Analytical and Sensory Characterization of the Stereoisomers of 3-Mercaptocycloalkanones and 3-Mercaptocycloalkanols

Article abstract of DOI:10.1021/acs.jafc.0c03113

3-Mercaptocycloalkanones and 3-mercaptocycloalkanols (chain lengths C5-C7) were obtained by addition of thioacetic acid to the respective 2-cycloalken-1-ones and subsequent enzyme-mediated hydrolysis and reduction with LiAlH4, respectively. The stereoisom

Full text of DOI:10.1021/acs.jafc.0c03113

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Chemistry and Biology of Aroma and Taste
Analytical and Sensory Characterization of the Stereoisomers
of 3-Mercaptocycloalkanones and 3-Mercaptocycloalkanols
Motoko Wakabayashi, Hidehiko Wakabayashi, Anja
Devenie Riegel, Wolfgang Eisenreich, and Karl-Heinz Engel
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c03113 • Publication Date (Web): 16 Jun 2020
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Analytical and Sensory Characterization of the Stereoisomers of 3-
Mercaptocycloalkanones and 3-Mercaptocycloalkanols
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Motoko Wakabayashi†*, Hidehiko Wakabayashi‡, Anja Devenie Riegel‡, Wolfgang
Eisenreich§ and Karl-Heinz Engel‡
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† College of Bioresource Sciences, Nihon University, Kameino 1866, 252-0880
Fujisawa, Japan
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‡ Technische Universität München, Lehrstuhl für Allgemeine Lebensmitteltechnologie,
Maximus-von-Imhof-Forum 2, D-85354 Freising-Weihenstephan, Germany
§ Technische Universität München, Lehrstuhl für Biochemie, Lichtenbergstraße 4, D-
85747 Garching, Germany
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CORRESPONDING AUTHOR FOOTNOTE.
* Author to whom correspondence should be addressed [Tel.: +81-466-84-3405. Fax:
+81-466-84-3405. E-mail wakabayashi.motoko@nihon-u.ac.jp
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ABSTRACT
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3-Mercaptocycloalkanones and 3-mercaptocycloalkanols (chain lengths C5-C7) were
obtained by addition of thioacetic acid to the respective 2-cycloalken-1-ones and
subsequent enzyme-mediated hydrolysis and reduction with LiAlH4, respectively. The
stereoisomers were separated via capillary gas chromatography using chiral
stationary phases. Their configurations were determined based on 1H NMR data and
enzyme-catalyzed kinetic resolutions. Odor thresholds and odor qualities were
assessed by capillary gas chromatography/olfactometry. Compared to the analogous
acyclic 4-mercapto-2-alkanones and 4-mercapto-2-alkanols, the cyclic polyfunctional
thiols lacked fruity, tropical notes; the perceived odor properties ranged from cooked,
roasted vegetable and meat-type to onion-related notes. The odor thresholds of the
enantiomers of the 3-mercaptocycloalkanones were mainly impacted by their ring
size rather than their configuration. For the 3-mercaptocycloalkanols, the (S)-
configuration at the stereogenic center bearing the thiol group and the relative
configuration of the second asymmetric center with the hydroxyl group were of
importance for low odor thresholds.
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Keywords
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3-mercaptocycloalkanones; 3-mercaptocycloalkanols; 3-acetylthiocycloalkanones; 3-
acetylthiocycloalkanols; chirality; absolute configuration; odor threshold; odor quality
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INTRODUCTION
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Among the volatile sulfur-containing constituents occurring in foods,1-3 polyfunctional
thiols are attractive flavor compounds with low odor thresholds and interesting odor
properties.4-6 A 1,3-oxygen sulfur functionality with a defined arrangement of
substituents is considered as an essential structural feature constituting a so-called
“tropical olfactophore”.6,7 Several of these polyfunctional thiols are chiral; accordingly,
studies on structure-odor relationships in homologous series increasingly take into
considerations not only the chain lengths and the positions of the functional groups
but also the impact of the configurations on the sensory properties.8,9 An example of
systematic investigations of these stereochemical aspects is the comparative
assessment of the sensory properties of homologous series of linear β-
mercaptoalkanones and β-mercaptoalkanols. For the different positional isomers, the
influence of the type and the spatial arrangements of the substituents at the
stereogenic centers has been demonstrated.10-16
The objective of the present study was to extend this knowledge gained for aliphatic
polyfunctional thiols to alicyclic representatives. As examples, the stereoisomers of 3-
mercaptocyclopentanone 1, 3-mercaptocyclohexanone 2, 3-mercaptocycloheptanone
3, and the corresponding 3-mercaptocycloalkanols 7-9 were investigated (Figure 1,
Table 1). The C5 and C6-homologues were chosen because their potential
precursors, i.e. 2-cyclopenten-1-one and 2-cyclohexen-1-one, have been reported as
Maillard reaction products17,18 as well as volatile constituents of various fruits.19-22
The C7-homologue was included to complement the homologous series and to allow
for a more comprehensive assessment of the impact of the ring size on the sensory
properties.
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In analogy to the aliphatic β-mercaptoalkanones and β-mercaptoalkanols, the
following approach was applied: (i) preparation of the 3-acetylthio-derivatives via
addition of thioacetic acid to the respective 2-cycloalkene-1-ones, (ii) formation of the
thiols by subsequent hydrolysis and reduction (for the 3-mercaptocycloalkanols); (iii)
establishment of a capillary gas chromatographic separation of the stereoisomers,
(iv) assignment of the configurations of the stereoisomers and their GC-order of
elution; and (v) determination of odor thresholds and odor properties via capillary gas
chromatography/olfactometry.
