Potential grape-derived contributions to volatile ester concentrations in wine

Article abstract of DOI:10.3390/molecules20057845

Grape composition affects wine flavour and aroma not only through varietal compounds, but also by influencing the production of volatile compounds by yeast. C₉ and C₁₂ compounds that potentially influence ethyl ester synthesis during fermentation were studied using a model grape juice medium. It was shown that the addition of free fatty acids, their methyl esters or acyl-carnitine and acyl-amino acid conjugates can increase ethyl ester production in fermentations. The stimulation of ethyl ester production above that of the control was apparent when lower concentrations of the C₉ compounds were added to the model musts compared to the C₁₂ compounds. Four amino acids, which are involved in CoA biosynthesis, were also added to model grape juice medium in the absence of pantothenate to test their ability to influence ethyl and acetate ester production. β-Alanine was the only one shown to increase the production of ethyl esters, free fatty acids and acetate esters. The addition of 1 mg·L^-1 β-alanine was enough to stimulate production of these compounds and addition of up to 100 mg·L^-1 β-alanine had no greater effect. The endogenous concentrations of β-alanine in fifty Cabernet Sauvignon grape samples exceeded the 1 mg·L^-1 required for the stimulatory effect on ethyl and acetate ester production observed in this study.

Full text of DOI:10.3390/molecules20057845

Molecules 2015, 20, 7845-7873; doi:10.3390/molecules20057845
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Potential Grape-Derived Contributions to Volatile Ester
Concentrations in Wine
1
,
2, †
2
1
1, ‡
Paul K. Boss *, Anthony D. Pearce , Yanjia Zhao , Emily L. Nicholson , Eric G. Dennis
and David W. Jeffery 2
1
CSIRO Agriculture Flagship, PMB 2, Glen Osmond, SA 5064, Australia;
E-Mails: emily.nicholson@csiro.au (E.L.N.); eric.dennis@ubc.ca (E.G.D.)
2
School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, PMB 1,
Glen Osmond, SA 5064, Australia; E-Mails: a_pearce@post.com (A.D.P.);
yanjia.zhao@student.adelaide.edu.au (Y.Z.); david.jeffery@adelaide.edu.au (D.W.J.)
†
‡
Present address: South Australian Research and Development Institute, GPO Box 397, Adelaide,
SA 5001, Australia
Present address: Department of Chemistry, The University of British Columbia, Okanagan Campus,
3333 University Way, Kelowna, BC V1V 1V7, Canada
*
Author to whom correspondence should be addressed; E-Mail: paul.boss@csiro.au;
Tel.: +61-8-830-38614.
Academic Editors: Helene Hopfer and Susan E. Ebeler
Received: 22 March 2015 / Accepted: 23 April 2015 / Published: 29 April 2015
Abstract: Grape composition affects wine flavour and aroma not only through varietal
compounds, but also by influencing the production of volatile compounds by yeast. C
9
and
C12 compounds that potentially influence ethyl ester synthesis during fermentation were
studied using a model grape juice medium. It was shown that the addition of free fatty acids,
their methyl esters or acyl-carnitine and acyl-amino acid conjugates can increase ethyl ester
production in fermentations. The stimulation of ethyl ester production above that of the
control was apparent when lower concentrations of the C compounds were added to the
9
model musts compared to the C12 compounds. Four amino acids, which are involved in CoA
biosynthesis, were also added to model grape juice medium in the absence of pantothenate
to test their ability to influence ethyl and acetate ester production. β-Alanine was the only
one shown to increase the production of ethyl esters, free fatty acids and acetate esters. The
−1
addition of 1 mg·L β-alanine was enough to stimulate production of these compounds and

Molecules 2015, 20
addition of up to 100 mg·L β-alanine had no greater effect. The endogenous concentrations
7846
−1
−1
of β-alanine in fifty Cabernet Sauvignon grape samples exceeded the 1 mg·L required for
the stimulatory effect on ethyl and acetate ester production observed in this study.
Keywords: grape; wine; ester; amino acid; CoA; yeast; fermentation
1
. Introduction
Wine is a complex solution containing abundant volatile compounds which contribute to wine aroma
and flavour, and consequently impact wine quality and appreciation. Wine aromas are broadly
categorised into three groups that reflect their source. Primary aromas are grape-derived volatiles that
pass through fermentation often unchanged, and are largely responsible for “varietal” aromas. Secondary
aromas, which are by far the greatest pool of volatile molecules, are produced through the winemaking
process, with the great majority produced by yeast during alcoholic fermentation as metabolism
byproducts [1,2]. Tertiary aromas develop in finished wine through storage and maturation, and result
from intermolecular chemical interactions and equilibrium effects as the wine matrix changes.
The volatile composition of most wines, or styles of wine, is very similar, and the varietal differences
that exist between wines made from different varieties of grapes are largely due to the relative ratios of
the volatile compounds contained within [3]. The types of volatile compounds are diverse, and include
esters, higher alcohols, aldehydes, ketones, lactones, acids, phenols, N-heterocycles, isoprenoids and
sulfur compounds [1,4]. Of these categories, higher alcohols represent the largest volatile pool in terms
of concentration, but esters have the largest number of contributing molecules [5].
In this work the term esters encompasses both ethyl and acetate esters, which have a significant effect
on wine aroma by contributing fruity and floral characteristics [6,7]. These esters are mainly produced
by yeast metabolism through fatty acid acyl- and acetyl-Coenzyme A (CoA) pathways [7,8]. CoA is a
critical cofactor for a large number of metabolic pathways and is used to activate intermediates during
the biosynthesis of medium chain fatty acids (MCFAs); the acyl-CoA intermediates formed are then
esterified with ethanol by esterase and transferase enzymes, forming MCFA ethyl esters. Acetate esters,
on the other hand, are produced through the condensation of yeast-derived higher alcohols with
acetyl-CoA, again under the control of ester-forming enzymes [7,8].
Ester production mainly occurs during fermentation when yeasts generate ample amounts of ethanol
and higher alcohols via sugar and amino acid metabolism, respectively [9], so CoA may play a significant
role in determining the ester content in wine [10]. For instance, it has been suggested that factors that
regulate the production or consumption of acetyl-CoA will in turn alter the amount of esters produced by
yeast, and there is evidence for the association of some specific amino acids with CoA
biosynthesis [11–13]. However, there are no reports directly studying the effect of CoA-related amino
acid composition (arising from the grapes) on CoA biosynthesis, and subsequently how a change in CoA
concentration might influence ester levels in wine.
Despite the role of acyl transferase enzymes in ester formation by yeast, Saerens et al. [14] indicated
that the level of gene expression is not the limiting factor for ester production, and that the availability
of MCFA precursors has an important role. Moreover, grape chemical composition has been highlighted

Molecules 2015, 20
7847
as being highly influential to the level of production of a suite of aroma compounds, and many grape
components are known to be depleted and converted to alcohols and esters through fermentation [15]. It
has been widely demonstrated that yeast can liberate volatile molecules from various grape-derived
conjugates, such as glycosidically bound and cysteine- and glutathione-conjugated volatile
compounds [16,17].
More recent investigations have sought to demonstrate the relationship between grape-derived
precursors and wine volatile aromas, such as a study involving ferments in model grape juice medium
spiked with varying amounts of natural grape juice to test the impact on wine aromas [18]. This
experiment revealed an array of complex interactions across the grape juice proportions added; however,
several fermentation esters were identified as having positive, largely linear correlations with grape juice
content in the ferments. Following this work, grape-derived aliphatic alcohols and aldehydes were
identified as precursors to acetate esters in wine [19]. In particular, the C compounds (E)-2-hexenal,
6
hexanal, (E)-2-hexen-1-ol, and hexan-1-ol were shown to be precursors to hexyl acetate, whereas
octan-1-ol and benzyl alcohol were identified as precursors to octyl acetate and benzyl acetate,
respectively [19]. These examples may also imply the presence of grape-derived MCFAs, or their
precursors, contributing to the pool of MFCA ethyl esters liberated during fermentation, which could
provide another important link between grape composition and wine volatile profile.
Ultimately, a deeper understanding of the sources and biosynthesis pathways of aroma compounds
involves linking viticultural practises, grape variety, grape chemical composition, yeast strain selection,
winemaking techniques and wine matrix interactions between aroma constituents with defined sensory
outcomes in the finished wine [20,21]. The aim of this study was to address one component of this
overall goal, and that was to identify links between potential grape components and wine ester
concentrations. A series of model fermentations were conducted with MCFAs and their putative
grape-derived precursors, as well as amino acids related to the biosynthesis of CoA, and esters arising
in these fermentations were quantified with GC-MS analysis.
2
. Results and Discussion
2.1. Potential Medium Chain Fatty Acid (MCFA)-Derived Precursors of Ethyl Esters
The first part of this study investigated potential grape-derived precursors of the ethyl esters of C
9
and C12 fatty acids (synthesised or commercially available). These two carbon lengths were chosen for
two reasons. First, odd-numbered fatty acids and their ethyl esters are found in low abundance in wine,
meaning that it is easier to follow the effect of the addition of odd-numbered potential precursors on
ethyl ester production as the endogenous concentrations are low. Second, ethyl dodecanoate is extracted
at moderate amounts by the SPME method employed in this study compared to shorter
even-numbered ethyl esters and hence does not saturate the detector [19]. This allowed the effects of C12
precursor addition to be readily detected and allowed the comparison of odd- and even-numbered
precursors to be tested in case they are metabolised differently during fermentation.
The potential grape-derived precursors tested included the C and C12 free fatty acids, as well as fatty
9
acids in various conjugated forms. Methyl esters were included because they have been identified in
grapes and represent possible contributors to the precursor pool [22]. Carnitine-fatty acid conjugates are

