370 J. Agric. Food Chem., Vol. 45, No. 2, 1997
Ha¨ring et al.
supplier should be taken into consideration) in the presence
of 800 units of catalase according to the procedure of Meister
(1952), yielding 68% 3: MS, m/ z (%) 128 (18), 119 (5), 102
layers were dried (anhydrous Na2SO4) and concentrated in a
stream of nitrogen. GC/MS analysis showed equimolar amounts
of 5 and 1.
(6), 101 (100), 100 (8), 74 (19), 73 (46), 56 (24), 55 (55), 45 (48),
Ethyl 5-Hydroxy-4-oxohexanoate (6). Method A (Enzymatic
Synthesis). Pyruvate decarboxylase (0.5 unit/mL) was pre-
saturated with thiamin diphosphate (0.05 mM) and MgSO4
(1.0 mM) in 1 mL of 0.2 M citrate buffer (pH 6.0). After 30
min of stirring, 2-oxoglutaric acid 5-ethyl ester (3) (10 mM)
and acetaldehyde (50 mM) were added and stirring was
continued for 3 days at room temperature. Saturation with
NaCl was followed by extraction with dichloromethane. The
organic layer was dried over anhydrous Na2SO4 and concen-
trated in a stream of dry nitrogen prior to GC/MS analysis.
Besides ketol 6 (4% yield) the decarboxylation product 4 was
found.
1
43 (15); H NMR (400 MHz, CDCl3) δ 1.26 (t, J 1′, ) 7.1 Hz,
2′
2′-H), 2.72 (t, J 3,4 ) 6.4 Hz, 4-H), 3.21 (t, 3-H), 4.15 (q, 1′-H);
13C NMR (100 MHz, CDCl3) δ 14.1 (C-2′), 28.0 (C-4), 32.7 (C-
3), 61.2 (C-1′), 159.8 (C-5), 172.1 (C-1), 194.2 (C-2).
Ethyl 4-Oxobutanoate (4). The reaction mixture contained
2-oxoglutaric acid 5-ethyl ester (30 mM), pyruvate decarboxy-
lase (0.17 unit/mL), thiamin diphosphate (5 mM), and MgSO4
(5 mM) in 1 mL of 0.2 M citrate buffer (pH 6.0). After 24 h at
room temperature, the reaction was terminated and the
solution was extracted with dichloromethane, dried over
anhydrous Na2SO4, and concentrated in a stream of dry
nitrogen for GC/MS analysis. A conversion rate of 70 % was
measured for the decarboxylation product 4. When the same
procedure was repeated with 2-oxoglutaric acid instead of ester
3, no decarboxylation product was detected.
Method B (Chemical Synthesis). The ethanolysis of 0.2
mmol of 4-oxo-5-hexanolide (7) in 3 mL of ethanol was
accomplished after 30 min of refluxing with catalytic amounts
of concentrated HCl. The solution was neutralized with CaCO3
and dried (anhydrous Na2SO4). Compound 6 was purified by
flash chromatography on silica gel (diethyl ether/pentane 5/5
to 9/1), and a colorless oil was received (34% yield): MS, m/ z
(%) 146 (1), 131 (2), 130 (2), 129 (30), 111 (6), 102 (32), 101
(50), 85 (13), 75 (5), 74 (24), 73 (20), 57 (14), 56 (17), 55 (34),
Accordingly to Suomalainen (1968) we decarboxylated 3 by
baker’s yeast, yielding 62% 4. Additionally, 4 was synthesized
from γ-butyrolactone (Banerji et al., 1987); its spectral proper-
ties were in good agreement with data previously published
(Smith et al., 1991; Kuehne and Pitner, 1989).
45 (100), 43 (59); 1H NMR (400 MHz, CDCl3) δ 1.26 (t, J 1′,2′
)
Ethyl 4-Hydroxy-5-oxohexanoate (5). Method A (Biotrans-
formation by Intact Cells, cf. Ohta et al., 1992). The fermenting
medium consisted of 2.0 g of dried baker’s yeast (prior to
incubation suspended in 10 mL of distilled water for 15 min),
ethyl 4-oxobutanoate (77 mM), sodium pyruvate (1.5 M), and
acetaldehyde (180 mM). After 24 h of stirring at room
temperature, the yeast was filtered off with Celite. The filtrate
was saturated with NaCl, extracted with dichloromethane,
dried over anhydrous Na2SO4, and concentrated to 1 mL at
40 °C in vacuo. Besides acetoin and γ-butyrolactone, minor
amounts of acyloin condensation product 5 (2%) were formed.
7.1 Hz, 2′-H), 1.42 (d, J 5,6 ) 7.0 Hz, 6-H), 2.61-2.77 (m, 2-H,
3-H), 4.13 (q, 1′-H), 4.31 (q, 5-H); 13C NMR (100 MHz, CDCl3)
δ 14.1 (C-2′), 19.8 (C-6), 28.0 (C-2), 32.2 (C-3), 60.8 (C-1′), 72.8
(C-5), 172.3 (C-1), 210.8 (C-4). GC/MS analysis of the crude
oil revealed about 40% 5 and 5% 1 as byproducts.