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MATERIALS AND METHODS
Chemicals. 2-Cyclopenten-1-one, 2-cyclohexen-1-one, 2-cyclohepten-1-one, lithium
aluminum hydride (LiAlH4), sodium borohyride (NaBH4), (E)-2-decenal (≥ 95%),
deuterochloroform (CDCl3), tetradeuteromethanol (CD3OD), anhydrous sodium
sulfate (Na2SO4), n-alkane standard solutions (C8-C40) and dichloromethane
(CH2Cl2) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
Thioacetic acid was obtained from Merck Schuchardt OHG (Hohenbrunn, Germany).
Silica gel (NormaSil60, 40−63 μm), n-hexane, 2-propanol, methanol and diethyl ether
were purchased from VWR Chemicals (Leuven, Belgium). Lipases from Candida
cylindracea (62316, ≥ 2 units (U)/mg; CCL) and Rhizopus oryzae (80612, ≥ 30 U/mg;
ROL) were purchased from Fluka Chemie (Buchs, Switzerland). Lipases from
Candida antarctica lipase B (L4777, > 5000 U/g, recombinant, expressed in
Aspergillus niger; CAL-B resin), porcine pancreas (L3126, 100-500 U/mg; PPL),
Pseudomonas cepacia (L9156, > 50 U/g; PCL) and an esterase from porcine liver
(E3019, ≥ 15 U/mg; PLE) were from Sigma-Aldrich Chemie GmbH (Germany).
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Syntheses.
3-Acetylthiocycloalkanones.
3-Acetylthiocyclopentanone
4,
3-
acetylthiocyclohexanone 5 and 3-acetylthiocycloheptanone 6 were synthesized by
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Michael-type addition of thioacetic acid (1.5 equiv.) to 2-cyclopenten-1-one, 2-
cyclohexen-1-one and 2-cyclohepten-1-one (10−20 mmol), respectively.10,13 The
mixture of 2-cycloalken-1-one and thioacetic acid was stirred for 1 h under ice cooling
and for another 20 h at room temperature (RT, 25 °C). After removing the excess of
thioacetic acid under reduced pressure using an aspirator at 40 °C, the crude
products were obtained. For further purification, they were dissolved in CH2Cl2 (5%
w/v); the solution was washed with saturated aqueous sodium chloride, dried over
anhydrous sodium sulfate, and the solvent was removed under reduced pressure
using an aspirator at RT. 3-Acetylthiocyclopentanone 4: mol yield: 81.2%, purity
(GC): 98.6%, linear retention index (LRI):23 2088 (DB-WAXetr), 1241 (DB-1); 3-
acetylthiocyclohexanone 5: mol yield: 84.5%, purity (GC): 98.8%, LRI: 2235 (DB-
WAXetr), 1355 (DB-1); 3-acetylthiocycloheptanone 7: mol yield: 90.7%, purity (GC):
92.2%, LRI: 2351 (DB-WAXetr), 1467 (DB-1). Mass spectrometric and NMR data of 4
- 6 (Supporting Information) were in agreement with those previously reported.24,25
3-Mercaptocycloalkanones. 3-Mercaptocycloalkanones 1-3 were obtained by
enzyme-catalyzed hydrolysis of the respective 3-acetylthiocycloalkanones 4-6. The 3-
acetylthiocycloalkanones (255 µmol) were dissolved in 3 mL of potassium phosphate
buffer (50 mM, pH 7.4), 50 mg of CAL-B resin was added, and the reaction was
performed at RT until more than 99% (GC) of the substrate was hydrolyzed (4: 6 h; 5:
6 h; 6: 76 h). The reaction mixture was extracted three times with CH2Cl2 (3 mL), the
organic phase was dried over anhydrous sodium sulfate, and the solvent was
removed under reduced pressure using an aspirator at RT. 3-
Mercaptocyclopentanone 1: mol yield: 47.1%, purity (GC): 98.6%, LRI: 1753 (DB-
WAXetr), 1018 (DB-1); 3-mercaptocyclohexanone 2: mol yield: 57.1%, purity (GC):
96.9%, LRI: 1875 (DB-WAXetr), 1098 (DB-1); 3-mercaptocycloheptanone 3: mol
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yield: 65.6%, purity (GC): 94.6%, LRI: 2008 (DB-WAXetr), 1222 (DB-1). Mass
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spectrometric data and NMR data of 1-3 (Supporting Information) corresponded to
those published.24,25
3-Mercaptocycloalkanols. 3-Mercaptocycloalkanols 7-9 were prepared by reduction
of the corresponding 3-acetylthiocycloalkanones 4-6 using LiAlH4. The reaction was
carried out under argon atmosphere and ice cooling. The 3-acetylthiocycloalkanones
(4: 0.63 mmol, 5: 0.58 mmol, 6: 0.136 mmol) were dissolved in 2 mL of distilled
diethyl ether. A suspension of LiAlH4 (2.82 mmol for 4, 2.57 mmol for 5, 0.57 mmol
for 6) in 5 mL of diethyl ether was added dropwise. After 1 h, the cooling was
removed and the mixture was stirred overnight at RT. The reaction was quenched
carefully by adding distilled water under ice cooling. The aqueous phase was
adjusted to pH 2 using hydrochloric acid (5%) and extracted twice with CH2Cl2 (5
mL). The pooled organic phases were dried with anhydrous Na2SO4, filtered and
evaporated under reduced pressure. 3-Mercaptocyclopentanol 7: 55.9 mg (0.47
mmol, mol yield: 56%, purity (GC): 99%, ratio of diastereoisomers: 44:56), LRI: 1882
(trans), 1878 (cis) on DB-WAXetr and 1024 (trans), 1023 (cis) on DB-1; 3-
mercaptocyclohexanol 8: 90.5 mg (0.69 mmol; mol yield: 66%, purity (GC): 92%, ratio
of diastereoisomers: 21:79), LRI: 2003 (trans), 2022 (cis) on DB-WAXetr and 1130
(trans), 1125 (cis) on DB-1; 3-mercaptocycloheptanol 9: 19.7 mg (0.135 mmol, mol
yield: 99%, purity (GC): 95%, ratio of diastereoisomers: 50:50), LRI: 2176 (trans),
2185 (cis) on DB-WAXetr and 1260 (trans and cis; not separated) on DB-1.
