Rates of formation of cis- and trans-oak lactone from 3-methyl-4- hydroxyoctanoic acid

Article abstract of DOI:10.1021/jf0496550

The rates of formation of both cis- and trans-oak lactone from the corresponding isomers of 3-methyl-4-hydroxyoctanoic acid have been measured in model wine at room temperature for a range of pH values. The half-life for formation of the trans-isomer at pH 2.9 was calculated to be 3.1 h, whereas that for the cis-isomer, at the same pH, was calculated to be 40.5 h. The k _trans/k_cis ratio in model wine was found to 12.86 ± 1.34 over the range of pH values employed. A reason for the more facile formation of the trans-isomer, based on conformational reasons, has been proposed. In acidic aqueous media the equilibrium between the oak lactones and their corresponding ring-opened analogues was found to favor the former entirely, with no evidence for the latter being found. Implications of the present study for the future analysis of oak samples, as well as for the interpretation of existing data, are discussed.

Full text of DOI:10.1021/jf0496550

J. Agric. Food Chem. 2004, 52, 4213−4218 4213
Rates of Formation of cis- and trans-Oak Lactone from
3-Methyl-4-hydroxyoctanoic Acid
KERRY L. WILKINSON,†,‡ GORDON M. ELSEY,*,†,‡ ROLF H. PRAGER,†
‡
ALAN P. POLLNITZ,‡ AND MARK A. SEFTON
School of Chemistry, Physics and Earth Sciences, Flinders University of South Australia,
P.O. Box 2100, Adelaide, South Australia 5001, Australia, and The Australian Wine Research
Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia
The rates of formation of both cis- and trans-oak lactone from the corresponding isomers of 3-methyl-
4-hydroxyoctanoic acid have been measured in model wine at room temperature for a range of pH
values. The half-life for formation of the trans-isomer at pH 2.9 was calculated to be 3.1 h, whereas
that for the cis-isomer, at the same pH, was calculated to be 40.5 h. The ktrans/kcis ratio in model wine
was found to 12.86 ( 1.34 over the range of pH values employed. A reason for the more facile
formation of the trans-isomer, based on conformational reasons, has been proposed. In acidic aqueous
media the equilibrium between the oak lactones and their corresponding ring-opened analogues was
found to favor the former entirely, with no evidence for the latter being found. Implications of the
present study for the future analysis of oak samples, as well as for the interpretation of existing data,
are discussed.
KEYWORDS: Oak; wine; oak lactone; whiskey lactone; kinetics; lactonization
INTRODUCTION
identified a galloylated glucoside of (3S,4S)-3-methyl-4-hy-
droxyoctanoic acid as a component of oakwood. Both this
compound 4 and the simple glucoside 3 are able to generate
cis-oak lactone during barrel-toasting, and perhaps also during
seasoning, but not during wine conservation in the absence of
enzymic activity (1).
The cis-isomer of the so-called oak lactones, (4S,5S)-5-butyl-
4-methyl-4,5-dihydro-2(3H)-furanone (1) (Figure 1), is regarded
as among the most important of the volatile components of
oakwood that are extracted into wine or spirits during barrel-
aging. Recent experiments (1) have shown that the nature-
identical enantiomer of cis-oak lactone had aroma detection
thresholds of 23 µg/L in a dry white wine and 46 µg/L in a red
wine, although there appeared to be a substantial variation in
the sensitivity of individual panelists to this compound.
There are many factors that influence the amount of cis- and
trans-oak lactone that can be extracted from oak. They include
the composition of the unseasoned wood, the seasoning and
toasting processes, and the length of time alcoholic beverages
spend in oak cooperage [the literature on cis- and trans-oak
lactone has been comprehensively reviewed (2)]. Such studies
indicate that one or more oak lactone precursors, present in
oakwood, are able to generate oak lactone during these
processes. Otsuka et al. (3) reported the isolation of a 3-methyl-
4-hydroxyoctanoic acid derivative, which they termed an oak
lactone precursor, and proposed the methylated gallic acid
derivative 2 (Figure 1) as the structure for this compound.
