.83 g (97%) of 3 as an off-white solid. m.p. 208.7-209.8 oC (lit. m.p. 214-216 oC). 235
Rf (10% MeOH/DCM): 0.31 1 H NMR: (600 MHz, Acetone-d6) δ: 7.62 (d, 1H, J = 16.0 Hz, H 7), 7.55 (app. d, 2H, J =
8.6 Hz, H 3,5 ), 6.90 (app. d, 2H, J = 8.6 Hz, H 2,6 ), 6.34 (d, 1H, J = 16.0 Hz, H 8). Physical and chemical properties were as previously reported. 214, 235
138
Chapter 6: Experimental
Ferulic acid (4) (2E)-3-(4-Hydroxy-3-methoxyphenyl)prop-2-enoic acid
With the same hydrolysis conditions as used above (for 3), reaction of trans -ethyl ferulate (12 ) (0.67 g, 3.01 mmol) gave 0.56 g (96%) of 4 as a yellow solid. m.p. 169.1-170.2 oC (lit. m.p. 168-169 oC). 236
Rf (10 % MeOH/DCM): 0.33 1 H NMR: (600 MHz, Acetone-d6) δ: 7.60 (d, 1H, J = 15.9 Hz, H 7), 7.33 (d, 1H, J = 2.0 Hz,
H3), 7.14 (dd, 1H, J = 8.1 and 2.0 Hz, H 5), 6.87 (d, 1H, J = 8.1 Hz, H 6), 6.38 (d, 1H, J =
15.9 Hz, H 8), 3.92 (s, 3H, OCH 3). Physical and chemical properties were as previously reported. 184, 235-236
1-O-Acetyl p-coumaric acid (19) (2E)-3-[4-(Acetyloxy)phenyl]prop-2-enoic acid
p-Coumaric acid ( 3) (0.68 g, 4.17 mmol) was dissolved in dry pyridine (5 mL) followed by the addition of acetic anhydride (2 mL, 21.10 mmol) and the mixture stirred at room temperature under a nitrogen atmosphere. After 52 hours the mixture was concentrated and the crude solid recrystallised from ethanol to furnish 0.71 g (83%) of 19 as colourless needles. m.p. 203.3-204.5 oC (lit. m.p. 200-205 oC). 237
Rf (10% MeOH/DCM): 0.33 1 H NMR: (400 MHz, DMSO-d6) δ: 7.74 (app. d, 2H, J = 8.6 Hz, H 3,5 ), 7.59 (d, 1H, J =
16.0 Hz, H 7), 7.18 (app. d, 2H, J = 8.6 Hz, H 2,6 ), 6.51 (d, 1H, J = 16.0 Hz, H 8), 2.28 (s, 3H,
OCOCH3). 1 H NMR: (400 MHz, Acetone-d6) δ: 7.73 (app. d, 2H, J = 8.7 Hz, H 3,5 ), 7.68 (d, 1H, J =
16.0 Hz, H 7), 7.20 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.51 (d, 1H, J = 16.0 Hz, H 8), 2.28 (s, 3H, 238 OCOCH3). Spectral properties correspond with that previously reported.
139
Chapter 6: Experimental
1-O-Acetyl ferulic acid (20) (2E)-3-[4-(Acetyloxy)-3-methoxyphenyl]prop-2-enoic acid
Ferulic acid ( 4) (1.06 g, 5.46 mmol) was reacted using the same conditions described for the preparation of 19 (above). Recrystallisation of the crude solid from ethanol afforded 1.02 g (79%) of 20 as an off-white solid. m.p. 196.3-197.2 oC (lit. m.p. 197-200 oC). 239
Rf (10% MeOH/DCM): 0.28 1 H NMR: (400 MHz, DMSO-d6) δ: 7.57 (d, 1H, J = 16.0 Hz, H 7), 7.48 (d, 1H, J = 1.8 Hz,
H3), 7.26 (dd, 1H, J = 8.1 and 1.8 Hz, H 5), 7.12 (d, 1H, J = 8.1 Hz, H 6), 6.59 (d, 1H, J =
16.0 Hz, H 8), 3.82 (s, 3H, OCH 3), 2.26 (s, 3H, OCOCH3). 1 H NMR: (600 MHz, Acetone-d6) δ: 7.65 (d, 1H, J = 16.0 Hz, H 7), 7.47 (d, 1H, J = 2.0 Hz,
H3), 7.26 (dd, 1H, J = 8.2 and 2.0 Hz, H 5), 7.12 (d, 1H, J = 8.2 Hz, H 6), 6.55 (d, 1H, J =
16.0 Hz, H 8), 3.91 (s, 3H, OCH 3), 2.25 (s, 3H, OCOCH 3). Spectral properties in DMSO were as previously reported. 239
Attempted synthesis of 1-O-chloroacetyl coumaric acid (21) (2E)-3-{4-[(Chloroacetyl)oxy]phenyl}prop-2-enoic acid
p-Coumaric acid ( 3) (151.5 mg, 0.92 mmol) was dissolved in dry pyridine (5 mL) followed by the addition of chloroacetic anhydride (0.73g, 4.26 mmol) and the mixture stirred at ambient temperature. After 16 hours the solid reaction mixture was dissolved in methanol (10 mL) and analysed by TLC which indicated no formation of the desired product.
140
Chapter 6: Experimental
1-O-Chloroacetyl p-coumaric acid (21) (2E)-3-{4-[(Chloroacetyl)oxy]phenyl}prop-2-enoic acid
Chloroacetyl chloride (0.90 mL, 11.30 mmol) was added to p-coumaric acid ( 3) (0.60 g, 3.68 mmol) dissolved in a minimal volume of 2M sodium hydroxide solution (8 mL) at 0 oC. The resulting suspension was stirred at room temperature for 5 minutes before being acidified with 2M hydrochloric acid solution. The precipitate was filtered, washed with cold water, dried and recrystallised from acetone/X4 to give 0.31 g (35%) of 21 as a crystalline white solid. m.p. 182.9-184.5 oC (lit. m.p. 186-187 oC). 144
Rf (10% MeOH/DCM): 0.41 1 H NMR: (400 MHz, Acetone-d6) δ: 7.78 (app. d, 2H, J = 8.7 Hz, H 3,5 ), 7.69 (d, 1H, J =
16.0 Hz, H 7), 7.27 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.53 (d, 1H, J = 16.0 Hz, H 8), 4.59 (s, 2H, 144 CH 2Cl). Physical and chemical properties were as previously reported.
1-O-Chloroacetyl ferulic acid (22) (2E)-3-{4-[(Chloroacetyl)oxy]-3-methoxyphenyl}prop-2-enoic acid
As described for 21 (above), reaction of ferulic acid ( 4) (0.60 g, 3.07 mmol) gave a crude mixture which was recrystallised from ethanol/water to give 0.54 g (65%) of 22 as a pale yellow crystalline solid. m.p. 148.3-149.6 oC (lit. m.p. 146-148 oC). 145
Rf (10% MeOH/DCM): 0.40 1 H NMR: (400 MHz, Acetone-d6) δ: 7.66 (d, 1H, J = 16.0 Hz, H 7), 7.51 (d, 1H, J = 1.9
Hz, H 3), 7.30 (dd, 1H, J = 8.2 and 1.9 Hz, H 5), 7.19 (d, 1H, J = 8.2 Hz, H 6), 6.56 (d, 1H, J
= 16.0 Hz, H 8), 4.58 (s, 2H, CH 2Cl), 3.93 (s, 3H, OCH 3). NMR data assignments made based on those of 21 , and physical properties were as previously reported. 145
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Chapter 6: Experimental
6.2.2 Synthesis of Hydroxycinnamoyl Tartrate Esters
Dibenzyl L-tartaric acid (24) Dibenzyl (2R,3R)-2,3-dihydroxybutanedioate
L-Tartaric acid (3.01 g, 20.04 mmol) was added to a mixture of benzyl alcohol (6.3 mL, 60.88 mmol) and p-toluene sulphonic acid (0.36 g, 2.06 mmol) in toluene (40 mL). Employing a Dean-Stark apparatus, the mixture was heated under reflux for 3 hours, in which 0.72 mL (100 % theoretical) of water was collected. The mixture was allowed to cool to room temperature, diluted with diethyl ether (30 mL), poured into saturated sodium bicarbonate solution (60 mL), and the product extracted with diethyl ether (3 x 30 mL), dried (MgSO 4) and concentrated. Trituration with X4/EtOAc (20:1) gave 6.24 g (94%) of 24 as a white solid. m.p. 49.8-51.0 oC (lit. m.p. 49-50 oC). 152
Rf (50% EtOAc/X4): 0.42
= +11.4 o (c 1.01, acetone) (lit. = +10.1) 148 1 H NMR: (400 MHz, CDCl 3) δ: 7.38-7.36 (m, 10H, ArH), 5.30 (d, 2H, J = 12.1 Hz,
CHaHbPh), 5.25 (d, 2H, J = 12.1 Hz, CH aHbPh), 4.63 (d, 2H, J = 7.7 Hz, CH), 3.35 (d, 2H, J = 7.7 Hz, OH). All physical and chemical properties were as previously reported. 148, 151- 152
General procedure for esterification with dibenzyl-L-tartrate The hydroxycinnamate (15 or 16 ) (0.50 mmol) and 24 (0.60 mmol) were dissolved in dry dichloromethane (15 mL) followed by the addition of trifluoroacetic anhydride (0.60 mmol) at 0 oC. The mixture was then stirred at ambient temperature for 6 hours before being poured onto saturated sodium bicarbonate solution (20 mL), extracted with dichloromethane (3 x 15 mL), washed with water (3 x 15 mL), dried (MgSO 4) and concentrated in vacuo . Purification by column chromatography (DCM-3% Et 2O/DCM) furnished the desired products ( 25 or 26 ), as well as a mixture of the mono- and di-ester.
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Chapter 6: Experimental
1-O-Benzyl p-coumaroyl dibenzyl L-tartaric acid (25) Dibenzyl (2R,3R)-2-({(2E)-3-[4-(benzyloxy)phenyl]prop-2-enoyl}oxy)-3- hydroxybutanedioate
From 15 (101.6 mg, 0.40 mmol), afforded 107.0 mg (47%) of 25 as a colourless gum, as well as 49.1 mg of a mixture of 25 and 27 (1:9).
Rf (50% EtOAc/X4): 0.56 = +12.6 o (c 0.7, chloroform) 1 H NMR: (400 MHz, CDCl 3) δ: 7.60 (d, 1H, J = 15.9 Hz, H 7), 7.45-7.15 (m, 17H, ArH and
H3,5 ), 6.98 (app. d, 2H, J = 8.8 Hz, H 2,6 ), 6.21 (d, 1H, J = 15.9 Hz, H 8), 5.63 (d, 1H, J = 2.3
Hz, H 2’ ), 5.27-5.02 (m, 6H, 3 x CH 2Ph,), 4.86 (dd, 1H, J = 2.3 and 7.5 Hz, H 3’ ), 3.28 (d, 1H, J = 7.5 Hz, OH). 13 C NMR: (400 MHz, CDCl 3) δ: 170.7 (C 4’), 166.8 (C 1’), 165.7 (C 9), 161.0 (C 1), 146.2
(C 7), 136.5, 135.1, 134.6 (3 x Ar), 130.0 (C 3,5 ), 130.3-127.0 (Ar), 115.2 (C 2,6 ), 113.2 (C 8),
73.7 ( CH2Ph), 72.8 (C 2’). 70.9 (C 3’), 70.2, 67.7 (2 x CH2Ph). IR (neat) ν: 3475, 2955, 1718, 1145, 732, 695. LRP (+EI) m/z (%): 566 (M +, 1), 475 ( 1), 325 ( 2), 254 ( 5), 237 ( 9), 181 ( 2), 164 ( 2), 147 (3), 107 ( 3), 91 ( 100 ), 65 ( 7). + HRMS calculated for C 34 H30 O8 [M] 566.1941, found 566.1948.
Di(1-O-benzyl coumaroyl)dibenzyl L-tartaric acid (27) Dibenzyl (2R,3R)-2,3-bis({(2E)-3-[4-(benzyloxy)phenyl]prop-2-enoyl}oxy)butanedioate
Isolated from the synthesis and purification of 25 .
143
Chapter 6: Experimental
Rf (50% EtOAc/X4): 0.69 1 H NMR: (600 MHz, CDCl 3) δ: 7.64 (d, 1H, J = 16.0 Hz, H 7), 7.45-7.15 (m, 12H, ArH and
H3,5 ), 6.99 (app. d, 2H, J = 8.8 Hz, H 2,6 ), 6.25 (d, 1H, J = 16.0 Hz, H 8), 5.91 (s, 1H, CH),
5.24 (d, 1H, J = 12.2 Hz, C HaHbPh), 5.14 (d, 1H, J = 12.2 Hz, CH aHbPh), 5.11 (app. s, 2H,
CH2Ph). Identification and characterisation based on reported data for the caffeoyl derivative. 154
1-O-Benzyl feruloyl dibenzyl L-tartaric acid (26) Dibenzyl (2R,3R)-2-({(2E)-3-[4-(benzyloxy)-3-methoxyphenyl]prop-2-enoyl}oxy)-3- hydroxybutanedioate
From 16 (185.9 mg, 0.65 mmol), yielded 197.0 mg (50%) of 26 as a colourless gum, as well as 67.5 mg of a mixture of 26 and 28 (1:19).
Rf (50% EtOAc/X4): 0.48 = +10.2 o (c 1.7, chloroform) 1 H NMR: (400 MHz, CDCl 3) δ: 7.59 (d, 1H, J = 15.9 Hz, H 7), 7.45-7.20 (m, 15H, ArH),
7.04 (d, 1H, J = 1.9 Hz, H 3), 7.01 (dd, 1H, J = 8.3 and 1.9 Hz, H 5), 6.88 (d, 1H, J = 8.3 Hz,
H6), 6.22 (d, 1H, J = 15.9 Hz, H8), 5.64 (d, 1H, J = 2.2 Hz, H 2’ ), 5.26-5.16 (m, 6H,
CH 2Ph), 4.86 (br. s, 1H, H 3’ ), 3.93 (s, 3H, OCH 3), 3.21 (d, 1H, J = 7.6 Hz, OH). 13 C NMR: (400 MHz, CDCl 3) δ: 170.7 (C 4’), 166.7 (C 1’), 165.6 (C 9), 150.7 (C 1), 149.8
(C 2), 146.6 (C 7), 136.5, 135.1, 135.0 (3 x Ar), 128.7-128.1 (Ar), 127.3 (C 4), 123.0 (C 5),
113.9 (C 8), 113.3 (C 6), 110.3 (C 3), 73.0 (C 2’ ), 70.9 ( CH2Ph), 70.8 (C 3’), 68.2, 67.7 (2 x
CH2Ph), 56.1 (OCH 3). IR (neat) ν: 3478, 2957, 1718, 1133, 731, 695. LRP (+EI) m/z (%): 596 (M +, 1), 505 ( 4), 355 ( 4), 284 ( 6), 267 ( 7), 194 ( 1), 149 ( 4), 107 (4), 91 ( 100 ), 65 ( 6). + HRMS calculated for C 35 H32 O9 [M] 596.2046, found 596.2035.
144
Chapter 6: Experimental
Di(1-O-benzyl feruloyl)dibenzyl L-tartaric acid (28) Dibenzyl (2R,3R)-2,3-bis({(2E)-3-[4-(benzyloxy)-3-methoxyphenyl]prop-2- enoyl}oxy)butanedioate
Isolated from the synthesis and purification of 26.