3-Acetylthiocycloalkanols. 3-Acetylthiocycloalkanols 10-12 were prepared by
reduction of the corresponding 3-acetylthiocycloalkanones 4-6 using NaBH4. The 3-
acetylthiocycloalkanones (4: 0.65 mmol, 5: 0.68 mmol, 6: 0.60 mmol) were dissolved
in 5 mL of methanol and 20 mL of potassium phosphate buffer (500 mM, pH 7.4). A
solution of NaBH4 (1.3 mmol) in 2 mL water was added dropwise under ice cooling.
After 30 min, the reaction was stopped by adjusting the pH to 5 using hydrochloric
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acid (5%). The aqueous phase was extracted twice with CH2Cl2 (10 mL), and the
pooled organic phases were dried with anhydrous Na2SO4, filtered and evaporated
under reduced pressure. 3-Acetylthiocyclopentanol 10: 95.9 mg (0.51 mmol, mol
yield: 84%, purity (GC): 87%, ratio of diastereoisomers: 39:61), LRI: 2283 (trans),
2269 (cis) on DB-WAXetr and 1281 (trans), 1277 (cis) on DB-1; 3-
acetylthiocyclohexanol 11: 113 mg (0.65 mmol; mol yield: 96%, purity (GC): 94%,
ratio of diastereoisomers: 10:90), LRI: 2378 (trans and cis; not separated) on DB-
WAXetr and 1377 (trans), 1362 (cis) on DB-1; 3-acetylthiocycloheptanol 12: 95.9 mg
(0.51 mmol, mol yield: 84%, purity (GC): 96%, ratio of diastereoisomers: 50:50), LRI:
2494 (trans), 2529 (cis) on DB-WAXetr and 1487 (trans), 1498 (cis) on DB-1. Mass
spectrometric and NMR data of 7-12 are provided in the Supporting Information.
Lipase-catalyzed Kinetic Resolutions. Enzyme Screenings. The 3-
acetylthiocycloalkanone (85 µmol) and the enzyme (ROL: 20 mg; CAL-B, PPL and
PCL: 10 mg; CCL: 2 mg; PLE: 0.4 mg) were added to 1 mL of potassium phosphate
buffer (50 mM, pH 7.4), and the mixture was stirred magnetically with a Teflon stir bar
at RT. Aliquots (5 µL), taken after 0.5, 1, 2, 4 and 8 h, were extracted with CH2Cl2
(100 µL), the organic phase was dried over anhydrous Na2SO4 and subjected to GC
analysis. The enantioselectivity (E) was calculated by using the enantiomeric
excesses of the product and the remaining substrate, based on GC peak areas.26
Preparation of enantiomerically enriched 3-acetylthiocyclohexanone. The enzyme-
catalyzed hydrolysis of 3-acetylthiohexanone was performed according to the
procedure described above, using PLE as biocatalyst and in double scale. After 4 h,
the reaction mixture was extracted three times with CH2Cl2 (4 mL), the combined
organic layers were dried over anhydrous sodium sulfate, filtered, and the solvent
was removed under reduced pressure using an aspirator at RT. The enantiomerically
enriched 3-acetylthiocyclohexanone was separated from the formed 3-
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mercaptocyclohexanone via column chromatography on silica gel by elution with n-
hexane/diethyl ether 2/1 (v/v). After removal of the solvent, the sample was subjected
to optical rotation measurement.
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NMR Spectroscopy. H NMR and C NMR spectra were recorded at 500 and 126
MHz, respectively, with Avance 500 spectrometers (Bruker, Rheinstetten, Germany).
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The samples were dissolved in CDCl3 or CD3OD. H-detected experiments were
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done with an inverse H/13C probehead. Direct C measurements were performed
with a QNP 13C/31P/29Si/19F/1H cryoprobe. The experiments were done in full
automation using standard parameter sets of the TOPSPIN 3.0 software package
(Bruker). 13C NMR spectra were recorded in proton-decoupled mode. The spectra
were recorded at 27 °C. All signals were assigned by proton−proton and
proton−carbon correlation experiments (e.g., COSY, HSQC, HMBC and NOESY).
Data processing was typically done with the MestreNova software, version 14.0.1
(Mestrelab Research, Santiago de Compostela, Spain).
HPLC Analyses. Normal phase HPLC separations of the diastereoisomers of 3-
acetylthiocycloalkanols were carried out on a Dionex HPLC system (UltiMate 3000
series, Dionex, Germering, Germany) equipped with a 3100 wavelength detector set
at 210 nm using a 250 × 4.6 mm i.d. Nucleosil 50−5 column (CS Chromatography,
Langerwehe, Germany); isocratic elution was performed at 25 °C with
hexane/isopropanol (95/5, v/v) at 0.7 mL/min. Separations of the enantiomers were
carried out with the same system using a 250 × 4.6 mm i.d. CHIRALCEL OD-H
column (DAICEL Corporation, Tokyo, Japan) or a 150 × 4.6 mm i.d. CHIRALPAK IA-
3 column (DAICEL Corporation, Tokyo, Japan) at 254 nm; isocratic elution was
performed at 25 °C with hexane/isopropanol (98/2, v/v) at 1 mL/min as described.25
GC Analyses. Capillary Gas Chromatography (GC). A 60 m x 0.32 mm i.d., 0.25 µm
df DB-1 column (J&W Scientific, Waldbronn, Germany) was installed into a 6890N
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instrument (Agilent Technologies, Waldbronn, Germany) equipped with a split-
splitless injector (230 °C, split ratio 1:10) and an FID (300 °C). The temperature
program was from 60 °C (5 min hold) to 300 °C (5 min hold) at 5 °C/min; hydrogen
was used as carrier gas (constant flow 1.8 mL/min).