However, an authentic sample of this structure synthesized in
our laboratory had completely different properties from the
isolated compound (4). More recently, Masson et al. (5)
Singleton (6) has described cases in which the oak aroma in
wine continued to intensify for some time after the removal of
oak chips. Were such intensification of aroma due to increases
in cis-oak lactone concentration, then this would also indicate
the presence in the wines of a precursor form other than the
glucoside 3 or its derivative 4, which are essentially unreactive
under wine conservation conditions. Pollnitz (7) has also
observed small increases in cis- (but not trans-) oak lactone
concentration in model wine extracts of oak chips following
the removal of the chips from the wine. A potential candidate
for the continued formation of cis-oak lactone in wine following
the removal of oak contact is the aglycon component of 3,
3-methyl-4-hydroxyoctanoic acid (6). This compound could
quite conceivably form in oakwood as a result of cleavage of
the glucose unit (from 3) or the galloylglucose unit (from 4),
during seasoning or toasting, via chemical or biological
processes.
In a study of wines fermented and then stored in oak
cooperage, Waterhouse and Towey (8) reported significant
differences in the ratio of cis/trans-oak lactone isomers between
wines stored in American compared with French barrels. These
authors discussed the factors that might be responsible for the
changes, observed by other authors, in the cis/trans ratio or in
* Corresponding author (telephone +61 8 8201 3071; fax +61 8 8201
2905; e-mail Gordon.Elsey@flinders.edu.au).
† Flinders University.
‡ The Australian Wine Research Institute.
10.1021/jf0496550 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/03/2004

4214 J. Agric. Food Chem., Vol. 52, No. 13, 2004
Wilkinson et al.
Figure 1. Structures of the oak lactones (1), proposed precursor 2, and isolated precursors 3 and 4.
stock solution therefore comprised 5.82 g/L trans- and 5.28 g/L cis-
potassium 3-methyl-4-hydroxyoctanoate. This corresponds to a total
concentration of 5 of 11.10 g/L.
the amounts of extractable oak lactone which result from toasting
or seasoning. Waterhouse and Towey speculated that such
observations are a result of chemical equilibria between the oak
lactones (1), their open-chain hydroxy acid equivalents 6, and
the ethyl esters of such hydroxy acids. Maga (2) has stated that
acceptance of this hypothesis would require the disregarding
of all previous data in relation to the roles played by cis- and
trans-oak lactone in alcoholic beverage flavor, because such
data “were subject to artifacts inherent in the analytical process”.
This study was undertaken to determine (i) the rate of
lactonization of 4-hydroxy-3-methyloctanoic acid; (ii) the
equilibria among oak lactone, the corresponding hydroxy acid,
and the ethyl ester of the hydroxy acid for each of the cis- and
trans-isomers; (iii) the effects of pH and ethanol concentration
in these studies; and (iv) the implications of the results for
carrying out analysis of oak extracts and interpreting the results
of previously published analyses.
Preparation of Oak Lactone Solution. A stock solution of oak
lactone was prepared by dissolving a 50:50 mixture of racemic cis-1
and racemic trans-1 (∼1.5 mg) in ethanol (250 mL). The total oak
lactone concentration was accurately measured as 5.32 mg/L, by gas
chromatography-mass spectrometry (GC-MS) quantification (9).
Determination of pKa of 3-Methyl-4-hydroxyoctanoic Acid (6).
An aqueous solution of potassium 3-methyl-4-hydroxyoctanoate (111.0
mg, 0.523 mmol) was prepared by diluting the stock solution of 5 (10
mL, 11.10 g/L) to a total volume of 100 mL with water. To this was
added aqueous hydrochloric acid (0.1M) with stirring, at 25 °C. The
pH of the solution was measured after the addition of 2.11 mL (0.4
equiv), 2.61 mL (0.5 equiv), and 3.11 mL (0.6 equiv). The pKa of 6
was measured in both model wine (12% ethanol) and model spirit (40%
ethanol) in an identical manner.
Effect of Sodium Bicarbonate Addition on Oak Lactone Con-
centration. Model wine (10% ethanol) solutions of 1 (532 µg/L) were
prepared by diluting the stock solution of 1 (1 mL, 5.32 mg/L) to a
total volume of 10 mL with water. To this solution was added saturated
sodium bicarbonate (5 mL). The solution was then sealed in glass
ampules (2 mL). Samples were analyzed (in duplicate) for oak lactone
content by GC-MS using SPME, after 1, 2, and 7 days at room
temperature.