Rf (50% EtOAc/X4): 0.58 1 H NMR: (400 MHz, CDCl 3) δ: 7.61 (d, 1H, J = 15.9 Hz, H 7), 7.45-7.15 (m, 10H, ArH),
7.05 (d, 1H, J = 1.8 Hz, H 3), 7.01 (dd, 1H, J = 8.4 and 1.8 Hz, H 5), 6.89 (d, 1H, J = 8.4 Hz,
H6), 6.26 (d, 1H, J = 15.9 Hz, H 8), 5.92 (s, 1H, H 2’ ), 5.26-5.13 (m, 4H, CH 2Ph), 3.93 (s,
3H, OCH 3). Identification and characterisation based on reported data for the caffeoyl derivative. 154
Attempted syntheses of feruloyl tartrate (8)
Attempt 1: 1-O-Benzyl feruloyl dibenzyl L-tartaric acid ( 26 ) (135.1 mg, 0.23 mmol) and 5% palladium on activated carbon (21.3 mg) were stirred in ethyl acetate (15 mL) followed by the addition of 1,4-cyclohexadiene (0.22 mL, 0.23 mmol) and the mixture was stirred at ambient temperature. After 3 hours TLC showed no formation of the desired product and further 1,4-cyclohexadiene (0.22 mL, 0.23 mmol) was added. After 24 hours analysis by TLC showed no change and the reaction mixture was filtered through celite, concentrated in vacuo and the crude mixture analysed by NMR, which suggested formation of the reduced analogue of the starting material ( 29 ).
Rf of reaction mixture (50% EtOAc/X4): 0.48
145
Chapter 6: Experimental
1 H NMR: (400 MHz, CDCl 3) δ: 5.54 (d, 1H, J = 2.2 Hz, H 2’ ), 4.82 (d, 1H, J = 2.2 Hz, H 3’ ), 1 2.84 (app. t, 2H, J = 7.9 Hz, H 7), 2.62 (m, 1H, H 8a ), 2.48 (m, 1H, H 8b ). Unobstructed H signals in addition to those of the remaining starting material ( 26 ).
Attempt 2: Palladium acetate (86.0 mg, 0.38 mmol) and triethylamine (0.05 mL, 0.36 mmol) were dissolved in dry dichloromethane (5 mL) and after 5 minutes of stirring at ambient temperature 1-O-benzyl feruloyl dibenzyl L-tartaric acid ( 26 ) (80.8 mg, 0.16 mmol) in dry dichloromethane (5 mL) was added dropwise. After a further 5 minutes triethylsilane (0.20 mL, 1.252 mmol) was added slowly and the mixture stirred at ambient temperature for 24 hours before being diluted with methanol (5 mL) filtered through celite and concentrated in vacuo . The crude oil was taken up in ethyl acetate/water (6:1, 20 mL), the aqueous layer separated and washed with ethyl acetate (2 x 10 mL). The combined organics were washed successively with phosphoric acid solution (1 M, 10 mL) and brine solution (10 mL) until a pH of 7 was achieved, then dried (MgSO 4) and concentrated in vacuo . The clear oil was dissolved in ethyl acetate and X4 added until a precipitate formed, which was filtered, washed with X4 and analysed by 1H NMR which suggested formation of the reduced analogue of the desired product (30 ).
Rf of crude mixture (50% EtOAc/X4): 0.00 1 H NMR: (400 MHz, CD 3OD) δ: 5.46 (d, 1H, J = 2.3 Hz, H 2’ ), 4.72 (d, 1H, J = 2.3 Hz, 1 H3’ ), 2.94 (app. t, 2H, J = 7.8 Hz, H 7), 2.71 (app. t, 2H, J = 7.8 Hz, H 8). Unobstructed H signals in the crude mixture.
Attempt 3: Debenzylation was attempted as described above, employing half the amount of triethylsilane (0.1 mL, 0.626 mmol). Analysis of the product by 1H NMR displayed the same peaks as above suggesting formation of the reduced product, with minor signals corresponding with formation of the desired product (8).
Rf of crude mixture (50% EtOAc/X4): 0.00
146
Chapter 6: Experimental
O,O’-Diacetyl L-tartaric anhydride (31) (3R,4R)-2,5-Dioxotetrahydrofuran-3,4-diyl diacetate
L-Tartaric acid (10.07 g, 67.08 mmol) in acetyl chloride (65 mL, 0.91 mol) was heated under reflux under a nitrogen atmosphere for 48 hours. The reaction mixture was allowed to cool to room temperature, concentrated in vacuo and the resulting oil recyrstallised from EtOAc/X4 to afford 11.82 g (82%) of 31 as a white crystalline solid. m.p. 135-136 oC (lit. m.p. 133-135 oC). 240 1 H NMR: (400 MHz, CDCl 3) δ: 5.68 (s, 1H, CH), 2.23 (s, 3H, OCH 3). 1 H NMR: (400 MHz, Acetone-d6) δ: 6.17 (s, 1H, CH), 2.19 (s, 3H, OCH 3). Spectral properties in chloroform were as previously reported. 161, 240-241
O,O’-Diacetyl L-tartaric acid (32) (2R,3R)-2,3-bis(Acetyloxy)butanedioic acid
O,O’ -Diacetyl L-tartaric anhydride ( 31) (1.03 g, 4.75 mmol) was dissolved in acetone (5 mL) followed by the addition of water (0.17 mL, 9.44 mmol) and the mixture was stirred at room temperature for 12 hours before being concentrated. Trituration with X4 gave 1.10 g (99%) of 32 as a white solid. m.p. 117-118 oC (lit. m.p. 118 oC). 242 1 H NMR: (400 MHz, Acetone-d6) δ: 5.72 (s, 1H, CH), 2.11 (s, 3H, OCH 3). Physical and chemical properties were as previously reported. 161, 242
O,O’-Diacetyl-di-tert-butyl L-tartrate (33) Di-tert-butyl (2R,3R)-2,3-bis(acetyloxy)butanedioate
147
Chapter 6: Experimental
Anhydrous magnesium sulphate (3.98 g) and sulphuric acid (0.5 mL) were stirred in dry dichloromethane (120 mL) for 15 minutes followed by the addition of O,O’ -diacetyl L- tartaric acid ( 32) (1.03 g, 4.42 mmol) and dry tert -butanol (4.0 mL, 41.82 mmol), and the reaction vessel was stoppered tightly. After 3 days the mixture was poured into saturated sodium bicarbonate solution (100 mL) and stirred until the MgSO 4 dissolved, then extracted in dichloromethane (3 x 50 mL), washed with brine (2 x 50 mL), dried (MgSO 4) and concentrated in vacuo . Column chromatography (20% EtOAc/X4) gave 0.76 g (49%) of 33 as a clear oil and 0.32 g of a mixture of 33 and the mono-acetate (80:20 as determined by proton integration).
Rf (50% EtOAc/X4): 0.66 1 H NMR: (400 MHz, CDCl 3) δ: 5.62 (s, 1H, CH), 2.16 (s, 3H, OCH 3), 1.44 (s, 9H, t-Bu). 1 H NMR: (400 MHz, DMSO-d6) δ: 5.51 (s, 1H, CH), 2.11 (s, 3H, OCH 3), 1.38 (s, 9H, t- Bu). Spectral properties in chloroform were as previously reported. 159
Mono-acetyl-di-tert-butyl L-tartrate (Tentative characterisation) Di-tert-butyl (2R,3R)-2-(acetyloxy)-3-hydroxybutanedioate
Obtained during the purification of 33.
Rf (50% EtOAc/X4): 0.60 1 H NMR: (400 MHz, CDCl 3) δ: 5.34 (d, 1H, J = 2.3 Hz, CH), 4.60 (dd, 1H, J = 7.1 and 2.3
Hz, CH), 3.09 (d, 1H, J = 7.1 Hz, OH), 2.12 (s, 3H, OCH 3), 1.49 (s, 9H, t-Bu), 1.46 (s, 9H, t-Bu). Data was extracted from the spectrum of the mixture.
Di-tert-butyl L-tartrate (34) Di-tert-butyl (2R,3R)-2,3-dihydroxybutanedioate
Diacetyl-di-tert -butyl L-tartrate ( 33) (3.50 g, 10.10 mmol) was dissolved in methanol (25 mL) followed by the addition of powdered potassium hydroxide (115.7 mg, 2.06 mmol) and the mixture stirred at room temperature. After 45 minutes the mixture was 148
Chapter 6: Experimental concentrated and purified by column chromatography (20% EtOAc/X4) yielding 0.79g (29%) of 34 as a white solid, m.p. 86.1-89.8 oC (lit. m.p. 91 oC), 159 as well as tert -butyl methyl L-tartrate as a clear oil.
Rf (40% EtOAc/X4): 0.49 = +11.0 o (c 1.0, acetone) 1 H NMR: (400 MHz, CDCl 3) δ: 4.36 (d, 1H, J = 6.9 Hz, CH), 3.09 (d, 1H, J = 6.9 Hz, OH), 1.52 (s, 9H, t-Bu). All physical and chemical properties were as previously reported. 159
tert-Butyl methyl L-tartrate tert-Butyl methyl (2R,3R)-2,3-dihydroxybutanedioate
Isolated during purification of 34.
Rf (40% EtOAc/X4): 0.27 1 H NMR: (400 MHz, CDCl 3) δ: 4.51 (dd, 1H, J = 7.6 and 1.8 Hz, CH), 4.41 (dd, 1H, J =
6.4 and 1.8 Hz, CH), 3.86 (s, 3H, OCH 3), 3.18 (d, 1H, J = 6.4 Hz, OH), 3.05 (d, 1H, J = 7.6 Hz, OH), 1.52 (s, 9H, t-Bu). Spectral properties were as previously reported. 159
General procedure for esterification with di-tert-butyl L-tartrate The hydroxycinnamate ( 19 -22 ) (0.50 mmol) was heated under reflux in dry benzene (10 mL) containing thionyl chloride (6.89 mmol). After 5 hours the mixture was allowed to cool to room temperature and then concentrated in vacuo . The crude residue was taken up in dry benzene (5 mL) and added dropwise to a solution of di-tert -butyl L-tartrate ( 34) (0.65 mmol) in dry pyridine (5 mL), then stirred at ambient temperature overnight. The mixture was concentrated and pyridine azeotropically removed with toluene. Purification with column chromatography (20% EtOAc/X4) gave the desired product ( 35-38 ).
149
Chapter 6: Experimental
1-O-Acetyl p-coumaroyl di-tert-butyl L-tartrate (35) Di-tert-butyl (2R,3R)-2-({(2E)-3-[4-(acetyloxy)phenyl]prop-2-enoyl}oxy)-3- hydroxybutanedioate
From 19 (101.4 mg, 0.49 mmol), and recrystallisation from 30% EtOAc/X4 gave 68.1 mg (31%) of white crystals. m.p. 143.6-144.2 oC.
Rf (50% EtOAc/X4): 0.57 = +2.04 o (c 0.5, acetone) 1 H NMR: (400 MHz, CDCl 3) δ: 7.73 (d, 1H, J = 16.0 Hz, H 7), 7.54 (app. d, 2H, J = 8.7 Hz,
H3,5 ), 7.13 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.45 (d, 1H, J = 16.0 Hz, H 8), 5.48 (d, 1H, J = 2.3
Hz, H 2’ ), 4.67 (dd, 1H, J = 6.9 and 2.3 Hz, H 3’ ), 3.20 (d, 1H, J = 6.9 Hz, OH), 2.31 (s, 3H,
OCOCH3), 1.51 (s, 9H, t-Bu 4), 1.44 (s, 9H, t-Bu 1). 13 C NMR: (600 MHz, CDCl 3) δ: 170.2 (C 4’ ), 169.3 (O COCH 3), 165.8 (C 9), 165.5 (C 1’ ),
152.4 (C 1), 145.5 (C 7), 131.9 (C 4), 129.6 (C 3,5 ), 122.3 (C 2,6 ), 116.7 (C 8), 84.0 ( C1(CH 3)3),
83.4 ( C4(CH 3)3), 73.5 (C 2’ ), 71.0 (C 3’ ), 28.1 (C4(CH3)3), 28.0 (C1(CH3)3), 21.3 (OCO CH3). IR (neat) ν: 2982, 1713, 1128, 1055, 1033, 1015. LRP (+EI) m/z (%): 450 (M +, <1 ), 408 ( 2), 352 ( 10 ), 338 ( 5), 321 ( 12 ), 296 ( 63 ), 278 ( 6), 251 ( 6), 206 ( 7), 189 ( 46 ), 164 ( 79 ), 147 ( 100 ), 119 ( 14 ), 57 ( 37 ), 41 ( 13 ). + HRMS calculated for C 23 H30 O9 [M] 450.1890, found 450.1891.
Details of crystal structure determination of 35
Crystal data for C23 H30 O9: M = 450.47, T = 100(2) K, orthorhombic, P212121, a = 3 5.7183(2), b = 8.7309(3), c = 46.9988(19) Å, V = 2346.46(15) Å , Z = 4, Dx = 1.275, F(000) = 960, µ = 0.822 mm -1, no. of unique data (Agilent Technologies SuperNova Dual diffractometer with Atlas detector using Cu Kα radiation so that θmax = 74.6°) = 4603, no. of parameters = 300, R (3842 data with I ≥ 2σ(I)) = 0.053, wR (all data) = 0.131. The structure was solved by direct-methods (SHELXS-97) and refined (anisotropic displacement parameters, C-bound H atoms in the riding model approximation, full 2 2 2 refinement of the hydroxyl-H atom, and a weighting scheme w = 1/[ σ (Fo ) + (0.067 P) ] 2 2 2 where P = ( Fo + 2 Fc )/3) with SHELXL-97 on F . The value of the Flack parameters = 0.0(2). 150
Chapter 6: Experimental
1-O-Acetyl feruloyl tert-butyl L-tartrate (36) Di-tert-butyl (2R,3R)-2-({(2E)-3-[4-(acetyloxy)-3-methoxyphenyl]prop-2-enoyl}oxy)-3- hydroxybutanedioate
From 20 (159.1 mg, 0.67 mmol), and recrystallisation from 30% EtOAc/X4 gave 154.0 mg (48%) of white crystals. m.p. 140.5-142.0 oC.
Rf (30% EtOAc/X4): 0.34 = -4.51 o (c 1.3, acetone) 1 H NMR: (600 MHz, CDCl 3) δ: 7.70 (d, 1H, J = 16.0 Hz, H 7), 7.12-7.11 (m, 2H, H 3,5 ),
7.05 (d, 1H, J = 8.6 Hz, H 6), 6.45 (d, 1H, J = 16.0 Hz, H 8), 5.50 (d, 1H, J = 2.3 Hz, H 2’ ),
4.68 (dd, 1H, J = 6.8 and 2.3 Hz, H 3’ ), 3.87 (s, 3H, OCH 3), 3.21 (d, 1H, J = 6.8 Hz, OH),
2.33 (s, 3H, OCOC H3), 1.51 (s, 9H, t-Bu 4), 1.44 (s, 9H, t-Bu 1). 13 C NMR: (600 MHz, CDCl 3) δ: 170.2 (C 4’ ), 168.9 (O COCH 3), 165.9 (C 1’ ), 165.5 (C 9),
151.5 (C 7), 145.9 (C 2), 141.8 (C 1), 133.2 (C 4), 123.4 (C 6), 121.8 (C 5), 116.8 (C 8), 111.3
(C 3), 84.1 ( C1(CH 3)3), 83.5 ( C4(CH 3)3), 73.5 (C 2’ ), 71.0 (C 3’ ), 56.1 (OMe), 28.1
(C4(CH3)3), 28.0 (C1(CH3)3), 20.8 (OCO CH3). IR (neat) ν: 1755, 1710, 1260, 1218, 1195, 1148, 1120, 1074, 1030, 981. Calc. C 59.99, H 6.71, O 33.30. Anal. C 59.79, H 6.73, O 33.48.
Details of crystal structure determination of 36
Crystal data for C24H32O10 : M = 450.47, T = 100(2) K, monoclinic, P21, a = 5.9894(1), b = 3 10.6483(1), c = 19.6676(2) Å, β = 96.324(1)º, V = 1246.71(3) Å , Z = 2, Dx = 1.280, F(000) = 512, µ = 0.837 mm -1, no. of unique data (Agilent Technologies SuperNova Dual diffractometer with Atlas detector using Cu Kα radiation so that θmax = 74.5°) = 4800, no. of parameters = 326, R (4762 data with I ≥ 2σ(I)) = 0.057, wR (all data) = 0.159. The structure was solved by direct-methods (SHELXS-97) and refined (anisotropic displacement parameters, all H atoms in the riding model approximation, and a weighting 2 2 2 2 2 scheme w = 1/[ σ (Fo ) + (0.098 P) + 0.941P] where P = ( Fo + 2 Fc )/3) with SHELXL-97 on F2. Two orientations, of equal weight, were discerned for a significant portion of the molecule. The aromatic rings were refined as hexagons (C–C = 1.39 Å), equivalent pairs of
151
Chapter 6: Experimental atoms were constrained to have identical anisotropic displacement parameters and these were constrained to be nearly isotropic. The value of the Flack parameters = 0.0(2).