Stereoselective Analysis. A 6890N gas chromatograph (Agilent Technologies) was
equipped with a split/splitless injector (230 °C, split ratio of 1:10) and an FID (300
°C); hydrogen was used as carrier gas (constant flow 1.6 mL/min). Separations of
enantiomers were performed using (I) a 30 m x 0.32 mm i.d., 0.25 µm df column
coated with octakis(2,3-di-O-n-butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin,27 (II)
a 25 m x 0.25 mm i.d., 0.25 µm df column with octakis-(2,6-di-O-pentyl-3-O-butyryl)-
γ-cyclodextrin (Lipodex E; MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany),
and (III) a 25 m x 0.25 mm i.d., 0.25 µm df column with heptakis(2,3-di-O-ethyl-6-O-
tert-butylsilyl)-β-cyclodextrin (MEGA-DEX DET-Beta; MEGA S.r.l., Legnano, Italy).
The used temperature programs are given in the Supporting Information.
Capillary Gas Chromatography/Olfactometry (GC/O). A Trace GC Ultra (Thermo
Fisher Scientific, Dreieich, Germany) equipped with a cold-on-column injector (oven
track mode), a heated sniffing port (200 °C) and an FID (250 °C) was used for the
sensory analyses. A deactivated fused silica pre-column was used (1 m x 0.53 mm
i.d.; BGB Analytik AG, Rheinfelden, Germany). The carrier gas was hydrogen
(constant pressure 75 kPa), the effluent was split 1:1 via a press-fit Y-splitter, and 30
cm x 0.25 mm i.d. deactivated fused silica capillaries (BGB Analytik AG) were used to
connect to both the sniffing port and the FID. The enantiomers of 1, 2, 4 and 5 were
assessed on chiral stationary phase (II), those of 3, 6, 7, 8, 10 and 11 on (I), and
those of 9 and 12 on (III); the used temperature programs are given in the Supporting
Information. Sensory assessments were performed by a total of six panelists (five
females, between 26 and 56 years old; one male, 59 years old). The stereoisomers
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of 1-6 and 7-12, respectively, were assessed by two sets of three panelists. Odor
thresholds in air were determined according to the previously described procedure
using (E)-2-decenal as internal standard with the reported odor threshold of 2.7 ng/L
in air.28,29 Known amounts of each compounds and of the internal standard were
dissolved in diethyl ether and diluted stepwise (1+1, (v/v)). Each dilution was
assessed three times by GC/O. A concentration level was considered as odor
threshold, if it was the lowest dilution step at which the odor was consistently
perceived in three consecutive GC/O-runs. The odor qualities were recorded at one
dilution step above the odor threshold. Flavor dilution factors (FD) were determined
by aroma extract dilution analysis (AEDA).30
Gas Chromatography-Mass Spectrometry (GC-MS). A 30 m x 0.25 mm i.d., 0.5
µm df DB-WAXetr fused silica capillary column (J&W Scientific) installed into a GC
Thermo Trace GC Ultra directly coupled to a Thermo ISQ mass spectrometer
(Thermo Fisher Scientific) with a split/splitless injector (220 °C, split ratio 1/50) was
used. The temperature program was from 40 °C (5 min hold) to 240 °C (30 min hold)
at 4 °C/min. Helium was used as carrier gas (constant pressure 75 kPa). Electron
impact mode (EI) mass spectra were measured at 70 eV in a scan range from m/z 30
to 250 (ion source temperature 250 °C, interface temperature 240 °C). For data
acquisition Xcalibur software version 2.1 (Thermo Fisher Scientific, Waltham, MA,
USA) was used.
Optical Rotation. A Polartronic-E polarimeter (Schmidt & Haensch, Berlin, Germany)
with a measuring cell (path length of 1 dm) and a sodium lamp (wavelength of 589
nm) was used. Chloroform was used as solvent, and measurements were performed
at 25 °C.
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RESULTS AND DISCUSSION
Syntheses and Determination of the GC-order of Elution of the Stereoisomers.
3-Mercapto- and 3-Acetylthiocycloalkanones. 3-Acetylthiocycloalkanones 4-6 (Figure
1) were synthesized by Michael-type addition of thioacetic acid to the corresponding
2-cycloalken-1-ones, in accordance with the procedure used to obtain the
corresponding acyclic β-acetylthioalkanones.10 Sufficient yields and purities were
achieved without the use of previously reported solvents and a catalyst.24 3-
Mercaptocycloalkanones 1-3 (Figure 1) were obtained by subsequent hydrolysis of
the 3-acetylthiocycloalkanones. Alkaline solvolysis resulted in various side-products;
therefore, enzyme-catalyzed hydrolyses under mild conditions using a lipase from
Candida antarctica (CAL-B) were employed.
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The GC-separation of the enantiomers of 1-6 was performed on octakis(2,3-di-O-
butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin as chiral stationary phase (Figure
2). Compared to octakis(2,6-di-O-pentyl-3-O-butyryl)-γ-cyclodextrin (Lipodex E),
previously reported for the separation of the enantiomers of 1, 2 and 4, 5,24 this
cyclodextrin derivative turned out to be superior for 2 and 5; furthermore, the
enantiomers of the C7-homologues 3 and 6 could only be separated on this
stationary phase.