Molecules 2015, 20
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potential ethyl ester precursors, as they are the intracellular transport molecule involved in the movement
of fatty acids across membranes as part of yeast metabolic activities [23]. Amino acid-fatty acid
conjugates have been reported in mammalian systems and in insects (e.g., [24,25]), but as yet have not
been described in plants. Nevertheless, fatty acid-amino acid conjugates can act as inducers of plant
defence responses when they exist in herbivorous insect saliva (e.g., [26]) and so amino acid conjugates
were included as putative precursors.
2
.1.1. Free Fatty Acids as Direct Precursors
and C12 fatty acids were added to model grape juice media (MGJM) fermentations across a range
C
9
intended to capture the concentrations reported in the literature for comparable molecules in
grapes [27,28]. No significant difference in the production of ethyl nonanoate was seen between the
control and the fermentations supplemented with 1 μM nonanoic acid (Figure 1A). However, there were
significant increases when 10 μM or 100 μM nonanoic acid was added to the MGJM, with ester
−1
concentrations reaching 1120 nmol·L . The addition of 1 mM nonanoic acid appeared to be toxic to the
yeast, as negligible mass was lost throughout the fermentation period. It has been shown in previous
studies that free fatty acids can be toxic to yeast at high concentrations [29,30].
No significant increase in the production of ethyl dodecanoate was observed after additions of up to
1
1
00 μM dodecanoic acid to the fermentations (Figure 1A). However, there was a large response when
mM dodecanoic acid was added, leading to a mean ethyl dodecanoate concentration of 8155 nM
−1
(
i.e., 1.86 mg·L , a sizeable quantity) compared to a mean of 118 nM in the control wine. An increased
lag phase was seen in the 1 mM samples, otherwise the fermentation kinetics appeared not to have been
impacted in the dodecanoic acid series of additions (data not shown). The esterification of the fatty acid
may be a detoxifying action that occurs above a particular toxicity threshold. The findings of Stevens
and Hofmeyr [31] support this, as esterification of C and C10 fatty acids reduced their toxicity to the
8
yeast. The results suggest that a concentration threshold for this toxicity exists, and that grape-derived
MCFAs could contribute to these concentrations. It would appear that the threshold is much lower for
nonanoic acid than for dodecanoic acid and, since most previous research has focused on the effects of
even chain MCFAs [8,32], the results of this study suggest a potential odd/even carbon number effect
that is worthy of further investigation. Alternatively, the stimulation of ethyl nonanoate production by a
lower concentration of nonanoic acid than that observed for dodecanoic acid and its corresponding ethyl
ester may reflect the size of the endogenous pool of substrate. As odd-numbered MCFAs are less
common than the even-numbered ones, a lower concentration would be required to increase the fatty
acid substrate concentration to a point where the production of the ethyl ester is significantly greater than
that in the control wine. This may also be the case if the fatty acids have differential abilities to diffuse
or be actively transported into the yeast where esterification occurs.

Molecules 2015, 20
7849
(
A) Free fatty acids
a’
10000
control 1µM 10µM 100µM 1mM
a
1
000
b
b’
b’
b’
b’
100
bc
c
c
1
0
1
Nonanoic acid
Dodecanoic acid
a’
(
B) Methyl esters
10000
control 1µM 10µM 100µM 1mM
a
1
000
b
b’
b’ b’ b’
1
00
c
d
10
d
1
Methyl nonanoate
Methyl dodecanoate
(
C) Acyl-carnitine conjugates
10000
control 1µM 10µM 100µM 1mM
a’
1000
b’
a
b’
c’
c’
b
100
c
1
0
1
c
c
Nonanoyl carnitine
Dodecanoyl carnitine
Figure 1. (A) Ethyl nonanoate and ethyl dodecanoate concentrations in model wines
produced after the addition of increasing amounts of nonanoic acid or decanoic acid to the
MGJM; (B) Ethyl nonanoate and ethyl dodecanoate concentrations in model wines produced
after the addition of increasing amounts of methyl nonanoate or methyl decanoate to the
MGJM; (C) Ethyl nonanoate and ethyl dodecanoate concentrations in model wines produced
after the addition of increasing amounts of nonanoyl carnitine or dodecanoyl carnitine to the
MGJM. Histograms represent the mean values (n = 3) and error bars show standard errors.
Different letters (e.g., a, b, c, d or aʹ, bʹ, cʹ) denote significant differences between treatments
at p < 0.05 using ANOVA followed by the Games-Howell post hoc test. Note that the y-axis
is in log scale.

Molecules 2015, 20
.1.2. Methyl Esters of MCFAs as Transesterification Substrates
The same concentrations of the methyl esters of nonanoic and dodecanoic acids as used for the free
7850
2
fatty acid experiments were added to MGJM to test the ability of these compounds to influence ethyl
ester production. Methyl nonanoate stimulated ethyl nonanoate production when added at concentrations
of 10 µM and greater (Figure 1B). The addition of 1 mM methyl nonanoate to the model must resulted
−1
in ethyl nonanoate production of 2304 nM (429 ng·L ) and did not have the toxic effect observed when
the free fatty acid was added to the must at this concentration. The addition of methyl dodecanoate
produced a similar response to that of the dodecanoic acid additions, with no significant increase in ethyl
dodecanoate at any concentration below 1 mM, but a significant increase of ethyl dodecanoate
production at that level of addition (Figure 1B). These observations could again be related to either a
toxicity response of the yeast above a certain threshold, a reflection of the size of the endogenous
substrate pools for these compounds in relation to the amount added to the must, or a consequence of
differential rates of transport into the yeast. The metabolism of methyl esters of fatty acids by yeast is
seemingly unknown; however, methyl esters of a number of MCFAs have been found in grapes [33],
and have been reported in model wines (e.g., [18]). Yeast-mediated transesterification, or alcoholysis, is
a probable route of formation of ethyl esters from methyl esters, although the concentrations of these
compounds in the fruit are likely to be too low to greatly influence ethyl ester concentrations in wine
based on the observations in this experiment.
2
.1.3. Acyl-Carnitine Conjugates, Putative Transport Forms of MCFAs
No significant difference was observed in the ethyl nonanoate concentrations of the control wines
and those produced after the addition of 1 and 10 μM nonanoyl carnitine. However, the addition of
00 μM nonanoyl carnitine to MGJM significantly increased the ethyl nonanoate concentration in the
1
resulting wine (Figure 1C). Increasing the concentration of nonanoyl carnitine from 100 μM to 1 mM
also caused significant increases in ethyl nonanoate production in the subsequent fermentations (Figure 1C).
The addition of 1 and 10 μM dodecanoyl carnitine to the MGJM caused a significant difference in ethyl
dodecanoate content of the model wines, and this was further enhanced when the model must was
supplemented with 100 μM dodecanoyl carnitine (Figure 1C). When 1 mM dodecanoyl carnitine was
added to the MGJM the yeast appeared to lose viability and fermentation of the MGJM did not occur,
denoted by no loss of weight and no flocculence in the must (data not shown).
The apparent toxicity of the high concentration of dodecanoyl carnitine compared to the free fatty
acid was unexpected. Fatty acids appear to be imported into yeast cells in a process that requires
activation of the molecule with CoA [34]. The carnitine conjugate may be transported more readily as it
is more soluble than the free fatty acid and because it is already activated. The increased transportation
of the carnitine conjugate into the cell, followed by liberation of the carnitine, may lead to toxic
intracellular levels of the free fatty acid that is not seen when the free fatty acid alone is added to the
model must. The mechanism for the transport of extracellular carnitine or conjugates into the cell in
Saccharomyces cerevisiae is still unclear.
Paradoxically, the nonanoyl carnitine conjugate did not inhibit fermentation when added to the must
at a concentration of 1 mM, whereas the free fatty acid did. This may reflect the difference in the fatty

