Precursors to damascenone: Synthesis and hydrolysis of isomeric 3,9-dihydroxymegastigma-4,6,7-trienes

Article abstract of DOI:10.1021/jf050327p

A series of four isomeric 3,9-dihydroxymegastigma-4,6,7-trienes, 8, has been prepared. The (3S,6R,9S) isomer of 8 proved to be identical to an isomer of this compound tentatively identified as an intermediate in the formation of damascenone from an allene triol. Each of the four isomers, when hydrolyzed independently of each other at pH 3.0 and 25°C, produced product mixtures in which the major product was damascenone (1). Contrary to expectation, 3-hydroxydamascone (5) was not observed in any of the hydrolyses. Consequently, the mechanism of formation of damascenone proposed earlier requires modification. In each hydrolysis, the product mixtures showed the presence of a second isomer of 8, produced by epimerization during hydrolysis. Chiral analysis on a Cyclosil B column revealed that this epimerization was occurring at C₃ in each of the hydrolyses.

Full text of DOI:10.1021/jf050327p

J. Agric. Food Chem. 2005, 53, 4895−4900 4895
Precursors to Damascenone: Synthesis and Hydrolysis of
Isomeric 3,9-Dihydroxymegastigma-4,6,7-trienes
CAROLYN J. PUGLISI,†,‡,§ MERRAN A. DANIEL,†,‡,§ DIMITRA L. CAPONE,‡,§
‡,§
GORDON M. ELSEY,*,†,‡,§ ROLF H. PRAGER,†,§ AND MARK A. SEFTON
The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064,
Australia; School of Chemistry, Physics and Earth Sciences, Flinders University, P.O. Box 2100,
Adelaide, South Australia 5001, Australia; and Cooperative Research Centre for Viticulture,
P.O. Box 154, Glen Osmond, South Australia 5064, Australia
A series of four isomeric 3,9-dihydroxymegastigma-4,6,7-trienes, 8, has been prepared. The
(3S,6R,9S) isomer of 8 proved to be identical to an isomer of this compound tentatively identified as
an intermediate in the formation of damascenone from an allene triol. Each of the four isomers, when
hydrolyzed independently of each other at pH 3.0 and 25 °C, produced product mixtures in which
the major product was damascenone (1). Contrary to expectation, 3-hydroxydamascone (5) was not
observed in any of the hydrolyses. Consequently, the mechanism of formation of damascenone
proposed earlier requires modification. In each hydrolysis, the product mixtures showed the presence
of a second isomer of 8, produced by epimerization during hydrolysis. Chiral analysis on a Cyclosil
B column revealed that this epimerization was occurring at C3 in each of the hydrolyses.
KEYWORDS: Damascenone; flavor precursors; wine; carotenoid metabolites; epimerization; acid
hydrolysis
INTRODUCTION
Glycoconjugated forms of both 4 (9) and 6 (10, 11) have been
isolated from various plants, and both have been shown to lead
to the production of damascenone.
Since its discovery in the extract of Bulgarian rose (Rosa
damascena) oil some 30 years ago, damascenone (1) has been
identified in many different types of plant material and has also
been one of the mainstays of the international perfume industry
(1). It is an extremely potent odorant, with measured aroma
detection thresholds of 50 ng L-1 in model wine (2) and as low
as 2 ng/L in water (3). Despite its importance to the flavor
industry, the biogenesis of naturally occurring damascenone
remains unclear. Evidence suggests that damascenone is formed
in vivo as the product of degradation of carotenoids such as
neoxanthin (2, Figure 1) (4). The allenic triol species 4, formally
derived by reduction of the C9 carbonyl in grasshopper ketone
(3), has previously been shown to produce, under mild acid
conditions, damascenone (1), 3-hydroxydamascone (5), and the
enyne diol 6, with 1 being formed only in very minor amounts
(6). The enyne 6 was also found to produce both 1 and 5, albeit
at a greatly reduced rate (7); hydrolytic data indicated that 6
produces, in one year, approximately the same quantity of
damascenone as the triol 4 produces in 1 day. Somewhat
surprisingly, perhaps, 5 has been found to be extremely resistant
toward dehydration, even under strongly acidic conditions (8),
indicating that it is a terminal product in this reaction sequence,
rather than an intermediate in the eventual formation of 1.
