Di-tert-butyl (2R,3R)-2-{[(2E)-3-(4-acet­yloxy-3-meth­oxy­phen­yl)prop-2-eno­yl]­oxy}-3-hy­droxy­butane­dioate

In the title mol­ecule, C24H32O10, one tert-butyl ester group is folded towards the central benzene ring while the other is directed away. The acetyl group is almost perpendicular to the benzene ring to which it is connected [C—C—O—C torsion angle = 90.4 (12)°]. The conformation about the ethene bond [1.313 (7) Å] is E. The atoms of the benzene ring and its attached ester group and part of the hy­droxy tert-butyl ester side chain are disordered over two sets of sites in a 50:50 ratio. Linear supra­molecular chains along the a axis mediated by hy­droxy–carbonyl O—H⋯O hydrogen bonds feature in the crystal packing. The same H atom also partakes in an intra­molecular O—H⋯O inter­action

1,4-Di-tert-butyl (2R,3R)-2-({(2E)-3-[4-(acet­yloxy)phen­yl]prop-2-eno­yl}­oxy)-3-hy­droxy­butane­dioate

The title compound, C23H30O9, has an approximate T-shape with the tert-butyl ester groups lying either side of the benzene ring. The acetyl group is almost perpendicular to the benzene ring to which it is connected [C—C—O—C torsion angle = −106.7 (3)°]. The conformation about the C=C double bond [1.331 (4) Å] is E. Linear supra­molecular chains along the a axis mediated by hy­droxy–carbonyl O—H⋯O hydrogen bonds feature in the crystal packing. The same H atom is also involved in an intra­molecular O—H⋯O inter­action

Multi-omic profiling reveals the ataxia protein sacsin is required for integrin trafficking and synaptic organization

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset cerebellar ataxia caused by mutations in SACS, which encodes the protein sacsin. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament disorganization, and progressive death of cerebellar Purkinje neurons. It is unclear why the loss of sacsin causes these deficits or why they manifest as cerebellar ataxia. Here, we perform multi-omic profiling in sacsin knockout (KO) cells and identify alterations in microtubule dynamics and mislocalization of focal adhesion (FA) proteins, including multiple integrins. Deficits in FA structure, signaling, and function can be rescued by targeting PTEN, a negative regulator of FA signaling. ARSACS mice possess mislocalization of ITGA1 in Purkinje neurons and synaptic disorganization in the deep cerebellar nucleus (DCN). The sacsin interactome reveals that sacsin regulates interactions between cytoskeletal and synaptic adhesion proteins. Our findings suggest that disrupted trafficking of synaptic adhesion proteins is a causal molecular deficit in ARSACS

Chemistry of hydroxycinnamate esters and their role as precursors to Dekkera produced off-flavour in wine.

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.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201

Exploiting Compositionally Similar Grape Marc Samples to Achieve Gradients of Condensed Tannin and Fatty Acids for Modulating In Vitro Methanogenesis

Ruminants produce large amounts of the greenhouse gas, methane, which can be reduced by supplementing feed with products that contain anti-methanogenic compounds, such as the solid winemaking by-product, grape marc. The aim of this study was to exploit compositional differences in grape marc to better understand the roles of condensed tannin and fatty acids in altering methanogenesis in a ruminant system. Grape marc samples varying in tannin extractability, tannin size and subunit composition, and fatty acid or tannin concentrations were selected and incubated in rumen fluid using an in vitro batch fermentation approach with a concentrate-based control. Four distinct experiments were designed to investigate the effects on overall fermentation and methane production. Generally, fatty acid concentration in grape marc was associated with decreased total gas volumes and volatile fatty acid concentration, whereas increased condensed tannin concentration tended to decrease methane percentage. Smaller, extractable tannin was more effective at reducing methane production, without decreasing overall gas production. In conclusion, fatty acids and tannin concentration, and tannin structure in grape marc play a significant role in the anti-methanogenic effect of this by-product when studied in vitro. These results should be considered when developing strategies to reduce methane in ruminants by feeding grape marc

Survey of the Variation in Grape Marc Condensed Tannin Composition and Concentration and Analysis of Key Compositional Factors