Molecules 2015, 20
7851
acid chain length which may allow more diffusion of the C
9
fatty acid into the cell compared to both
nonanoyl carnitine and free dodecanoic acid. Certainly the stimulation of ethyl nonanoate production
was 10-fold lower after the addition of 10 or 100 µM nonanoyl carnitine compared to the equivalent
amounts of nonanoic acid (Figure 1). Alternatively, the odd number of carbons in the nonanoyl carnitine
conjugate may inhibit any further metabolism of this compound into something that may be toxic to the
yeast (for example, the free acid) compared to the even-numbered dodecanoyl carnitine.
The concentration of acyl carnitine conjugates in grape berries is unknown. A study that quantified
free carnitine and carnitine esters in many different foods found that fleshy fruits such as apples, guavas
−1
and oranges contain concentrations of total carnitine esters of between 0.05–1.45 mg·kg [35]. The
grape seed, being a source of lipids, may contain more carnitine esters than the rest of the berry, but further
work is required to determine if these conjugates from grapes can alter ester composition in wines.
2
.1.4. Acyl-Amino Acid Conjugates Potentially Exploiting Permeases
Addition of nonanoyl alanine to the MGJM at a concentration of 100 μM resulted in a significant
increase in the production of ethyl nonanoate in the resulting wine (Figure 2A). Similar to when the free
fatty acid was added to the musts, 1 mM nonanoyl alanine appeared to be toxic to the yeast as there was
negligible weight loss in these samples; however, ethyl nonanoate concentrations were significantly
higher than the control. Nonanoyl isoleucine addition also caused an increase in ethyl nonanoate
production when added in concentrations at 100 μM and above (Figure 2B). The toxicity seen when
1
mM nonanoyl alanine was added to the MGJM was not observed after the addition of nonanoyl
isoleucine to the MGJM (Figure 2A,B).
Ethyl dodecanoate concentrations were significantly higher only in the wine produced after the
addition of 1 mM dodecanoyl alanine to the must, which led to a mean wine concentration of almost
−1
−1
1
0,000 nmol·L , or 2.24 mg·L (Figure 2A). On the contrary, no significant difference was seen in
ethyl dodecanoate production at any level of dodecanoyl isoleucine addition (Figure 2B).
The transport mechanism for extracellular alanine and isoleucine is via the general yeast amino acid
permeases or other permeases with a less broad specificity [36]. However, the conjugated MCFAs are
perhaps more likely to enter the cell in a manner similar to MCFAs. It would appear that the alanine
conjugates are transported more easily into the cell as the addition of both the nonanoyl and dodecanoyl
variants to the musts resulted in wines with greater concentrations of their respective ethyl esters than
did the equivalent addition of the isoleucine conjugates (Figure 2). Alternatively, the alanine conjugates
could be more easily esterified or hydrolysed into the free fatty acid, which would increase the available
substrate for esterification. Increased transport or hydrolysis of the conjugate could also explain the
increased toxicity of the nonanoyl alanine conjugate at 1 mM compared to the isoleucine conjugate. The
identification and quantification of acyl-amino acid conjugates in plants has not been reported although
it is only recently that such compounds have been described in mammals and insects [24,25]. Future
targeted studies may reveal their presence in the plant kingdom as well.
2
.2. Amino Acids Involved in CoA Biosynthesis
Four amino acids known to be involved in CoA synthesis (Figure 3) were selected for studies to
determine their effect on ester production in MGJM. Valine is associated with CoA generation as it is

Molecules 2015, 20
7852
converted to 2-oxoisovalerate, a precursor of pantothenate [12]. Arginine is the precursor for the
biosynthetic production of spermine [13], from which β-alanine is synthesised by S. cerevisiae via
polyamine degradation. β-Alanine then acts as the intermediate for pantothenic acid biosynthesis [13].
The later steps of CoA synthesis require the reaction of pantothenate with cysteine and ATP (Figure 3).
(
A) Acyl-alanine conjugates
a’
10000
Control 1μM 10μM 100μM 1mM
1000
a
b
b’
b’
c
b’ b’
100
c
10
c
1
Nonanoyl alanine
Dodecanoyl alanine
(
B) Acyl-isoleucine conjugates
1
0000
Control 1μM 10μM 100μM 1mM
1000
a
b
1
00
c
10
c
c
1
Nonanoyl isoleucine
Dodecanoyl isoleucine
Figure 2. (A) Ethyl nonanoate and ethyl dodecanoate concentrations in model wines
produced after the addition of increasing amounts of acyl-alanine conjugates to the MGJM.
(
B) Ethyl nonanoate and ethyl dodecanoate concentrations in model wines produced after
the addition of increasing amounts of acyl-isoleucine conjugates to the MGJM. Histograms
represent the mean values (n = 3) and error bars show standard errors. Different letters (e.g.,
a, b, c or aʹ, bʹ) denote significant differences between treatments at p < 0.05 using ANOVA
followed by the Games-Howell post hoc test. Note that the y-axis is in log scale.
Since grape juice contains various chemical compounds including pantothenic acid and other
precursors that could affect ester syntheses, it is necessary to eliminate these factors when studying the
role of these four amino acids in ester production during fermentation. Using a chemically defined
MGJM containing a simple mixture of sugars and nutrients (excluding nitrogen sources) can imitate
winemaking conditions and is more suitable when gauging the effects of changing one or more variables.
However, supplementing model musts with certain amino acids at different levels will also contribute to

Molecules 2015, 20
7853
differences in total nitrogen contents, which can influence ester synthesis during fermentation (e.g.,
37]). Hence, total yeast assimilable nitrogen (YAN) was normalised across the experiments by the
addition of inorganic ammonium in the form of ammonium chloride (NH Cl). It was also necessary to
[
4
eliminate pantothenic acid from the normal MGJM as it is through this pathway that we hypothesised
that the amino acids may have an effect on ester production.
Figure 3. Simplified pathway of CoA biosynthesis in the yeast S. cerevisiae to emphasise
the potential role of the four amino acids (boxed) used in this study.
2
.2.1. Stimulation of Ester Production by β-Alanine.
A set of model fermentations were conducted containing one CoA-related amino acid at two
concentrations (50 and 100 mg·L−1 β-alanine; 10 and 30 mg·L−1 L-cysteine; 330 and 650 mg·L−1
L-arginine; 30 and 120 mg·L−1 L-valine). The cysteine, arginine and valine concentrations fall within, or
are slightly higher than, the typical concentrations of these amino acids found in grape juices [38]. The
β-alanine concentrations used were slightly higher than those previously reported for V. vinifera
grapes [39]. Negative control fermentations did not contain amino acids, and positive control
fermentations included all four amino acids at the lowest level added in the individual treatments

Molecules 2015, 20
7854
described above (i.e., 50 mg·L−1 β-alanine; 10 mg·L−1 L-cysteine; 330 mg·L−1 L-arginine and
3
0 mg·L−1 L-valine).
The different treatments affected the length of the fermentation lag phase. The model must
supplemented with the mixed amino acids had a lag phase of approximately two days, whereas all the
other fermentations had a lag phase of four days, despite them having the same total YAN (data not
shown). This could be due to the differences in structural complexity of the nitrogen source [40].
It was hypothesised that the four amino acids of interest would influence ester production through
their role in CoA biosynthesis (Figure 3). Therefore, the concentrations of these amino acids could
theoretically influence the level of CoA produced by yeast during fermentations, and CoA bearing
acetyl/acyl groups would impact on the synthesis of ethyl/acetate esters.
The β-alanine-supplemented wines had significantly more ethyl hexanoate, ethyl octanoate and ethyl
−
1
−1
−1
decanoate (115–125 µg·L ; 114–122 µg·L ; 92–103 µg·L , respectively) than the control wine
−1
−1
−1
(
16 µg·L ; 39 µg·L ; 34 µg·L ) or those produced after supplementation of the must with the other
three amino acids (Figure 4). However, there were no significant differences between the ethyl ester
−1
−1
concentrations in those wines produced from must with either 50 mg·L or 100 mg·L β-alanine added.
This suggests that the effect of the addition of this compound on ethyl ester production may be already
−1
saturated at 50 mg·L . The wines that were produced after the addition of all four amino acids to the
must also contained significantly more ethyl esters than the control, but in all three cases the
concentrations in the wines were less than those observed in those produced after the addition of
−1
5
0 mg·L β-alanine alone (Figure 4). This may be due to the difference in the time it took for the
fermentations to reach dryness, as this happened approximately two days earlier in the musts with all
four amino acids added compared to the other treatments. Volatilisation or metabolism of the ethyl esters
may have occurred in these wines while the other treatments were still reaching dryness. Individual
additions of arginine, cysteine and valine did not have an impact on ester concentrations as they were
not significantly different from the negative control in all cases. This suggests that the yeast is not limited
in its ability to produce ethyl esters during fermentation by the supply of arginine, cysteine or valine.
−1
−1
Significantly increased concentrations of MCFAs (hexanoic acid 1522–1732 µg·L vs. 681 µg·L ;
−1
−1
−1
−1
octanoic acid 3826–5119 µg·L vs. 1804 µg·L ; decanoic acid 545–1195 µg·L vs. 174 µg·L ) were
observed in fermentations spiked with 50 and 100 mg·L−1 β-alanine or with all four amino acids
compared to the control wine (Figure 5). Therefore, the increase in ethyl ester production may be linked
to an increase in the production of MCFAs rather than an increase in activation of existing free fatty
acids via an increase in CoA concentration. The lack of any significant difference in hexanoic and
octanoic acid content between the wines produced after the addition of four amino acids and those made
after β-alanine addition alone may reflect the lower volatility of the MCFAs, compared to the ethyl
esters, which reduces their loss from the wine in the two-day time difference between the completions
of the fermentations. However, this was not the case for decanoic acid, which was found in significantly
lower concentrations when all four amino acids were added compared to those spiked with β-alanine
alone (Figure 5C).

Molecules 2015, 20
7855
(
(
(
A)
1
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
a
0
0
0
0
0
0
0
0
0
a
b
c
c
c
c
c
c
c
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(
50) (100) (330) (650) (10) (30) (30) (120)
B) 0.8
a
a
0
0
0
0
0
0
0
.7
.6
.5
.4
.3
.2
.1
0
b
c
c
c
c
c
c
c
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(
50) (100) (330) (650) (10) (30) (30) (120)
C) 0.6
a
0
0
0
0
0
.5
.4
.3
.2
.1
0
ab
b
c
c
c
c
c
c
c
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(50) (100) (330) (650) (10) (30) (30) (120)
Figure 4. The concentration of (A) ethyl hexanoate, (B) ethyl octanoate and (C) ethyl
decanoate in model wines produced after the addition of amino acids as indicated on the
−1
x-axis. The number in brackets represents the concentration added in mg·L . Histograms
represent the mean values (n = 3) and error bars show standard errors. Different letters (e.g.,
a, b, c) denote significant differences between treatments at p < 0.05 using ANOVA followed
by Tukey’s post hoc test.