Earlier work in our laboratories has shown the effect of
glycoconjugation on the reactivity of activated hydroxyl func-
tions. For example, the enyne diol 6 produced both 1 and 5 in
a ratio of 1:17 when heated at 50 °C in a pH 3.0 buffer (12). In
contrast, when the corresponding C9 â-D-glucopyranoside of 6
was heated under identical conditions, the proportion of dama-
scenone formed increased to 1:11. Furthermore, the rate of
1
hydrolysis of the glucoside was approximately /10 that of the
aglycon (7). These results have been interpreted as showing that
the acid-catalyzed ionization of an activated hydroxyl function
is suppressed when that position is glycoconjugated. Conse-
quently, other processes might become competitive, in this case,
loss of the C3 hydroxyl leading to formation of 1. Thus, the
site of glycosylation might strongly influence the formation of
1 from precursors such as 4.
These earlier studies also provided tentative evidence for the
intermediacy of two new compounds, the dienyne 7 and the
allenic diol 8, in the formation of 1 from 4 (6). On the basis of
this evidence, a mechanism for the formation of the hydrolysis
products from 4 was proposed as shown in Figure 2. Loss of
the C5 alcohol concomitant with rearrangement produces 6,
whereas direct elimination of the C5 hydroxyl would produce
the allenic diol 8. From this point it could be anticipated that
reaction might proceed through either the C3 or C9 hydroxyls.
Loss of the former would be expected to produce damascenone,
whereas loss of the latter would be expected to furnish 5. The
* 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.
§ Cooperative Research Centre for Viticulture.
10.1021/jf050327p CCC: $30.25
© 2005 American Chemical Society
Published on Web 04/30/2005

4896 J. Agric. Food Chem., Vol. 53, No. 12, 2005
Puglisi et al.
Figure 1. Proposed pathway for the formation of 1 from neoxanthin (2), including the structures of two tentatively identified intermediates, 7 and 8, and
the generic numbering scheme employed for megastigmane derivatives (5).
Figure 2. Proposed mechanism for hydrolysis of triol 4.
ratio of products formed would then be dependent upon the
relative reactivity of the two alcohols toward acid-catalyzed
ionization.
mation of its identity from the earlier study and, second, a
thorough investigation into the hydrolytic behavior of this
compound. It was anticipated that these authentic allene diols
would produce mixtures consisting principally of damascenone
and its hydroxylated derivative 5 and that the ratio of these two
products would be determined by the relative reactivity toward
ionization of the hydroxyl functional groups located at either
C3 or C9.
Recently we reported the synthesis of an authentic sample
of 7 and confirmed its identity as one of the intermediates
observed in the hydrolysis of 4 (4). In addition, the product of
its hydrolysis proved to be almost exclusively (>90%) dama-
scenone 1, confirming 7 as the immediate precursor to naturally
formed damascenone. The remainder (<10%) comprised two
compounds, which had previously been observed in the hy-
drolysis of damascenone precursors and which were assigned
as the C9 hydrate of damascenone 13 and the C9 ethanol adduct
14.
Comparisons between the allenic triol 4 and the diol 8 are
revealing: in the former, the C3 position is not activated, unlike
the other two oxygenated positions, and the major hydrolysis
products of 4 (i.e., 5 and 6) both retain the C3 hydroxyl. In the
diol 8, on the other hand, both C3 and C9 are activated, and one
would expect formation of damascenone from 8 to be competi-
tive with the formation of 5.
MATERIALS AND METHODS
Materials. Chemicals were purchased from Sigma-Aldrich. (S)-
Phorenol (ee > 96%) was provided by Dr. K. Puntener, Hoffmann-La
Roche International. All solvents used were of pesticide grade from
OmniSolv (Darmstadt, Germany). All organic solvent solutions were
dried over anhydrous sodium sulfate before being filtered. pH measure-
ments were made with an EcoScan pH 5/6 meter (Eutech Instruments,
Singapore), which was calibrated before use. Column chromatography
was performed using silica gel 60 (230-400 mesh) from Merck. Optical
rotations were measured on a Polaar 21 polarimeter operating at 20
1
°C. Routine H and 13C NMR spectra were recorded (in CDCl3) with
a Varian Gemini spectrometer at operating frequencies of 300 and 75.5
MHz, respectively. Mass spectra were recorded on a Hewlett-Packard
(HP) 6890 gas chromatograph fitted with a liquid HP 6890 series
Thus, our aims in conducting this study were twofold: first,
synthesis and characterization of the allenic diol 8 and confir-

Hydrolysis of Damascenone Precursors
J. Agric. Food Chem., Vol. 53, No. 12, 2005 4897
injector and coupled to a HP 5973 mass spectrometer, with the
instrument set up as described by Janusz et al. (17). Buffer solutions
were prepared by saturating a 10% ethanol solution with potassium
hydrogen tartrate and adding 10% tartaric acid solution until the required
value was reached.