4-Oxo-5-hexanolide (7). An aqueous solution of 1 mg of
hydroxy ester 6 was mixed with catalytic amounts of concen-
trated H2SO4 and stirred for 16 h at room temperature. The
mixture was extracted with CH2Cl2, and the combined organic
layers were dried over Na2SO4 and concentrated in a stream
of dry nitrogen. The product distribution was monitored by
GC/MS showing 60% 7, 30% 1, and 10% 5. Following a
simplified procedure of Georgiadis et al. (1991), the δ-lactone
7 was additionally synthesized starting from 2-acetylfuran:
MS, m/ z (%) 128 (10), 100 (6), 85 (2), 84 (3), 60 (2), 57 (6), 56
(100), 55 (7), 45 (17), 44 (9), 43 (50), 42 (25); 13C NMR (100
MHz, CDCl3) δ 15.9 (C-6), 28.3 (C-2), 33.2 (C-3), 79.5 (C-5),
169.8 (C-1), 205.2 (C-4). 1H NMR data were identical with
those from Georgiadis et al. (1991).
Method B (Enzymatic Synthesis). Pyruvate decarboxylase
(0.1 unit/mL) was presaturated with thiamin diphosphate (0.05
mM) and MgSO4 (1.0 mM) in 1.0 mL of 0.2 M citrate buffer
(pH 6.0). After 30 min of stirring, ethyl 4-oxobutanoate (10
mM) and sodium pyruvate (30 mM) were added and stirring
was continued for 3 days at room temperature. Saturation
with NaCl was followed by extraction with dichloromethane.
The organic layer was dried over anhydrous Na2SO4 and
concentrated in a stream of dry nitrogen. Subsequent GC/
MS analysis revealed a conversion rate of 4% to ketol 5. When
the same procedure was repeated with 4-oxobutyric acid
instead of ester 4, no acyloin condensation product was
detected.
RESULTS AND DISCUSSION
Biom im et ic Syn t h esis of Soler on e. We applied
pyruvate decarboxylase (EC 4.1.1.1) (PDC) as key
enzyme for the biomimetic synthesis elucidating the
formation of solerone 1 (Figure 1). The thiamin diphos-
phate dependent enzyme from S. cerevisiae is respon-
sible for the decarboxylation of pyruvate during the
course of alcoholic fermentation. After loss of carbon
dioxide from 2-oxoacids, the resulting aldehyde is
released. Alternatively, the cofactor-bound decarboxyl-
ation product can react further with an another alde-
hyde. By the latter acyloin condensation a new carbon-
carbon bond will be formed, thus opening a biosynthetic
way to R-hydroxy carbonyl compounds (Koga, 1995;
Kren et al., 1993).
While in the presence of 2-oxoglutaric acid neither
decarboxylation nor acyloin condensation has been
observed, as one could expect from results published
previously (Suomalainen et al., 1969), we succeeded in
the enzymatic conversion of the mono ethyl ester 3 to
ethyl 4-oxobutanoate (4), using both whole yeast cells
(S. cerevisiae) and purified PDC. The oxo ester 4 served
as substrate for a second reaction catalyzed by PDC.
Formation of a new carbon-carbon bond was ac-
complished in the presence of pyruvic acid, which acted
as donor of a C2 unit. Thus, ethyl 4-hydroxy-5-oxo-
hexanoate (5) was obtained for the first time as the
Method C (Chemical Synthesis). To a solution of 3.9 mmol
(500 mg) of solerone in 1.7 mL of ethanol was added 0.017
mmol of concentrated H2SO4. After 19 h of stirring at room
temperature, the pH was adjusted to 7.0 by 0.034 mmol of
CaCO3. Stirring was continued for another hour, the suspen-
sion was filtered off, and the solvent was evaporated. The
yellowish oil (204 mg) still contained about 10% solerone and
other byproducts. Samples for spectroscopic purposes were
purified by HPLC on a silica column (Eurosphere Si 100,
Knauer, Berlin, Germany, 250 × 4 mm, 5 µm, methyl tert-
butyl ether/pentane 7/3 to 10/0 in 30 min; detection at 275
nm): MS, m/ z (%) 131 (13), 129 (10), 111 (4), 88 (5), 85 (100),
60 (5), 57 (25), 55 (14), 45 (14), 44 (10), 43 (76), 40 (24); 1H
NMR (400 MHz, CDCl3) δ 1.27 (t, J 1′,2′ ) 7.1 Hz, 2′-H), 1.71-
1.85 (m, 3-H), 2.26 (s, 6-H), 2.44-2.58 (m, 2-H), 4.15 (q, 1′-H),
4.21-4.27 (m, 4-H); 13C NMR (100 MHz, CDCl3) δ 14.1 (C-2′),
25.2 (C-6), 28.5/29.5 (C-2/C-3), 60.6 (C-1′), 75.7 (C-4), 173.2 (C-
1), 209.4 (C-5).
Solerone (1). Method A. (S)-Solerone (1) was prepared from
L-glutamic acid as published previously (Berti et al., 1983).
The spectroscopic data of 1 did correspond to those published
by Mosandl and Hollnagel (1989).
Method B. An aqueous solution of 10 mg of hydroxy ester
5 was mixed with catalytic amounts of concentrated H2SO4
and stirred for 15 h at room temperature. The mixture was
extracted with dichloromethane, and the combined organic