Chemistry of hydroxycinnamate esters and their role as precursors to Dekkera produced off-flavour in wine.
Date
2012
Authors
Hixson, Josh L.
Editors
Advisors
Elsey, Gordon Michael
Curtin, Christopher D.
Sefton, Mark Aidan
Taylor, Dennis Kenwyn
Curtin, Christopher D.
Sefton, Mark Aidan
Taylor, Dennis Kenwyn
Journal Title
Journal ISSN
Volume Title
Type:
Thesis
Citation
Statement of Responsibility
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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 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 transethyl- 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.
School/Discipline
School of Agriculture, Food and Wine
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2012
Provenance
Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.