Methods. (S)-4-tert-Butyldimethylsilyloxy-2,6,6-trimethylcyclohex-
2-enone [(S)-10]. To a solution of (S)-phorenol (9) (1.0 g, 6.40 mmol)
in pyridine (10 mL) was added tert-butyldimethylsilyl chloride (1.47
g, 9.60 mmol). The mixture was stirred at room temperature overnight,
quenched with water, and extracted with ethyl acetate. The organic
layer was washed with brine and dried (Na2SO4) before being
concentrated in vacuo to yield material that was used without further
purification (1.60 g, 92%): [r]D -49.0 (c 0.40, CHCl3) [lit. (18) -57
(c 0.44, CHCl3]; δH 6.49 (1H, m, H3), 4.56 (1H, m, H4), 1.99 (1H,
ddd, J ) 13.0, 5.5, and 1.9 Hz, H5a), 1.87 (1H, dd, J ) 13.0 and 9.6
Hz, H5b), 1.78-1.76 (3H, m, H9), 1.14, 1.11 (6H, 2s, H7,8), 0.92 (9H,
s, t-Bu), 0.13, 0.11 (6H, 2s, SiMe).
(3S,9S)-Dihydroxymegastigma-4,6,7-triene [(3S,9S)-8]. These reac-
tions were first optimized using both 10 and but-3-yn-2-ol in racemic
form. The products of these reactions were necessarily mixtures of
diastereomers, but gave entirely satisfactory spectroscopic and mi-
croanalytical data: Anal. (11) Calcd for C19H34O3Si: C, 67.4; H, 10.1.
Found: C, 67.2; H, 10.0. Anal. (12) Calcd for C19H34O2Si: C, 70.7; H,
10.6. Found: C, 70.7; H, 10.7.
(S)-But-3-yn-2-ol (0.31 g, 4.50 mmol) in ether (200 mL) was treated
with n-BuLi (2.5 M, 3.5 mL, 8.6 mmol) at 0 °C and stirred at room
temperature for 2 h. (S)-10 (0.40 g, 1.6 mmol) was then added, and
the mixture was stirred at room temperature for 48 h before being
quenched with saturated NH4Cl solution. The residue was extracted
with diethyl ether, washed with brine, dried (Na2SO4), and concentrated
in vacuo. The crude material was purified by column chromatography
(30% ethyl acetate/hexanes) to yield (3S,9S)-11 as a colorless oil (0.35
g, 69%). NMR analysis revealed the product to be a mixture of two
diastereomers.
(3S,9S)-11 (0.25 g, 0.73 mmol) in ether (10 mL) was treated with
LiAlH4 (0.14 g, 3.7 mmol) and the mixture stirred at reflux for 4 h.
The reaction was quenched by the addition of a solution of saturated
sodium sulfate, and the product was extracted with ethyl acetate. The
organic extracts were then washed with 10% NaOH solution and brine,
before being dried (Na2SO4) and concentrated in vacuo. The residue
was purified by column chromatography (10% ethyl acetate/hexane)
to yield (3S,9S)-12 as a pair of diastereomers (85 mg, 36%). The product
was treated with tert-butylammonium fluoride (t-BAF) (0.53 mL of
1.0 M solution in THF, 0.53 mmol) in dichloromethane (10 mL)
overnight at room temperature, before being quenched with saturated
NaHCO3 solution and extracted with ethyl acetate. The extract was
washed with brine, dried, and concentrated in vacuo to yield 80 mg of
crude material. Purification by chromatography (40% ethyl acetate,
hexane) yielded pure SS-1 isomer (12 mg, 23%), followed by a mixed
fraction (27 mg, 50%), followed by a fraction containing pure SS-2
isomer (8 mg, 14%).
mmol) and (S)-10 (0.40 g, 1.6 mmol). Identical workup gave (3S,9R)-
11 as a colorless oil (0.35 g, 69%), again as a pair of diastereomers.