Effect of Sodium Bicarbonate Addition on 3-Methyl-4-hydroxy-
octanoic Acid Concentration. Model wine (10% ethanol) solutions
of 6 (10 mL, 412 µg/L) were prepared by diluting the stock solution
of 5 (50 µL, 11.21 mg/mL) to a total volume of 10 mL with ethanol (1
mL) and water. To this solution was added saturated sodium bicarbonate
(5 mL). The solution was then sealed in glass ampules (2 mL). Samples
were analyzed (in duplicate) for oak lactone content by GC-MS using
SPME, after 1, 2, and 7 days at room temperature.
Determination of Lactonization Rates for the Conversion of 6
into 1. Model wine (12% ethanol) solutions of 6 (cis, 441 µg/L; trans,
479 µg/L) were prepared by diluting the stock solution of 5 (1 mL;
cis, 5.37 g/L; trans, 5.84 g/L) with ethanol (12 mL) and a buffer solution
of the appropriate pH to a total volume of 100 mL. For a desired pH
of 2.9, it was found that a buffer at pH 2.8 was required, with the final
pH being obtained after addition of the ethanol solution of 5. This
relationship between added buffer pH and the final obtained pH was
consistent over the pH range utilized in this study. Aliquots (5 mL)
were taken (in duplicate) over time, quenched with a saturated solution
of sodium bicarbonate (5 mL), and analyzed using SPME by GC-MS
for oak lactone content as described by Pollnitz et al. (9). Additionally,
the extent of lactonization of 6 was investigated in model wine at pH
1.0 (accurate rates were not determined) and showed that both isomers
reached completion within 2 h. The rates of lactonization in model
spirit (40% ethanol) were measured in an analogous manner. For this
medium, a buffer solution of pH 5.0 was employed, which corresponded
to a final pH of 5.5 after dilution.
MATERIALS AND METHODS
Chemicals were purchased from Sigma-Aldrich. All solvents used
were of HPLC grade from OmniSolv (Darmstadt, Germany). All
organic solvent solutions were dried over anhydrous sodium sulfate
before being filtered. Oak lactone was quantified by the SIDA method
reported previously (9). pH measurements were made with an EcoScan
pH 5/6 meter (Eutech Instruments, Singapore), which was calibrated
before use.
Preparation of Potassium 3-Methyl-4-hydroxyoctanoate Stock
Solution for Lactonization Study. To a 50:50 mixture of racemic cis-1
and racemic trans-1 (1.0061 g, 6.45 mmol) in ethanol (10 mL) was
added a solution of potassium hydroxide (347 mg, 6.19 mmol) in water
(20 mL). After 16 h at room temperature, water (50 mL) was added,
followed by extraction with diethyl ether (3 × 25 mL) to remove
residual oak lactone. The combined ether extracts were quantified for
oak lactone and revealed the presence of cis-oak lactone (108 mg) and
trans-oak lactone (74 mg). The aqueous solution [containing trans-5
(584 mg) and cis-5 (537 mg)] was diluted with water to 100 mL. This
stock solution therefore comprised 5.84 g/L trans- and 5.37 g/L cis-
potassium 3-methyl-4-hydroxyoctanoate. This corresponds to a total
concentration of 5 of 11.21 g/L.
Preparation of Potassium 3-Methyl-4-hydroxyoctanoate Stock
Solution for pKa Determinations. To a 50:50 mixture of racemic cis-1
and racemic trans-1 (1.0134 g, 6.49 mmol) in ethanol (4 mL) was added
a solution of potassium hydroxide (303 mg, 5.40 mmol) in water (20
mL). After 72 h at room temperature, water (50 mL) was added,
followed by extraction with diethyl ether (3 × 25 mL) to remove
residual oak lactone. The combined ether extracts were quantified for
oak lactone and revealed the presence of cis-oak lactone (118 mg) and
trans-oak lactone (78 mg). The aqueous solution [containing trans-5
(582 mg) and cis-5 (528 mg)] was diluted with water to 100 mL. This

Formation of cis- and trans-Oak Lactone
J. Agric. Food Chem., Vol. 52, No. 13, 2004 4215
Figure 2. Formation of oak lactones (1) from the open-chain carboxylate species 5.