Attempted synthesis of p-coumaroyl L-tartrate (7)
1-O-Acetyl p-coumaroyl tert -butyl L-tartrate ( 35) (68.1 mg, 0.15 mmol) was dissolved in dry dichloromethane (5 mL) followed by the addition of trifluoroacetic acid (0.30 mL, 3.92 mmol) and the mixture was stirred at ambient temperature. After disappearance of the starting material (as shown by TLC) the mixture was concentrated in vacuo and taken up in acetone/3 M hydrochloric acid (3:1, 6 mL) and heated under reflux for 3 hours before being allowed to cool to ambient temperature. The mixture was diluted with ethyl actetate 1 (20 mL), washed with brine (3 x 20 mL), dried (MgSO4) and concentrated in vacuo . H NMR of the crude product indicated formation of the desired product with major impurities, and attempted purification using reverse-phase chromatography (linear gradient from 0.1% formic acid/water to 0.1% formic acid/acetonitrile) failed to separate the by- products. To protonate any potentially occuring salts, the crude product was taken up in methanol (5 mL), the pH adjusted to 1 with 2M HCl and the mixture stirred at ambient temperature for 16 hours before being concentrated in vacuo and analysed by 1H NMR which showed no change in the ratio of product and impurities.
Rf of mixture (50% EtOAc/X4): 0.00
1-O-Chloroacetyl p-coumaroyl tert-butyl L-tartrate (37) Di-tert-butyl (2R,3R)-2-{[(2E)-3-{4-[(chloroacetyl)oxy]phenyl}prop-2-enoyl]oxy}-3- hydroxybutanedioate
From 21 (91.4 mg, 0.38 mmol), gave 54.7 mg (30%) of 37 (m.p 126.5-127.2 oC), as well as a mixture of 37 and di-ester in an 84:16 ratio as determined by proton integration.
152
Chapter 6: Experimental
Rf (50% EtOAc/X4): 0.65 = -3.22 o (c 1.6, acetone) 1 H NMR: (400 MHz, CDCl 3) δ: 7.72 (d, 1H, J = 16.0 Hz, H 7), 7.55 (app. d, 2H, J = 8.7 Hz,
H3,5 ), 7.16 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.45 (d, 1H, J = 16.0 Hz, H 8), 5.48 (d, 1H, J = 2.1
Hz, H 2’ ), 4.67 (d, 1H, J = 2.1 Hz, H 3’ ), 4.31 (s, 2H, OCH 2Cl), 3.25 (br. s, 1H, OH), 1.50 (s,
9H, t-Bu 4), 1.43 (s, 9H, t-Bu 1). 13 C NMR: (600 MHz, CDCl 3) δ: 170.2 (C 4’ ), 165.8 (O COCH 3), 165.7 (C 9), 165.4 (C 1’ ),
152.0 (C 1), 145.1 (C 7), 132.5 (C 4), 129.8 (C 3,5 ), 121.8 (C 2,6 ), 117.1 (C 8), 84.0 ( C1(CH 3)3),
83.5 ( C4(CH 3)3), 73.8 (C 2’ ), 70.7 (C 3’ ), 40.9 (OCO CH2Cl), 28.1 (C4(CH3)3), 27.6
(C1(CH3)3). IR (neat) ν: 3460, 2975, 2929, 1718, 1127, 839. LRP (+EI) m/z (%): 484 (M +, <1 ), 428 ( 3), 372 ( 8), 355 ( 12 ), 327 ( 31 ), 296 ( 18 ), 278 ( 8), 240 ( 19 ), 223 ( 100 ), 206 ( 2), 164 ( 94 ), 147 ( 94 ), 119 ( 15 ), 57 ( 74 ), 41 ( 17). + HRMS calculated for C 23 H29 ClO 9 [M] 484.1500, found 484.1494.
1-O-Chloroacetyl feruloyl tert-butyl L-tartrate (38) Di-tert-butyl (2R,3R)-2-{[(2E)-3-{4-[(chloroacetyl)oxy]-3-methoxyphenyl}prop-2- enoyl]oxy}-3-hydroxybutanedioate
From 22 (50.1 mg, 0.19 mmol), gave 32.0 mg (34%) of 38 as a white solid (m.p 106.0- 106.8 oC), as well as 19.1 mg (24%) of the dechloroacetylated product ( 40).
Rf (50% EtOAc/X4): 0.60 = -6.69 o (c 1.5, acetone) 1 H NMR: (600 MHz, CDCl 3) δ: 7.70 (d, 1H, J = 16.0 Hz, H 7), 7.12-7.08 (m, 3H, H 3,5,6 ),
6.45 (d, 1H, J = 16.0 Hz, H 8), 5.50 (d, 1H, J = 2.3 Hz, H 2’ ), 4.68 (br. s, 1H, H 3’ ), 4.34 (s,
2H, CH 2Cl), 3.86 (s, 3H, OCH 3), 3.22 (br. s, 1H, OH), 1.51 (s, 9H, t-Bu 4), 1.44 (s, 9H, t-
Bu 1). 13 C NMR: (600 MHz, CDCl 3) δ: 170.2 (C 4’ ), 165.8 (O COCH 3), 165.4 (C 1’ ), 165.3 (C 9),
151.3 (C 7), 145.5 (C 2), 141.2 (C 1), 133.7 (C 4), 123.2 (C 6), 121.7 (C 5), 116.9 (C 8), 111.6
153
Chapter 6: Experimental
(C 3), 84.0 ( C1(CH 3)3), 83.5 ( C4(CH 3)3), 73.8 (C 2’ ), 70.8 (C 3’ ), 56.5 (OMe), 40.7
(OCO CH2Cl), 28.3 (C4(CH3)3), 27.6 (C1(CH3)3). IR (neat) ν: 3479, 2925, 1717, 1259, 1130, 845, 815, 764. LRP (+EI) m/z (%): 514 (M +, 1), 458 ( 1), 402 ( 2), 382 ( 2), 357 ( 6), 326 ( 14 ), 308 ( 4), 270 (10 ), 253 ( 16 ), 236 ( 13 ), 194 ( 100 ), 177 ( 26 ), 145 ( 9), 133 ( 6), 117 (4), 89 ( 4), 77 ( 6), 57 (8), 41 ( 6). + HRMS calculated for C 24 H31 ClO 10 [M] 514.1606, found 514.1601.
p-Coumaroyl tert-butyl L-tartrate (39) Di-tert-butyl (2R,3R)-2-hydroxy-3-{[(2E)-3-(4-hydroxyphenyl)prop-2- enoyl]oxy}butanedioate
1-O-Chloroacetyl p-coumaric acid ( 21 ) (0.21 g, 0.86 mmol) was heated under reflux in dry benzene (10 mL) containing thionyl chloride (0.50 mL, 6.89 mmol). After 5 hours the mixture was allowed to cool to ambient temperature and concentrated in vacuo . The crude residue was taken up in dry benzene (5 mL) and added dropwise to a solution of di-tert - butyl L-tartrate ( 34) (0.33 g, 1.24 mmol) in dry pyridine (5 mL), then stirred at room temperature for 45 hours. The mixture was concentrated and pyridine azeotropically removed with toluene. Purification by column chromatography (20% EtOAc/X4) gave 68.6 mg (19 %) of 39 as a white solid (m.p 162.3-163.7 oC), as well as 47.1 mg (11%) of 37 .
Rf (50% EtOAc/X4): 0.38 = -5.57 o (c 1.0, acetone) 1 H NMR: (400 MHz, CDCl 3) δ: 7.60 (d, 1H, J = 15.9 Hz, H 7), 7.29 (app. d, 2H, J = 8.6 Hz,
H3,5 ), 6.84 (app. d, 2H, J = 8.6 Hz, H 2,6), 6.18 (d, 1H, J = 15.9 Hz, H 8), 5.51 (d, 1H, J = 2.3
Hz, H 2’ ), 4.68 (d, 1H, J = 2.3 Hz, H 3’ ), 3.34 (br. s, 1H, OH), 1.52 (s, 9H, t-Bu 4), 1.44 (s,
9H, t-Bu 1). 13 C NMR: (400 MHz, CDCl 3) δ: 170.2 (C 4’ ), 166.8 (C 1’ ), 166.2 (C 9), 159.0 (C 1), 146.8
(C 7), 130.4 (C 3,5 ), 126.4 (C 4), 116.2 (C 2,6 ), 113.2 (C 8), 84.2 ( C1(CH 3)3), 84.0 ( C4(CH 3)3),
73.4 (C 2’ ), 71.1 (C 3’ ), 28.2 (C4(CH3)3), 28.0 (C1(CH3)3).
154
Chapter 6: Experimental
IR (neat) ν: 3389, 2977, 1712, 1145, 1129, 988, 831. LRP (+EI) m/z (%): 408 (M +, 1), 352 ( 6), 296 ( 64 ), 279 ( 16 ), 251 ( 17 ), 206 ( 1), 164 ( 61 ), 147 ( 100 ), 119 ( 18 ), 91 ( 11 ), 57 ( 20 ), 41 ( 12 ). + HRMS calculated for C 21 H28 O8 [M] 408.1784, found 408.1783.
Feruloyl tert-butyl L-tartrate (40) Di-tert-butyl (2R,3R)-2-hydroxy-3-{[(2E)-3-(4-hydroxy-3-methoxyphenyl)prop-2- enoyl]oxy}butanedioate
1-O-Chloroacetyl ferulic acid ( 22 ) (0.21 g, 0.76 mmol) was submitted to the same reaction conditions as described for 39 (above). This afforded 92.7 mg (28%) of 40 as a white solid (m.p 172.6-174.0 oC), as well as 24.8 mg (6%) of 38 .
Rf (50 % EtOAc/X4): 0.38 = -8.87 o (c 1.0, acetone) 1 H NMR: (600 MHz, CDCl 3) δ: 7.68 (d, 1H, J = 15.9 Hz, H 7), 7.07 (dd, 1H, J = 8.1 and
1.8 Hz, H 5), 7.04 (d, 1H, J = 1.8 Hz, H 3), 6.92 (d, 1H, J = 8.1 Hz, H 6), 6.35 (d, 1H, J = 15.9
Hz, H 8), 5.50 (d, 1H, J = 2.3 Hz, H 2’ ), 4.67 (dd, 1H, J = 6.9 and 2.3 Hz, H 3’ ), 3.93 (s, 3H,
OCH 3), 3.20 (d, 1H, J = 6.9 Hz, OH), 1.51 (s, 9H, t-Bu 4), 1.44 (s, 9H, t-Bu 1). 13 C NMR: (400 MHz, CDCl 3) δ: 170.3 (C 4’ ), 166.0 (C 1’ ), 165.9 (C 9), 148.4 (C 1), 146.9
(C 2), 146.7 (C 7), 126.8 (C 4), 123.7 (C 5), 114.8 (C 6), 113.8 (C 8), 109.3 (C 3), 84.0
(C1(CH 3)3), 83.3 ( C4(CH 3)3), 73.3 (C 2’ ), 71.0 (C 3’ ), 56.1 (OCH 3), 28.1 (C4(CH3)3), 28.0
(C1(CH3)3). IR (neat) ν: 3465, 2929, 1726, 1146, 1120, 981, 844. + HRMS calculated for C 22 H30 O9 [M + Na] 461.1788, found 461.1772.
155
Chapter 6: Experimental p-Coumaroyl L-tartrate (7) (2R,3R)-2-Hydroxy-3-{[(2E)-3-(4-hydroxyphenyl)prop-2-enoyl]oxy}butanedioic acid
p-Coumaroyl tert -butyl L-tartrate ( 39 ) (46.2 mg, 0.11 mmol) was dissolved in dry dichloromethane (5 mL) followed by the addition of trifluoroacetic acid (0.18 mL, 2.29 mmol) and the mixture stirred at room temperature under a nitrogen atmosphere for 24 hours before being concentrated. Purification by reversed-phase chromatography (C18, eluted with acetonitrile/H 2O/formic acid, 30:69:1) gave 7 as an amorphous solid, 27.5 mg (82%).
Rf (20% MeOH/DCM): 0.00 1 H NMR: (400 MHz, CD 3OD) δ: 7.74 (d, 1H, J = 15.9 Hz, H 7), 7.48 (app. d, 2H, J = 8.7
Hz, H 3,5 ), 6.81 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.38 (d, 1H, J = 15.9 Hz, H 8), 5.55 (d, 1H, J =
2.3 Hz, H 2’ ), 4.77 (d, 1H, J = 2.3 Hz, H 3’ ). 13 C NMR: (400 MHz, CDCl 3) δ: 174.0 (C 4’ ), 170.8 (C 1’ ), 168.0 (C 9), 161.5 (C 1), 147.8
(C 7), 131.4 (C 3,5 ), 127.1 (C 4), 116.8 (C 2,6 ), 114.1 (C 8), 74.9 (C 2’ ), 71.7 (C 3’). All physical and chemical properties were as previously reported. 243
Feruloyl L-tartrate (8) (2R,3R)-2-Hydroxy-3-{[(2E)-3-(4-hydroxy-3-methoxyphenyl)prop-2- enoyl]oxy}butanedioic acid
Feruloyl tert -butyl L-tartrate ( 40) (35.9 mg, 0.082 mmol) was dissolved in dry dichloromethane (2 mL) followed by the addition of trifluoroacetic acid (0.13 mL, 1.63 mmol) and the mixture stirred at room temperature under a nitrogen atmosphere for 24 hours before being concentrated. Purification by reversed-phase chromatography (C18, eluted with acetonitrile/H 2O/formic acid, 30:69:1) gave 8 as an off-white amorphous solid, 11.0 mg (41%).
Rf (20% MeOH/DCM): 0.00
156
Chapter 6: Experimental
1 H NMR: (400 MHz, CD 3OD) δ: 7.73 (d, 1H, J = 16.0 Hz, H 7), 7.20 (d, 1H, J = 1.9 Hz,
H3), 7.10 (dd, 1H, J = 8.2 and 1.9 Hz, H 5), 6.82 (d, 1H, J = 8.2 Hz, H 6), 6.41 (d, 1H, J =
16.0 Hz, H 8), 5.57 (d, 1H, J = 2.4 Hz, H 2’ ), 4.78 (d, 1H, J = 2.4 Hz, H 3’ ), 3.89 (s, 3H,
OCH 3). 13 C NMR: (400 MHz, CDCl 3) δ: 173.8 (C 4’ ), 170.6 (C 1’ ), 167.9 (C 9), 150.8 (C 1), 149.4
(C 2), 148.1 (C 7), 127.6 (C 4), 124.3 (C 5), 116.4 (C 6), 114.3 (C 8), 111.7 (C 3), 74.9 (C 2’ ), 71.7
(C 3’), 56.5 (OCH 3). All physical and chemical properties were as previously reported. 243-244
6.2.3 Synthesis of Hydroxycinnamoyl Glucose Esters Chloroacetyl chloride (46)
Chloroacetic acid (100.25 g, 1.06 mol) and thionyl chloride (75 mL, 1.03 mol) were heated under reflux for 2 hours under a nitrogen atmosphere. Distillation under nitrogen gave chloroacetyl chloride (45-55% yield over a number of attempts) as a clear liquid, b.p. 102- 106 oC (lit. b.p. 106 oC), 245 and left chloroacetic anhydride as a clear solid, m.p. 49-50 oC (lit. m.p 48-60 oC)246 which could be separated from residual chloroacetic acid by kugelrohr distillation under reduced pressure (105 oC at 20 mmHg). (Chloroacetic acid lit. b.p. 189 oC, 120-123 oC at 20 mmHg). 246
1,2,3,4,6-Penta-O-chloroacetyl-D-glucopyranoside (41) 1,2,3,4,6-Pentakis-O-(chloroacetyl)-D-glucopyranose
D-Glucose (4.02 g, 22.4 mmol) was dissolved in dry dichloromethane (90 mL) and dry pyridine (10 mL). Chloroacetyl chloride ( 46 ) (17.6 mL, 221.0 mmol) was added dropwise at 0 oC, following which the reaction mixture was heated under reflux for 24 hours. The reaction mixture was poured onto ice water (100 mL), the organics were separated then washed with 2 M HCl solution (3 x 100 mL), saturated sodium bicarbonate solution (3 x
157
Chapter 6: Experimental
100 mL), saturated brine solution (2 x 100 mL), dried (MgSO 4) and concentrated. The crude mixture was purified by column chromatography (DCM) to give 11.42 g (91 %) of 41 as a pale yellow gum.
