Stereoselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol by lipase-catalyzed hydrolysis of 3-acetylthioesters

Article abstract of DOI:10.1021/jf030166u

The enantioselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol, two potent sulfur-containing aroma compounds, by lipase-catalyzed hydrolysis of the corresponding 3-acetylthioesters was investigated. The stereochemical course of the kinetic resolutions was followed by capillary gas chromatography using modified cyclodextrins as chiral stationary phases. The enzyme preparations tested varied significantly in terms of activity and enantioselectivity (E). The highest E value (E = 36) was observed for the hydrolysis of 3-acetylthiohexanal catalyzed by lipase B from Candida antarctica resulting in (S)-configured thiol products. Immobilization of the enzyme (E = 85) and the use of tert-butyl alcohol as cosolvent (E = 49) improved the enantioselectivity. Modification of the acyl moiety of the substrate (3-benzoylthiohexanal) had no significant impact. The sulfur-containing compounds investigated possess attractive odor properties, and only one of the enantiomers exhibits the pleasant citrus type note.

Full text of DOI:10.1021/jf030166u

J. Agric. Food Chem. 2003, 51, 4349−4355 4349
Stereoselectivity of the Generation of 3-Mercaptohexanal and
-Mercaptohexanol by Lipase-Catalyzed Hydrolysis of
-Acetylthioesters
3
3
†
†
HIDEHIKO WAKABAYASHI, MOTOKO WAKABAYASHI,
WOLFGANG EISENREICH,‡ AND KARL-HEINZ ENGEL*
,†
Lehrstuhl f u¨ r Allgemeine Lebensmitteltechnologie, Technische Universit a¨ t M u¨ nchen, Am Forum 2,
D-85350 Freising-Weihenstephan, Germany, and Lehrstuhl f u¨ r Organische Chemie und Biochemie,
Technische Universit a¨ t M u¨ nchen, Lichtenbergstrasse 4, D-85748 Garching, Germany
The enantioselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol, two potent
sulfur-containing aroma compounds, by lipase-catalyzed hydrolysis of the corresponding 3-acetyl-
thioesters was investigated. The stereochemical course of the kinetic resolutions was followed by
capillary gas chromatography using modified cyclodextrins as chiral stationary phases. The enzyme
preparations tested varied significantly in terms of activity and enantioselectivity (E). The highest E
value (E ) 36) was observed for the hydrolysis of 3-acetylthiohexanal catalyzed by lipase B from
Candida antarctica resulting in (S)-configured thiol products. Immobilization of the enzyme (E ) 85)
and the use of tert-butyl alcohol as cosolvent (E ) 49) improved the enantioselectivity. Modification
of the acyl moiety of the substrate (3-benzoylthiohexanal) had no significant impact. The sulfur-
containing compounds investigated possess attractive odor properties, and only one of the enantiomers
exhibits the pleasant citrus type note.
KEYWORDS: Thioester; thiol; lipase; enantioselectivity; 3-acetylthiohexanal; 3-acetylthiohexanol
INTRODUCTION
various plants, especially tropical fruits (25). For example,
passion fruit’s flavor is typically determined by sulfur-containing
compounds (26). 3-Mercaptohexanol, first identified in yellow
passion fruits (27) and later also described as a volatile
constituent of wine (28) plays an important sensory role. The
corresponding aldehyde, 3-mercaptohexanal, had been described
as a synthetic intermediate (29). Later, it was reported as a flavor
compound in cooked liver and was described as imparting
Lipases are well-established biocatalysts, which are widely
used for regioselective and enantioselective biotransformations
(1, 2). For esters, alcohols, and acids, many examples of kinetic
resolutions of enantiomers via hydrolysis, transesterification, and
esterification have been described (3, 4). Analogous reactions
have been reported for thio acids and esters (5-8). Apart from
a first communication on the lipase-catalyzed hydrolysis of
“
tropical fruit” type aroma notes (30). Synthesis via combina-
3
-acetylthiocycloheptene (9), the exploitation of the stereo-
torial approach and sensory evaluation by gas chromatography/
olfactometry (GC/O) revealed this mercaptoaldehyde to have a
citrus peel note (31).
selectivity of enzyme-catalyzed reactions of sulfur-containing
esters started rather late (10, 11). In the meantime, various
approaches have been described (12-14), many of them
focusing on the enzymatic resolution of 2-arylpropionates, an
important class of nonsteroidal antiinflammatory drugs (15-17).