(3S,9R)-11 (0.25 g, 0.73 mmol) was treated with LiAlH4 (0.14 g,
3.7 mmol) in ether (10 mL) as described above. Workup gave, after
chromatography (3S,9R)-12 (64 mg, 23%) as a colorless oil. Depro-
tection with t-BAF (0.40 mL of 1.0 M solution in THF, 0.40 mmol)
provided 74 mg of crude material, which was chromatographed as
before to give, in order of elution, pure SR-1 isomer (16 mg, 40%), a
mixed fraction (19 mg, 47%), and pure SR-2 isomer (4 mg, 10%).
SR-1 isomer, (3S,6S,9R)-8: [r]D +12.8 (c 0.25, CHCl3); δH 5.66
(1H, br d, J ) 5.4 Hz, H8), 5.60 (1H, m, H4), 4.41-4.29 (2H, m, H3,9),
1.91 (1H, ddd, J ) 12.5, 5.8, and 1.1 Hz, H2a), 1.75 (3H, app t, J ∼
1.6 Hz, H13), 1.62 (2H, br s, OH), 1.42 (1H, dd, J ) 12.5 and 9.6 Hz,
H2b), 1.31 (3H, d, J ) 6.3 Hz, H10), 1.11, 1.07 (6H, 2s, H11,12); δC
199.7, 130.1, 128.0, 116.0, 101.5, 66.2, 65.8, 45.8, 33.7, 29.6, 28.2,
23.5, 20.9; MS, m/z (%) 208 (5), 193 (10), 190 (23), 175 (45), 157
(15), 149 (29), 146 (30), 131 (100), 121 (30), 115 (39), 109 (26), 105
(27), 91 (45), 77 (28), 69 (22).
SR-2 isomer, (3S,6R,9R)-8: [r]D +35.2 (c 0.06, CHCl3); δH 5.62-
5.55 (2H, m, H4,8), 4.41-4.30 (2H, m, H3,9), 1.92 (1H, ddd, J ) 12.3,
5.7, and 1.1 Hz, H2a), 1.74 (3H, dd, J ) 1.6 and 1.3 Hz, H13), 1.61
(2H, br s, OH), 1.45 (1H, dd, J ) 12.3 and 9.6 Hz, H2b), 1.31 (3H, d,
J ) 6.4 Hz, H10), 1.13, 1.05 (6H, 2s, H11,12); δC 199.9, 130.2, 128.0,
116.2, 101.5, 66.4, 65.9, 46.1, 33.9, 30.0, 27.6, 23.5, 20.8; MS, m/z
(%) 208 (4), 193 (3), 190 (15), 175 (30), 157 (9), 149 (25), 146 (34),
131 (100), 121 (20), 115 (37), 109 (22), 105 (23), 91 (48), 77 (27), 69
(20).
General Hydrolysis Procedure. Solutions of 8 (1 mg/L) in buffered
10% aqueous ethanol at pH 3.0 were sealed in ampules and heated at
25 °C in a water bath for 24 h. After this time, ampules were removed
and opened, and the contents were extracted with ether and subjected
to GC-MS analysis, on either an achiral DB-1701 column or a chiral
Cyclosil B column, using the conditions reported by Wilkinson et al.
(19). Several ampules of each were retained in the water bath for 6
months, after which time they were opened and analyzed.
Hydrolysis of 8 (Details GiVen for the SS-2 Isomer; Others
Conducted in an Identical Manner). A solution of (3S,6R,9S)-8 (1 mg/
L, 10 mL aliquots) was prepared, heated, and extracted as described
above. The product mixture was shown to contain 1, 7, hydrate 13,
ethanol adduct 14, unreacted SS-2 isomer, a second isomer of 8 that
proved to be indistinguishable from the synthetic SR-1 isomer, and
two isomers of a compound for which mass spectra were in accord
with the assigned structure 15; isomer 1, m/z (%) 236 (24), 221 (100),
203 (10), 191 (21), 177 (50), 149 (26), 147 (31), 146 (30), 137 (94),
131 (77), 121 (35), 109 (63), 105 (41), 91 (45), 77 (28), 69 (21); isomer
2, m/z (%) 236 (23), 221 (100), 203 (10), 191 (19), 177 (49), 149 (26),
147 (28), 146 (23), 137 (91), 131 (57), 121 (34), 109 (60), 105 (38),
91 (41), 77 (25), 69 (21).