Molecules 2015, 20
7856
(
A) 18
16
14
12
10
8
6
4
2
0
a
a
a
b
b
b
b
b
b
b
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(50) (100) (330) (650) (10) (30) (30) (120)
(
B) 45
a
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
a
a
b
b
b
b
b
b
b
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(50) (100) (330) (650) (10) (30) (30) (120)
(
C) 9
a
8
7
6
5
4
3
2
1
0
a
b
c
c
c
c
c
c
c
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(50) (100) (330) (650) (10) (30) (30) (120)
Figure 5. The concentration of (A) hexanoic acid, (B) octanoic acid and (C) decanoic acid
in model wines produced after the addition of amino acids as indicated on the x-axis. The
−1
number in brackets represents the concentration added in mg·L . Histograms represent the
mean values (n = 3) and error bars show standard errors. Different letters (e.g., a, b, c) denote
significant differences between treatments at p < 0.05 using ANOVA followed by Tukey’s
post hoc test.
The concentration of three abundant acetate esters was also determined in the model wines produced
after amino acid additions to the model must (Figure 6). Both ethyl acetate and phenylethyl acetate

Molecules 2015, 20
7857
production was higher in the wines produced after β-alanine addition to the musts, either alone or in
combination with the other three amino acids, compared to control wine. However, isoamyl acetate
concentrations were not significantly higher in the wines produced after β-alanine addition compared to
the control. This suggests that isoamyl alcohol concentrations may be limiting production of the
corresponding acetate ester, but the concentration of the alcohol moiety of the acetate ester is not limiting
for ethyl acetate and phenylethyl acetate production. Presumably, increased CoA synthesis caused by the
presence of β-alanine in the must can then increase production of these acetate esters during fermentation.
(A) 140
1
20
a
1
00
ab
80
60
40
20
0
b
c
c
c
c
c
c
c
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(50) (100) (330) (650) (10) (30) (30) (120)
(
B)
0
.2
a
a
0
.18
.16
.14
.12
0
0
0
b
0
.1
c
c
c
c
0
0
0
0
.08
.06
.04
.02
0
c
c
c
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(
50) (100) (330) (650) (10) (30) (30) (120)
(C)
0.5
0
.45
a
ab
0
.4
.35
.3
.25
.2
.15
.1
0
0
0
0
ab
0
ab ab
ab
0
ab
ab
b
ab
0
.05
0
Cont
β
b-Ala
β
b-Ala Arg
Arg
Cys
Cys
Val
Val
All
(50) (100) (330) (650) (10) (30) (30) (120)
Figure 6. The concentration of (A) ethyl acetate, (B) isoamyl acetate and (C) phenylethyl
acetate in model wines produced after the addition of amino acids as indicated on the
−1
x-axis. The number in brackets represents the concentration added in mg·L . Histograms
represent the mean values (n = 3) and error bars show standard errors. Different letters (e.g.,
a, b, c) denote significant differences between treatments at p < 0.05 using ANOVA followed
by Tukey’s post hoc test.

Molecules 2015, 20
.2.2. β-Alanine Concentrations in Grapes are Sufficient to Stimulate Ester Production
7858
2
−1
The previous experiment demonstrated that additions of 50 and 100 mg·L β-alanine could increase
ester concentrations in model fermentations, but cysteine, arginine and valine did not have a significant
effect. The concentrations of β-alanine added to the must were above the maximum reported in the
−1
−1
literature for V. vinifera (33 mg·L ; [39]) or Vitis labruscana (13 mg·L ; [41]). A collection of
0 Cabernet Sauvignon grape parcels obtained from eight growing regions across two vintages was
5
analysed for β-alanine content to confirm that similar concentrations would be found in
Australian-grown fruit. The concentration of β-alanine in these samples ranged from 14 to 66 mg·L−1
with no apparent pattern based on vintage or growing region. Given that some of these values were
−1
below the lowest concentration of β-alanine tested in the experiment above (50 mg·L ), lower
concentrations of β-alanine were assessed for their ability to stimulate ester production.
The results indicated that a significant stimulation of ethyl hexanoate production was achieved with
−1
the addition of 1 mg·L β-alanine to the model must, and any further increase did not significantly
increase ethyl ester production further (Figure 7A). This result was the same for ethyl octanoate and
ethyl decanoate production (data not shown). Hexanoic acid, octanoic acid and decanoic acid also
−1
accumulated to higher concentrations in the wines produced after the addition of 1 mg·L or more
β-alanine—data is shown for hexanoic acid (Figure 7B). Similarly, ethyl acetate was present in higher
concentrations in wines produced after any β-alanine addition compared to the control treatment
(
Figure 7C), although the accumulation of phenylethyl acetate and isoamyl acetate was not significantly
different from the control treatments for some of the low β-alanine concentrations (data not shown).
−1
This suggests that 1 mg·L or less of β-alanine is sufficient to stimulate the production of greater
amounts of MCFAs and their corresponding ethyl esters, but the addition of more β-alanine does not
−1
significantly increase this production further. It is possible that 1 mg·L of β-alanine is enough to
account for the requirements of the yeast for maximum fatty acid anabolism and any extra is utilised for
other forms of nitrogen metabolism. The effect of β-alanine additions on acetate ester production is
probably complicated by the limitation of the alcohol moiety of the ester in the case of isoamyl acetate
and phenylethyl acetate; in the case of ethyl acetate, where the alcohol is not limiting, the accumulation
is similar to that seen for the ethyl esters (Figure 7).
2
.2.3. β-Alanine or Pantothenate Supplementation Have a Similar Effect on Ester Production
To test whether the effect of β-alanine addition to the model must had a similar effect as adding
pantothenate, another set of fermentations was prepared with either no additions, addition of 100 mg·L−1
−1
β-alanine (1.12 mM), addition of 267 mg·L panthothenate (1.12 mM), and addition of both β-alanine
and pantothenate (same concentration as above).

Molecules 2015, 20
7859
(
A) 0.9
0
0
0
0
0
0
0
0
.8
.7
.6
.5
.4
.3
.2
.1
0
a
a
a
a
a
a
a
b
Cont
1
2.5
5
10
25
50
100
β-alanine (mg/L)
(
B) 20
18
16
14
12
10
8
6
4
2
0
a
a
a
a
a
a
a
b
Cont
1
2.5
5
10
25
50
100
β-alanine (mg/L)
(
C) 250
a
a
a
2
1
1
00
50
00
a
a
a
a
b
5
0
0
Cont
1
2.5
5
10
25
50
100
β-alanine (mg/L)
Figure 7. The concentration of (A) ethyl hexanoate, (B) hexanoic acid and (C) ethyl acetate
in model wines produced after the addition of increasing amounts of β-alanine as indicated
on the x-axis. Histograms represent the mean values (n = 3) and error bars show standard
errors. Different letters (e.g., a, b) denote significant differences between treatments at
p < 0.05 using ANOVA followed by Tukey’s post hoc test.

Molecules 2015, 20
7860
(
A) 0.9
0
0
0
0
0
0
0
0
.8
.7
.6
.5
.4
.3
.2
.1
0
a
a
a
b
Cont
β-Ala
Panto
β-Ala/Panto
(
B) 18
a
1
1
1
1
6
4
2
0
8
6
4
2
0
a
a
b
Cont
β-Ala
Panto
β-Ala/Panto
(
C) 250
a
2
1
1
00
50
00
a
b
c
5
0
0
Cont
β-Ala
Panto
β-Ala/Panto
Figure 8. The concentration of (A) ethyl hexanoate, (B) hexanoic acid and (C) ethyl acetate
in model wines produced after the addition of β-alanine (β-Ala), pantothenate (Panto) or both
compounds (β-Ala/Panto) as indicated on the x-axis. Histograms represent the mean values
(
n = 3) and error bars show standard errors. Different letters (e.g., a, b, c) denote significant
differences between treatments at p < 0.05 using ANOVA followed by Tukey’s post hoc test.
Ethyl hexanoate production in the fermentations was stimulated to the same degree by the addition
of β-alanine, panthothenate or both compounds to the MGJM (Figure 8A). This was also observed for
ethyl decanoate, although ethyl octanoate concentrations were not significantly different to the control
when only pantothenate was added to the must (data not shown). Hexanoic acid concentrations were
significantly greater in the wines produced from all three treatments compared to the control (Figure 8B)