Considering the importance of sulfur-containing compounds
in flavor chemistry, it is not surprising that enzyme-catalyzed
reactions have also been proposed as strategies to obtain
flavoring compounds. In addition to the nonhydrolytic liberation
of potent volatiles from precursors using â-lyases (18, 19),
lipase-catalyzed syntheses (20, 21) as well as hydrolyses of
thioesters (22, 23) have been described as useful approaches.
Sulfur-containing volatiles are not only generated during the
thermal treatment of foods (24) but are also biosynthesized in
Recently, the potential to use porcine liver esterase for the
generation of 3-mercaptohexanal by hydrolysis of 3-acetylthio-
hexanal has been reported (32). However, the stereochemical
course of the reaction had not been followed. The aim of this
study was to screen lipases from different sources for their
potential to generate 3-mercaptohexanal and 3-mercaptohexanol
by hydrolysis of the corresponding thioesters and to investigate
the degree of enantiodiscrimination related to these kinetic
resolutions.
MATERIALS AND METHODS
Materials. E-2-Hexenal, thioacetic acid, thiobenzoic acid, p-chloro-
mercuribenzoic acid, and L-cysteine (∼99%) were obtained from Fluka
Chemie AG (Germany).
Lipases from Aspergillus niger (catalog number, 62301; abbreviation,
ANL), Aspergillus oryzae (95184; AOL), Candida antarctica (62299;
*
To whom correspondence should be addressed. Tel: +49-8161-714250.
Fax: +49-8161-714259. E-mail: K.H.Engel@lrz.tu-muenchen.de.
†
Lehrstuhl f u¨ r Allgemeine Lebensmitteltechnologie.
Lehrstuhl f u¨ r Organische Chemie und Biochemie.
‡
1
0.1021/jf030166u CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/18/2003

4350 J. Agric. Food Chem., Vol. 51, No. 15, 2003
Wakabayashi et al.
CAL), C. antarctica lipase A (62287; CAL-A), C. antarctica lipase B
1002; SE-54, 1032; DB-WAX, 1359. GC-MS (m/z (relative inten-
(
62288; CAL-B), Penicillium roqueforti (62308; PRL), and Rhizopus
sity)): 55 (100), 41 (70), 42 (62), 70 (40), 61 (34), 81 (25), 57 (24),
43 (23), 80 (22), 114 (16), 99 (16), 132 (M ; 17).
+
•
oryzae (80612; ROL) were purchased from Fluka Chemie AG. Lipases
from C. antarctica lipase B (L4777; CAL-B resin), Candida rugosa
Enzyme-Catalyzed Hydrolysis. A 50 µmol amount of the thioester
substrate (8.7 mg of 3-acetylthiohexanal, 11.8 mg of 3-benzoylthio-
hexanal, and 8.8 mg of 3-acetylthiohexanol, respectively) was added
to 500 µL of 50 mM potassium phosphate buffer (pH 7.4). The enzyme
preparation (ROL, 10 mg; ANL, 50 mg; WGL, 25 mg; MJL, 25 mg;
PRL, 50 mg; MML, 5 mg; PCL, 25 mg; PPL, 2 mg; CRL, 25 mg;
PLE, 0.4 mg; AOL, 20 mg; TTL, 100 mg; CAL, 10 mg; CAL-A, 10
mg; CAL-B, 10 mg; CAL-B resin, 10 mg) was added, and the mixture
was stirred magnetically (300 rpm) with a Teflon bar at 25 °C. After
2 h, an aliquot of 20 µL of the reaction mixture was extracted with
500 µL of dichloromethane. The organic phase was dried over
anhydrous sodium sulfate and subjected to GC analysis. For the
experiments on the cosolvent effects, 10-40 vol % of acetone and
tert-butyl alcohol, respectively, was added to the buffer solution.
Separation of Remaining Substrate/Product and Conversion into
3-Mercaptohexanol. Kinetic resolution of 3-acetylthiohexanal (350
µmol substrate; reaction time 8 h) was performed as described above
using CAL-B as the catalyst. The organic extract obtained was
concentrated to 100 µL by using a nitrogen stream and added to 10
mL of ice-cooled 0.1 M potassium phosphate buffer (pH 7.4). After
sodium borohydride (3.7 mg, 98 µmol in 1 mL of distilled water) was
added, the solution was stirred continuously for 1 h under ice-cooling.
The pH was adjusted to 3 using 2 N sulfonic acid, and the solution
was extracted two times with 10 mL of diethyl ether. The combined
extracts were washed (two times with 10 mL of distilled water) and
dried over anhydrous sodium sulfate.
Separation of 3-acetylthiohexanol and 3-mercaptohexanol was
achieved by using p-hydroxymercuribenzoate (35). The organic solution
was extracted three times (each for 5 min) with 10 mL of an aqueous
solution (pH 8.5) of p-hydroxymercuribenzoate (2.5 mM) prepared from
p-chloromercuribenzoate (36). The organic phase was washed with 10
mL of distilled water and dried over anhydrous sodium sulfate.