Compounds 1,6 7,4 13,14 and 1414 were identified by comparison of
retention times and mass spectra with those of authentic samples.
SS-1 isomer, (3S,6S,9S)-8: [r]D -22.6 (c 0.02, CHCl3); δH 5.66 (1H,
br d, J ) 5.3 Hz, H8), 5.60 (1H, m, H4), 4.42-4.28 (2H, m, H3,9), 1.91
(1H, ddd, J ) 12.5, 5.8, and 1.1 Hz, H2a), 1.74 (3H, app t, J ∼ 1.6 Hz,
H13), 1.60 (2H, br s, OH), 1.43 (1H, dd, J ) 12.5 and 9.8 Hz, H2b),
1.31 (3H, d, J ) 6.3 Hz, H10), 1.12, 1.07 (6H, 2s, H11,12); δC 199.9,
130.3, 128.0, 116.3, 101.6, 66.2, 65.9, 45.9, 33.8, 29.7, 28.3, 23.5, 20.9;
MS, m/z (%) 208 (4), 193 (5), 190 (22), 175 (43), 157 (16), 149 (35),
146 (24), 131 (100), 121 (26), 115 (35), 109 (32), 105 (30), 91 (53),
77 (26), 69 (24).
SS-2 isomer, (3S,6R,9S)-8: [r]D +45.0 (c 0.02, CHCl3); δH 5.61
(1H, m, H4), 5.58 (1H, br d, J ) 6.0 Hz, H8), 4.42-4.28 (2H, m, H3,9),
1.92 (1H, ddd, J ) 12.3, 5.8, and 1.1 Hz, H2a), 1.75 (3H, app t, J ∼
1.6 Hz, H13), 1.60 (2H, br s, OH), 1.45 (1H, dd, J ) 12.3 and 9.6 Hz,
H2b), 1.31 (3H, d, J ) 6.3 Hz, H10), 1.12, 1.05 (6H, 2s, H11,12); δC
200.1, 130.1, 127.9, 116.1, 101.4, 66.4, 65.9, 46.1, 33.9, 30.1, 27.7,
23.5, 20.8; MS, m/z (%) 208 (3), 193 (4), 190 (11), 175 (20), 157 (12),
149 (20), 146 (28), 131 (100), 121 (16), 115 (29), 109 (28), 105 (19),
91 (44), 77 (21), 69 (16).
RESULTS AND DISCUSSION
Synthesis of Allenes 8. The synthesis of the allenic diols
(Figure 3) was based on a strategy that involved as little
stereochemical manipulation as was practicable. By using a
starting reagent with fixed (S) stereochemistry at C3, the
stereochemistry at C9 can be introduced by selective use of
optically active 3-butyn-2-ol. This would allow the preparation
of four of the possible eight diastereomers of 8, necessary for
a proper evaluation of their hydrolytic behavior.
The hydroxyl in (S)-phorenol (9) was protected as its TBS
ether and then treated with the dilithio derivative of either (S)-
3-butyn-2-ol or the enantiomeric (R) analogue (13). Each
reaction produced a pair of isomers of 11, which differed in
their stereochemistry at C6. The individual diastereomers were
not isolated for either reaction, but rather each was treated with
LAH to produce a pair of isomers epimeric at the allenic position
(20). Neither the pair of isomers of 12 produced from the (S)-
(3S,9R)-Dihydroxymegastigma-4,6,7-triene [(3S,9R)-8]. The above
reaction sequence was repeated using (R)-but-3-yn-2-ol (0.31 g, 4.5

4898 J. Agric. Food Chem., Vol. 53, No. 12, 2005
Puglisi et al.
Figure 3. Synthesis of target allenes from (S)-phorenol (9).
3-butyn-2-ol series of reactions nor the pair from the corre-
sponding (R)-3-butyn-2-ol series could be adequately separated
at this stage. However, after removal of the silyl protecting
group, each series of compounds was separable by column
chromatography. The two isomers of 8 produced from the (S)-
3-butyn-2-ol series of reactions were labeled SS-1 and SS-2,
with the numerical descriptor referring to the order of elution
during column chromatography on silica. The assignment of
C6 stereochemistry to these two isomers is discussed in the next
section. Similarly, the two isomers of 8 produced from the (R)-
3-butyn-2-ol series of reactions were labeled SR-1 and SR-2.