Oak Lactone Stability in Model Wine. Model wine (10% ethanol)
Table 1. Measured pH and pK Values for 6 in a Variety of Media
a
solutions of 1 (532 µg/L) were prepared by diluting the stock solution
of 1 (1 mL, 5.32 mg/L) to a total volume of 10 mL with either pH 1
or pH 2.8 buffer solutions. Samples were analyzed (in duplicate) for
oak lactone content by GC-MS using SPME, after 0, 2, 6, and 336 h
at room temperature.
Studies on the Formation of Ethyl 3-Methyl-4-hydroxyoctanoate
in Model Wine. Model wine (10% ethanol) solutions of 1 (532 µg/L),
prepared by diluting the stock solution of 1 (10 mL, 5.32 mg/L) to a
total volume of 100 mL with pH 2.8 buffer solution, and 6 (cis, 441
µg/L; trans, 479 µg/L), prepared by diluting the stock solution of 5 (1
mL; cis, 5.37 g/L; trans, 5.84 g/L) to a total volume of 100 mL with
ethanol (10 mL) and pH 2.8 buffer solution, were analyzed by GC-
MS using SPME, in both SCAN and selected ion monitoring (SIM)
modes, after 28 days at room temperature.
a
medium
HCl addedb
water
model wine
model spirit
0.4
0.5d
0.6
4.45
4.26
4.06
4.77
4.57
4.34
5.51
5.29
5.04
c
pH
a Model wine, 12% (v/v) EtOH in H O; model spirit, 40% (v/v) EtOH in H O.
2
2
b Molar equivalents of HCl added relative to 5. c Mean values from two replicates.
d pH ) pK at this point, according to Henderson−Hasselbach equation.
a
Table 2. Calculated Rate Constants, Half-Lives, and Rate Ratios for
the Conversion of 6 into 1 at 25 °C
cis-1
trans-1
103k
t1/2
3.1
RESULTS AND DISCUSSION
a
b
e
medium
pH
103kc,d
t1/2
ktrans/kcis
Methodology Employed. The approach taken to determine
the equilibrium constants and reaction rates for the processes
shown in Figure 2 was based on two assumptions. First, under
acidic conditions, the first step (protonation of the carboxylate
species 5) occurs rapidly. As the pKa of the resulting acid 6 is
expected to be similar to that of other carboxylic acids, it is
unlikely to undergo significant reionization. The pKa of 6 can
then be determined according to the Henderson-Hasselbach
equation by direct measurement of the pH at 50% neutralization,
that is, when the concentrations of acid and base are exactly
equal:
model wine
2.9
3.1
3.3
3.5
3.7
17.1
10.3
8.0
4.2
1.8
40.5
67.3
86.6
165.0
385.1
222.5
139.0
104.8
44.4
13.0
13.5
13.1
10.6
5.0
6.6
15.6
27.4
25.3
14.0
12.86 ± 1.34f
model spirit
5.5
0.1
6931.0
2.1
330.1
21.0
a Model wine, 12% (v/v) EtOH in H O; model spirit, 40% (v/v) EtOH in H O.
.
b pH error, ±0.05. c Units of k, h-1 d Correlation coefficients were >0.99 in all cases,
except for cis-oak lactone formation in model spirit, for which the value was 0.978.
e Units of t1/2, h. Error expressed as standard deviation.
2
2
f
pH ) pKa - log10([HA]/[A-])
by solid-phase microextraction (SPME) of the headspace, rather
than by liquid-liquid extraction.
The second assumption was that the second step from Figure
2, cyclization to produce oak lactone, would be relatively slow.
This would allow the rate of lactonization to be determined by
“freezing” the reaction at various intervals and measuring the
amount of oak lactone formed. We proposed to quench the
lactonization reaction by neutralizing the mixture, through the
addition of saturated sodium bicarbonate solution. That the
quenching procedure itself would not influence the concentration
of oak lactone was established by adding an excess of saturated
sodium bicarbonate to model wine solutions of either 1 or the
open-chain derivative 6. The oak lactone content of these
solutions was then monitored by GC-MS after 1, 2, and 7 days.