Molecules 2015, 20
7861
and octanoic and decanoic acid responded in the same manner to the supplementation of the must
data not shown). Of the acetate esters, ethyl acetate production was significantly increased in response
(
to the addition of pantothenate to the model must, but even greater production was observed when
β-alanine was added (Figure 8C). When isoamyl acetate and phenylethyl acetate were examined, only
those wines produced after the addition of β-alanine to the must had greater amounts of these esters
compared to the control (data not shown).
In general, both β-alanine and panthothenate could stimulate ester production in the model
fermentation system used in these experiments. However, in some cases, β-alanine was more efficient
than pantothenate at increasing ester concentrations. This observation seems counterintuitive given that
pantothenate is produced from pantoate and β-alanine during CoA biosynthesis (Figure 3). However,
there may be differences in the ability of yeast to transport these compounds into the cell which in turn
could influence CoA and ester production. It is known that pantothenate is transported by the Fen2p
symporter, whereas β-alanine is transported mainly by the general amino acid permease Gap1p, although
other transporters are involved [42]. The redundancy in the β-alanine transport system may make its
transport more efficient and hence the concentration in the yeast cytoplasm may be greater than that of
pantothenate when both are at the same concentration in the media. Alternatively, β-alanine may be used
in other metabolic pathways that can also stimulate ester production independent of CoA biosynthesis.
A survey of panthothenate concentrations in grapes grown in the Pacific northwest of the USA found
that they ranged from 0.18 to 1.26 mg·L−1 [43]. This is lower than the values reported for
β-alanine (this manuscript, [39]) which would suggest that β-alanine may have a more important role in
CoA production during fermentation of grapes than pantothenate.
3
3
3
. Experimental Section
.1. Material
.1.1. Chemicals
Commercially available compounds used for fermentation spiking were purchased from
Sigma-Aldrich (Castle Hill, NSW, Australia) and used without further preparation. Putative precursor
compounds were synthesised from commercially available reagents and other chemicals outlined below,
such as amino acids, salts and sugars, were analytical reagent grade (Sigma-Aldrich). All solvents used
were analytical reagent (Sigma-Alrich) or HPLC grade (Merck, Kilsyth, VIC, Australia) and water was
obtained from a Milli-Q purification system (Millipore, North Ryde, NSW, Australia). Solutions were
%
v/v with the balance made up with Milli-Q water, unless otherwise noted.
3
.1.2. Nuclear Magnetic Resonance (NMR)
1
13
Proton ( H) and carbon ( C) NMR spectra were recorded with a Bruker Avance III spectrometer
operating at 600 MHz for proton and 150 MHz for carbon nuclei. Chemical shifts were recorded as δ
values in parts per million (ppm). Spectra were acquired in methanol-d at ambient temperature, using
4
the solvent residual peak as internal reference, and resonances were assigned by routine 2D
correlation experiments.

Molecules 2015, 20
.1.3. High-Resolution Mass Spectrometry (HR-MS)
Spectra were obtained on a Bruker microTOF-Q II with electrospray ionization (ESI) in positive
7862
3
−1
mode. Samples dissolved in 2:1 methanol/water at concentrations of approximately 1−2 mg·L were
analysed by flow injection.
3
.1.4. HPLC-MS Instrumentation
Analysis of synthesised compounds was undertaken using a ThermoFinnigan Surveyor HPLC
connected to a ThermoFinnigan LCQ Deca XP Plus mass spectrometer. Electrospray ionisation in
positive ion mode was used and data acquisition and processing were performed using Xcalibur software
(
version 1.3, Thermo Scientific, San Jose, CA, USA).
3.1.5. GC-MS Instrumentation
Analyses of synthetic standards and fermentation esters were conducted with an Agilent (Palo Alto,
CA, USA) 7890A gas chromatograph, equipped with a Gerstel (Mülheim an der Ruhr, Germany) MP2
autosampler fitted with a DVB/CAR/PDMS SPME fibre (2 cm, 50/30 μm, Supelco, Bellefonte, PA,
USA), coupled to an Agilent 5975C mass spectrometer as described previously [19]. Selected ion
monitoring (SIM) was used for detection of analytes for quantitation and scan mode (m/z 35−350; scan
rate, 4.45 scans/s) was used for synthesised standards. Compounds were identified by comparing their
mass spectra to spectral libraries and from the analysis of authentic reference compounds.
3.1.6. Melting Points
A Buchi melting point B-540 unit was used, and melting points were uncorrected.
.2. Synthesis of Compounds
3
3
.2.1. d -Ethyl Nonanoate
5
To a stirred solution of d
room temperature was added pyridine (614 μL, 7.59 mmol) followed by 4-dimethylaminopyridine
DMAP) (0.106 g, 0.868 mmol). After dissolution, nonanoyl chloride (1.50 mL, 8.32 mmol) was added
drop-wise to the solution at 0 °C, causing the formation of a white precipitate. Additional anhydrous
CH Cl (3 mL) was added and the solution was stirred overnight at room temperature, before being
quenched with NaHCO (10 mL). The phases were separated, and the aqueous phase was extracted with
CH Cl (3 × 20 mL), and the combined organic phases were washed with brine (20 mL), dried (NaSO ),
and filtered before being concentrated in vacuo to yield a yellow/brown oil. Purification by column
chromatography on silica (85% hexane/EtOAc, R = 0.45) gave the title compound (1.41 g, 7.37 mmol,
9%) as a colorless oil (97% pure by GC-MS). H-NMR (600 MHz, CD OD): δ 2.29 (2H, t, J = 7.6 Hz,
); 1.62 (2H, app q, J = 7.4 Hz, H ); 1.35–1.22 (10 H, m, H4-8); 0.88 (3H, t, J = 7.0 Hz, H
150 MHz, MeOH-d ): δ 173.97 (C ); 59.40 (JC-D = 22 Hz, CD ); 34.39 (C ); 31.79 (C ); 29.21 (C
9.13 (C ); 29.11 (C ); 24.97 (C ); 22.63 (C ); 14.09 (C ); 13.21 (JC-D = 19.4 Hz, CD ). ESI-HRMS
6
-ethanol (500 μL, 8.64 mmol) in anhydrous CH
2
Cl
2
(4 mL) under N
2
at
(
2
2
3
2
2
4
f
1
8
3
1
3
H
2
3
9
); C-NMR
);
(
4
1
2
2
7
4
2
5
6
3
8
9
3

Molecules 2015, 20
7863
+
+
+
(
(
m/z): Calcd for C11
H D
18 5
O ([M+H] ), 192.2006; found 192.2022. EI-MS (m/z) (%) 191 (M , 3), 162
2
12), 148 (13), 141 (19), 120 (6), 106 (39), 93 (100), 84 (6), 74 (28), 61 (20), 57 (13), 55 (17).
3
.2.2. d -Ethyl Dodecanoate
5
This compound was prepared using the procedure described for d
chloride (1.85 mL, 8.29 mmol), d -ethanol (500 μL, 8.64 mmol), pyridine (614 μL, 7.6 mmol), and
DMAP (105.6 mg, 864 μmol) in CH Cl (13 mL). Purification by column chromatography on silica
5
-ethyl nonanoate, using dodecanoyl
6
2
2
(
85% hexane/EtOAc, R
f
= 0.59) gave the title compound (1.85 g, 7.93 mmol, 96%) as a colourless oil,
1
which was >99% pure (GC-MS). H-NMR (MeOH-d
4
) δ 2.29 (2H, t, J = 7.6 Hz, H
2
); 1.62 (2H, app q,
) δ 173.97 (C );
); 31.88 (C10); 29.58 (C4,5); 29.44 (C ); 29.32 (C );
); 22.67 (C11); 14.11 (C12); 13.31 (C14); 13.2 (JC-D = 19.5 Hz, CD ).
([M+H] ), 234.2467; found, 234.2476. EI-MS (m/z) (%) 233
13
J = 7.2 Hz, H
3
); 1.35–1.22 (16H, m, H4-8); 0.88 (3H, t, J = 7.0 Hz, H12); C-NMR (MeOH-d
9.39 (JC-D = 22.2Hz, CD ); 58.88 (C13); 34.38 (C
9.25 (C ); 29.13 (C ); 24.97 (C
4
1
5
2
2
2
6
7
8
9
3
3
+
+
ESI-HRMS (m/z): Calcd for C14
H
D O
24 5 2
+
(M , 6), 204 (5), 190 (12), 183 (12), 162 (15), 148 (8), 120 (5), 106 (52), 98 (5), 94 (12), 93 (100), 84 (6),
8
3 (5), 74 (24), 69 (7), 63 (6), 61 (11), 57 (10), 55 (16), 43 (16), 41 (16), 34 (6), 32 (9), 29 (6), 28 (33).
.2.3. (2R/S)-3-Carboxy-2-(nonanoyloxy)-N,N,N-trimethylpropan-1-aminium (N-Nonanoyl carnitine)
Carnitine derivatives were prepared based on the method of Bøhmer and Bremer [44]. To a stirred
3
solution of trifluoroacetic acid (TFA) (500 μL) at room temperature containing (±)-carnitine chloride
1.00 g, 5 mmol) was added dropwise nonanoyl chloride (1.77 g, 10 mmol), at which time a white
(
precipitate formed. The reaction was maintained at 50 °C with stirring overnight. After cooling to room
temperature, acetone (5.5 mL) was added, stirring was maintained for 1 hour and the precipitate that
formed was removed via centrifugation. Diethyl ether (~3 mL) was added dropwise to incipient
cloudiness, followed by cooling to 0 °C. Additional Et
well underway. Once crystallisation was complete, centrifugation and resuspension in Et
used to wash the crystals (repeated two more times), followed by isolation and drying. Purification by
2
O (10 mL) was added once crystallisation was
2
O (40 mL) was
recrystallisation in MeOH/Et
2
O produced the known [45] title compound (1.16 g, 3.43 mmol, 69%) as
1
a white crystalline solid, which was >99% pure (HPLC-MS), mp 155.2–157.7 °C. H-NMR (MeOH-d
4
)
δ 5.63 (1H, app q, J = 6.9 Hz); 4.92 (1H, bs); 3.49 (1H, dd, J = 14.4, 8.6 Hz); 3.75 (1H, dd, J = 14.4,
0
0
3
.90 Hz); 3.23 (9H, s); 2.79–2.76 (2H, m); 2.39 (2H, m); 1.63 (2H, q, J = 7.35 Hz); 1.38–1.25 (10H, m);
1
3
.90 (3H, t, J = 7.1 Hz). C-NMR (MeOH-d
4
) δ 174.27, 172.54, 69.50, 66.35, 54.68, 37.93, 35.22,
3.11, 30.50, 30.39, 30.31, 25.83, 23.84, 14.60.
3
.2.4. (2R/S)-3-Carboxy-2-(dodecanoyloxy)-N,N,N-trimethylpropan-1-aminium
(
N-Dodecanoyl carnitine)
Using the same reaction and isolation procedure as that for N-nonanoyl carnitine above,
N-dodecanoyl carnitine was prepared using (±)-carnitine chloride (1.00 g, 5 mmol) and dodecanoyl
chloride (2.19 g, 10 mmol) in (TFA) (500 μL). Purification by recrystallisation in MeOH/Et O produced
2
the known [45] title compound (1.32 g, 3.47 mmol, 69%) as a white crystalline solid, which was 93%
1
pure (HPLC-MS), mp 167.5–175.3 °C. H-NMR (MeOH-d
4
) δ 5.63 (1H, app q, J = 6.9 Hz); 3.89 (1H,