Rf (50% EtOAc/X4): 0.56 1 H NMR: (600 MHz, CDCl 3) δ: 6.43 (d, 0.55H, J = 3.7 Hz, H 1α), 5.82 (d, 0.45H, J = 8.2
Hz, H 1β), 5.59 (dd, 0.55H, J = 9.9 Hz, H 3α), 5.40 (dd, 0.45H, J = 9.5 Hz, H 3β), 5.27-5.22
(m, 2H, H 2,4 ), 4.40-4.36 (m, 1H, H 6a), 4.33-4.30 (m, 1.45H, H 6b,5 β), 4.25 (ddd, 0.55H, J =
10.3, 3.9 and 2.3 Hz, H 5α), 4.19-4.00 (m, 10H, OCH 2Cl). Spectral properties were as previously reported. 142, 182 ααα-Anomer Isolated as the remaining starting material in the synthesis of 42 (below). 1 H NMR: (400 MHz, CDCl 3) δ: 6.43 (d, 1H, J = 3.7 Hz, H 1), 5.58 (dd, 1H, J = 10.0 and
9.6 Hz, H 3), 5.28-5.21 (m, 2H, H 2,4 ), 4.38 (dd, 1H, J = 12.6 and 4.0 Hz, H 6a ), 4.31 (dd, 1H,
J = 12.6 and 2.3 Hz, H 6b ), 4.25 (ddd, 1H, J = 10.2, 4.0 and 2.3 Hz, H 5), 4.18, 4.12 (2 x app.s, 2H, OCH 2Cl), 4.04, 4.03 (2 x app. s, 1H, OCH 2Cl), 4.01, 4.00 (2 x app. s, 2H,
OCH 2Cl).
2,3,4,6-Tetra-O-chloroacetyl-D-glucopyranoside (42) 2,3,4,6-Tetrakis-O-(chloroacetyl)-D-glucopyranose
1,2,3,4,6-Penta-O-chloroacetyl-D-glucopyranoside ( 41) (11.15 g, 19.82 mmol) was dissolved in THF (150 mL) followed by the addition of hydrazine acetate (1.83 g, 19.88 mmol) and the reaction mixture stirred at ambient temperature. After 5 hours the reaction mixture was concentrated in vacuo and the crude product purified by column chromatography (DCM - 5% Et 2O/DCM) to afford 5.32 g (55 %) of 42 as an amorphous solid.
Rf (50% EtOAc/X4): 0.36 1 H NMR: (400 MHz, CDCl 3) δ: 5.66 (dd, 0.7H, J = 10.0 and 9.7 Hz, H 3α), 5.54 (d, 0.7H, J
= 3.6 Hz, H 1α), 5.38 (dd, 0.3H, J = 9.7 and 9.6 Hz, H 3β), 5.19 (dd, 0.3H, J = 9.7 and 9.7
Hz, H 4β), 5.18 (dd, 0.7H, J = 9.7 and 9.6 Hz, H 4α), 5.02-4.98 (m, 1H, J = 3.6 and 10.0 Hz, 158
Chapter 6: Experimental
H2), 4.85 (d, 0.3H, J = 8.0 Hz, H 1β), 4.40-4.32 (m, 3H, H 5,6a,6b), 4.14-4.00 (m, 8H, 142, 182 OCH 2Cl). Spectral properties were as previously reported.
2,3,4,6-Tetra-O-chloroacetyl-D-glucopyranosyltrichloroacetimidate (43) 2,3,4,6-Tetrakis-O-(chloroacetyl)-1-O-(2,2,2-trichloroethanimidoyl)-D-glucopyranose
2,3,4,6-Tetra-O-chloroacetyl-D-glucopyranoside ( 42) (5.04 g, 10.36 mmol) was dissolved in dry dichloromethane (100 mL) followed by the addition of trichloroacetonitrile (10.38 mL, 103.52 mmol), DBU (0.31 mL, 2.07 mmol) and the reaction mixture stirred at ambient temperature. After 4 hours the reaction mixture was concentrated and the crude product purified by column chromatography (DCM - 2% Et 2O/DCM) to give 4.92 g (75%) of 43 as a pale yellow amorphous solid.
Rf (30 % EtOAc/X4): 0.27 1 H NMR: (600 MHz, CDCl 3) δ: 8.79 (s, 0.33H, NH β), 8.77 (s, 0.67H, NH α), 6.60 (d,
0.67H, J = 3.7 Hz, H 1α), 5.94 (d, 0.33H, J = 7.6 Hz, H 1β), 5.68 (dd, 0.67H, J = 9.8 and 9.8
Hz, H 3α), 5.42 (dd, 0.33H, J = 8.4 and 8.4 Hz, H 3β), 5.30-5.24 (m, 2H, H 2,4 ), 4.39-4.31 (m,
3H, H 5,6a,6b), 4.13-3.99 (m, 8H, OCH 2Cl). Spectral properties were as previously reported. 142, 144
General procedure for hydroxycinnamate glycosylation 2,3,4,6-Tetra-O-chloroacetyl-D-glucopyranosyltrichloroacetimidate (43) (0.55 mmol) was dissolved in dry dichloromethane (10 mL) containing 4 Å molecular sieves, followed by the addition of the hydroxycinnamate ( 3, 4, 15 , 16 , 19 -22 , 47 ) (0.45 mmol). After 20 minutes of stirring at ambient temperature trimethylsilyl triflate (0.33 mmol) was added slowly. The reaction mixture was stirred at room temperature for a further 4 hours, after which it was quenched with saturated sodium bicarbonate solution (15 mL), washed with water (3 x
15 mL), then brine (2 x 15 mL), dried (MgSO 4) and concentrated in vacuo . Purification with column chromatography (20-30% EtOAc/X4) yielded the product ( 44, 45, 48 -50, 54, 159
Chapter 6: Experimental
55, 57 , 58 ). Analogues that were achieved as a gum could be solidified via trituration with methanol, and the melting points quoted are the point at which the solid reverted back to a gum.
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl cinnamate (48) 2,3,4,6-Tetrakis-O-(chloroacetyl)-1-O-[(2E)-3-phenylprop-2-enoyl]-βββ-D-glucopyranose
From 47 (122.3 mg, 0.83 mmol), gave 0.27 g (54 %) of 48 as a white solid.
Rf (50 % EtOAc/X4): 0.53 1 H NMR: (400 MHz, CDCl 3) δ: 7.77 (d, 1H, J = 16.0 Hz, H 7), 7.56-7.53 (m, 2H, H 3,5 ),
7.42-7.41 (m, 3H, H 1,2,6 ), 6.41 (d, 1H, J = 16.0 Hz, H 8), 5.91 (d, 1H, J = 8.2 Hz, H 1’ ), 5.44
(dd, 1H, J = 9.5 and 9.4 Hz, H 3’ ), 5.34 (dd, 1H, J = 9.5 and 8.2 Hz, H 2’ ), 5.27 (dd, 1H, J =
10.0 and 9.4 Hz, H 4’ ), 4.42 (dd, 1H, J = 12.6 and 4.2 Hz, H 6a’ ), 4.33 (dd, 1H, J = 12.6 and
2.3 Hz, H 6b’ ), 4.12 (s 2H, OCH 2Cl), 4.05 (d, 1H, J = 14.5 Hz, OC HaHbCl), 4.03-3.99 (m,
5H, 2x OCH 2Cl and H 5’ ), 4.02 (d, 1H, J = 14.5 Hz, OCH aHbCl). Spectral properties were as previously reported. 144
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl 1-O-benzyl p-coumarate (44) 1-O-{(2E)-3-[4-(Benzyloxy)phenyl]prop-2-enoyl}-2,3,4,6-tetrakis-O-(chloroacetyl)-βββ-D- glucopyranose
From 15 (106.9 mg, 0.42 mmol), yielded 158.4 mg (52%) of 44 as a white solid. m.p 126.2-127.7 oC.
Rf (50% EtOAc/X4): 0.74 = -16.99 o (c 1.5, chloroform)
160
Chapter 6: Experimental
1 H NMR: (400 MHz, CDCl 3) δ: 7.71 (d, 1H, J = 16.0 Hz, H 7), 7.49 (app. d, 2H J = 8.8 Hz,
H3,5 ), 7.44-7.32 (m, 5H, ArH), 6.99 (app. d, 2H, J = 8.8 Hz, H 2,6 ), 6.26 (d, 1H, J = 16.0 Hz,
H8), 5.91 (d, 1H, J = 8.2 Hz, H1’ ), 5.44 (dd, 1H, J = 9.5 and 9.5 Hz, H 3’ ), 5.33 (dd, 1H, J =
9.5 and 8.2 Hz, H 2’ ), 5.26 (dd, 1H, J = 9.7 and 9.5 Hz, H 4’ ), 5.10 (s, 2H, CH 2Bn), 4.41 (dd,
1H, J = 12.5 and 4.3 Hz, H 6a’ ), 4.32 (dd, 1H, J = 12.5 and 2.3 Hz, H 6b’ ), 4.12 (app. s, 2H,
OCH 2Cl), 4.04-3.99 (m, 7H, 3x OCH 2Cl and H 5’ ). 13 C NMR: (400 MHz, CDCl 3) δ: 167.1, 167.0, 166.4, 166.3 (4 x O COCH 2Cl), 164.9 (C 9),
161.4 (C 1), 148.1 (C 7), 136.4 (Ar), 130.6 (C 3,5 ), 129.0 (2 x Ar), 128.5 (Ar), 127.7 (2 x Ar),
126.8 (C 4), 115.5 (C 2,6 ), 113.0 (C 8), 91.5 (C 1’ ), 73.9 (C 3’ ), 72.0 (C 5’ ), 71.4 (C 2’ ), 70.3
(CH2Ph), 69.2 (C 4’ ), 62.9 (C 6’ ), 40.9, 40.7, 40.4, 40.3 (4 x O CH2Cl). IR (neat) ν: 2960, 1754, 1149, 1101, 1064, 826, 744. + HRMS calculated for C 30H28 Cl 4O12 [M + Na] 745.0203, found 745.0163.
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl 1-O-benzyl ferulate (45) 1-O-{(2E)-3-[4-(Benzyloxy)-3-methoxyphenyl]prop-2-enoyl}-2,3,4,6-tetrakis-O- (chloroacetyl)-βββ-D-glucopyranose
From 16 (127.0 mg, 0.45 mmol), gave 183.1 mg (54%) of 45 as a white residue. Addition of methanol followed by evaporation under reduced pressure gave a white solid. m.p 55.7- 59.9 oC.
Rf (50% EtOAc/X4): 0.66 = -8.85 o (c 1.1, chloroform) 1 H NMR: (400 MHz, CDCl 3) δ: 7.68 (d, 1H, J = 15.9 Hz, H 7), 7.44-7.42 (m, 2H, ArH),
7.39-7.36 (m, 2H, ArH), 7.33-7.30 (m, 1H, ArH), 7.09-7.04 (m, 2H, H 3,5 ), 6.88 (d, 1H, J =
8.2 Hz, H 6), 6.26 (d, 1H, J = 15.9 Hz, H 8), 5.90 (d, 1H, J = 8.2 Hz, H 1’ ), 5.43 (dd, 1H, J =
9.5 and 9.5 Hz, H 3’ ), 5.33 (dd, 1H, J = 9.5 and 8.2 Hz, H 2’ ), 5.26 (dd, 1H, J = 9.6 and 9.5
Hz, H 4’ ), 5.20 (s, 2H, CH 2Ph), 4.42 (dd, 1H, J = 12.5 and 4.4 Hz, H 6a’ ), 4.32 (dd, 1H, J =
12.5 and 2.3 Hz, H 6b’ ), 4.12 (app. s, 2H, OCH 2Cl), 4.04 and 4.03 (2 x app. s, 2 x 1H, O
CH 2Cl), 4.01 and 4.01 (m, 5H, 2 x OCH 2Cl and H 5’ ), 3.93 (s, 3H, OCH 3).
161
Chapter 6: Experimental
13 C NMR: (400 MHz, CDCl 3) δ: 167.3, 167.1, 166.6, 166.5 (4 x O COCH 2Cl), 165.0 (C 9),
151.3 (C 1), 150.1 (C 2), 148.4 (C 7), 136.6 (Ar), 129.0 (2 x Ar), 128.4 (Ar), 127.5 (2 x Ar),
127.2 (C 4), 123.6 (C 5), 113.5 (C 6), 113.4 (C 8), 110.5 (C 3), 91.6 (C 1’ ), 73.5 (C 3’ ), 72.2 (C 5’ ),
71.1 (C 2’ ), 70.7 ( CH2Bn), 69.3 (C 4’ ), 62.8 (C 6’ ), 56.2 (OCH 3), 40.8, 40.5, 40.5, 40.5 (4 x
OCH 2Cl). IR (neat) ν: 2958, 1754, 1135, 1068, 1000, 792, 697. + HRMS calculated for C 31 H30 Cl 4O13 [M + Na] 775.0309, found 775.0284.
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl p-coumarate (49) 2,3,4,6-Tetrakis-O-(chloroacetyl)-1-O-[(2E)-3-(4-hydroxyphenyl)prop-2-enoyl]-βββ-D- glucopyranose
From 3 (53.5 mg, 0.33 mmol), afforded 60.0 mg (29 %) of a mixture of 49 , and a minor unidentified impurity.
Rf (50% EtOAc/X4): 0.53 1 H NMR: (400 MHz, CDCl 3) δ: 7.70 (d, 1H, 15.8 Hz, H 7), 7.45 (app. d, 2H, J = 8.7 Hz,
H3,5 ), 6.86 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.52 (d, 1H, J = 8.2 Hz, H 1’ ), 4.43-4.30 (m, 2H,
H6a’,6b’ ), 4.12-3.99 (4 x OCH 2Cl). Assignment and identification was based on unobstructed proton shifts, and the known spectrum of the feruloyl analogue ( 50 ).
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl ferulate (50) 2,3,4,6-Tetrakis-O-(chloroacetyl)-1-O-[(2E)-3-(4-hydroxy-3-methoxyphenyl)prop-2- enoyl]-βββ-D-glucopyranose
From 4 (157.4 mg, 0.81 mmol), trituration of the columned product with methanol gave 207.6 mg (39%) of 50 as a white solid. m.p. 157.8-158.7 oC. 162
Chapter 6: Experimental
Rf (50 % EtOAc/X4): 0.48 1 H NMR: (400 MHz, CDCl 3) δ: 7.69 (d, 1H, J = 15.9 Hz, H 7), 7.10 (dd, 1H, J = 8.2 and
1.9 Hz, H 5), 7.04 (d, 1H, J = 1.9 Hz, H 3), 6.93 (d, 1H, J = 8.2 Hz, H 6), 6.25 (d, 1H, J = 15.9
Hz, H 8), 5.90 (d, 1H, J = 8.2 Hz, H 1’ ), 5.43 (dd, 1H, J = 9.6 and 9.5 Hz, H 3’ ), 5.33 (dd, 1H,
J = 9.5 and 8.2 Hz, H 2’ ), 5.26 (dd, 1H, J = 9.7 and 9.6 Hz, H 4’ ), 4.42 (dd, 1H, J = 12.6 and
4.2 Hz, H 6a’ ), 4.32 (dd, 1H, J = 12.6 and 2.4 Hz, H 6b’ ), 4.12 (app. s, 2H, OCH 2Cl), 4.04-
3.99 (m, 7H, 3 x OCH 2Cl and H 5’ ), 3.95 (s, 3H, OCH 3). Spectral properties were as previously reported. 183
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl 1-O-acetyl p-coumarate (54) 1-O-{(2E)-3-[4-(Acetyloxy)phenyl]prop-2-enoyl}-2,3,4,6-tetrakis-O-(chloroacetyl)-βββ-D- glucopyranose
From 19 (133.7 mg, 0.65 mmol) , gave 146.3 mg (40 %) of 54 as a white residue. Addition of methanol followed by evaporation under reduced pressure gave a white solid. m.p 55.6- 58.7 oC.