3-Acetylthiohexanol contained in this solution was converted to
3-mercaptohexanol by alkaline hydrolysis according to the procedure
described above and subjected to GC on the chiral stationary phase.
The aqueous phase was washed with 10 mL of dichloromethane
followed by addition of L-cysteine (0.91 g; 7.5 mmol) dissolved in 10
mL of distilled water. After 10 min equilibrium at room temperature,
the pH was adjusted to 6 by addition of 5% hydrochloric acid. The
released 3-mercaptohexanol was isolated by extraction with dichloro-
methane (two times 20 mL; each for 5 min). The combined extracts
were washed with 10 mL of distilled water, dried over anhydrous
sodium sulfate, and also subjected to GC on the chiral stationary phase.
GC Analysis. Capillary GC was performed on the following four
GC systems. (I) A Carlo Erba MEGA2 gas chromatograph equipped
with FID and FPD. Parallel detection was achieved by dividing the
effluent of the column (DB-WAX, J&W; 60 m × 0.32 mm i.d.; film
thickness 0.25 µm) via a chrom-fit connector and short pieces of
deactivated fused silica capillaries to the two detectors. Split injection
was performed at 215 °C, and column temperature was programmed
from 40 (5 min hold) to 230 °C (25 min hold) at 4 °C/min. The detector
temperature was 240 °C for FID and 140 °C for FPD. Hydrogen was
used as the carrier gas at 105 kPa.
(
L1754; CRL), Mucor jaVanicus (L8906; MJL), Mucor miehei (L9031;
MML), porcine pancreas (L3126; PPL), Pseudomonas cepacia (L9156;
PCL), Thermomyces lanuginosus (L0902; TLL), wheat germ (L3001;
WGL), and the esterase from porcine liver (E3019; PLE) were from
Sigma-Aldrich Chemie GmbH (Germany).
Synthesis of the Thioester Substrates. Thioesters were synthesized
by Michael type addition of thiocarboxylic acid to R,â-unsaturated
carbonyls (33, 34).
3
-Acetylthiohexanal. A mixture of E-2-hexenal (2.3 mL, 20 mmol)
and thioacetic acid (2.1 mL, 30 mmol) was stirred for 2 h under ice-
cooling and for another 24 h at room temperature (25 °C). After the
excess of thioacetic acid was removed under reduced pressure at 40
°
C, 3.48 g (20 mmol) of a pale yellow, sticky liquid was obtained (mol
yield from E-2-hexenal, 100%; purity, 95% by GC). GC retention
indices: DB-1, 1231; SE-54, 1266; DB-WAX, 1845. GC-MS (m/z
(relative intensity)): 43 (100), 131 (18), 41 (18), 55 (15), 99 (10), 103
+
•
1
(
9), 89 (7), 70 (7), 69 (7), 114 (4), 174 (M ; 2). H NMR (500 MHz,
): δ, ppm 0.89 (3H, t, 7.2 Hz, H-6), 1.37 (4H, m, H-4, H-5),
.30 (3H, s, CH -CO), 2.69 (2H, m, H-2), 3.91 (1H, qui, 6.8 Hz, H-3),
.67 (1H, t, 1.8 Hz, H-1). C NMR (125.6 MHz): δ, ppm 200.5 (CO-
), 195.6 (C-1), 49.2 (C-2), 38.7 (C-3), 36.9 (C-4), 32.9 (CO-CH ),
0.5 (C-5), 14.9 (C-6).
-Benzoylthiohexanal. A mixture of E-2-hexenal (2.3 mL, 20 mmol)
CDCl
2
9
CH
2
3
3
13
3
3
3
and thiobenzoic acid (3.5 mL, 30 mmol) was stirred for 2 h under ice-
cooling and for another 24 h at room temperature (25 °C). After the
reaction mixture was dissolved in 20 mL of dichloromethane, the
solution was washed with 10 mL of 0.1 M sodium phosphate buffer
(
pH 8.5) and two times with 10 mL of distilled water. After it was
dried over anhydrous sodium sulfate, dichloromethane was removed
under reduced pressure at 40 °C. A 4.96 g (21 mmol) amount of a
yellowish, sticky liquid was obtained (mol yield from E-2-hexenal,
1
5
05%; purity, 79% by GC). GC retention indices: DB-1, 1835; SE-
4, 1860. GC-MS (m/z (relative intensity)): 105 (100), 77 (30), 51
+
•
(
10), 106 (8) 139 (5), 41 (4), 208 (1), 131 (1), 114 (1), 236 (M ; 1).