Molecules 2015, 20
7864
dd, J = 14.3, 8.6 Hz); 3.74 (1H, d, J = 14.3 Hz); 3.22 (9H, s); 2.79–2.76 (2H, m); 2.39 (2H, app t,
1
3
J = 7.5 Hz); 1.63 (2H, q, J = 7.3 Hz); 1.37–1.24 (16H, m); 0.90 (3H, t, J = 7.0 Hz). C-NMR
MeOH-d ) δ 174.13, 172.41, 69.37, 66.21, 37.77, 35.08, 33.07, 30.74, 30.59, 30.48, 30.42, 30.18, 25.71,
3.75, 14.48.
(
4
2
3
.2.5. Methyl 2-(Nonanoylamino)propanoate (N-Nonanoyl Alanine Methyl Ester)
Amide coupling was performed according to the method of Staab [46] and Staab et al. [47] using
carbonyl diimidazole (CDI). To a stirred solution of nonanoic acid (322.9 μL, 2 mmol) in anhydrous
THF (4.39 mL) under N was added CDI (324.3 mg, 2 mmol), causing effervescence from liberated
CO . Stirring was continued at room temperature for 2 h and methyl L-alaninate (279.2 mg, 2 mmol)
2
2
was added, resulting in separation of a brown oily residue. Stirring at room temperature continued
overnight, by which time a white precipitate had formed. The solvent was removed in vacuo, resulting
in the formation of white crystals in an oily residue, which were then taken up in Et
2
O (10 mL), EtOAc
(
20 mL), and H
successively with H
dried (MgSO
.27 mmol, 113%) which was used crude in the subsequent reaction. A small portion was purified for
characterisation by column chromatography on silica (80% CH Cl /EtOAc, R = 0.51) to give the title
2
SO
4
(1 M, 15 mL). The phases were separated and the organic phase was washed
2
SO (1 M, 3 × 15 mL), water (20 mL), and brine (20 mL). The organic phase was
4
4
), filtered, and concentrated in vacuo, producing a white crystalline solid, (551.3 mg,
2
2
2
f
2
0
compound as a pale yellow powder, which was 98% pure (HPLC-MS), mp 36.9–38.8 °C. [α]
D
= +4.8
1
(
c 0.620, CHCl
3
). H-NMR (MeOH-d
4
) δ 6.05–6.12 (1H, m, NH); 4.61 (1H, app q, J = 7.2 Hz, H11);
); 1.63 (2H, app q, J = 7.4 Hz, H ); 1.40 (3H, d, J = 7.1 Hz, H12); 1.35–1.22
10H, m, H4-8); 0.87 (3H, t, J = 7.0 Hz, H12); C-NMR (MeOH-d ) δ 173.72 (C10); 172.65 (C ); 52.42
); 22.60 (C );
2
.20 (2H, t, J = 7.6 Hz, H
2
3
1
3
(
(
4
1
C
13); 47.81 (C11); 36.54 (C
2
); 31.77 (C
7
); 29.25 (C
4
); 29.17 (C
5
); 29.09 (C
6
); 25.53 (C
3
8
26NO +
([M+H] ), 244.1985; found, 244.1907.
+
1
8.56 (C12); 14.06 (C ). ESI-HRMS (m/z): Calcd for C13
9
H
3
3
.2.6. 2-(Nonanoylamino)propanoic acid (N-nonanoyl alanine)
To a stirred solution of THF (24.4 mL) containing N-nonanoyl methyl ester (501.3 mg, 1.24 mmol)
was added aqueous NaOH (1 M, 9.76 mL) and the solution was stirred at room temperature for 5 hours.
After adjustment to pH 3 with HCl (1 M), the solution was extracted with CH Cl (3 × 20 mL). The
combined organic fractions were washed with water (20 mL) and brine (20 mL), before being dried
MgSO ), filtered and concentrated in vacuo to produce a colourless oil. Purification by crystallisation in
MeOH/H O gave the title compound (414.8 mg, 1.81 mmol, 90%) as a colourless oil, which was >98%
pure (HPLC-MS). [α]
1H, m, NH); 4.58 (1H, app q, J = 7.1 Hz, H11); 2.24 (2H, t, J = 7.7 Hz, H
); 1.46 (3H, d, J = 7.2 Hz, H ); 1.35–1.22 (10H, m, H4-8); 0.88 (3H, t, J = 7.0 Hz, H
MeOH-d ) δ 175.93 (C10); 174.11 (C ); 48.21 (C11); 36.40 (C ); 31.76 (C ); 29.22 (C ); 29.13 (C
); 25.54 (C ); 22.60 (C ); 18.06 (C12); 14.06 (C ). ESI-HRMS (m/z): Calcd for C12
2
2
(
4
2
2
D
0
1
= −17.2 (c 0.580, CHCl
3
). H-NMR (MeOH-d
4 2
) δ 8.35 (1H, bs, CO H); 6.36–6.28
(
2
); 1.63 (2H, app q, J = 7.2 Hz,
1
3
H
3
2
9
); C-NMR
);
(
4
1
2
7
4
5
2
9.09 (C
6
3
8
9
H
24NO +
3
+
([M+H] ), 230.1748; found, 230.1751.

Molecules 2015, 20
.2.7. Methyl 2-(Dodecanoylamino)propanoate (N-dodecanoyl alanine methyl ester)
7865
3
Using the same reaction and isolation procedure as that for N-nonanoyl alanine methyl ester above,
N-dodecanoyl alanine methyl ester was prepared using dodecanoic acid (817.6 μL, 4 mmol), CDI
648.6 mg, 4 mmol) and methyl L-alaninate (558.3 mg, 4 mmol) in THF (8.79 mL), producing a white,
waxy solid (1.108 g, 3.88 mmol, 97%). Purification by recrystallisation was attempted in CH Cl /hexane
(
2
2
and EtOH/hexane, but the product formed an organogel, so the crude product underwent the
saponification procedure described below. A small portion was purified for characterisation by column
chromatography on silica (80% CH
2
Cl
2
/EtOAc, R = 0.47) to give the title compound as a white
f
2
0
crystalline solid, which was >99% pure (HPLC-MS), mp 58.8–61.2 °C. [α]
D
= +5.0 (c 0.600, CHCl ).
3
1
H-NMR (MeOH-d
2H, t, J = 7.7 Hz, H
m, H4-11); 0.88 (3H, t, J = 7.0 Hz, H12); C-NMR (MeOH-d
7.94 (C14); 36.67 (C ); 31.04 (C10); 29.56 (C4-9); 25.67 (C
Melting point and spectral data were in complete accord with those previously reported [48].
4
) δ 6.10–6.02 (1H, m, NH); 4.61 (1H, app q, J = 7.2 Hz, H14); 3.75 (3H, s, H16); 2.20
); 1.63 (2H, app q, J = 7.4 Hz, H ); 1.40 (3H, d, J = 7.1 Hz, H15); 1.35–1.20 (16H,
) δ 173.87 (C13); 172.83 (C ); 52.56 (C16);
); 22.77 (C11); 18.70 (C15); 14.06 (C12).
(
3
2
1
3
4
1
4
2
3
3
.2.8. 2-(Dodecanoylamino)propanoic acid (N-dodecanoyl alanine)
Using the same reaction and isolation procedure as that for N-nonanoyl alanine above, N-dodecanoyl
alanine was prepared using N-dodecanoyl alanine methyl ester (1.05 g, 3.69 mmol), THF (64.7 mL), and
aqueous NaOH (1 M, 25.9 mL) to produce a white powder. Purification by recrystallisation in
MeOH/H
2
O gave the title compound (928.8 mg, 3.42 mmol, 86%) as a white powder, which was >98%
1
pure (HPLC-MS), mp 82.8–83.7 °C. H-NMR (MeOH-d
4
) δ 6.30–6.23 (1H, m, NH); 4.57 (1H, app q,
); 1.62 (2H, app q, J = 7.3 Hz, H ); 1.46 (3H, d, J = 7.2 Hz,
15); 1.35–1.21 (16H, m, H4-11); 0.88 (3H, t, J = 7.0 Hz, H12); C-NMR (MeOH-d ) δ 175.62 (C13);
74.06 (C ); 48.22 (C14); 36.41 (C ); 31.88 (C10); 29.67 (C ); 29.57 (C ); 29.44 (C ); 29.31 (C ); 29.27
); 29.15 (C ); 25.53 (C ); 22.66 (C11); 18.00 (C15); 14.10 (C12). Melting point and spectral data were
in complete accord with those previously reported [48].
J = 7.1 Hz, H14); 2.24 (2H, t, J = 7.6 Hz, H
2
3
1
3
H
4
1
1
2
4
5
6
7
(
C
8
9
3
3
.2.9. Methyl 2-(Nonanoylamino)-3-methylpentanoate (N-nonanoyl isoleucine methyl ester)
Using the same reaction and isolation procedure as that for N-nonanoyl alanine methyl ester above,
N-nonanoyl isoleucine methyl ester was prepared using nonanoic acid (645.9 μL, 4 mmol), CDI (648.6
mg, 4 mmol) and methyl L-isoleucinate (726.6 mg, 4 mmol) in THF (8.79 mL), producing a pale brown
oil (1.022 g, 3.58 mmol, 90%). A small portion was purified for characterisation by column
chromatography on silica (85% CH
2
Cl
2
/EtOAc, R
f
= 0.57) producing the title compound as a pale brown
) δ 5.92–5.99 (1H, m, NH); 4.62 (1H, dd,
); 1.92–1.84 (1H, m, H12); 1.64 (2H, app q, J = 7.3 Hz,
); 1.27 (11H, m, H4-8); 1.17 (1H, dqd, J = 14.8, 7.4, 7.3 Hz,
1
oil, which was >98% pure (HPLC-MS). H-NMR (MeOH-d
4
J = 8.6, 5.0 Hz, H11); 2.22 (3H, t, J = 7.6 Hz, H
2
H
3
); 1.43 (1H, dqd, J = 14.8, 7.4, 4.7 Hz, H
13); 0.92 (3H, t, J = 7.4 Hz, H14); 0.90 (3H, d, J = 6.8 Hz, H15); 0.88 (3H, t, J = 7.0 Hz, H
) δ 172.87 (C ); 172.73 (C10); 56.11 (C11); 52.06 (C16); 37.97 (C12); 36.70 (C ); 31.78 (C
); 29.21 (C ); 29.12 (C ); 15.38 (C13); 14.08 (C ); 11.55
14). ESI-HRMS (m/z): Calcd for C16
3
1
3
H
9
). C-NMR
);
(MeOH-d
4
1
2
7
2
9.26 (C
4
5
6
); 25.64 (C
3
); 25.18 (C15); 22.61 (C
8
9
32NO +
([M+H] ), 286.2368; found, 286.2377.
+
(
C
H
3