The solution of 1 was unaffected by added sodium bicarbonate
after 2 days, although a small decrease (e10%) was observed
after 7 days. Furthermore, only trace amounts of oak lactone
could be detected in the solutions of 6, even 7 days postquench-
ing. Therefore, samples taken during the lactonization rate study
were quenched with saturated sodium bicarbonate solution and
subsequently analyzed by GC-MS within 24 h, without excep-
tion. Because the quantification method utilized (9) allows the
concentrations of both cis- and trans-oak lactone to be in-
dependently measured during a single analysis, the rate study
was performed on an oak lactone sample comprising a 50:50
mixture of racemic cis- and trans-oak lactone. Additionally, to
avoid artifactual formation of oak lactone in the GC-MS injector
block, as observed by Pollnitz et al. (9), samples were analyzed
Measurement of pKa Values of 3-Methyl-4-hydroxy-
octanoic Acid (6). The pKa of a 50:50 mixture of both threo-
and erythro-3-methyl-4-hydroxyoctanoic acid (6) was deter-
mined by measuring the pH of an aqueous solution of the
potassium salt 5, upon the addition of hydrochloric acid. The
pH and pKa values are collected in Table 1. The value for 6 in
aqueous solution, 4.26, is similar to that for acetic acid, 4.76
(10), supporting the first assumption outlined above. As can be
observed in Table 1, as the ethanol content of the medium
increases, there is a concomitant increase in the value of pKa.
This has been observed before, in connection with the pKa of
substituted aryl carboxylic acids (11), and is almost certainly a
reflection of the lower ionizing power of the solution as the
aqueous content is reduced.
Measurements of Rate Constants for Conversion of 6 into
1. A model wine solution of 6 was prepared from a solution of
5 and a potassium hydrogen tartrate buffer solution adjusted to
the required pH. Aliquots were taken at various intervals,
quenched by the addition of saturated sodium bicarbonate, and
analyzed by GC-MS to determine the concentrations of both
oak lactones. The pH values, rate constants, half-lives, and trans/
cis ratios are collected in Table 2. The solution showed good
first-order kinetics for the formation of both cis-oak lactone and
trans-oak lactone, as evidenced by the excellent r2 values

4216 J. Agric. Food Chem., Vol. 52, No. 13, 2004
Wilkinson et al.
for the cis-isomer, lactonization must proceed from a conformer
such as D or E, but the preferred conformer is actually F. Ring
closure is now occurring from the least populated conformer
and would therefore be expected to be slower, relative to the
trans-isomer. An alternative explanation is that there is an
unfavorable steric interaction between the methyl and butyl
groups in both the transition state and product for formation of
the cis- but not the trans-isomer.
Implications for Previous Studies. Finally, we wished to
address the points raised by Waterhouse and Towey (8). To
investigate the equilibria between oak lactone (1), the corre-
sponding hydroxy acid 6, and the ethyl ester of 6 for each of
the cis- and trans-isomers, aqueous ethanol solutions of oak
lactone at pH 1.0 and 2.9 were prepared, and the concentration
of oak lactone was monitored over time. The oak lactone content
of these solutions was found to remain constant over 28 days,
suggesting that ring opening of the lactone is unlikely under
such conditions. Additionally, solutions of 6 at pH 1.0 reached
the theoretical maximum values (of both cis- and trans-oak
lactone) within 2 h. The potential formation of ethyl 3-methyl-
4-hydroxyoctanoate, as a result of ethanolysis reactions, as
proposed by Waterhouse and Towey (8), was also investigated.
Model wine solutions of both 1 and 6 were analyzed by GC-
MS in both scan and SIM modes. However, despite comparison
with mass spectrometric data from a sample synthesized in an
analogous manner to the corresponding isopropyl ester (12),
we found no evidence to support the formation of the ethyl
esters, even though these esters could be seen easily by GC-
MS. Therefore, even in an aqueous environment, the oak
lactones will be found almost entirely in the ring-closed form
at equilibrium.
Figure 3. Plot of ln k versus pH for formation of both cis- and trans-oak
lactone.
obtained. As expected for this acid-mediated reaction, the rate
is strongly influenced by the pH of the medium (Figure 3).