1
H NMR (500 MHz, CDCl ): δ, ppm 0.93 (3H, t, 7.2 Hz, H-6), 1.45
3
(
(
(
2H, m, H-5), 1.72 (2H, qua, 7.5 Hz, H-4), 2.81 (2H, m, H-2), 4.16
1H, qui, 6.8 Hz, H-3), 7.42 (2H, m, m-Ph), 7.55 (1H, m, p-Ph), 7.92
1
3
2H, m, o-Ph), 9.74 (1H, t, 1.8 Hz, H-1). C NMR (125.6 MHz): δ,
ppm 200.2 (CO-Ph), 191.3 (C-1), 136.8, 133.5, 128.6, 127.2 (Ph),
9.0 (C-2), 38.3 (C-3), 36.6 (C-4), 20.3 (C-5), 13.7 (C-6).
-Acetylthiohexanol. A solution of 3-acetylthiohexanal (112 mg, 0.64
4
3
mmol) in 5 mL of methanol was added to 20 mL of 0.5 M potassium
phosphate buffer (pH 7.4). After sodium borohydride (50 mg, 1.3 mmol
dissolved in 2 mL of water) was added dropwise to the stirred solution
under ice-cooling, the solution was stirred for another 30 min. The pH
was adjusted to 5 using 2 N sulfonic acid, and the solution was extracted
with dichloromethane (two times 10 mL). After the combined extracts
were washed (10 mL of distilled water) and dried (anhydrous sodium
sulfate), dichloromethane was removed under reduced pressure at 40
°
(
C. A 97.3 mg (0.55 mmol) amount of a transparent liquid was obtained
mol yield from 3-acetylthiohexanal, 86%; purity, 95% by GC). GC
retention indices: DB-1, 1293; DB-WAX, 2090. GC-MS (m/z (relative
intensity)): 43 (100), 55 (52), 88 (27), 41 (25), 83 (19), 82 (18), 116
(II) A Carlo Erba GC 6000 gas chromatograph with FID; the columns
used were DB-1 (30 m × 0.25 mm i.d.; film thickness 1 µm) and SE-
54 (15 m × 0.25 mm i.d.; film thickness 0.15 µm), respectively. Split
injection was performed at 220 °C, and the column temperature was
programmed from 50 (2 min hold) to 300 °C (5 min hold) at 4 °C/
min. Hydrogen was used as the carrier gas at 80 kPa for DB-1 and 45
kPa for SE-54.
(III) Two Fisons GC 8000 gas chromatographs equipped with FID
detectors and coupled via a moving capillary stream switching (MCSS)
system (37) were used. A DB-5 fused silica column (28 m × 0.25 mm
i.d.; film thickness 0.25 µm; J&W) was used as the precolumn, and a
fused silica column (30 m × 0.25 mm i.d.) coated with 33% heptakis-
(per-O-ethyl)-â-cyclodextrin in OV1701-vi (PerEt-â-CD) was used as
the main column. Split injection was performed at 205 °C, and the
precolumn temperature was programmed from 40 (5 min hold) to 115
+
•
1
(
15), 133 (7), 101 (7), 158 (1), 176 (M ; 1). H NMR (500 MHz,
CDCl ): δ, ppm 0.89 (3H, t, 7.3 Hz, H-6), 1.5-1.4 (4H, m, H-4, H-5),
.34 (3H, s, CH -CO), 1.98 (2H, m, H-2), 3.62 (1H, nd, H-3), 3.62
3
2
3
13
(
(
1H, nd, H-1). C NMR (125.6 MHz): δ, ppm 198.6 (CO-CH
3
), 60.2
C-1), 41.6 (C-2), 39.2 (C-3), 37.6 (C-4), 31.1 (CO-CH ), 20.6 (C-5),
3
1
0
4.1 (C-6).
Synthesis of 3-Mercaptohexanal. 3-Acetylthiohexanal (113 mg,
.65 mmol) dissolved in 1 mL of methanol was added to 10 mL of 0.5
N sodium hydroxide aqueous solution, and the mixture was stirred for
0 min under ice-cooling. The pH was adjusted to 2 using 2 N sulfonic
acid, and the solution was extracted with dichloromethane (two times
0 mL). After it was washed (two times 10 mL of distilled water) and
3
1
dried (anhydrous sodium sulfate), the dichloromethane solution was
subjected to GC and GC/MS analysis. GC retention indices: DB-1,

Enantioselective Hydrolysis of Thioesters
C at 3 °C/min and from 115 to 240 °C (15 min hold) at 15 °C/min.