All four synthetic isomers were distinguishable from one another
by GC-MS, with the order of elution on a DB-1701 column
being SR-2, SS-2, SS-1, and SR-1.
Hydrolysis of Allenes 8. The first issue to be resolved
concerned the identity of the compound tentatively identified
as 8, from the original hydrolysate of 4. The original triol 4
was synthesized by Skouroumounis et al. (15) from diastereo-
merically pure [3′R*,1R*,4S*,6S*]-1-(3′-hydroxybut-1′-ynyl)-
2,2,6-trimethylcyclohexane-1,4-diol, which was fractionally
crystallized from a mixture and for which relative stereochem-
istry was established by X-ray crystallography (16). This
diastereomer was used for the conversion to 4, with the relative
stereochemistry at C3 and C9 remaining unaltered.
(retention time, mass spectrum, and co-injection), the second
of these isomers (SS-2) provided an exact match with the
compound tentatively identified as 8 from the hydrolysate of
4, and we are thus able to assign stereochemistry to the (3S,9S)
allenes: SS-1 as (3S,6S,9S)-8 and SS-2 as (3S,6R,9S)-8.
Each of the four allene diols 8 was subjected to acid
hydrolysis, and the products were examined after 24 h and 6
months of reaction time. Some general features were observed
in the products; in the case of the long-term hydrolysis, the
products were damascenone (1) and the addition products 13
and 14, in a ratio similar to that observed previously in the
hydrolysis of 7 (4). However, when the short-term hydrolyses
were examined, the major products (at the same oxidation level
as damascenone) were damascenone 1, intermediate 7, unreacted
8 (plus epimer), two compounds tentatively identified as epimers
of 15, and minor amounts of damascenone addition products
13 and 14 (Figure 4). Minor amounts (<10%) of several
oxidation products were also observed. Noteworthy in each case
was the complete absence of 3-hydroxydamascone (5), even in
trace amounts. This result demanded reappraisal of the original
mechanism proposed by Skouroumounis and Sefton for the
formation of 1, 5, and 6 (7).
Each allene diol 8 showed, after 24 h, considerable reaction
progress (∼40% consumption of 8), with unconsumed allene
diol now accompanied by an equal amount of a compound that
was clearly epimeric, on the basis of mass spectrometric
analysis. Each isomer of 8 gave only a single new epimer of 8;
in the case of the SS-2 isomer, this epimer proved to be
indistinguishable from the SR-1 isomer by achiral GC. This
Given that the original hydrolysis experiments were conducted
on material that was diastereomerically homogeneous and that
the relative stereochemistry at all positions was known, then a
match with one of the authentic (3S,9S) allene isomers 8 would
be expected. Accordingly, when compared by GC-MS analysis

Hydrolysis of Damascenone Precursors
J. Agric. Food Chem., Vol. 53, No. 12, 2005 4899
Figure 4. Products of the interrupted hydrolysis of allenes 8.
Table 1. Absolute Stereochemistries Expected for Epimerization at either C9 or C3 during Hydrolysis of 8 and Retention Times Observed on a Chiral
Stationary Phase
after epimerizationa
GC retention times of
starting isomer
after epimerizationb
at C9
at C3
isomers of 8c
SS-1 (
SS-2 (
SR-1
)
3S,6S,9S)
3S,6R,9S)
SS-1
SS-2
SR-1
SR-2
+
+
+
+
SR-2d
SR-1d
SS-2d
SS-1d
(3S,6S,9S)
+
+
+
+
(3S,6S,9R)
(3S,6S,9S)
+
+
+
+
(3R,6S,9S)
each 18.09
18.10 and 18.17
each 18.13
)
(3S,6R,9S)
(3S,6R,9R)
(3S,6S,9R)
(3S,6R,9R)
(3S,6R,9S)
(3S,6S,9S)
(3S,6R,9S)
(3S,6S,9R)
(3S,6R,9R)
(3R,6R,9S)
(3R,6S,9R)
(3R,6R,9R)
SR-2
18.07 and 18.15
a Stereochemistries expected after epimerization at either C3 or C9. b Isomers of 8 present after hydrolysis interrupted at 24 h, as determined on an achiral (DB-1701)
GC column. c Retention times (minutes) of isomers of 8 present after hydrolysis interrupted at 24 h, as determined on a chiral (Cyclosil B) GC column. d Or its enantiomer.
pairing of the SS-2 isomer with the SR-1 isomer was observed
in the converse reaction also. Similarly, the SS-1 isomer gave
an epimer indistinguishable from the SR-2 isomer, and vice
versa. Table 1 lists the epimeric pairs obtained after hydrolysis
of each of the four allene diastereomers.