For the assignment of the absolute configurations, the initial aim was to employ
lipase- or esterase-catalyzed kinetic resolutions of the 3-acetylthiocycloalkanones 4-6
to obtain optically enriched enantiomers. According to a previous study, lipases from
Candida rugosa and Aspergillus niger did not show enantioselectivity.24 Screening of
other commercially available enzyme preparations revealed porcine liver esterase
(PLE) to exhibit an enantioselectivity (E=25) sufficient for a preparative kinetic
resolution of 5 (Table 2). After 4h, the PLE-catalyzed hydrolysis of 3-
acetylthiocyclohexanone 5 resulted in enantiomeric excesses (ee) of 88% and 52%
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for the remaining substrate and the formed thiol, respectively. Upon separation of
product and remaining substrate via silica gel column chromatography, partial
racemization of 3-acetylthiocyclohexanone occurred. However, the resulting
enantiomeric excess (48%) of the remaining substrate was sufficient to subject this
material to polarimetry. The comparison of the measured specific rotation with data
available from literature25,31 enabled to assign the (S)-configuration of 5 remaining
after the kinetic resolution (Supporting Information).
Owing to the low enantioselectivity of the PLE-catalyzed hydrolyses of 4 and 6 (Table
2) and the partial racemizations of (S)-4 and (S)-6 upon chromatography using silica
gel, the resulting enantiomeric excesses were not high enough to allow for the
assignment of their configurations via comparison of the optical rotations to those
previously reported.25 Therefore, as alternative approach, the enantiomers of 4 and 6
were separated via HPLC using a chiral stationary phase.25 Subsequent
investigations of the separated enantiomers via capillary GC revealed the order of
elution as (S) before (R) on octakis(2,3-di-O-butyryl-6-O-tert-butyldimethylsilyl)-γ-
cyclodextrin (Supporting Information).
3-Mercapto- and 3-Acetythiocycloalkanols. The 3-acetylthiocycloalkanones 4-6 were
transformed into 3-mercaptocycloalkanols 7-9 by reduction with LiAlH4,12,14 and into
12
3-acetylthiocycloalkanols 10-12 by reduction with NaBH4. The GC-separation of the
resulting stereoisomers is shown in Figure 3. For the homologues with chain lengths
C5 and C6, separations of the four stereoisomers were achieved using octakis(2,3-di-
O-n-butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin as chiral stationary phase
(Figure 3A). The stereoisomers of the C7 homologue could not be completely
separated. Among various stationary phases tested, heptakis(2,3-di-O-ethyl-6-O-tert-
butylsilyl)-β-cyclodextrin showed the best results; however, there remained
coelutions of the two stereoisomers of cis-9 and 12, respectively (Figure 3B).
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For the assignment of the order of elution of the trans- and cis-diastereoisomers of 7-
9 and 10-12, the diastereoisomers of the 3-acetylthiocycloalkanols 10-12 were
separated via normal phase HPLC and subsequently subjected to NMR analysis. As
depicted exemplarily for 3-acetylthiocyclohexanol 11 in Figure 4, the NOESY signals
showed a correlation between the protons in the positions 1 (H-1) and 3 (H-3) of the
second eluting diastereoisomer DII-11, whereas for the first eluting diastereoisomer
DI-11, no NOESY signals between the protons in positions 1 (H-1) and 3 (H-3) were
observed (Supporting Information).
Subsequent solvolysis of DI and DII, respectively, with NaOH/MeOH yielded the
corresponding diastereoisomers of 3-mercaptocyclohexanol (DI-8 and DII-8). The
application of the same procedure to the C5- and C7-homologues resulted in a GC
order of elution of the diastereoisomers of 3-mercaptocycloalkanols 7-9 and 3-
acetylthiocycloalkanols 10-12 as trans before cis for 7 and 10 and cis before trans
for 8 and 11 on octakis(2,3-di-O-n-butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin
and trans before cis for 9 and 12 on heptakis(2,3-di-O-ethyl-6-O-tert-butylsilyl)-β-
cyclodextrin chiral stationary phase (Figure 3).
A procedure based on enzyme-catalyzed kinetic resolution was applied to obtain
enantiomerically enriched thiols to assign the order of elution of the individual
stereoisomers of each homologue 7-9 and 10-12 on the employed chiral stationary
phases. The approach is exemplary shown for 3-mercaptocyclohexanol 8 and 3-
acetylthiocyclohexanol 11 in Figure 5. The first step was the hydrolysis of racemic 3-
acetylthiocyclohexanone 5 with PLE. After 4 h, 3-(R)-mercaptocyclohexanone 2a´
was the preferentially formed product, and 3-(S)-acetylthiocyclohexanone 5a was the
main enantiomer of the remaining substrate (Figure 5A). Their subsequent reduction
with NaBH4 resulted in the corresponding excesses of the (3R)-configured
diastereoisomers
of
3-mercaptocyclohexanol
13
and
the
(3S)-configured
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diastereoisomers of 3-acetylthiocyclohexanol (Figure 5B). Taking into account the
order of elution of the cis- and trans-diastereoisomers, the comparison of this elution
pattern of the compounds enantiomerically enriched in position 3 with the GC
separation of the stereoisomers of the racemic mixtures of 3-mercaptocyclohexanol
and 3-acetylthiocyclohexanol allowed to assign their order of elution (Figure 5C). The
application of the same procedure to the C5- and C7-homologues resulted in the
order
of
elution
and
on
octakis(2,3-di-O-n-butyryl-6-O-tert-butyldimethylsilyl)-γ-
heptakis(2,3-di-O-ethyl-6-O-tert-butylsilyl)-β-cyclodextrin,
cyclodextrin
respectively, shown in Figure 3. The observed inconsistencies in the order of elution
of the cis- and trans-stereoisomers of the different homologues underline that the
GC-order of elution of stereoisomers of a homologous series has to be determined
individually and cannot be extrapolated from one homologue to the other.