J. Agric. Food Chem., Vol. 51, No. 15, 2003 4351
°
Table 1. Enzyme-Catalyzed Kinetic Resolution of 3-Acetylthiohexanal
For the main column, it was from 60 (20 min hold) to 110 °C at 1
C/min and 110 to 150 °C (5 min hold) at 15 °C/min. Hydrogen was
°
enantiomeric
enantio-
used as the carrier gas at 150 kPa. Transfers onto the main column
using cut time intervals were 15.4-16.1 min for 3-mercaptohexanal
and 27.2-27.7 min for 3-acetylthiohexanal. The data recording as well
as the control of the MCSS system were managed with Chrom-Card
for Windows software (Fisons Instruments) on a work station.
excess (%)
conversiona
selectivity
preferred
enzyme
ees
eep
(%)
E
enantiomer
ROL
ANL
WGL
MJL
PRL
MML
PCL
PPL
CRL
PLE
AOL
TLL
CAL
0.9
21.0
17.8
0.5
0.9
0.5
2.1
4.3
8.2
32.0
5.6
30.4
2.1
21.1
51.1
36.5
18.4
27.2
37.1
4.1
2.3
9.1
29.7
29.6
50.2
41.2
55.0
76.3
45.0
66.1
91.1
96.7
4.9
43.5
32.4
2.2
4.1
2.1
1.5
2
3
1.1
1.1
1.2
2
2
3
3
4
10
3
6
36
85
R
R
R
S
S
S
S
S
S
S
S
S
S
R
S
S
(IV) A Carlo Erba GC 6000 gas chromatograph equipped with FID;
the column used was a fused silica column (30 m × 0.25 mm i.d.)
coated with a 0.25 µm film of 50% heptakis(2,3-di-O-methyl-6-O-tert-
butyldimethylsilyl)-â-cyclodextrin in OV1701-vi (DiMe-â-CD). Split
injection was performed at 200 °C, and the column temperature was
programmed from 50 (2 min hold) to 200 °C (5 min hold) at 3 °C/
min. Hydrogen was used as the carrier gas at 80 kPa. For the analysis
of 3-benzoylthiohexanal, the column temperature was programmed from
6.7
12.6
14.1
43.7
9.3
28.5
4.6
24.2
35.9
27.4
1
20 to 150 °C at 2 °C/min and from 150 to 180 °C (5 min hold) at 0.2
C/min.
Capillary GC/O. GC/O was performed on the following two GC
systems. (I) A DB-WAX column (55 m × 0.32 mm i.d.; film thickness
.25 µm; J&W) installed into Carlo Erba GC4200 gas chromatograph
°
CAL-A
CAL-B
CAL-Bb
0
a
Reaction time, 2 h; other conditions, see Materials and Methods. b Immobilized
enzyme adsorbed on a macroporous resin.
equipped with a split/splitless injector and FID. At the end of the
column, the effluent was split 1:1 for FID and sniffing port with chrom-
fit connector and deactivated fused silica tube. The column temperature
was programmed from 60 (5 min hold) to 230 °C at 4 °C/min. The
injector temperature was 215 °C, and the detector temperature was 240
Enzyme-Catalyzed Hydrolysis of 3-Acetylthiohexanal.
Commercially available enzyme preparations (15 lipases and
one esterase) of various origins (microbial, plant, and mam-
malian) were tested for their suitability to hydrolyze the thioester
bond in 3-acetylthiohexanal. The conversion rate and stereo-
chemical course of the reaction were followed by means of
capillary GC. As shown in Table 1, 3-acetylthiohexanal (1) was
accepted as substrate by all enzymes tested. The retention index
and MS spectrum of the generated 3-mercaptohexanal (2) were
identical to data from literature (30) and to those obtained from
a synthesized reference compound. Formation of the product
by chemical cleavage of substrate could be ruled out by
incubation under the same conditions without enzymes.
Using heptakis(per-O-ethyl)-â-cyclodextrin (PerEt-â-CD) and
heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-â-cyclo-
dextrin (DiMe-â-CD) as chiral stationary phases, the enanti-
omers of 3-mercaptohexanal (2a,b), 3-acetylthiohexanal (1a,b),
and 3-acetylthiohexanol (3a,b) could be separated for the first
time by means of capillary GC (Figure 1). This enabled the
determination of the enantiomeric excesses of product (eep) and
remaining substrate (ees) on the basis of the peak areas. The
data obtained were used to calculate conversion rates (c) and
enantioselectivities (E) applying equations previously described
for kinetic resolutions (38).