Compounds 15 were assigned as being ethyl ethers of 8,
primarily on the basis of their mass spectra. The masses (m/z
236, M+) and fragmentation patterns are entirely consistent with
this assignment. Additional evidence comes from the epimer-
ization of 8 itself; protonation and ionization at C3 of 8 followed
by capture by either water or ethanol provide an entirely
reasonable pathway to these compounds.
The fact that epimerization takes place only at C3 under the
conditions used in this study (conditions chosen for their
similarity to normal wine maturation) has implications for the
mechanism of formation of damascenone. The original proposal
(Figure 2) was consistent with the available data but needs to
be revised in light of the new findings. The suggestion that both
5 and 7 are formed from a common intermediate, 8, is
contradicted by the finding that the hydrolysate produced from
authentic 8 is completely devoid of 5. This compound, the major
product in the hydrolysis of allenic triol 4, appears to be formed
directly from 4, via a process as yet undetermined.
Whether this epimerization is taking place at C3 or C9 is
impossible to determine from these data alone. For instance, if
epimerization is occurring at C9 of the SS-2 isomer, then the
new epimer will have (3S,6R,9R) stereochemistry. Conversely,
if this epimerization is occurring at C3, the new epimer will
have (3R,6R,9S) stereochemistry and will be the enantiomer of
the synthetic isomer having (3S,6S,9R) stereochemistry. Thus,
the ultimate assignment of stereochemistry to the SR-1 and SR-2
isomers is reliant upon our being able to distinguish between
C3 and C9 epimerization. Table 1 also shows the absolute
stereochemistry of the products that would be formed from each
of the two processes (i.e., C3 or C9 epimerization). If epimer-
ization takes place at C9, then the pair of products from the
SS-2 isomer of 8 would have the same absolute stereochemistry
as the pair formed from the SR-1 isomer, whereas these pairs
of products would have differing absolute stereochemistries if
epimerization were at C3. Similarly, the SS-1 and SR-2 isomers
would give pairs of products with the same absolute stereo-
chemistry only for C9 epimerization.
Whereas the four diastereomers of 8 cannot be distinguished
from their corresponding enantiomers by achiral GC, chiral GC
offers scope for resolving such isomers. The retention times
for gas chromatography on a chiral Cyclosil B column of the
isomers of 8 formed in the hydrolysates are also shown in Table
1 and demonstrate that epimerization took place at C3. That
these results were not due to minor variation in chromatographic
behavior of the isomers was demonstrated by rerunning all of
the samples in a different order, on a separate occasion, with
identical outcomes. We are thus able to confirm the absolute
stereochemistries for the two (3S,9R) allenes: SR-1 as
(3S,6S,9R)-8 and SR-2 as (3S,6R,9R)-8.
ACKNOWLEDGMENT
We are very grateful to Dr. K. Puntener, Hoffmann-La Roche
International, for supplying a sample of authentic (S)-phorenol
(ee > 96%). We thank Profs. I. S. Pretorius and P. B. Høj, as
well as Dr. E. J. Waters, for support and feedback.
LITERATURE CITED
(1) Pickenhagen, W. In FlaVor ChemistrysThirty Years of Progress;
Teranishi, E. L. W., Hornstein, I., Eds.; Kluwer Academic/
Plenum Publishers: New York, 1999; pp 75-87.
(2) Guth, H. Quantitation and sensory studies of character impact
odorants of different white wine varieties. J. Agric. Food Chem.
1997, 45, 3027-3032.
(3) Buttery, R. G.; Teranishi, R.; Ling, L. Identification of dama-
scenone in tomato volatiles. Chem. Ind. 1988, 238.
(4) Puglisi, C. J.; Elsey, G. M.; Prager, R. H.; Skouroumounis, G.
K.; Sefton, M. A. Identification of a precursor to naturally
occurring â-damascenone. Tetrahedron Lett. 2001, 42, 6937-
6939.

4900 J. Agric. Food Chem., Vol. 53, No. 12, 2005
Puglisi et al.