Sensory Properties of the Stereoisomers. Odor thresholds of the stereoisomers
were determined by three panelists via GC/O.28 The panelists also recorded the odor
qualities perceived at one dilution step above the odor thresholds.
3-Mercapto- and 3-Acetylthiocycloalkanones. Only for the enantiomers of 3-
mercaptocyclopentanone 1a and 1a´, the odor thresholds determined by panelists 1
and 3 differed by factors of 14 and 20, respectively (Table 3). In all other cases, the
odor thresholds determined by the panelists were either the same or differed by
factors of 2-8. This corresponds to 1-3 dilution steps and reflects the inter-panelists
variability of the employed sensory assessment procedure.
The ring size turned out to be a major factor impacting the odor thresholds.
Compared to both enantiomers of 3-mercaptocyclopentanone 1 and 3-
mercaptocyclohexanone 2, the thresholds of the respective enantiomers of the C7
homologue 3-mercaptocycloheptanone 3 were consistently higher. For the 3-
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acetylthio-derivatives, this effect of the ring size was less pronounced and only
observed for the (S)-enantiomers 4a-6a.
Differences between the enantiomers were also dependent on the ring size. Among
the 3-mercaptocycloalkanones, only the (R)-enantiomer of the C6 homologue 2
showed consistently lower odor thresholds than the antipode. For the acetylthio-
derivatives, differences between the enantiomers were more pronounced; for the C5
and C6 homologues 4 and 5, the (S)-enantiomers consistently exhibited lower
thresholds.
Regarding the odor qualities of the investigated 3-mercaptocycloalkanones and 3-
acetylthiocycloalkanones, the panelists often used the descriptors “sulfury” and
“cooked vegetable” (Table 4). “Fruity” and “tropical” notes were only reported for 3-
(R)-mercaptocyclohexanone 2a´ by one panelist. There were no obvious correlations
between odor descriptions and ring size or configurations.
3-Mercapto- and 3-Acetylthiocycloalkanols. The variability of the odor thresholds of
the 3-mercaptocycloalkanols 7-9 and the 3-acetylthiocycloalkanols 10-12 among the
three panelists was quite low (Table 5). Except for the assessment of stereoisomer
10a´, the values determined for the other stereoisomers were either the same or
differed only by factors ranging from 3 to maximum 5 between the panelists.
There was no clear impact of the ring size on differences in odor thresholds between
the
stereoisomers
of
the
3-mercaptocycloalkanols.
Among
the
3-
acetylthiocycloalkanols, a similar phenomenon as for the 3-mercaptoalkanones was
observed, i.e. the odor thresholds of the stereoisomers of the C7 homologue 12 were
consistently higher than those of 10 and 11.
Except for the cis-stereoisomer 7a´ of homologue C5, the odor thresholds of the cis-
stereoisomers of the 3-mercaptocycloalkanols were consistently higher than those of
the trans-stereoisomers. For each chain length, one of the four 3-
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mercaptocycloalkanol stereoisomers exhibited the lowest odor threshold. Two of the
stereoisomers with the minimum thresholds, i.e. 8b and 9b, possess a trans-(1S,3S)-
configuration. For the C5 homologue, the stereoisomer 7a´ with the lowest odor
threshold has a cis-(1R,3S)-configuration.
In contrast to the odor qualities reported for stereoisomers of the linear 4-mercapto-2-
alkanols,14 “fruity” or “tropical” notes were lacking (Table 6). There was no consistent
correlation between ring size and odor qualities of the 3-mercaptocycloalkanols;
however, configuration-dependent clusters of certain descriptors were observed. For
example, the 8b´and 9b´ (1R,3R)-stereoisomers (chain lengths C6 and C7) were
associated with “cooked or roasted meat”-notes, and for the 7a-9a (1S,3R)-
stereoisomers “baked potato” “cooked/grilled vegetable” and “nutty” notes were
reported. It is also noteworthy that the 7a´ (1R,3S)- and the 9b (1S,3S)-
stereoisomers which showed the lowest odor thresholds among the stereoisomers of
the respective chain length (Table 5) were both characterized by “onion”-related
descriptors. For the acetylthio-derivatives, ”cooked vegetable”, “burnt” and “rubbery”
notes prevailed; there was also no correlation between ring size and odor properties.
Comparison of the Sensory Data to Those of Acyclic β-Mercaptoalkanones and β-
Mercaptoalkanols with the Same Chain Lengths. Except for the previously reported
high odor threshold of (S)-4-mercapto-2-hexanone,11 the odor thresholds of the 3-
mercaptocycloalkanones 1 and 2 were in the same order of magnitude as those of
the corresponding acyclic 4-mercapto-2-alkanones11 and 2-mercapto-4-alkanones,15
and there were no significant differences between the enantiomers (Figure 6A). For
the C7 homologue 3, there were also no differences between the enantiomers, but
the odor thresholds were one order of magnitude higher than those of 4-mercapto-2-
heptanone11 and 2-mercapto-4-heptanone.15
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It is noteworthy that in contrast to the linear 3-mercaptoalkanones the acetylation of
the mercapto group of the 3-mercaptocycloalkanones did not result in a significant
increase of the odor thresholds. In particular for the chain lengths C5 and C6, the
odor thresholds of the cyclic acetyl-derivatives were clearly lower than those of the
corresponding acyclic 4-mercapto-2-alkanones11 and 2-mercapto-4-alkanones15
(Figure 6B).