The most pronounced enantiodiscrimination was observed for
Candida antarctica lipase. The optically enriched product and
remaining substrate obtained by the kinetic resolution using
CAL-B (nonimmobilized) as catalyst were used to assign the
absolute configurations. The order of elution of the enantiomers
of 3-mercaptohexanol on DiMe-â-CD had been determined
previously (39). Thus, the orders of elution for the enantiomers
of 3-mercaptohexanal, 3-acetylthiohexanal, and 3-acetythiohex-
anol could be assigned by transforming the enantiomerically
enriched compounds into 3-mercaptohexanol, using the series
of reactions outlined in Figure 2. Because of the nonenanti-
oselective course of these reactions, the enantiomeric ratios
determined after each step were in accordance with the starting
ratios (apart from a slight racemization observed for the alkaline
hydrolysis). 3-Mercaptohexanol obtained by reduction of the
mixture obtained after the enzyme-catalyzed resolution with
sodium borohydride and subsequent selective extraction of the
thiol using p-hydroxymercuribenzoate proved to be the (S)
enantiomer 4b (ee 60%). The enantiomer 4a obtained by alkaline
°
C for FID and 200 °C for sniffing port. Makeup flow of 20 mL/min
nitrogen was used for the sniffing port. Hydrogen was used as the carrier
gas at 100 kPa.
(II) A DiMe-â-CD column installed into HP5890II gas chromato-
graph equipped with a cooled-on-column injector and FID. The effluent
was split to FID and a sniffing port as described above. The typical
temperature program was from 40 (2 min hold) to 80 °C at 40 °C/min,
8
0 to 140 °C at 2 °C/min, and 140 to 200 °C (5 min hold) at 20 °C/
min. The injector temperature was controlled as oven tracking mode,
and the detector temperature was 220 °C for FID and 200 °C for the
sniffing port. Hydrogen was used as the carrier gas at 70 kPa.
GC-MS. DB-WAXETR fused silica column (30 m × 0.25 mm i.d.;
film thickness 0.5 µm; J&W) installed into a Finnigan GC8000 gas
chromatograph equipped with a split/splitless injector and a Voyager
mass spectrometer were used. The mass spectrometer was operated at
scan mode at 20-400 amu, and the ionization voltage was 70 eV. The
column temperature was programmed from 40 (5 min hold) to 240 °C
(15 min hold) at 4 °C/min. The injector temperature was 215 °C, and
the transfer line temperature was 230 °C. The injector was used as
split mode, and the split flow was 27 mL/min. Helium was used as the
carrier gas at 75 kPa.
For the analysis of 3-benzoylthiohexanal, SE-54 fused silica column
(
15 m × 0.25 mm i.d.; film thickness 0.15 µm) was used, and the
temperature program was from 50 (2 min hold) to 250 °C (5 min hold)
at 4 °C/min. Helium was used as the carrier gas at 25 kPa.
NMR Spectroscopy. H NMR and 13C NMR spectra were recorded
at 500.13 and 125.6 MHz with an AVANCE 500 spectrometer (Bruker
Instruments, Germany). Two-dimensional COSY, HMQC, and HMBC
experiments were performed according to standard Bruker software
1
(XWINNMR 3.0). Samples were measured in 0.5 mL of [D]-
chloroform. The sample temperature was 25 °C. Composite pulse
1
3
decoupling was used in the C NMR experiments. All signals were
assigned by proton-proton and proton-carbon correlation experiments
(COSY, HMQC, and HMBC).
RESULTS AND DISCUSSION
Synthesis of 3-Acetylthioesters. 3-Acetylthiohexanal (1) was
synthesized by Michael type addition of thioacetic acid to E-2-
hexenal. 3-Acetylthiohexanol (3) was obtained by subsequent
reduction with sodium borohydride under controlled pH condi-
tions to prevent alkaline hydrolysis of the thioester moiety. The
identities of the compounds were confirmed by means of GC-
MS and NMR.

4352 J. Agric. Food Chem., Vol. 51, No. 15, 2003
Wakabayashi et al.
Figure 1. Capillary GC separation of (A) racemic 3-acetylthiohexanal (1) and 3-mercaptohexanal (2) (GC system III); (B) racemic 3-acetylthiohexanol
3) and 3-mercaptohexanol (4) (GC system IV); (C,D) enantiomerically enriched compounds obtained after kinetic resolution (CAL-B, 8 h, c ) 42% (C)
(
and 41% (D)). Enantiomers a, (R)-configuration; enantiomers b, (S)-configuration.
may be explained by the structure of the substrate. According
to a rule established by Kazlauskas et al. (40, 41) for esters of
secondary alcohols, the substrates resolved most efficiently by
lipase-catalyzed hydrolyses are those with substituents that differ
significantly in size, and the enantiomer preferred by the enzyme
can be predicted. This rule could also be confirmed for
hydrolysis and interesterification, respectively, of corresponding
esters of secondary thiols (11, 13). For 3-acetylthiohexanal, the
only slight differences in size between the substituents at the
stereocenter do not allow a clear categorization and prediction
according to the rule of Kazlauskas. The enantioselectivity
observed for CAL-B must be based on factors other than solely
the difference in size of the substituents.