Rf (50% EtOAc/X4): 0.60 = -11.58 o (c 1.6, chloroform) 1 H NMR: (400 MHz, CDCl 3) δ: 7.74 (d, 1H, J = 16.0 Hz, H 7), 7.56 (app. d, 2H, J = 8.7 Hz,
H3,5), 7.15 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.36 (d, 1H, J = 16.0 Hz, H 8), 5.90 (d, 1H, J = 8.3
Hz, H 1’ ), 5.44 (dd, 1H, J = 9.5 and 9.5 Hz, H 3’ ), 5.33 (dd, 1H, J = 9.5 and 8.3 Hz, H 2’ ), 5.26
(dd, 1H, J = 9.6 and 9.5 Hz, H 4’ ), 4.42 (dd, 1H, J = 12.5 and 4.2 Hz, H 6a’ ), 4.33 (dd, 1H, J
= 12.5 and 2.2 Hz, H 6b’ ), 4.12 (app. s, 2H, OCH 2Cl), 4.04-4.00 (m, 7H, 3 x OCH 2Cl and
H5’ ), 2.32 (s, 3H, OCOCH 3). 13 C NMR: (400 MHz, CDCl 3) δ: 169.2 (O COCH 3), 167.1, 167.0, 166.4, 166.3 (4 x
OCOCH 2Cl), 164.5 (C 9), 152.8 (C 1), 147.0 (C 7), 131.5 (C 4), 129.9 (C 3,5 ), 122.5 (C 2,6 ),
115.9 (C 8), 91.6 (C 1’ ), 72.3 (C 3’ ), 72.1 (C 5’ ), 71.3 (C 2’ ), 69.2 (C 4’ ), 62.8 (C 6’ ), 40.7, 40.4,
40.4, 40.3 (4 x O CH2Cl), 20.8 (OCOCH3). IR (neat) ν: 2959, 1757, 1147, 1069, 1005, 912, 791. + HRMS calculated for C 25 H24 Cl 4O13 [M + Na] 696.9839, found 696.9810. 163
Chapter 6: Experimental
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl 1-O-acetyl ferulate (55) 1-O-{(2E)-3-[4-(Acetyloxy)-3-methoxyphenyl]prop-2-enoyl}-2,3,4,6-tetrakis-O- (chloroacetyl)-βββ-D-glucopyranose
From 20 (132.4 mg, 0.56 mmol), afforded 168.7 mg (44%) of 55. Addition of methanol followed by evaporation under reduced pressure gave a white solid. m.p 59.8-62.3 oC.
Rf (50 % EtOAc/X4): 0.50 = -4.63 o (c 1.1, chloroform) 1 H NMR: (400 MHz, CDCl 3) δ: 7.72 (d, 1H, J = 16.0 Hz, H 7), 7.14 (dd, 1H, J = 8.1 and
1.8 Hz, H 5), 7.11 (d, 1H, J = 1.8 Hz, H 3), 7.08 (d, 1H, J = 8.1 Hz, H 6), 6.35 (d, 1H, J = 16.0
Hz, H 8), 5.90 (d, 1H, J = 8.3 Hz, H 1’ ), 5.43 (dd, 1H, J = 9.6 and 9.5 Hz, H 3’ ), 5.34 (dd, 1H,
J = 9.5 and 8.3 Hz, H 2’ ), 5.26 (dd, 1H, J = 9.7 and 9.6 Hz, H 4’ ), 4.42 (dd, 1H, J = 12.5 and
4.2 Hz, H 6a’ ), 4.33 (dd, 1H, J = 12.5 and 2.2 Hz, H 6b’ ), 4.13 (app. s, 2H, OCH 2Cl), 4.04-
4.00 (m, 7H, 3 x OCH 2Cl and H 5’ ), 3.88 (s, 3H, OCH 3), 2.33 (s, 3H, OCOCH 3). 13 C NMR: (400 MHz, CDCl 3) δ: 168.9 (O COCH 3), 167.1, 167.0, 166.4, 166.3 (4 x
OCOCH 2Cl), 164.4 (C 9), 151.7 (C 1), 147.5 (C 7), 142.3 (C 2), 132.7 (C 4), 123.6 (C 6), 122.0
(C 5), 115.9 (C 8), 111.5 (C 3), 91.5 (C 1’ ), 73.9 (C 3’ ), 72.3 (C 5’ ), 71.2 (C 2’ ), 69.2 (C 4’ ), 62.8
(C 6’ ), 56.1 (OCH 3), 40.7, 40.4, 40.3, 40.3 (4 x O CH2Cl), 20.8 (OCO CH3). IR (neat) ν: 2959, 1756, 1147, 1070, 1004, 791. + HRMS calculated for C 26 H26 Cl 4O14 [M + Na] 726.9945, found 726.9936.
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl 1-O-chloroacetyl p-coumarate (57) 2,3,4,6-Tetrakis-O-(chloroacetyl)-1-O-[(2E)-3-{4-[(chloroacetyl)oxy]phenyl}prop-2- enoyl]-βββ-D-glucopyranose
From 21 (210.6 mg, 0.88 mmol), gave 0.30 g (48%) of 57 as a white honeycomb. m.p 48.0-51.0 oC. 164
Chapter 6: Experimental
Rf (50% EtOAc/X4): 0.59 = -4.60 o (c 0.9, chloroform) 1 H NMR: (400 MHz, CDCl 3) δ: 7.73 (d, 1H, J = 16.0 Hz, H 7), 7.58 (app. d, 2H, J = 8.7 Hz,
H3,5 ), 7.20 (app. d, 2H, J = 8.7 Hz, H 2,6 ), 6.37 (d, 1H, J = 16.0 Hz, H 8), 5.90 (d, 1H, J = 8.2
Hz, H 1’ ), 5.44 (dd, 1H, J = 9.6 and 9.5 Hz, H 3’ ), 5.33 (dd, 1H, J = 9.5 and 8.2 Hz, H 2’ ), 5.26
(dd, 1H, J = 9.7 and 9.6 Hz, H 4’ ), 4.42 (dd, 1H, J = 12.5 and 4.3 Hz, H 6a’ ), 4.33 (m, 3H,
ArOCOCH 2Cl and H 6b’ ), 4.12 (app. s, 2H, OCH 2Cl), 4.04-4.01 (m, 7H, 3 x OCH 2Cl and
H5’ ). 13 C NMR: (400 MHz, CDCl 3) δ: 167.1, 166.9, 166.4, 166.2 (4 x Glc-OCOCH 2Cl), 165.6
(ArO COCH 2Cl), 164.3 (C 9), 152.3 (C 1), 146.7 (C 7), 132.0 (C 4), 129.9 (C 3,5 ), 122.0 (C 2,6 ),
116.4 (C 8), 91.5 (C 1’ ), 73.9 (C 3’ ), 72.2 (C 5’ ), 71.4 (C 2’ ), 69.2 (C 4’ ), 62.8 (C 6’), 40.9
(ArOCOCH2Cl), 40.6, 40.3, 40.3, 40.3 (4 x Glc-OCO CH2Cl). IR (neat) ν: 2958, 1752, 1138, 1069, 1002, 926, 792, 754. + HRMS calculated for C 25 H23 Cl 5O13 [M + Na] 730.9449, found 730.9445.
2,3,4,6-Tetra-O-chloroacetyl-βββ-D-glucopyranosyl 1-O-chloroacetyl ferulate (58) 2,3,4,6-Tetrakis-O-(chloroacetyl)-1-O-[(2E)-3-{4-[(chloroacetyl)oxy]-3- methoxyphenyl}prop-2-enoyl]-βββ-D-glucopyranose
From 22 (224.0 mg, 0.83 mmol), afforded 0.38 g (64%) of 58 as a pale yellow honeycomb. m.p 62.0-64.7 oC.
Rf (40% EtOAc/X4): 0.46 1 H NMR: (400 MHz, CDCl 3) δ: 7.71 (d, 1H, J = 16.0 Hz, H 7), 7.15 (dd, 1H, J = 8.1 and
1.8 Hz, H 5), 7.12 (d, 1H, J = 1.8 Hz, H 3), 7.11 (d, 1H, J = 8.1 Hz, H 6), 6.36 (d, 1H, J = 16.0
Hz, H 8), 5.90 (d, 1H, J = 8.2 Hz, H 1’ ), 5.44 (dd, 1H, J = 9.5 and 9.4 Hz, H 3’ ), 5.33 (dd, 1H,
J = 9.5 and 8.2 Hz, H 2’ ), 5.26 (dd, 1H, J = 9.9 and 9.4 Hz, H 4’ ), 4.42 (dd, 1H, J = 12.5 and
4.3 Hz, H 6a’ ), 4.33 (m, 3H, ArOCH 2Cl and H 6b’ ), 4.12 (app, s. 2H, OCH 2Cl), 4.04-4.01 (m,
7H, 3 x OCH 2Cl and H 5’ ), 3.88 (s, 3H, OCH 3). 13 C NMR: (400 MHz, CDCl 3) δ: 167.1, 166.9, 166.4, 166.3 (4 x Glc-OCOCH 2Cl), 165.2
(ArO COCH 2Cl), 164.3 (C 9), 151.4 (C 1), 147.2 (C 7), 141.6 (C 2), 133.2 (C 4), 123.2 (C 6), 165
Chapter 6: Experimental
121.9 (C 5), 116.4 (C 8), 111.7 (C 3), 91.5 (C 1’ ), 73.9 (C 3’ ), 72.3 (C 5’ ), 71.4 (C 2’ ), 69.2 (C 4’ ),
62.8 (C 6’ ), 56.2 (OCH 3), 40.6-40.3 (5 x OCH 2Cl). + HRMS calculated for C 26 H25 Cl 5O14 [M + Na] 760.9555, found 760.9523. Physical and chemical properties were as previously reported. 183
General procedure for de-chloroacetylation (ambient light) 2,3,4,6-Tetra-O-chloroacetyl-β-D-glucopyranosyl hydroxycinnamate (48 -50) (100.0 mg) was dissolved in pyridine/water (1:1, 10 mL) and stirred at room temperature for 4 hours. The reaction mixture was concentrated and the crude mixture purified using XAD-8 resin
(eluted with 60% MeOH/H 2O) to give a mixture of cis - and trans -β-D-glucopyranosyl hydroxycinnamate as a colourless residue ( 53 , 9, 10 ).
1-O-βββ-D-Glucopyranosyl cinnamate (53) 1-O-(3-Phenylacryloyl)-βββ-D-glucopyranose
From 48 (95.6 mg, 0.16 mmol), gave 10 mg (21%) of 53 as a mixture of cis /trans -isomers.
Rf (20% MeOH/DCM): 0.40 1-O-βββ-D-Glucopyranosyl trans-cinnamate 1-O-[(2E)-3-Phenylprop-2-enoyl]-βββ-D-glucopyranose 1 H NMR: (400 MHz, CD 3OD) δ: 7.81 (d, 1H, J = 16.0 Hz, H 7), 7.64-7.62 (m, 2H, ArH),
7.44-7.40 (m, 3H, ArH), 6.58 (d, 1H, J = 16.0 Hz, H 8), 5.60 (d, 1H, J = 7.7 Hz, H 1’ ), 3.86
(dd, 1H, J = 12.1 and 2.0 Hz, H 6a’ ), 3.70 (dd, 1H, J = 12.1 and 4.8 Hz, H 6b’ ), 3.50-3.35 (m,
4H, H 2’,3’,4’,5’ ). 1-O-βββ-D-Glucopyranosyl cis-cinnamate 1-O-[(2Z)-3-Phenylprop-2-enoyl]-βββ-D-glucopyranose 1 H NMR: (400 MHz, CD 3OD) δ: 7.69-7.66 (m, 2H, ArH), 7.35-7.33 (m, 3H, ArH), 7.10
(d, 1H, J = 12.7 Hz, H 7), 6.02 (d, 1H, J = 12.7 Hz, H 8), 5.54 (d, 1H, J = 8.1 Hz, H 1’ ), 3.87-
3.83 (m, 1H, H 6a’ ), 3.71-3.66 (m, 1H, H 6b’ ), 3.49-3.34 (m, 4H, H 2’,3’,4’,5’ ).
166
Chapter 6: Experimental
The spectrum of each isomer was extracted from the mixture. Spectral properties for the trans -isomer were as previously reported. 247-248 Assignment and identification of the cis - isomer was performed using the known trans -isomer ( trans -53 ) and the data for the cis - aglycone ( cis -47 ). 235
1-O-βββ-D-Glucopyranosyl p-coumarate (9) 1-O-[3-(4-Hydroxyphenyl)acryloyl]-βββ-D-glucopyranose
From 49 (60.0 mg, 0.10 mmol), yielded 10.3 mg (33%) of 9 as a mixture of cis /trans - isomer, as well as 9.6 mg (25%) of 51.
Rf (20% MeOH/DCM): 0.29 1-O-βββ-D-Glucopyranosyl trans-p-coumarate 1-O-[(2E)-3-(4-Hydroxyphenyl)prop-2-enoyl]-βββ-D-glucopyranose 1 H NMR: (400 MHz, CD 3OD) δ: 7.73 (m, 1H, H 7), 7.48 (app. d, 2H, J = 8.5 Hz, H 3,5 ), 6.82
(app. d, 2H, J = 8.5 Hz, H 2,6 ), 6.37 (d, 1H, J = 15.9 Hz, H 8), 5.58 (d, 1H, J = 8.0 Hz, H 1’ ),
3.85 (m, 1H, H 6a’ ), 3.68 (m, 1H, H 6b’ ), 3.47-3.32 (m, 4H, H 2’,3’,4’,5’ ). 1-O-βββ-D-Glucopyranosyl cis-p-coumarate 1-O-[(2Z)-3-(4-Hydroxyphenyl)prop-2-enoyl]-βββ-D-glucopyranose 1 H NMR: (400 MHz, CD 3OD) δ: 7.73 (m, 2H, H 3,5 ), 6.94 (d, 1H, J = 12.9 Hz, H 7), 6.82
(app. d, 2H, J = 8.8 Hz, H 2,6 ), 5.82 (d, 1H, J = 12.9 Hz, H 8), 5.55 (d, 1H, J = 8.0 Hz, H 1’ ),
3.85 (m, 1H, H 6a’), 3.68 (m, 1H, H 6b’ ), 3.47-3.32 (m, 4H, H 2’, 3’, 4’, 5’ ). The spectrum of each isomer was extracted from the mixture. Spectral properties for the trans -isomer were as previously reported. 108, 179 Assignment and identification of the cis - isomer was performed using the known trans -isomer ( trans -9) and the data for the cis - aglycone ( cis -3). 214, 235
167
Chapter 6: Experimental
6-O-Chloroacety-βββ-D-glucopyranosyl trans-p-coumarate (51) 6-O-(Chloroacetyl)-1-O-[(2E)-3-(4-hydroxyphenyl)prop-2-enoyl]-βββ-D-glucopyranose
Isolated during the synthesis of 9.
Rf (20% MeOH/DCM): 0.58 1 H NMR: (400 MHz, CD 3OD) δ: 7.73 (d, 1H, J = 15.9, H 7), 7.49 (app. d, 2H, J = 8.6 Hz,
H3,5 ), 6.82 (app. d, 2H, J = 8.6 Hz, H 2,6 ), 6.37 (d, 1H, J = 15.9 Hz, H 8), 5.55 (d, 1H, J = 7.9
Hz, H 1’ ), 4.49 (dd, 1H, J = 12.0 and 2.2 Hz, H 6a’ ), 4.33 (dd, 1H, J = 12.0 and 5.6 Hz, H 6b’ ),
4.23 (s, 2H, OCH 2Cl), 3.49-3.36 (m, 4H, H 2’,3’,4’,5’).
1-O-βββ-D-Glucopyranosyl ferulate (10) 1-O-[3-(4-Hydroxy-3-methoxyphenyl)acryloyl]-βββ-D-glucopyranose
From 50 (219.3 mg, 0.33 mmol), gave 24.1 mg (20%) of 10 as a mixture of cis /trans - isomer.