Molecules 2015, 20
.2.10. 2-(Nonanoylamino)-3-methylpentanoic acid (N-nonanoyl isoleucine)
7866
3
Using the same reaction and isolation procedure as that for N-nonanoyl alanine above, N-nonanoyl
isoleucine was prepared using N-nonanoyl isoleucine methyl ester (0.972 g, 3.41 mmol), THF
4.25 mL), and aqueous NaOH (1 M, 3.4 mL) to produce a white solid. Recrystallisation in 1:1
EtOAc/hexane produced the title compound (590 mg, 2.17 mmol, 64%) as fine white crystals that were
(
2
0
1
9
8% pure (HPLC-MS). [α]
CO2H); 6.24–6.16 (1H, m, NH); 4.63 (1H, dd, J = 8.5, 4.7 Hz, H11); 2.31–2.22 (2H, m, H
1H, m, H12); 1.63 (2H, app q, J = 7.4 Hz, H ); 1.50 (1H, dqd, J = 13.4, 7.4, 4.5 Hz, H ); 1.35–1.21 (10H,
m, H4-8); 1.26–1.17 (1H, m, H13); 0.95 (3H, d, J = 6.8 Hz, H15); 0.94 (3H, t, J = 7.4 Hz, H14); 0.88 (3H,
D
= +26.9 (c 1.135, CHCl
3
). H-NMR (MeOH-d
4
) δ 8.45–7.72 (1H, bs,
2
); 1.99–1.92
(
3
3
1
3
t, J = 7.0 Hz, H
); 31.77 (C ); 29.23, 29.17, 29.11 (C4,5,6); 25.69 (C
1.57 (C14). ESI-HRMS (m/z): Calcd for C15
9
). C-NMR (MeOH-d
4
) δ 175.40 (C ); 174.03 (C10); 56.41 (C11); 37.62 (C12); 36.62
1
(
C
2
7
3
); 25.03 (C15); 22.61 (C
8
); 15.37 (C13); 14.07 (C );
9
30NO +
([M+H] ), 272.2215; found, 272.2220.
+
1
H
3
3
.2.11. Methyl 2-(dodecanoylamino)-3-methylpentanoate (N-dodecanoyl isoleucine methyl ester)
Using the same reaction and isolation procedure as that for N-nonanoyl alanine methyl ester above,
N-dodecanoyl isoleucine methyl ester was prepared using dodecanoic acid (801.6 mg, 4 mmol), CDI
648.6 mg, 4 mmol), methyl L-isoleucinate (726.6 mg, 4 mmol) in THF (8.79 mL), producing a white
solid (1.269 g, 3.85 mmol, 96%). A small portion was purified for characterisation by column
(
chromatography on silica (85% CH
2
Cl
2
/EtOAc, R
f
= 0.59) producing the title compound as white
2
0
1
powder, which was 98% pure (HPLC-MS). [α]
D
= +24.1 (c 0.76, CHCl
3
). H-NMR (MeOH-d
4
) δ
5
.99–5.94 (1H, m, NH); 4.62 (1H, dd, J = 8.6, 5.0 Hz, H14); 2.22 (2H, app t, J = 7.6 Hz, H
2
); 1.92–1.84
(
1H, m, H15); 1.63 (2H, app q, J = 7.3 Hz, H3); 1.44 (1H, dqd, J = 14.8, 7.4, 4.7 Hz, H16); 1.36–1.21
(
16H, m, H4-11); 1.16 (1H, dqd, J = 14.8, 7.4, 7.3 Hz, H13); 0.92 (3H, t, J = 7.4 Hz, H17); 0.91 (3H, d,
1
3
J = 6.9 Hz, H18); 0.88 (3H, t, J = 7.0 Hz, H
9
). C-NMR (MeOH-d
); 31.87 (C10), 30.92, 29.58, 29.46, 29.30, 29.21 (C4,5,6,7,8,9);
); 25.18 (C18); 22.66 (C11); 15.38 (C16); 14.10 (C12); 11.55 (C17). ESI-HRMS (m/z): Calcd for
4
) δ 172.91 (C
1
); 172.74 (C
3
); 56.12
(
C
14); 52.07 (C19); 37.97 (C15); 36.71 (C
2
2
5.65 (C
3
+
+
C
19
H38NO
3
([M+H] ), 328.2838; found, 328.2846.
3
.2.12. 2-(Dodecanoylamino)-3-methylpentanoic acid (N-dodecanoyl isoleucine)
Using the same reaction and isolation procedure as that for N-nonanoyl alanine above, N-dodecanoyl
isoleucine was prepared using N-dodecanoyl isoleucine methyl ester (1.96 g, 5.98 mmol), THF
4.25 mL), and aqueous NaOH (1 M, 6.1 mL) to produce a white solid. Purification by recrystallisation
in 9:1 MeOH/H O overnight at −4 °C produced an organogel, which dissolved at room temperature, and
vacuum filtration at 0 °C afforded the title compound (737 mg, 2.35 mmol, 61%) as very fine white
(
2
2
0
1
crystals that were >98% pure (HPLC-MS). [α]
.18–6.09 (1H, m, NH); 4.63 (1H, dd, J = 8.3, 4.7 Hz, H14); 2.22–2.30 (2H, m, H
15); 1.64 (2H, app q, J = 7.1 Hz, H ); 1.55–1.47 (1H, m, H16); 1.36–1.25 (16H, m, H4-11), 1.25–1.17
1H, m, H16); 0.96 (3H, d, J = 6.8 Hz, H18); 0.94 (3H, t, J = 7.4 Hz, H17); 0.88 (3H, t, J = 7.0 Hz, H12).
13C-NMR (MeOH-d
) δ 175.48 (C13); 173.97 (C13); 56.40 (C14); 37.59 (C15); 36.65 (C ); 31.88 (C10),
D
= +22.5 (c 1.02, CHCl
3
). H-NMR (MeOH-d
4
) δ
6
2
); 1.99–1.92 (1H, m,
H
3
(
4
2