It is noteworthy that the four commercial preparations of
Candida antarctica lipase employed as catalysts differed
significantly in their enantioselectivities. This yeast produces
two different lipases (A and B), which have been purified and
characterized (42). Both have been cloned and expressed in A.
oryzae (43). The original lipase preparation from the yeast
CAL) exhibited only low selectivity for the (S)-configured
substrate (E ) 3). This preference was significantly enhanced
E ) 36) when using CAL-B obtained from recombinant A.
oryzae. In contrast, the heterologously expressed lipase A (CAL-
A) showed preference for the opposite (R) enantiomer (E ) 6).
(
(
The most pronounced discrimination (E ) 85) was observed
for the enzyme preparation with CAL-B immobilized on a
macroporous acrylic resin. This enhancement of enantioselec-
tivity may be explained by the increased rigidity of the enzyme
conformation due to interactions with the polymer. Similar
phenomena have been described for the lipase from Candida
cylindracea immobilized on agarose and silica gel (44).
Figure 2. Sequence of reactions applied to convert product and substrate
of the kinetic resolution into 3-mercaptohexanol in order to determine their
absolute configurations.
hydrolytic cleavage of the remaining thioester 3a (ee 92%) was
shown to have the (R) configuration (ee 86%).
As shown in Table 1, the enzyme preparations tested differed
strongly in terms of degree of enantiodiscrimination. The fact
that there was no consistent preference of the same enantiomer
A partial adsorption of the thiol product on the resin (from
20 to 75%, depending on enzyme and substrate concentrations)
turned out to be a disadvantage of using the enzyme in the
immobilized form. Extraction of the removed resin with

Enantioselective Hydrolysis of Thioesters
J. Agric. Food Chem., Vol. 51, No. 15, 2003 4353
of the hydrolysis of 3-acetylthiohexanal remains unclear. NMR
studies of CAL-B applied to the hydrolysis of 3-chloro-1-
phenylmethoxy-2-propyl butanoate had shown that the confor-
mation of the enzyme most likely remains unchanged upon
addition of up to 50% acetone (45). The increased solubility of
the liberated alcohol resulting from the addition of acetone to
the reaction mixture had been discussed as possible explanation
of the phenomenon (46).
Influence of Structural Modifications on Enantioselectiv-
ity. The effects of the replacement of the aldehyde function in
the thioester substrate by an alcoholic group on enzyme activities
and enantioselectivities are summarized in Table 3. The
preference of enantiomers remained the same as observed for
the hydrolysis of 3-acetylthiohexanal. However, the lipases from
TLL and CAL exhibited conversion rates as well as enantio-
selectivities significantly lower than those for the aldehyde
substrate. CAL-A even showed no enantiodiscrimination when
catalyzing the hydrolysis of 3-acetylthiohexanol.
Table 2. Effects of Cosolvents on the Enantioselectivity of the
Hydrolysis of 3-Acetylthiohexanal Catalyzed by CAL-B
enantiomeric
enantio-
selectivityb
E
excess (%)
ees eep
1.1
conversiona
(%)
concentration
(vol %)
cosolvent
acetone
5
91.1
92.5
89.2
87.1
93.5
93.8
91.8
35.9
28.2
17.3
10.2
33.5
32.9
19.5
36
35
20
16
47
49
29
10
36.3
18.6
9.9
47.0
45.9
22.2
2
0
4
0
tert-butanol
10
2
0
40
a
Reaction time, 2 h; other conditions, see Materials and Methods. b Preferred
enantiomer, S.