A clear difference in rates of lactonization is evident between
the isomers. The ratio of the rates changes little, irrespective of
the medium or the pH. In the case of the rates measured in the
model spirit, the value for cis-oak lactone is based on data
collected over substantially less than 1 half-life (24 days of
monitoring, compared with a calculated half-life of ≈300 days).
The correlation coefficient for this particular measurement is
slightly lower than the others, allowing one to suggest that the
slightly higher ratio for this medium (ktrans/kcis ) 21.0) is a
manifestation of the less accurate measurement of the rate
constant for cis-oak lactone in this solution. This difference in
reactivity between the isomers was also observed during the
synthesis of the glycoconjugates of 6 (1). One possible reason
for the more facile ring closure to the trans-isomer can be seen
by considering the Newman projections for each isomer of 6
(Figure 4). For this isomer, lactonization must proceed from a
conformer such as A or C, that is, where the C1 carbonyl and
C4 hydroxyl groups are in close proximity. Conformer A is
favored, having only two gauche interactions, whereas conform-
ers B and C each have three such interactions. Ring closure,
therefore, occurs from the most populated conformer. However,
Waterhouse and Towey (8) discussed data from Chatonnet
(13) which showed that the ratio of cis/trans-oak lactone in
extracts of toasted wood increased with increasing toasting
levels. They hypothesised that the open-chain form of oak
lactone closes during toasting and “the cis-lactone, for kinetic
or thermodynamic reasons, dehydrates to a greater extent under
these conditions, and the ratio of cis to trans isomers increases
as well”. However, as shown in Table 2, the trans-lactone is
formed in aqueous solution more rapidly than the cis-isomer
from their respective hydroxy acids at all pH values. Further-
more, hydrolytic studies indicate that the trans-isomer, as
expected from steric considerations, is also the thermodynami-
cally more favorable product (1). Another explanation for the
Figure 4. Conformers available to the different isomers of 6.

Formation of cis- and trans-Oak Lactone
J. Agric. Food Chem., Vol. 52, No. 13, 2004 4217
findings of Chatonnet (13), supported by the data in Table 2,
is that the conditions of extraction he employed (4 weeks of
maceration at 18 °C, pH 3.5) were sufficient for any (3S,4R)-
hydroxy acid that might have been present to ring-close to the
trans-isomer but were likely to be insufficient for the corre-
sponding (3S,4S)-hydroxy acid (measured half-life of ≈7 days
at 25 °C) to completely lactonize to the cis-form. If most of the
oak lactone were in the open-chain form in unheated wood but
ring-closed in heated samples, then this could explain the
observed increases of cis-oak lactone in the latter. An alternative
explanation for Chatonnet’s data is that the results are a real
reflection of total oak lactone in the wood and that the increased
concentration in cis-oak lactone is a result of decomposition of
the recently identified galloyl glucoside 4 of the hydroxy acid
6.
undergo complete lactonization during barrel maturation. Like-
wise, although the half-life for formation of cis-oak lactone was
determined to be almost 300 days in model spirit at pH 5.5,
given that most spirits are barrel-aged for several years, near-
complete lactonization is likely to be achieved.
The addition of oak staves, chips, shavings, or powder to
wine, as a more rapid and economical method of oak treatment
(17), allows the incorporation of oak flavors and interaction of
wood and wine constituents without traditional barrel maturation.
The increased surface area of chips or shavings (as compared
with barrels) results in greater rates of extraction, and so wines
typically receive only several days of contact with oak,
depending on the preference of the winemaker. Given that the
half-life for formation of cis-oak lactone was determined to be
≈14 days at pH 3.7, the formation of cis-oak lactone after the
oak has been removed is possible. This provides a plausible
explanation for the intensification of oak aroma even after the
removal of oak, as reported by Singleton (6).
The assertion of Waterhouse and Towey (8) that when the
ratio of cis- to trans-oak lactone “is determined from wine stored
in barrels for 8 months, the ratio is always near the range 1-2
regardless of toasting levels...” becomes questionable when one
examines the original source of these data (13). The ratios quoted
by Waterhouse and Towey are actually composites for wines
stored in barrels made from two different species of oak from
two different regions (Allier and Limousin). There was a trend
for the cis/trans ratio in wines in the Limousin oak to increase
with increasing toasting level (from 1.4 to 4.9 for light to very
heavy toast). For the wine in Allier barrels, the ratios varied in
a nonsystematic way from 0.8 to 3.1, albeit with the highest
ratio at the highest toast level. Although the ratios for the higher
toast levels were not as high as those observed for the laboratory
experiments, this could simply be because the latter were
obtained from shavings of the outer 3 mm of wood, rather than
from intact staves.