Molecules 2015, 20
9.58, 29.46, 29.31, 29.29, 29.19 (C4,5,6,7,8,9); 25.68 (C
7867
2
3
); 25.04 (C18); 22.67 (C11); 15.40 (C16); 14.11
36NO +
([M+H] ), 314.2675; found, 314.2690.
+
(
C12); 11.58 (C17). ESI-HRMS (m/z): Calcd for C18
H
3
3.3. Yeast
Fermentations were carried out with the commercial Saccharomyces cerevisiae strain EC1118 (Prise
de Mousse, AB Mauri, Australia) with preparation based on the protocol reported by Dennis et al. [19].
3
.4. Fermentation Conditions
3
.4.1. Trials Involving Fatty Acids and Precursors
Triplicate fermentations were conducted using model grape juice media (MGJM), prepared as
reported previously [19] and sterile filtered prior to use (0.20 μm disposable sterile filter units, Nalgene,
Rochester, NY, USA). Controlled fermentations (50 mL) were carried out with individual putative
precursor compounds (stock solutions prepared volumetrically using acetone as solvent) spiked into
MGJM at 0 μM, 1 μM, 10 μM, 100 μM, and 1 mM and then inoculated with yeast starter culture (1 mL).
Air locks were used to maintain an anaerobic environment. Fermentations were conducted in darkness,
at ~20 °C, until mass loss stabilised. Yeast cells were removed from the finished fermentations by
centrifugation (2300 rcf for 5 min) and the clarified wines were then stored in glass under N
2
at 4 °C
prior to analysis.
3
.4.2. Trials Involving Amino Acids and Pantothenate
MGJM was prepared according to the procedure adapted from Henschke and Jiranek [49] and
summarised by McBryde et al. [50] with some modifications. The medium contained D-glucose
−
1
−1
−1
−1
−1
(
100 g·L ), D-fructose (100 g·L ), 3 g·L L-malic acid, 0.2 g·L citric acid, 1.14 g·L K
2
HPO ,
4
−
1
.7H
−1
.4H
−1
−1
, 13.6 μg·L−1
1
.23 mg·L MgSO
4
2
O, 198.2 μg·L MnCl
2
2
O, 135.5 μg·L ZnCl
2
, 32 μg·L FeCl
2
−1
−1
.6H
.2H
−1
MoO .
−1
CuCl
2
, 5.7 μg·L boric acid, 29.1 μg·L Co(NO
3
)
2
2
O, 24.2 μg·L Na
2
4
2H
2
O, 10.8 μg·L KI,
−1
O .
−1
−1
−1
3
.12 g·L KNaC
4
H
4
6
4H
2
O, 0.44 g·L CaCl
2
2
O, 100 mg·L myo-insitol, 2 mg·L pyridoxide
−1
−1
−1
−1
HCl, 2 mg·L nicotinic acid, 0.5 mg·L thiamine, 0.2 mg·L p-aminobenzoic acid, 125 μg·L biotin,
−
1
−1
−1
0
.2 mg·L folic acid, 0.2 mg·L riboflavin, and 10 mg·L ergosterol dispersed in 0.5 mL Tween 80
and dissolved in 0.5 mL hot ethanol. The pH of the medium was corrected to 3.4 and sterile filtered (as
specified above). The final yeast assimilable nitrogen (YAN) content of each fermentation was adjusted
−
1
to 450 mg·L by additions of NH
4
Cl solution and target amino acids prepared as follows: NH Cl was
4
−1
dissolved in water at a concentration of 250 g·L and sterilised by filtration; amino acids were prepared
as individual stock solutions by dissolving 50 mg of L-cysteine, 320 mg of β-alanine, 500 mg of
L-arginine and 200 mg of L-valine in 10 mL sterile water, and 2.5 g of D-pantothenic acid hemicalcium
salt was dissolved in 10 mL sterile water.
3
.5. Quantitative GC-MS Analysis of Esters and Acids
SPME-GC-MS was utilised with the conditions described above under GC-MS instrumentation for
the analysis of fermentation treatments. SIM ions are presented in Table 1. A sample of the fermentation

Molecules 2015, 20
7868
medium (1 mL) was diluted volumetrically with sterile water to give a final volume of 10 mL, which
was transferred to an SPME vial. NaCl (3 g) was added into each vial along with 10 μL of an ethanolic
−
1
−1
solution containing d13-hexanol (920 mg·L ), d11-hexanoic acid (930 mg·L ), d
5
-ethyl nonanoate
-linalool (1.73 mg·L ), and 10 μL of an ethanolic solution
-hexyl acetate (8.76 mg·L ) prior to analysis. For quantifying the analytes, triplicate
−1
−1
−1
(
9.2 mg·L ), d16-octanal (82.1 mg·L ) and d
3
−1
containing d
3
standards in model wine (12% aqueous ethanol, pH adjusted to 3.2 with tartaric acid) were prepared at
evenly spaced concentrations across the range. The highest standard concentration for each analyte was
approximately 150% of the highest concentration observed in the model wines. Calibrations were linear
2
throughout the range with R = 0.97–0.99. All calibration samples were prepared and analysed according
to the protocols outlined above.
3
.6. Determination of β-Alanine Content of Grape Juice
β-Alanine was quantified in grape juice using an AccQ Fluor Reagent Kit (Waters Corporation,
Milford, MA, USA) and a method based on that of Cohen and Michaud [51]. An SPE step was added
prior to derivatisation to reduce chromatographic background [52]. The grape juice was obtained from
a 200 mg aliquot of frozen ground grape material that had been prepared from approximately 25 g of
berries. The samples were all from the Cabernet Sauvignon variety and were obtained from commercial
vineyards in the Barossa Valley, Clare Valley, Coonawarra, Eden Valley, Langhorne Creek, McLaren
Vale, Riverland and Wrattonbully grapegrowing regions of South Australia in the 2013 and 2014 vintages.
Table 1. Qualifier and quantifier ions used for GC-MS analysis of fermentation volatiles in
SIM mode.
Analyte (Internal Standard)
Ethyl acetate
SIM Ions (Quantifier in Bold)
43, 61, 70
Ethyl hexanoate
Ethyl octanoate
Ethyl nonanoate
Ethyl decanoate
Ethyl dodecanoate
Isoamyl acetate
Phenyl ethyl acetate
Hexanoic acid
43, 60, 88
88, 101, 127
73, 88, 101, 141, 186
73, 88, 101, 157
43, 88, 101, 157, 183
43, 55, 70, 87
39, 43, 65, 91, 104
41, 60, 73, 87
Octanoic acid
43, 55, 60, 73, 101
57, 60, 73, 101, 129
46, 59, 84
Decanoic acid
(
d
3
-Hexyl Acetate)
-Ethyl Nonanoate)
11-Hexanoic Acid)
(
d
5
41, 60, 93
(
d
63, 71
3
.7. Data Analysis
Results are presented as the mean of triplicate fermentation samples. The results were analysed by
one-way analysis of variance (ANOVA) followed by the Games-Howell (fatty acid experiments) or
Tukey’s (amino acid experiments) post hoc tests using SPSS version 20 (IBM; Armonk, NY, USA).

Molecules 2015, 20
. Conclusions
Understanding the links between grape composition and concentrations of volatile compounds in
7869
4
wine has important implications with respect to viticultural and winemaking parameters and the impact
of these on wine style and quality. Based on previous studies and an examination of yeast biochemical
pathways, known and putative grape-derived precursors were assessed for their ability to produce ethyl
and acetate esters upon fermentation in chemically defined model media. Significant stimulating effects
of MCFAs and their derivatives were revealed, as was the apparent toxicity of some compounds at the
highest concentrations tested. MCFAs, their methyl esters and carnitine conjugates were able to
contribute to ethyl ester production, albeit with variable influences of odd and even chain length MCFAs.
Amino acid conjugates of MCFAs also produced the corresponding ethyl esters, but with differences
evident between C and C12 MCFAs and for alanine compared to isoleucine; a toxic effect was noted at
9
higher concentrations of nonanoyl alanine, whereas dodecanoyl isoleucine had no significant effect on
ester production.
Investigation of four amino acids related to CoA production by yeast revealed the exclusive effect of
β-alanine on the formation of a range of MFCAs and their ethyl esters, along with acetate esters. Ethyl
−1
hexanoate, octanoate and decanoate significantly increased with 50 and 100 mg·L of β-alanine, as did
their MCFA counterparts, along with ethyl and phenylethyl acetates, but not isoamyl acetate. The results
implied a stimulatory effect on MCFA synthesis, leading to corresponding increases in esters, but
isoamyl acetate levels were deemed to be limited by isoamyl alcohol concentrations. Additional
experiments with lower concentrations of β-alanine, and β-alanine along with pantothenate, showed that
levels at or below the usual grape concentrations of these compounds were able to stimulate production
of a range of MCFAs and their ethyl esters, as well as ethyl, isoamyl and phenylethyl acetate esters. Both
β-alanine and panthothenate promoted ester production but β-alanine was more effective in
some circumstances.
Overall, a range of putative and known grape-derived precursors were shown to have important
effects on the production by yeast of MCFAs, and ethyl and acetate esters. These interesting outcomes
open the way for additional investigations of odd versus even MCFA chain length, MCFA carnitine
derivatives and the importance of β-alanine relative to pantothenate in actual juices or musts, which may
reveal even greater insights into the relationship between nonvolatile grape components and wine aroma
constituents. Ultimately, being able to predict and control wine aroma based on grape composition and
winemaking inputs could be realised as a result of such studies.
Acknowledgments
The authors wish to thank Sue Maffei, Annette Böttcher, Michelle Walker and Tommaso Liccioli for
technical assistance. Martin Johnston and Clark Nash are acknowledged for their assistance with NMR
analyses. This work was partly funded by CSIRO and The University of Adelaide. A.D.P. was supported
through The University of Adelaide by an Ian Cocks Memorial Scholarship. We thank Treasury Wine
Estates, Yalumba Wines and CCW Co-operative Limited for access to vineyards for grape samples. This
project was partly funded by Australia’s grapegrowers and winemakers through their investment body
the Australian Grape and Wine Authority with matching funding from the Australian Federal

Molecules 2015, 20
7870
Government. CSIRO Agriculture Flagship and The University of Adelaide are partners in the Wine
Innovation Cluster.
Author Contributions
P.K.B., E.G.D. and D.W.J. conceived and designed the experiments; all authors performed the
experiments; A.D.P., Y.Z., D.W.J., E.L.N. and P.K.B. analysed the data; all authors wrote and edited
the paper.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available.
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