Rf (20 % MeOH/DCM): 0.32 βββ-D-Glucopyranosyl trans-ferulate 1-O-[(2E)-3-(4-Hydroxy-3-methoxyphenyl)prop-2-enoyl]-βββ-D-glucopyranose 1 H NMR: (400 MHz, CD 3OD) δ: 7.73 (d, 1H, J = 15.9 Hz, H 7), 7.21 (d, 1H, J = 1.9 Hz,
H3), 7.10 (dd, 1H, J = 8.2 and 1.9 Hz, H 5), 6.82 (d, 1H, J = 8.2 Hz, H 6), 6.41 (d, 1H, J =
15.9 Hz, H8), 5.58 (d, 1H, J = 7.5 Hz, H 1’ ), 3.90 (s, 3H, OCH 3), 3.86 (m, 1H, H 6a’ ), 3.70
(m, 1H, H 6b’ ), 3.49-3.35 (m, 4H, H 2’, 3’,4’ ,5’ ). βββ-D-Glucopyranosyl cis-ferulate 1-O-[(2Z)-3-(4-Hydroxy-3-methoxyphenyl)prop-2-enoyl]-βββ-D-glucopyranose 1 H NMR: (400 MHz, CD 3OD) δ: 7.87 (d, 1H, J = 1.9 Hz, H 3), 7.17 (dd, 1H, J = 8.3 and
1.9 Hz, H 5), 6.94 (d, 1H, J = 13.0 Hz, H 7), 6.77 (d, 1H, J = 8.3 Hz, H 6), 5.83 (d, 1H, J =
168
Chapter 6: Experimental
13.0 Hz, H 8), 5.56 (d, 1H, J = 7.8 Hz, H 1’ ), 3.88 (s, 3H, OCH 3), 3.88-3.84 (m, 1H, H 6a’ ),
3.72-3.66 (m, 1H, H 6b’ ), 3.49-3.35 (m, 4H, H 2’,3’,4’,5’ ). The spectrum of each isomer was extracted from the mixture. Spectral properties for the trans -isomer were as previously reported. 108, 179, 183 Assignment and identification of the cis -isomer was performed using the known trans -isomer ( trans -10 ) and the data for the cis - aglycone ( cis -4). 184, 235
General procedure for de-chloroacetylation (red light) 2,3,4,6-Tetra-O-chloroacetyl-β-D-glucopyranosyl hydroxycinnamate (54, 57 , 58 ) (300.0 mg) was dissolved in pyridine/water (1:1, 20 mL) and stirred at room temperature in the dark for 4 hours (54) or for 6 hours ( 57 and 58 ). Only being exposed to red light, the reaction mixture was concentrated and the crude mixture purified with XAD-8 resin
(eluted with 60% MeOH/H 2O) to give a trans -β-D-glucopyranosyl hydroxycinnamate as a colourless residue.
1-O-βββ-D-Glucopyranosyl 1-O-acetyl p-coumarate (56) 1-O-{(2E)-3-[4-(Acetyloxy)phenyl]prop-2-enoyl}-βββ-D-glucopyranose
From 54 (113.8 mg, 0.17 mmol), gave 12.7 mg (20%) of 56 as a white residue containing minor impurities of 9, as well as 2.3 mg (4%) of 9.
Rf (20 % MeOH/DCM): 0.39 1 H NMR: (400 MHz, CD 3OD) δ: 7.80 (d, 1H, J = 16.0 Hz, H 7), 7.67 (app. d, 2H J = 8.5
Hz, H 3,5 ), 7.17 (app. d, 2H, J = 8.5 Hz, H 2,6 ), 6.56 (d, 1H, J = 16.0 Hz, H 8), 5.59 (d, 1H, J =
8.0 Hz, H 1’ ), 3.86 (dd, 1H, J = 12.1 and 1.9 Hz, H 6a’ ), 3.70 (dd, 1H, J = 12.1 and 4.8 Hz,
H6b’ ), 3.47-3.38 (m, 4H, H 2’,3’,4’,5’ ), 2.29 (s, 3H, OCOCH 3).
169
Chapter 6: Experimental
1-O-βββ-D-Glucopyranosyl trans-p-coumarate (9) 1-O-[(2E)-3-(4-Hydroxyphenyl)prop-2-enoyl]-βββ-D-glucopyranose
From 57 (261.7 mg, 0.37 mmol), gave 51.5 mg (43%) of 9 as a white residue, determined to have undergone acyl migration, largely consisting of the 1-O-β-ester (approx. 80%). The migrated mixture was found to revert back to the 1-O-β-ester after standing in pH 3.5 model wine media.
Rf (20 % MeOH/DCM): 0.29 1 H NMR: (400 MHz, CD 3OD) δ: 7.73 (d, 1H, J = 15.9 Hz, H 7), 7.48 (app. d, 2H, J = 8.5
Hz, H3,5 ), 6.82 (app. d, 2H, J = 8.5 Hz, H 2,6 ), 6.37 (d, 1H, J = 15.9 Hz, H 8), 5.57 (d, 1H, J =
7.9 Hz, H 1’ ), 3.85 (dd, 1H, J = 12.1 and 1.8 Hz, H 6a’ ), 3.69 (dd, 1H, J = 12.1 and 4.6 Hz,
H6b’ ), 3.45-3.38 (m, 4H, H 2’,3’,4’,5’ ). MS (-EI) m/z (%): 325.7 (M -, 100 ), 265.5 ( 7), 187.7 ( 8), 163.4 ( 21 ), 145.2 ( 44 ). Physical and chemical properties for the 1-O-β-ester were as previously reported. 108, 131, 179
1-O-βββ-D-Glucopyranosyl trans-ferulate (10) 1-O-[(2E)-3-(4-Hydroxy-3-methoxyphenyl)prop-2-enoyl]-βββ-D-glucopyranose
From 58 (501.8 mg, 0.68 mmol), 47.5 mg (20%) of 10 as an off-white residue, determined to have undergone acyl migration, largely consisting of the 1-O-β-ester (approx. 90%). The migrated mixture was found to revert back to the 1-O-β-ester after standing in pH 3.5 model wine media.
Rf (20% MeOH/DCM): 0.32 1 H NMR: (400 MHz, CD 3OD) δ: 7.73 (d, 1H, J = 15.9 Hz, H 7), 7.21 (d, 1H, J = 1.9 Hz,
H3), 7.10 (dd, 1H, J = 8.2 and 1.9 Hz, H 5), 6.82 (d, 1H, J = 8.2 Hz, H 6), 6.41 (d, 1H, J =
15.9 Hz, H 8), 5.58 (d, 1H, J = 7.5 Hz, H 1’ ), 3.90 (s, 3H, OCH 3), 3.86 (dd, 1H, J = 12.1 and
2.1 Hz, H 6a’ ), 3.70 (dd, 1H, J = 12.1 and 4.5 Hz, H 6b’ ), 3.49-3.35 (m, 4H, H 2’,3’,4’,5’ ). 170
Chapter 6: Experimental
MS (-EI) m/z (%): 355.3 (M -, 100 ), 295.5 ( 8), 217.2 ( 20), 193.6 ( 25), 175.4 ( 32 ). Physical and chemical properties for the 1-O-β-ester were as previously reported. 108, 179, 183, 200
171
Chapter 6: Experimental
6.3 Experimental Procedures for Chapter 3.
Theoretical studies into the thermodynamics of glucose ester migration The ten possible glucose esters (1/2/3/4/6-O-α/β-) were drawn for both p-coumaroyl glucose ( 9) and feruloyl glucose ( 10 ) using the equilibrium geometry optimised in four different solvents (water, dichloromethane, ethanol and toluene), and the energies given in Hartrees (a.u.) were converted to kJ/mol using a factor of 2625.5. The final ester energies were calculated relative to the 1-O-β-ester in each case. The raw data is displayed in Appendix 1.
Theoretical studies into the kinetics of glucose ester migration The four intermediates for each migration were drawn and the equilibrium geometry optimised for three different conditions (vacuum, water and dichloromethane). The energies calculated were converted to kJ/mol and expressed relative to intermediate 1. The raw data is displayed in Appendix 2.
Wine samples for analysis One white wine (Stanley Classic Dry White) and one red wine (Yalumba 1997 Shiraz) were extracted with and without spikes of p-coumaroyl and feruloyl glucose (5 mg/L). Liquid-liquid extraction was performed with 50 mL of wine, extracting alternatively with diethyl ether (3 x 50 mL) and ethyl acetate (3 x 50 mL) before being concentrated under reduced pressure at 30 oC and taken up in methanol (2 mL). Solid-phase extraction was performed using XAD-8 resin, where 50 mL of wine was loaded, washed with water, eluted with 25%, 50%, 75% methanol in water and then 100% methanol, fractions were individually concentrated and taken up in methanol (2 mL). Concentrated wine samples were prepared from 50 mL of wine at 30 oC under reduced pressure until the volume had reduced to 5 mL. Neat wine samples were passed through a 45 µm syringe filter and analysed directly. Standards of p-coumaroyl glucose ( 9) and feruloyl glucose (10 ) were prepared to determine retention times and response factors (in methanol 10 mg/L, 100 mg/L), extraction efficiencies were estimated using the prepared 3 point calibration curve, along with the 2 point curves produced by analysing spiked and unspiked wine samples. 172
Chapter 6: Experimental
HPLC analysis of wine samples Analyses were performed on an Agilent 1100 instrument (Agilent, Forest Hill, Vic, Australia) equipped with a quaternary pump and diode array detector (DAD). The column was a 250 x 4.6 mm, 3 µm, 100 Å Luna C18, operated at 25 ºC and protected by a C18 guard column (4 x 2 mm) (Phenomenex, Lane Cove, NSW, Australia). The eluents were formic acid/water (0.5:99.5 v/v, Eluent A), formic acid/acetonitrile/water (0.5:25.0:74.5 v/v, Eluent B) and methanol (Eluent C) with a flow rate of 1 mL/min. A gradient was applied as follows: 20% to 30% B linear from 0 to 20 minutes; 30% to 50% B linear from 20 to 50 minutes; 50% B to 100% C linear from 50 to 60 minutes; 100% C to 20% B from 60 to 65 minutes. The column was equilibrated with 20% B for 10 minutes prior to an injection. A 20 µL injection volume was used for each sample and DAD signals were recorded at all available wavelengths for compound identification, and quantified using 280 and 320 nm. Compounds in each sample were identified by comparison of their retention times and UV/Vis spectra with those of authentic standards.
LCMS analysis of wine samples HPLC-MS or MS/MS analysis was carried out using a 4000 Q TRAP hybrid tandem mass spectrometer interfaced with a Turbo V ion source for elecrospray ionization (AB Sciex AB Sciex, Foster City, CA), combined with an Agilent 1200 HPLC system equipped with a binary pump, degasser, autosampler, column oven, and photodiode array (PDA) detector.
HPLC conditions: A 10 µL aliquot of the samples was injected and chromatographed using the same column and elution profile as described for HPLC, above. The column temperature was maintained at 25˚C during the HPLC run. The eluent from the HPLC was split by use of a splitter (a tee) and delivered at a follow rate of 0.45 mL/min to the mass spectrometer and at 0.55 mL/min to the PDA detector with monitoring wavelengths at 290, 320 and 370 nm with a slit width of 4 and a bandwidth of 16 nm.
Electrospray and mass spectrometric conditions: All mass spectrometric data were obtained in negative ion mode. Nitrogen gas was used for the curtain, nebulizer, turbo and collision gases. The Turbo V ion source parameter were set at -3500 V for the ion spray potential, -60 V for the declustering potential, -10 V 173
Chapter 6: Experimental for the entrance potential, 50 psi for gas 1 (nebulizer) and gas 2 (turbo), 15 psi for the curtain gas, and 500 °C for the turbo gas (gas 2) temperature.
For tandem mass spectrometry, the collision potential was set in an appropriate range from -15 to -25 V and the collision gas pressure was set at high. Product ion spectra of m/z 325 for p-coumaroyl glucose and m/z 355 for feruloyl glucose were recorded in a mass range from m/z 50 to 400 with a scan time of 1 s and a step mass of 0.1. For selected reaction monitoring, the following mass transitions were monitored with a dwell time of 50 ms; m/z 325 119, 145, 163 and 187 for p-coumaroyl glucose, and m/z 355 119, 175, 193 and 217 for feruloyl glucose.
174
Chapter 6: Experimental
6.4 Experimental Procedures for Chapter 4.
cis-Ethyl coumarate (cis-11) Ethyl (2Z)-3-(4-hydroxyphenyl)prop-2-enoate
cis -Ethyl coumarate ( cis -11 ) was achieved as a minor product from the aforementioned Wittig reaction (Synthesis of 11 , Chapter 6.2.1), or by irradiation under ultra-violet light (365 nm), whereby isolation using column chromatography (10% EtOAc/X4) yielded a white solid. m.p. 72.3-74.4 oC (lit. m.p.73-74 oC). 185
Rf (50% EtOAc/X4): 0.51 1 H NMR: (400 MHz, CDCl 3) δ: 7.63 (app. d, 2H, J = 8.6 Hz, H 3,5 ), 6.85 (d, 1H, J = 12.7
Hz, H 7), 6.80 (app. d, 2H, J = 8.6 Hz, H 2,6 ), 5.83 (d, 1H, J = 12.7 Hz, H 8), 4.21 (q, 2H, J =
7.1 Hz, O CH 2CH 3), 1.29 (t, 3H, J = 7.1 Hz, OCH 2CH 3). All physical and chemical properties were as previously reported. 185
cis-Ethyl ferulate (cis-12) Ethyl (2Z)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate
cis -Ethyl ferulate ( cis -12 ) was achieved as a minor product from the aforementioned Wittig reaction (Synthesis of 12, Chapter 6.2.1), or by irradiation under ultra-violet light (365 nm), whereby isolation using column chromatography (10% EtOAc/X4) yielded a colourless oil.
Rf (50% EtOAc/X4): 0.55 1 H NMR: (400 MHz, CDCl 3) δ: 7.77 (d, 1H, J = 1.9 Hz, H 3), 7.11 (dd, 1H, J = 8.5 and 1.9
Hz, H 5), 6.88 (d, 1H, J = 8.5 Hz, H 6), 6.79 (d, 1H, J = 12.9 Hz, H 7), 5.81 (d, 1H, J = 12.9
Hz, H 8), 4.21 (q, 2H, J = 7.1 Hz, O CH 2CH 3), 3.92 (s, 3H, OCH 3), 1.29 (t, 3H, J = 7.1 Hz,
OCH 2CH 3). All physical and chemical properties were as previously reported. 185
175
Chapter 6: Experimental cis-p-Coumaric acid (cis-3) (2Z)-3-(4-Hydroxyphenyl)prop-2-enoic acid
cis -Ethyl coumarate ( cis -11 ) (0.11 g, 0.59 mmol) was dissolved in 1:1 aqueous ethanol (v/v, 10 mL) followed by the addition of potassium hydroxide (0.10 g, 1.78 mmol), then the reaction mixture was stirred at room temperature for 3 days. The mixture was then diluted with water (5 mL), unwanted organics extracted with diethyl ether (2 x 10 mL), the aqueous layer acidified to pH 3 with 2 M hydrochloric acid solution and extracted with ethyl acetate (2 x 10 mL). Concentration at reduced pressure gave 97 mg (99%) of a 80:20 mixture of trans - and cis -p-coumaric acid as an off-white solid. 1 H NMR: (400 MHz, DMSO-d6) δ: 9.55 (br. s, 0.80H, trans -COOH), 9.84 (br. s, 0.20H, cis -COOH), 7.63 (app. d, 0.40H, J = 8.6 Hz, cis -H3,5 ), 7.51 (app. d, 1.60H, J = 8.6 Hz, trans -H3,5 ), 7.49 (d, 0.80H, J = 15.9 Hz, trans -H7), 6.79 (app. d, 1.60H, J = 8.6 Hz, trans -
H2,6), 6.78-6.73 (m, 0.60H, cis -H2,6,7 ), 6.28 (d, 0.80H, J = 15.9 Hz, trans -H8), 5.72 (d,
0.20H, J = 12.8 Hz, cis -H8). Spectral properties were as previously reported. 214, 235
cis-p-Coumaric acid by UV irradiation trans -p-Coumaric acid (3) was dissolved in acetone and exposed to UV light at 365 nm for 4 days. The resulting mixture of isomers was concentrated and analysed by NMR to show a trans :cis -ratio of approximately 61:39.
cis-Ferulic acid (cis-4) (2Z)-3-(4-Hydroxy-3-methoxyphenyl)prop-2-enoic acid
Using the same procedure as described above (for cis -3), reaction of cis -ethyl ferulate ( cis - 12 ) (0.31 g, 1.39 mmol) gave 268 mg (99%) of a 65:35 mixture of trans - and cis -ferulic acid as a yellow solid.