ACKNOWLEDGMENT
We thank Profs. P. B. Høj, I. S. Pretorius, and W. D. Lawrance
for their advice and encouragement.
LITERATURE CITED
(1) Wilkinson, K. L.; Elsey, G. M.; Prager, R. H.; Tanaka, T.; Sefton,
M. A. Precursors to oak lactone II: Synthesis, separation and
cleavage of several â-D-glucopyranosides of 3-methyl-4-hy-
droxyoctanoic acid. Tetrahedron 2004, 60, in press.
(2) Maga, J. A. Oak lactones in alcoholic beverages. Food ReV. Int.
1996, 12, 105-130.
(3) Otsuka, K.; Zenibayashi, Y.; Itoh, M.; Totsuka, A. Presence and
significance of two diastereomers of â-methyl-γ-octalactone in
aged distilled liquors. Agric. Biol. Chem. 1974, 38, 485-490.
(4) Raunkjær, M.; Sejer Pederson, D.; Elsey, G. M.; Sefton, M. A.;
Skouroumounis, G. K. Precursors to oak lactone I: Synthesis
of gallate ester derivatives of 3-methyl-4-hydroxyoctanoic acid.
Tetrahedron Lett. 2001, 42, 8717-8719.
As further support for their hypothesis, Waterhouse and
Towey (8) quoted the results of a seasoning trial by Sefton and
colleagues (14), in which the levels of oak lactone were observed
to increase under Australian climatic conditions. However, these
seasoning results cannot be a manifestation of the equilibrium
between oak lactone and its open-chain forms. As the present
study shows, the extraction conditions employed by Sefton et
al. (14) (model wine, pH 3.2, 50 °C, 7 days) are more than
sufficient to ensure the near-complete conversion of any open-
chain form into oak lactone.
(5) Masson, E.; Baumes, R.; Le Guerneve´, C.; Puech, J.-L. Identi-
fication of a precursor of â-methyl-γ-octalactone in the wood
of sessile oak (Quercus petraea (Matt.) Liebl.). J. Agric. Food
Chem. 2000, 48, 4306-4309.
Any free hydroxyacid present in oak or oak extracts is going
to be subject to lactonization to produce oak lactone. Our results
show that this process is essentially irreversible at wine pH.
Thus, any aglycon extracted into wine will be completely
converted to the lactone form over time. The extraction and
analysis of wine/oak samples could potentially lead to mislead-
ing results if adequate time for lactonization is not taken into
consideration. Oak extracts are often prepared by extraction with
ethanol/water or acetone/water at room temperature, overnight
(i.e., 16 h) (15, 16). Under such conditions lactonization of any
hydroxy acid 6 that might be present is likely to be incomplete
and could result in the underestimation of cis-oak lactone
concentration. For analyses specific to oak lactone, we recom-
mend that the extracting medium be kept acidic (pH ≈3.0), and
subsequently heated (≈50 °C) for several days, to ensure
complete lactonization of any 3-methyl-4-hydroxyoctanoic acid
which might be present.
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The results from the present study indicate that lactonization
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days at room temperature, whereas lactonization of the cis-
isomer could take up to several weeks, depending on the pH of
the wine. Therefore, it is anticipated that both isomers would

4218 J. Agric. Food Chem., Vol. 52, No. 13, 2004
Wilkinson et al.
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Received for review March 1, 2004. Revised manuscript received April
26, 2004. Accepted April 28, 2004. This project was supported by
Australia’s grapegrowers and winemakers through their investment
body, the Grape and Wine Research and Development Corporation
(GWRDC), with matching funds from the Australian government. This
work was conducted by The Australian Wine Research Institute and
Flinders University as part of their collaborative research program.
We thank the GWRDC for financial assistance in the form of a research
scholarship to K.L.W.
JF0496550