(5) Aasen, A. J.; Kimland, B.; Almqvist, S.-O.; Enzell, C. R. Tobacco
chemistry. 15. New tobacco constituents. Structures of five
isomeric megastigmatrienones. Acta Chem. Scand. 1972, 26,
2573-2576.
(6) Skouroumounis, G. K.; Massy-Westropp, R. A.; Sefton, M. A.;
Williams, P. J. Precursors of damascenone in fruit juices.
Tetrahedron Lett. 1992, 33, 3533-3536.
(7) Skouroumounis, G. K.; Sefton, M. A. Acid-catalyzed hydrolysis
of alcohols and their â-D-glucopyranosides. J. Agric. Food Chem.
2000, 48, 2033-2039.
(15) Skouroumounis, G. K. â-Damascenone precursors in grapes and
wine. Ph.D. Thesis, University of Adelaide, 1991.
(16) Massy-Westropp, R. A.; Skouroumounis, G.; Snow, M. R.;
Tiekink, E. R. T. Crystal structure of [3′R*,1R*,4S*,6S*]-1-(3′-
hydroxybut-1′-ynyl)-2,2,6-trimethylcyclohexane-1,4-diol, C13H22O3.
Z. Kristallogr. 1992, 198, 151-154.
(17) Janusz, A. J.; Capone, D. L.; Puglisi, C. J.; Perkins, M. V.; Elsey,
G. M.; Sefton, M. A. (E)-1-(2,3,6-Trimethylphenyl)buta-1,3-
diene: a potent grape-derived odorant in wine. J. Agric. Food
Chem. 2003, 51, 7759-7763.
(8) Ohloff, G.; Rautenstrauch, V.; Schulte-Elte, K. H. Model
reactions for the biosynthesis of damascone compounds and their
preparative application. HelV. Chim. Acta 1973, 56, 1503-1513.
(9) Naf, R.; Velluz, A.; Thommen, W. Isolation of a glucosidic
precursor of damascenone from Lycium halimifolium mil.
Tetrahedron Lett. 1990, 31, 6521-6522.
(10) Baderschneider, B.; Skouroumounis, G. K.; Winterhalter, P.
Isolation of two glucosidic precursors of â-damascenone from
Riesling wine. Nat. Prod. Lett. 1997, 10, 111.
(11) Straubinger, M.; Knapp, H.; Oka, N.; Watanabe, N.; Winterhalter,
P. Isolation of a glucosidic â-damascenone precursor from rose
petals. J. Agric. Food Chem. 1997, 45, 4053-4056.
(12) Skouroumounis, G. K.; Massy-Westropp, R. A.; Sefton, M. A.;
Williams, P. J. In Progress in FlaVour Precursor Studiess
Analysis, Generation and Biotechnology; Schreier, P., Winter-
halter, P., Eds.; Allured Publishing: Carol Stream, IL, 1993; pp
275-278.
(13) Demole, E.; Enggist, P. Novel synthesis of 3,5,5-trimethyl-4-
(2-butenylidene)cyclohex-2-en-1-one, a major constituent of
Burley tobacco flavor. HelV. Chim. Acta 1974, 57, 2087-2091.
(14) Daniel, M. A.; Elsey, G. M.; Capone, D. L.; Perkins, M. V.;
Sefton, M. A. Fate of damascenone in wine: the role of SO2. J.
Agric. Food Chem. 2004, 52, 8127-8131.
(18) Tanake, A.; Yamamoto, H.; Oritani, T. Lipase-catalyzed asym-
metric synthesis of (R)- and (S)-4-tert-butyldimethylsilyloxy-
2,6,6-trimethyl-2-cyclohexenone and their dihydro derivatives.
Tetrahedron: Asymmetry 1995, 6, 1273-1278.
(19) 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, 6091-6100.
(20) Eliel, E. L.; Wilen, S. H. In Stereochemistry of Organic
Compounds; Wiley-Interscience: New York, 1994; pp 1122-
1133.
Received for review February 14, 2005. Revised manuscript received
April 5, 2005. Accepted April 7, 2005. This project is supported by
Australia’s grapegrowers and winemakers through their investment
body, the Grape and Wine Research and Development Corporation,
with matching funds from the Australian government, and by the
Commonwealth Cooperative Research Centres Program. The work was
conducted by The Australian Wine Research Institute and Flinders
University as part of the research program of the Cooperative Research
Centre for Viticulture.
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