The sensory data obtained for the 3-mercaptocycloalkanols confirmed the role of the
substituent R4 in the model for a „tropical olfactophore“,6,7 as discussed previously
based on the investigations of structure-odor relationships for 2-mercapto-4-
alkanones15 and 2-mercapto-4-alkanols.16 When applying the model of Rowe6 to the
3-mercaptocycloalkanols 7-9, the chain of the respective substituent R4 is longer
than methyl and thus comparable to the substituent R4 in the respective linear 2-
mercapto-4-alkanols.15 The closure of the resulting ring is not at position 2, as
foreseen by the model of Rowe6, but at position 3. This is reflected in the fact that the
ranges of odor thresholds of the stereoisomers of 3-mercaptocyclohexanol 8 (1.4 - 45
ng/L) and 3-mercaptocycloheptanol 9 (2.3 - 23 ng/L) are in similar orders of
magnitude as those reported for the acyclic 2-mercapto-4-hexanol (2.4 - 21 ng/L) and
2-mercapto-4-heptanol (0.2 – 51 ng/L) with the same chain lengths.16 In contrast,
they are orders of magnitude higher than those of the corresponding acyclic
positional isomers 4-mercapto-2-hexanone and 4-mercapto-2-heptanone.14 They are
also significantly higher than the odor thresholds described for racemates of the
respective 3-mercapto-1-alkanols,32 another class of β-mercaptoalkanols meeting the
requirements of the model developed by Rowe6 regarding the substituent R4. For the
stereoisomers of 3-mercaptocyclopentanol 7, the range of the odor thresholds is also
higher than the range determined for 4-mercapto-2-pentanol (structurally identical to
2-mercapto-4-pentanol)14 and those reported for racemic 3-mercapto-1-pentanol.32
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When ranking the odor thresholds of the individual stereoisomers, it is striking that for
each chain length the 3-mercaptocycloalkanol stereoisomer with the lowest odor
threshold possesses (S)-configuration at the stereogenic center bearing the thiol
group (Figure 7). The trans-(1S,3S)-configuration of the 3-mercaptocycloheptanol
stereoisomer 9b with the lowest odor threshold corresponds to the anti-(2S,4S)-
configuration of the analogous linear 2-mercapto-4-heptanol stereoisomer exhibiting
the lowest odor threshold.16 For both compound classes, this spatial arrangement of
the substituents seems to be essential for a low threshold, because a change of the
configuration at the carbon atom bearing the hydroxyl group from (S) to (R) leads to
drastic increases of the odor thresholds for the resulting cis-3-mercaptocycloheptanol
(Table 5) and the syn-stereoisomer of 2-mercapto-4-heptanol.16 For the positional
isomer 4-mercapto-2-heptanol, anti-configuration is also a prerequisite for the lowest
odor threshold; however, for this compound class having a methyl substituent as
substituent R4 and thus being in accordance with the Rowe model,6 the enantiomeric
anti-(2R,4R)-stereoisomer showed the lowest threshold.
For 3-mercaptocyclohexanol, also the trans-(1S,3S)-stereoisomer 8b showed the
lowest odor threshold; a reversal of the configuration at the stereogenic center
bearing the hydroxy group resulting in the cis-(1R,3S)-sterooisomer leads to an
increase of the odor threshold by a factor 30 (Table 5). For the linear 2-mercapto-4-
hexanol, the (2S)-configuration at the center bearing the thiol group appears to be
the key property for a low odor threshold; both the anti-(2S,4S)- and the syn-(2S,4R)
stereoisomers showed threshold minima among the four possible streoisomers.16 For
the isomeric 4-mercapto-2-hexanol, comparable to 3-mercaptocyclohexanol an anti-
stereoisomer shows the lowest odor threshold, but (R)-configuration is required at the
carbon atom bearing the thiol group.14
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For 3-mercaptocyclopentanol, the (1R,3S)-stereoisomer 7a´ showed the lowest
threshold; the linear comparator 4-mercapto-2-pentanol meets the requirements of
the Rowe model,6 and in agreement with the data described for the chain lengths C6
and C7 the configuration required for the lowest threshold is the mirror image, i.e.,
(2S,4R).14
In summary, the assessments demonstrate that the odor thresholds of the
enantiomers of the investigated 3-mercaptocycloalkanones are mainly influenced by
their ring size rather than their configuration. On the other hand, for the
corresponding 3-mercaptocycloalkanols the (S)-configuration at the stereogenic
center bearing the thiol group is a key structural feature required to result in the
lowest odor threshold among the stereoisomers. In addition, the relative configuration
of the second asymmetric center with the hydroxyl group is of importance. Owing to
the cyclic structures, the chain at this stereogenic center, corresponding to the
substituent R4 in the model established for “tropical olfactophores”,6 is longer than
methyl. Accordingly, configuration-odor relationships of the 3-mercaptocycloalkanols
8 and 9 are similar to those of the corresponding linear 2-mercapto-4-alkanols16 and
differ from those of the isomeric 4-mercapto-2-alkanols.14 In the future, a combination
of the sensory data obtained for the stereoisomers with comparative molecular field
analysis might assist in creating quantitative structure activity relationship models
predicting odor thresholds and properties of these compound classes.
The stereoisomers of the cyclic polyfunctional thiols exhibit a wide array of interesting
odor properties, ranging from cooked, roasted vegetable and meat-type to onion-type
notes. Considering the natural occurrence of their potential precursors, the respective
2-cycloalken-1-ones,17-22 3-mercaptocycloalkanones and –alkanols of chain lengths
C5 and C6 might exist in natural systems. The chromatographic and sensory data
elaborated in this study provide a basis to assist in respective future investigations.
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ASSOCIATED CONTENT
Supporting Information
Structures of all investigated stereoisomers. GC, MS and NMR data of synthesized
compounds. Chromatographic data for the GC- and HPLC-separations of
stereoisomers using chiral stationary phases.