176
Chapter 6: Experimental
1 H NMR: (400 MHz, DMSO-d6) δ: 9.55 (br. s, 0.65H, trans -COOH), 9.46 (br. s, 0.35H, cis -COOH), 7.65 (d, 0.35H, J = 2.0 Hz, cis -H3), 7.48 (d, 0.65H, J = 15.9 Hz, trans -H7),
7.27 (d, 0.65H, J = 1.9 Hz, trans -H3), 7.15 (dd, 0.35H, J = 8.3 and 2.0 Hz, cis -H5), 7.08
(dd, 0.65H, J = 8.2 and 1.9 Hz, trans -H5), 6.79 (d, 0.65H, J = 8.2 Hz, trans -H6), 6.75 (d,
0.35H, J = 12.9 Hz, cis -H7), 6.75 (d, 0.35H, J = 8.2 Hz, cis -H6), 6.36 (d, 0.65H, J = 15.9
Hz, trans -H8), 5.73 (d, 0.35H, J = 12.9 Hz, cis -H8), 3.81 (s, 1.95H, trans -OCH 3), 3.75 (s,
1.05H, cis -OCH 3). Spectral properties were as previously reported. 184, 235
cis-Ferulic acid by UV irradiation trans -Ferulic acid ( 4) was dissolved in acetone and exposed to UV light at 365 nm for 4 days. The resulting mixture of isomers was concentrated and analysed by NMR to show a trans :cis -ratio of approximately 50:50.
Stability of cis/trans-p-coumaric acid mixture Portions of cis/trans-p-coumaric acid ( 3) (61:39 cis:trans ratio, 5 mg) either as a solid, or dissolved in acetone (5 mL) were stored under four different conditions. Both a solid and liquid sample was stored at ambient temperature in the dark, at ambient temperature under ambient light conditions, at 4 oC or at -20 oC. Samples stored at 4 or -20 oC were not exposed to light. After two weeks of storage, liquid samples were concentrated in vacuo and all samples were analysed by 1H NMR and the isomeric ratio determined by integration of the signals. The ratios determined are shown in Table 4.2.
Effect of group 10 metals on ethyl coumarate isomerisation Experiment 1: Into four separate flasks were placed: 1) trans -Ethyl coumarate ( 11 ) (60.3 mg, 0.31 mmol) and dichloromethane (3 mL). 2) trans -Ethyl coumarate ( 11 ) (56.0 mg, 0.29 mmol), dichloromethane (3 mL) and 10% palladium on activated carbon (15.7 mg, 0.015 mmol of Pd, 5% by moles). 3) trans -Ethyl coumarate ( 11 ) (42.4 mg, 0.22 mmol), dichloromethane (3 mL) and 10% palladium on activated carbon (311.9 mg, 0.29 mmol of Pd, 132% by moles). 177
Chapter 6: Experimental
4) trans -Ethyl coumarate ( 11 ) (31.0 mg, 0.16 mmol), dichloromethane (3 mL) and 10% platinum on activated carbon (4.9 mg, 0.002 mmol of Pt, 1.6% by moles).
The four mixtures were irradiated under 365 nm light for 18 hours before being filtered through celite and concentrated in vacuo . Analysis of the crude mixtures by 1H NMR allowed for determination of the isomeric ratios by integration, which existed as follows: 1) 68:32, trans:cis -ethyl coumarate. 2) 74:26, trans:cis -ethyl coumarate. 3) 100 % trans -ethyl coumarate. 4) 70:30, trans:cis -ethyl coumarate.
Experiment 2: Into two separate flasks were placed: 1) cis -Ethyl coumarate ( cis -11 ) (12.8 mg, 0.07 mmol) and dichloromethane (4 mL). 2) cis -Ethyl coumarate ( cis -11 ) (10.3 mg, 0.05 mmol), dichloromethane (4 mL) and palladium acetate (7.8 mg, 0.03 mmol of Pd).
Both mixtures were stirred at ambient temperature under ambient light conditions for 48 hours before being concentrated in vacuo and subjected to analysis by 1H NMR. Analysis of mixture 1 indicated minor conversion from cis - to trans -ethyl coumarate while mixture 2 contained only trans -ethyl coumarate.
Theoretical studies into the photoisomerisation of hydroxycinnamates
Energy profiles were initially calculated in a vacuum at the S 0 and T 1 state using either a dynamic dihedral constraint between 180 and 0 o with the geometry optimised every 10 o, or by manually constraining the dihedral in 10 o increments with the geometry optimised for the resulting 19 structures. Energies were calculated in Hartrees and converted to kJ/mol, o with the energy displayed relative to the S 0 180 dihedral geometry. Solvated energy profiles were produced in an analogous fashion optimising the geometries in water. The energy profile geometries were initially calculated from the MMFF geometry, then from the MMFF conformer. The raw data is displayed in Appendix 3.
178
Chapter 6: Experimental
Vertical excitation energies were calculated by optimising the unconstrained trans - configuration, followed by single-point energy calculation at the T 1 level. HOMO-LUMO gaps were calculated by comparing the orbitals in the S 0 state. The raw data is displayed in Appendix 4.
179
Chapter 6: Experimental
6.5 Experimental Procedures for Chapter 5.
6.5.1 General Procedures for Chapter 5 Media for yeast growth YPD – Yeast extract (1% w/v), peptone (2% w/v), D-glucose (2% w/v) in Milli-Q water was autoclaved and stored at room temperature. YNB – US Biologicals Yeast Nitrogen Base in Milli-Q water (6.76 g/L) was supplemented with glucose (20 g/L), the pH adjusted to 3.5 with 10% HCl solution, sterile filtered (Stericap TM PLUS 0.22 µm) and stored at room temperature.
Starter cultures Yeasts were obtained from the AWRI culture collection on MYPG plates, transferred to YPD broth and stored at 28 oC with constant shaking (150 r.p.m.) until the cell count surpassed 1 x 10 8 cells/mL as determined by haemocytometry.
Fermentation experiments Fermentations were performed in triplicate in 250 mL fermentation flasks equipped with a gas-lock. YNB media (200 mL) was spiked with the specified compound and inoculated with 1 x 10 6 cells/mL of yeast from a starter culture. Where specified, control flasks containing media and the spike were established in the same manner, without inoculation, and all experiments were conducted at 28 oC. The experiments were concluded after the yeast stationary phase, as determined by the optical density.
Ferment sampling An aliquot of 5 mL was taken from each ferment and from this the yeast growth was determined. The remaining sample was centrifuged (4000 r.p.m. for 5 minutes at 25 oC), the supernatant decanted from the yeast pellet, and stored at -20 oC until required for analysis. Sampling was initially performed every two days, with additional samples taken near the completion of fermentation (as the results specify).
180
Chapter 6: Experimental
Yeast growth Was determined from fermentation samples by optical density as measured with a Beckman Coulter DU 530 Life Sciences UV/Vis Spectrophotometer. Aliquots (100 µL) were diluted with water (10 x) and the yeast growth determined by measuring the absorbance at 600 nm using water as a blank.
Model wine A saturated solution of potassium hydrogen tartrate in demineralised water was acidified to pH 3.5 with tartaric acid, stored at 4 oC overnight, then decanted from the precipitate.
4-Ethylphenol / 4-ethylguaiacol analysis Analysis of 4-ethylphenol and 4-ethylguaiacol was performed as described by Pollnitz et al. 63 The concentration of 4-ethylphenol and 4-ethylguaiacol in the fermentation samples were measured in µg/L and the percentage conversion calculated using a full molar conversion from substrate to ethylphenol. The average percentage conversion across the three replicates is displayed in Appendix 5, which was used to produce figures as shown in Chapter 5.
Results for analysis of uninoculated controls are not shown, but all were found to contain no traces of ethylphenols.
181
Chapter 6: Experimental cis / trans-Ferulic acid analysis An unpublished HPLC method developed by the AWRI was utilised, with the solvent parameters altered to achieve maximum resolution of isomers.
Analyses were performed on an Agilent 1100 instrument (Agilent, Forest Hill, VIC, Australia) equipped with a quaternary pump and diode array detector (DAD). The column consisted of a mixed-mode RP/weak anion exchange (WAX) stationary phase based on N- (10-undecenoyl)-3-aminoquinuclidine, bonded to thiol-functionalised silica (150 x 2 mm, 5
µm, 100 Å VDS Optilab, Berlin, Germany), operated at 25 ºC and protected by an NH 2 guard column (4 x 2 mm) (Phenomenex, Lane Cove, NSW, Australia). The eluents were formic acid/water (0.1:99.9 v/v, Eluent A), and formic acid/acetonitrile (0.1:99.9 v/v, Eluent B) with a flow rate of 0.400 mL/min. Isocratic elution was performed using 70% A and 30% B with run time of 7 minutes. A 50% aqueous acetonitrile solution wash was used as the column wash eluent. A 20 µL injection volume was used for each sample and DAD signals were recorded at 280, 320, 353, 370 and 520 nm. Compounds in each sample were identified by comparison of their retention times and UV/Vis spectra with those of standards, with quantifications calculated using absorbance at 280 and 320 nm only. trans -Ferulic acid was dissolved in ethanol and serial dilutions were prepared (75 mg/L, 62.5 mg/L, 50 mg/L, 25 mg/L, 10 mg/L, 5 mg/L and 1 mg/L). The produced trans -ferulic acid calibration curve was used to quantify trans -ferulic acid in prepared isomeric mixtures of cis /trans -ferulic acid (50:50 cis /trans -ferulic acid, concentrations used as above, equating to 37.5 mg/L, 31.25 mg/L, 25 mg/L, 12.5 mg/L, 5 mg/L, 2.5 mg/L and 0.5 mg/L). The calibration was checked by running cis /trans -mixtures of known concentration and ratio (25:75 cis /trans -ferulic acid, 10 mg/L, 20 mg/L; 12.5:87.5 cis /trans -ferulic acid, 10 mg/L, 20 mg/L).
cis / trans-Ethyl hydroxycinnamate analysis Analysis of trans -ethyl ferulate and trans -ethyl coumarate was performed using the method described by Sleep. 141, 249 Quantification of cis -ethyl coumarate and cis -ethyl ferulate was achieved by determining the differences in extraction efficiency and mass spectral responses from the trans -isomers through extracting and analysing mixtures of known concentration and ratio (1:1, 5 mg/L and 10 mg/L). 182
Chapter 6: Experimental
Rapid GCMS quantifications were performed using the same method, quantified using a 4 point calibration curve of the trans -internal standard and cis /trans -ethyl esters (0 mg/L, 5 mg/L, 10 mg/L and 15 mg/L).
HPLC quantifications of the trans -isomers were performed using the same chromatographic conditions described in Chapter 6.3 for analysis of wine samples, employing external standards of trans -ethyl ferulate and trans -ethyl coumarate prepared at 5, 10, 15 and 20 mg/L. The produced calibration curve was used to quantify trans -ethyl ferulate and trans -ethyl coumarate in the fermentation samples, which were syringe filtered (0.45 µm) and analysed without dilution.
183
Chapter 6: Experimental
6.5.2 Fermentation of trans -Hydroxycinnamate Esters Fermentation of ethyl hydroxycinnamates Stock solutions of ethyl coumarate (1 mg/mL) and ethyl ferulate (1 mg/mL) were made up in ethanol, and the fermentations spiked at 10 mg/L. These were inoculated with AWRI 1499, and along with uninoculated controls, were stored and mixed manually at each sampling point. Substrate selectivity was tested in a similar manner, inoculating with either AWRI 1499, AWRI 1608 or AWRI 1613 and stored with occasional shaking.
Fermentation of hydroxycinnamoyl tartrate esters Stock solutions of p-coumaroyl tartrate (1 mg/mL) and feruloyl tartrate (1 mg/mL) were made up in ethanol, and the fermentations spiked at 10 mg/L. These were inoculated with AWRI 1499, and along with uninoculated controls, were stored with constant shaking. Hydroxycinnamoyl tartrate ester strain dependence was performed in a similar manner, with fermentations inoculated with either AWRI 1499, AWRI 1608 or AWRI 1613 and stored with constant shaking.
Fermentation of hydroxycinnamoyl glucose esters Stock solutions of p-coumaroyl glucose (1.03 mg/mL) and feruloyl glucose (0.95 mg/mL) were made up in ethanol under red light and stored in the dark. The fermentations were spiked at 10 mg/L and inoculated with AWRI 1499. These, along with uninoculated controls, were covered in foil to maintain isomeric purity, stored and mixed manually at each sampling point with.
6.5.3 Stereoselectivity of D. bruxellensis Enzyme Activities Decarboxylase stereoselectivity Stock solutions of trans -ferulic acid (5 mg/mL), cis /trans -ferulic acid (50:50 ratio, 5 mg/mL), trans -p-coumaric acid (5 mg/mL) and cis /trans -p-coumaric acid (39:61 ratio, 5 mg/mL) were made up in ethanol. Ferulate and p-coumarate fermentations were performed separately, being spiked with either trans -acid (50 mg/L) or cis /trans -acid (50 mg/L), inoculated with AWRI 1499, stored and mixed manually at each sampling point.
184
Chapter 6: Experimental
NMR spectra of uninoculated flasks containing cis /trans -acid (50 mg/L) were used to monitor the isomeric ratio over the fermentation period. NMR samples were produced by extraction of the entire 200 mL ferment with ethyl acetate (3 x 50 mL), then removal of the solvent by rotary evaporation. The ratio was determined by the integration of either the H 8 or the H 3 signals.
Ethyl esterase stereoselectivity Stock solutions cis -ethyl coumarate (1 mg/mL) and cis -ethyl ferulate (1 mg/mL) were made up in ethanol under red light and stored in the dark. The fermentations were spiked with both esters at 10 mg/L, inoculated with AWRI 1499, wrapped in alfoil to maintain isomeric purity and along with uninocluated controls, were stored and mixed manually at each sampling point. Samples for analysis were taken under red light.
185
Appendix 1: Data for Migration Thermodynamics
Appendix 1: Data for Migration Thermodynamics.