Grape marc contains a number of compounds with potential antimethanogenic activity in ruminants, including condensed tannins (CTs). Using direct phloroglucinolysis, a survey of CT chemistry across 66 grape marc samples showed diversity in concentration (6.9 to 138.8 g/kg of dry matter). Concentration was found to be independent of CT composition, although all compositional variables were significantly correlated (<i>P</i> < 0.0001). Twenty samples diverse in CT were selected from a cluster analysis and analyzed for compounds relevant to ruminant digestion and methanogenesis, including metabolizable energy (6.6–12.0 MJ/kg DM), crude protein (3.2–14.4% DM), neutral detergent fiber (18.4–61.4% DM), and ethanol soluble carbohydrates (2.0–40.6% DM). Fatty acid concentrations varied throughout the 20 samples (5.2–184.5 g/kg DM), although fatty acid profile showed two distinct groups. Grape marc varies widely in nutritional value, and in compounds that have been linked with changes in ruminant digestion and methane emissions

Hydroxycinnamoyl Glucose and Tartrate Esters and Their Role in the Formation of Ethylphenols in Wine

Synthesized <i>p</i>-coumaroyl and feruloyl l-tartrate esters were submitted to Brettanomyces bruxellensis strains AWRI 1499, AWRI 1608, and AWRI 1613 to assess their role as precursors to ethylphenols in wine. No evolution of ethylphenols was observed. Additionally, <i>p</i>-coumaroyl and feruloyl glucose were synthesized and submitted to B. bruxellensis AWRI 1499, which yielded both 4-ethylphenol and 4-ethylguaiacol. Unexpected chemical transformations of the hydroxycinnamoyl glucose esters during preparation were investigated to prevent these in subsequent synthetic attempts. Photoisomerization gave an isomeric mixture containing the <i>trans</i>-esters and undesired <i>cis</i>-esters, and acyl migration resulted in a mixture of the desired 1-<i>O</i>-β-ester and two additional migrated forms, the 2-<i>O</i>-α- and 6-<i>O</i>-α-esters. Theoretical studies indicated that the photoisomerization was facilitated by deprotonation of the phenol, and acyl migration is favored during acidic, nonaqueous handling. Preliminary LC-MS/MS studies observed the migrated hydroxycinnamoyl glucose esters in wine and allowed for identification of feruloyl glucose in red wine for the first time

Effects of Feeding either Red or White Grape Marc on Milk Production and Methane Emissions from Early-Lactation Dairy Cows

Globally, annual production of grape marc (GM), the residue of skins, seeds and stems remaining after making wine, has been estimated to be approximately nine million tonnes. No previous studies have compared effects on milk production and methane emissions when GM from either red or white grapes was fed to dairy cows. This experiment examines the effects of partial replacement of a perennial ryegrass (Lolium perenne L.) based diet with GM from either red or white grapes on yield and composition of milk and methane emissions. Thirty-two Holstein dairy cows in early lactation were offered either a control diet containing 15.0 kg dry matter (DM) of freshly harvested perennial ryegrass and 5.2 kg of a concentrate mix, or a diet similar to the control diet but with 5 kg DM of ryegrass replaced with 5 kg DM of GM from red grapes (RGM), or a diet similar to the RGM diet except the GM was from white grapes (WGM). Individual cow feed intakes, milk yields, and methane emissions were measured. Both diets containing GM decreased milk yields by approximately 10% and methane emissions by 15%. When fed to dairy cows, GM reduces methane emissions but at the cost of decreased milk production

Theoretical studies for ozonide formation during the ozonolysis of bicyclic endoperoxides

Theoretical investigations on the treatment of bicyclic endoperoxides (1,2-dioxines) with ozone at the HF/6-31G*, MP2/6-31G*or 6-311G*, and DFT(B3LYP)/6-31G*levels of theory indicate that the estimated activation energies for formation of the possible endo-endo, endo-exo, exo-endo, or exo-exo transition states along with the formation of the primary ozonides and product ozonides are very sensitive to effects of electron correlation and basis set. This study suggests that MP2/6-311G*is the best level of theory for evaluating such systems. At the MP2/6-311G*level of theory it was found that the transition state for primary ozonide formation was lowest in energy when ozone approaches in an endo facial fashion with a further 3kJmol-1 stabilisation seen when the central oxygen within the primary ozonide protrudes outwards (exo) as opposed to inwards (endo). The product ozonides are predicted to be more stable than the combined starting materials by some 380-580kJmol-1 depending on the level of theory, clearly highlighting the substantive exothermic nature of this type of ozonolysis reaction.Nicole M. Cain, Josh L. Hixson, and Dennis K. Taylo