Table 3. Enzyme-Catalyzed Kinetic Resolution of 3-Acetylthiohexanol
and 3-Benzoylthiohexanal
enantiomeric
enantio-
Menthylbenzoate has been reported as suitable starting
material to obtain (-)-menthol via hydrolysis catalyzed by CRL
(47). To study the effect of a bulkier acyl residue on the kinetic
resolution, 3-benzoylthiohexanal was employed as substrate. The
synthesis was performed by addition of thiobenzoic acid to E-2-
hexenal. Capillary GC separation of the enantiomers was
achieved on DiMe-â-CD as chiral stationary phase (R, 1.02;
K1, 42.3; R, 1.13; 145 °C isothermal; hydrogen 31.4 cm/s). When
using CAL, CAL-A, CAL-B, TLL, PPL, and WGL as biocata-
lysts, the conversion rates observed after 2 h were negligible
excess (%)
conversiona
selectivity
preferred
enantiomer
enzyme
ees
eep
(%)
E
3-Acetylthiohexanol
ANL
WGL
TLL
PPL
PLE
CAL
CAL-A
CAL-B
93.9
82.0
1.3
2.9
64.8
0.7
6.3
38.0
19.1
52.4
24.5
35.9
6.8
93.8
68.4
6.2
5.2
72.6
1.9
3
5
2
3
3
R
R
S
S
S
S
2
1
0.2
3.5
27.1
81.1
25.1
12
S
(<0.1%) as compared to the data obtained for ANL, PLE, and
3-Benzoylthiohexanal
CRL (Table 3). The replacement of the acetyl moiety by a bulky
group drastically reduced the conversion rates without significant
impact on the enantioselectivities.
ANL
PLE
CRL
0.1
6.9
0.7
2.9
71.3
54.6
0.9
10.1
1.2
1
6
3
S
S
Odor Descriptions. Odor descriptions of 3-acetylthiohexanal,
3-acetylthiohexanol, and 3-mercaptohexanal were determined
by means of GC/O. As shown in Table 4, the sulfur-containing
volatiles exhibited attractive citrus type notes. The odors of the
stereoisomers differed significantly, and only one of the
enantiomers possessed the pleasant fruity note. Quantitative
studies on differences in odor thresholds of the enantiomers are
in progress.
The enantiomers of 3-mercaptohexanol had been reported to
possess the same odor properties (48). Structural modifications
at the hydroxy moiety, e.g., 3-mercaptohexyl alkanoates (49)
and 1-methoxyhexane-3-thiol (50), and at the thio group, e.g.,
3-methylthiohexanol (48), resulted in significant sensory dif-
ferences between enantiomers.
a
Reaction time, 2 h; other conditions, see Materials and Methods.
dichloromethane revealed that the adsorbed 3-mercaptohexanol
had the same enantiomeric composition as the portion still
present in the buffer solution. Therefore, the increased enantio-
selectivity obtained by using immobilized CAL-B is not due
to enantiodiscriminating phenomena involved in adsorption/
desorption.
Influence of Cosolvents on Enantioselectivity. It had been
reported that the enantioselectivity of CAL-B in the hydrolysis
of esters can be enhanced by addition of water miscible organic
solvents, in particular acetone and tert-butyl alcohol (45). As
shown in Table 2, the enantioselectivity of the hydrolysis of
3
-acetylthiohexanal was not influenced by the presence of
These sensory data demonstrate that it is worthwhile to invest
in methods to obtain enantiomers of this group of sulfur-
containing flavor compounds and to exploit the enantioselec-
tivity of enzyme-based approaches.
acetone at a level of 10 vol %; higher concentrations of this
cosolvent actually resulted in a decrease of E. Addition of tert-
butyl alcohol significantly improved the enantiodiscrimination
up to a level of 20 vol %; higher proportions again resulted in
lower enantioselectivity. These results are not fully consistent
with the formerly reported effects of acetone and tert-butyl
alcohol on the enantioselectivity of CAL-B (45). The mechanism
underlying the influence of cosolvents on the enantioselectivity
ACKNOWLEDGMENT
We thank M. Dregus and E. Takahisa for analytical assistance
and advice.
Table 4. Odor Properties of 3-Acetylthiohexanal, 3-Acetylthiohexanol, and 3-Mercaptohexanal, Determined by GC/O
compound
racemic mixture
(R)-enantiomer
(S)-enantiomer
3
-acetylthiohexanala
grapefruit, citrus peel, sweet
citrus peel, sulfurous, fruity
sulfurous, citrus peel
sulfurous, roasted, citrus peel
fruity, grapefruit, sulfurous
sulfurous, rubberlike
fruity, sweet, grapefruit
sulfurous, roasted, rubberlike
green, citrus peel, fruity
b
3-acetylthiohexanal
c
3-mercaptohexanal
a
Amounts at GC sniffing port: 0.1 (racemic mixture) and 1.0 µg (enantiomers). b Amounts at GC sniffing port: 0.07 (racemic mixture) and 0.3 µg (enantiomers).
c
Amounts at GC sniffing port: 0.01 (racemic mixture) and 0.04 µg (enantiomers). GC/O systems I (racemic mixture) and II (enantiomers) were used.

4354 J. Agric. Food Chem., Vol. 51, No. 15, 2003
Wakabayashi et al.
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JF030166U