Table A1.1: Energies of p-coumaroyl and feruloyl glucose esters in water, using DFT B3LYP 6-31G*. p -Coumaroyl glucose Feruloyl glucose Hartrees (a.u.) kJ/mol Relative to 1- O -βββ Hartrees (a.u.) kJ/mol Relative to 1- O -βββ 1- O -βββ -1184.20519 -3109130.3 0.0 -1298.72015 -3409789.3 0.0 1- O -ααα -1184.18255 -3109070.8 59.4 -1298.70484 -3409749.1 40.2 2- O -βββ -1184.19626 -3109106.8 23.4 -1298.71136 -3409766.2 23.1 2- O -ααα -1184.20248 -3109123.2 7.1 -1298.71770 -3409782.8 6.4 3- O -βββ -1184.19781 -3109110.9 19.4 -1298.71293 -3409770.3 19.0 3- O -ααα -1184.19715 -3109109.2 21.1 -1298.71205 -3409768.0 21.3 4- O -βββ -1184.19503 -3109103.6 26.7 -1298.71037 -3409763.6 25.7 4- O -ααα -1184.19211 -3109095.9 34.3 -1298.71263 -3409769.5 19.7 6- O -βββ -1184.19020 -3109090.9 39.4 -1298.70643 -3409753.2 36.0 6- O -ααα -1184.20156 -3109120.7 9.5 -1298.71677 -3409780.4 8.9
Table A1.2: Energies of p-coumaroyl and feruloyl glucose esters in dichloromethane, using DFT B3LYP 6-31G*.
p -Coumaroyl glucose Feruloyl glucose Hartrees (a.u.) kJ/mol Relative to 1- O -βββ Hartrees (a.u.) kJ/mol Relative to 1- O -βββ 1- O -βββ -1184.20123 -3109119.9 0.0 -1298.71642 -3409779.5 0.0 1- O -ααα -1184.18389 -3109074.4 45.5 -1298.70860 -3409758.9 20.5 2- O -βββ -1184.19515 -3109103.9 16.0 -1298.71055 -3409764.1 15.4 2- O -ααα -1184.20189 -3109121.6 -1.7 -1298.71734 -3409781.9 -2.4 3- O -βββ -1184.19669 -3109108.0 11.9 -1298.71201 -3409767.9 11.6 3- O -ααα -1184.19673 -3109108.1 11.8 -1298.71190 -3409767.6 11.9 4- O -βββ -1184.19374 -3109100.2 19.7 -1298.70910 -3409760.2 19.2 4- O -ααα -1184.19117 -3109093.5 26.4 -1298.71229 -3409768.6 10.8 6- O -βββ -1184.19349 -3109099.6 20.3 -1298.70875 -3409759.3 20.1 6- O -ααα -1184.20361 -3109126.1 -6.2 -1298.71927 -3409786.9 -7.5
186
Appendix 1: Data for Migration Thermodynamics
Table A1.3: Energies of p-coumaroyl and feruloyl glucose esters in ethanol, using DFT B3LYP 6-31G*.
p -Coumaroyl glucose Feruloyl glucose Hartrees (a.u.) kJ/mol Relative to 1- O -βββ Hartrees (a.u.) kJ/mol Relative to 1- O -βββ 1- O -βββ -1184.21194 -3109148.0 0.0 -1298.72783 -3409809.4 0.0 1- O -ααα -1184.19874 -3109113.3 34.7 -1298.72248 -3409795.4 14.0 2- O -βββ -1184.20851 -3109139.0 9.0 -1298.72467 -3409801.1 8.3 2- O -ααα -1184.21514 -3109156.4 -8.4 -1298.73133 -3409818.6 -9.2 3- O -βββ -1184.21175 -3109147.5 0.5 -1298.72781 -3409809.4 0.1 3- O -ααα -1184.21154 -3109146.9 1.1 -1298.72747 -3409808.5 0.9 4- O -βββ -1184.20785 -3109137.3 10.7 -1298.72393 -3409799.2 10.2 4- O -ααα -1184.20617 -3109132.8 15.1 -1298.72677 -3409806.6 2.8 6- O -βββ -1184.20733 -3109135.9 12.1 -1298.72339 -3409797.8 11.7 6- O -ααα -1184.21677 -3109160.7 -12.7 -1298.73306 -3409823.2 -13.7
Table A1.4: Energies of p-coumaroyl and feruloyl glucose esters in toluene, using DFT B3LYP 6-31G*. p -Coumaroyl glucose Feruloyl glucose Hartrees (a.u.) kJ/mol Relative to 1- O -βββ Hartrees (a.u.) kJ/mol Relative to 1- O -βββ 1- O -βββ -1184.20094 -3109119.1 0.0 -1298.71557 -3409777.2 0.0 1- O -ααα -1184.18122 -3109067.3 51.8 -1298.70599 -3409752.1 25.2 2- O -βββ -1184.19381 -3109100.4 18.7 -1298.70870 -3409759.2 18.0 2- O -ααα -1184.20068 -3109118.4 0.7 -1298.71557 -3409777.2 0.0 3- O -βββ -1184.19488 -3109103.2 15.9 -1298.70959 -3409761.5 15.7 3- O -ααα -1184.19458 -3109102.4 16.7 -1298.70918 -3409760.5 16.8 4- O -βββ -1184.19246 -3109096.9 22.3 -1298.70733 -3409755.6 21.6 4- O -ααα -1184.18910 -3109088.0 31.1 -1298.71038 -3409763.6 13.6 6- O -βββ -1184.19104 -3109093.1 26.0 -1298.70580 -3409751.6 25.7 6- O -ααα -1184.20182 -3109121.4 -2.3 -1298.71679 -3409780.4 -3.2
Table A1.5: Energies of p-coumaroyl glucose esters in different solvents, relative to the 1- O-α-ester.
Energy (kJ/mol) relatve to the 1- O -ααα -ester Water Dichloromethane Ethanol Toluene St. Dev. 1- O -βββ -59.44 -45.53 -34.66 -51.77 10.48 2- O -βββ -36.00 -29.56 -25.65 -33.06 4.47 2- O -ααα -52.33 -47.26 -43.06 -51.09 4.18 3- O -βββ -40.07 -33.61 -34.16 -35.86 2.92 3- O -ααα -38.33 -33.71 -33.61 -35.08 2.20 4- O -βββ -32.77 -25.86 -23.92 -29.51 3.93 4- O -ααα -25.10 -19.11 -19.51 -20.69 2.75 6- O -βββ -20.09 -25.20 -22.55 -25.78 2.62 6- O -ααα -49.91 -51.77 -47.34 -54.09 2.86
187
Appendix 2: Data for Migration Kinetics
Appendix 2: Data for Migration Kinetics.
188
Appendix 2: Data for Migration Kinetics
189
Appendix 3: Data for Energy Profiles
Appendix 3: Data for Energy Profiles.
Table A3.1: Data for p-coumaric acid energy profile in a vacuum.
S0 T1 o o Dihedral Angle Hartrees (a.u.) kJ/mol Relative to S 0 180 Hartrees (a.u.) kJ/mol Relative to S 0 180 180 -573.429091 -1505537.9 0.0 -573.341720 -1505308.5 229.4 170 -573.428809 -1505537.1 0.7 -573.342894 -1505311.6 226.3 160 -573.427103 -1505532.6 5.2 -573.344340 -1505315.3 222.5 150 -573.424026 -1505524.6 13.3 -573.345986 -1505319.7 218.2 140 -573.419539 -1505512.8 25.1 -573.347703 -1505324.2 213.7 130 -573.413554 -1505497.1 40.8 -573.349252 -1505328.2 209.6 120 -573.406317 -1505478.1 59.8 -573.350502 -1505331.5 206.3 110 -573.397875 -1505455.9 82.0 -573.351420 -1505333.9 203.9 100 -573.388198 -1505430.5 107.4 -573.351946 -1505335.3 202.5 90 -573.372181 -1505388.4 149.4 -573.352043 -1505335.6 202.3 80 -573.383155 -1505417.3 120.6 -573.352070 -1505335.6 202.2 70 -573.393105 -1505443.4 94.5 -573.351687 -1505334.6 203.2 60 -573.401892 -1505466.4 71.4 -573.350894 -1505332.6 205.3 50 -573.409397 -1505486.2 51.7 -573.349653 -1505329.3 208.6 40 -573.415538 -1505502.3 35.6 -573.347991 -1505324.9 212.9 30 -573.420179 -1505514.5 23.4 -573.345932 -1505319.5 218.3 20 -573.423281 -1505522.6 15.3 -573.343416 -1505312.9 224.9 10 -573.424826 -1505526.7 11.2 -573.340433 -1505305.1 232.8 0 -573.424840 -1505526.7 11.2 -573.337018 -1505296.1 241.7
Table A3.2: Data for ethyl coumarate energy profile in a vacuum.
S0 Dynamic Forwards S0 Manual S0 Dynamic Backwards T1 o o o o Dihedral Angle kJ/mol Relative to S 0 180 kJ/mol Relative to S 0 180 kJ/mol Relative to S 0 180 kJ/mol Relative to S 0 180 180 -1712001.0 0.0 -1711999.3 0.0 -1711770.6 230.4 170 -1711998.9 2.1 -1711997.5 1.8 -1711770.5 230.4 160 -1711993.2 7.8 -1711992.0 7.3 -1711771.0 229.9 150 -1711983.7 17.3 -1711982.9 16.3 -1711772.1 228.9 140 -1711970.2 30.8 -1711970.2 29.1 -1711774.1 226.9 130 -1711952.9 48.0 -1711953.8 45.5 -1711776.5 224.5 120 -1711932.4 68.6 -1711933.9 65.4 -1711778.6 222.4 110 -1711908.4 92.6 -1711910.7 88.6 -1711780.5 220.5 100 -1711881.6 119.4 -1711884.3 115.0 -1711781.6 219.4 90 -1711852.3 148.6 -1711855.1 144.2 -1711835.2 165.8 -1711782.1 218.9 80 -1711820.9 180.1 -1711822.6 176.7 -1711862.8 138.2 -1711781.5 219.5 70 -1711787.8 213.2 -1711787.6 211.6 -1711887.6 113.3 -1711780.1 220.9 60 -1711750.4 248.9 -1711908.2 92.8 -1711777.9 223.0 50 -1711924.3 76.7 -1711774.6 226.4 40 -1711958.0 41.3 -1711936.3 64.7 -1711770.6 230.4 30 -1711959.8 41.2 -1711965.2 34.1 -1711944.8 56.2 -1711765.6 235.3 20 -1711968.1 32.9 -1711969.7 29.6 -1711950.5 50.5 -1711759.4 241.6 10 -1711972.6 28.4 -1711971.7 27.6 -1711952.4 48.6 -1711752.4 248.6 0 -1711973.1 27.9 -1711971.4 27.9 -1711953.9 47.1 -1711744.5 256.5
190
Appendix 3: Data for Energy Profiles
Table A3.3: Data for p-coumaroyl glucose energy profile in a vacuum.
S0 Dynamic Forwards S0 Dynamic Backwards T1 o o o Dihedral Angle kJ/mol Relative to S 0 180 kJ/mol Relative to S 0 180 kJ/mol Relative to S 0 180 180 -3109035.5 0.0 -3108809.4 226.2 170 -3109033.9 1.7 -3108809.4 226.1 160 -3109028.3 7.2 -3108809.5 226.1 150 -3109019.1 16.4 -3108809.7 225.8 140 -3109006.3 29.3 -3108810.1 225.4 130 -3108989.9 45.6 -3108810.3 225.2 120 -3108969.9 65.6 -3108813.0 222.6 110 -3108946.5 89.0 -3108814.2 221.3 100 -3108919.9 115.7 -3108815.1 220.4 90 -3108890.4 145.2 -3108909.1 126.5 -3108815.1 220.4 80 -3108934.5 101.0 -3108814.3 221.2 70 -3108955.9 79.7 -3108812.9 222.6 60 -3108972.9 62.7 -3108810.9 224.6 50 -3108985.7 49.8 -3108807.8 227.8 40 -3108995.9 39.7 -3108803.3 232.3 30 -3109003.1 32.4 -3108797.5 238.0 20 -3109004.3 31.2 -3108790.7 244.8 10 -3109006.6 28.9 -3108783.4 252.1 0 -3109006.6 28.9 -3108790.3 245.3
Table A3.4: Data for T 1 energy profiles in water.
T1 p -Coumaric acid T1 p -Coumaroyl glucose o o Dihedral Angle kJ/mol Relative to S 0 180 kJ/mol Relative to S 0 180
S0 180 -1505539.2 -3109133.9 180 -1505309.7 229.4 -3108912.2 221.7 170 -1505307.3 231.8 -3108911.8 222.1 160 -1505316.6 222.5 -3108910.5 223.4 150 -1505320.9 218.2 -3108909.2 224.7 140 -1505325.4 213.8 -3108908.4 225.6 130 -1505329.5 209.6 -3108907.5 226.4 120 -1505332.9 206.3 -3108908.8 225.2 110 -1505335.3 203.9 -3108909.4 224.5 100 -1505336.6 202.5 -3108910.2 223.7 90 -1505336.8 202.3 -3108909.9 224.0 80 -1505336.3 202.9 -3108909.0 224.9 70 -1505334.5 204.6 60 -1505331.5 207.7 -3108905.1 228.8 50 -1505327.1 212.1 -3108902.3 231.7 40 -1505321.5 217.6 -3108897.7 236.2 30 -1505315.0 224.2 -3108892.4 241.5 20 -1505307.8 231.3 -3108886.8 247.1 10 -1505300.3 238.9 -3108882.1 251.8 0 -1505293.1 246.1 -3108883.2 250.7
191
Appendix 4: Data for Vertical Excitations and HOMO-LUMO Gaps
Appendix 4: Data for Vertical Excitations and HOMO-LUMO Gaps.
192
Appendix 4: Data for Vertical Excitations and HOMO-LUMO Gaps
193
Appendix 4: Data for Vertical Excitations and HOMO-LUMO Gaps
194
Appendix 4: Data for Vertical Excitations and HOMO-LUMO Gaps
195
Appendix 5: Data from Ethylphenol Analyses
Appendix 5: Data from Ethylphenol Analyses.
Table A5.1: Percentage conversion from trans -ethyl esters ( 11 and 12 ) to ethylphenols in fermentations with AWRI 1499.
Fermentation Progress (Days) 2 4 6 8 9 4-EP 0.1 ± 0.1 8.8 ± 0.4 28.8 ± 1.2 45.8 ± 1.5 51.4 ± 2.4 4-EG 0.0 ± 0.0 0.5 ± 0.0 1.9 ± 0.1 3.3 ± 0.1 4.0 ± 0.2
Table A5.2: Percentage of ethyl esters ( 11 and 12 ) remaining in fermentations.
Fermentation Progress (Days) 2 4 6 8 9 Ethyl coumarate 96.0 ± 5.4 85.7 ± 3.7 65.3 ± 6.9 44.3 ± 3.3 33.3 ± 1.3 Ethyl ferulate 93.7 ± 5.9 95.0 ± 4.6 89.7 ± 10.8 90.7 ± 9.0 72.3 ± 3.3
Table A5.3: Percentage conversion from trans -ethyl esters ( 11 and 12 ) to ethylphenols in fermentations with different strains of D. bruxellensis .
AWRI 1499 AWRI 1608 AWRI 1613 4-EP 67.0 ± 0.1 55.6 ± 0.8 N.D 4-EG 7.7 ± 0.2 3.0 ± 0.1 N.D
Table A5.4: Percentage conversion from hydroxycinnamoyl glucose esters ( 9 and 10 ) to ethylphenols in fermentations with AWRI 1499.
Fermentation Progress (Days) 2 4 6 8 4-EP 6.4 ± 2.7 12.6 ± 1.0 15.8 ± 1.1 20.6 ± 1.7 4-EG 10.3 ± 3.0 14.6 ± 0.6 18.1 ± 1.4 24.2 ± 2.9
10 12 14 16 4-EP 33.3 ± 2.9 34.4 ± 1.8 34.3 ± 0.6 36.0 ± 0.7 4-EG 33.5 ± 2.4 38.6 ± 0.8 33.7 ± 1.7 36.1 ± 1.4
196
Appendix 5: Data from Ethylphenol Analyses
Table A5.5: Percentage conversion from trans- and cis/trans -ferulic acid ( 4) to 4- ethylguaiacol in D. bruxellensis fermentations.
Fermentation Progress (Days) 2 3 4 5 6 7 9 trans -ferments 3.0 ± 1.2 17.2 ± 1.9 46.8 ± 4.5 67.1 ± 4.0 78.2 ± 5.2 75.3 ± 2.5 69.8 ± 4.5 cis/trans -ferments 1.7 ± 0.5 8.2 ± 0.6 22.6 ± 1.6 30.8 ± 1.7 37.3 ± 2.4 38.4 ± 0.7 39.6 ± 0.3
Table A5.6: Percentage conversion from trans - and cis/trans -p-coumaric acid ( 3) to 4- ethylphenol in D. bruxellensis fermentations. Fermentation Progress (Days) 2 4 6 8 10 12 trans -ferments 7.9 ± 0.6 44.8 ± 1.1 56.4 ± 1.6 56.7 ±2.6 66.8 ± 1.6 61.7 ± 3.6 cis/trans -ferments 5.2 ± 0.4 29.9 ± 1.8 37.3 ± 1.2 37.3 ± 2.7 42.4 ± 0.5 40.6 ± 2.4
Table A5.7: Percentage conversion from cis -ethyl ferulate ( cis -12 ) and cis -ethyl coumarate (cis -11 ) to ethylphenols in D. bruxellensis fermentations.
Fermentation Progress (Days) 2 4 6 8 10 4-EP 0.00 ± 0.00 0.07 ± 0.12 0.51 ± 0.04 0.74 ± 0.04 0.86 ± 0.02 4-EG 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.13 ± 0.11 0.25 ± 0.03
197
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