This material is available free of charge via the Internet at http://pubs.acs.org.
ACKNOWLEDGEMENTS
We thank Christine Schwarz for recording the NMR, and Viktoriia Dudko, Markéta
Rynešová and Iulia Poplacean for participating in the GC/O assessments.
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REFERENCES
491
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505
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509
510
511
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1. Boelens, M. H.; van Gemert, L. J. Volatile Character-Impact Sulfur Compounds
and their Sensory Properties. Perfum. Flavor. 1993, 18, 29–39.
2. Mussinan, C. J.; Keelan, M. E. Sulfur Compounds in Foods. - An Overview. In
Sulfur Compounds in Foods; Mussinan, C.J., Keelan, M.E., Eds. ACS
Symposium Series 564; American Chemical Society: Washington, DC, 1994; pp
1–6.
3. Blank, I. Sensory Relevance of Volatile Organic Sulfur Compounds in Food. In
Heteroatomic Aroma Compounds; Reineccius, G. A., Reineccius, T. A., Eds.;
ACS Sympoium Series 826; American Chemical Society: Washington, 2002; pp.
25-53.
4. Vermeulen, C.; Guyot-Declerck, C.; Collin, S. Combinatorial Synthesis and
Sensorial Properties of Mercapto Primary Alcohols and Analogues. J. Agric. Food
Chem. 2003, 51, 3618–3622.
5. Vermeulen, C.; Gijs, L.; Collin, S. Sensorial Contribution and Formation
Pathways of Thiols in Foods: A Review. Food Rev. Int. 2005, 21, 69–137.
6. Rowe, D. J. High Impact Aroma Chemicals. In Advances in Flavours and
Fragrances - From the Sensation to the Synthesis; Swift, K. A. D., Ed.; The Royal
Society of Chemistry: Cambridge, U.K., 2002; pp 202–226.
7. Robert, F.; Héritier, J.; Quiquerez, J.; Simian, H.; Blank, I. Synthesis and
Sensorial Properties of 2-Alkylalk-2-enals and 3-(Acetylthio)-2-alkyl Alkanals. J.
Agric. Food Chem. 2004, 52, 3525–3529.
8. Schoenauer, S.; Polster, J.; Schieberle, P. Influence of structural modification
and chirality on the odor potency and odor quality of thiols. In: Engel, K.-H.;
Takeoka, G. (Eds.) Importance of Chirality to Flavor Compounds. ACS
21
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Page 22 of 41
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
Symposium Series 1212, American Chemical Society, Washington, DC, 2015;
pp. 135-146.
9. Schoenauer, S.; Buergy, A.; Kreissl, J.; Schieberle, P. Structure/Odor Activity
Studies on Aromatic Mercaptans and Their Cyclohexane Analogues Synthesized
by Changing the Structural Motifs of Naturally Occurring Phenyl Alkanethiols. J.
Agric. Food Chem. 2019, 67, 2598-2606.
10. Wakabayashi, M.; Wakabayashi, H.; Eisenreich, W.; Morimitsu, Y.; Kubota, K.;
Engel, K.-H. Determination of the absolute configurations of 4-mercapto-2-
alkanones using the 1H NMR anisotropy method and enzyme-catalyzed kinetic
resolution of the corresponding 4-acetylthio-2-alkanones. Eur. Food Res.
Technol. 2011, 232, 753–760.
11. Wakabayashi, M.; Wakabayashi, H.; Nörenberg, S.; Kubota, K.; Engel, K.-H.
Comparison of odour thresholds and odour qualities of the enantiomers of 4-
mercapto-2-alkanones and 4-acetylthio-2-alkanones. Flavour Fragr. J. 2015, 30,
171–178.
12. Nörenberg, S.; Reichardt, B.; Andelfinger, V.; Eisenreich, W.; Engel, K.-H.
Influence of the Stereochemistry on the Sensory Properties of 4-Mercapto-2-
Heptanol and its Acetyl-Derivatives. J. Agric. Food Chem. 2013, 61, 2062–2069.
13. Kiske, C.; Nörenberg, S.; Ecker, M.; Ma, X.; Taniguchi, T.; Monde, K.; Eisenreich,
W.; Engel, K.-H. Reinvestigation of the Absolute Configurations of Chiral β-
Mercaptoalkanones Using Vibrational Circular Dichroism and 1H NMR Analysis.
J. Agric. Food Chem. 2016, 64, 8563–8571.
14. Nörenberg, S.; Kiske, C.; Reichardt, B.; Andelfinger, V.; Pfeiffer, A.; Schmidts, F.;
Eisenreich, W.; Engel, K. H. Analysis and Sensory Evaluation of the
Stereoisomers of a Homologous Series (C5-C10) of 4-Mercapto-2-alkanols. J.
Agric. Food Chem. 2017, 65, 8913–8922.
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541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
15. Kiske, C.; Riegel, A. D.; Hopf, R.; Kvindt, A.; Poplacean, I.; Taniguchi, T.;
Swamy, M. M. M.; Monde, K.; Eisenreich, W.; Engel, K. H. Determination of the
Absolute Configurations and Sensory Properties of the Enantiomers of a
Homologous Series (C6-C10) of 2-Mercapto-4-alkanones. J. Agric. Food Chem.
2019, 67, 1187–1196.
16. Riegel, A.D.; Kiske, C.; Dudko, V.; Poplacean, I.; Eisenreich, W.; Engel, K.-H.
Absolute Configurations and Sensory Properties of the Stereoisomers of a
Homologous Series (C6-C10) of 2-Mercapto-4-alkanols. J. Agric. Food Chem.
2020, 68, 2738-2746
17. Baltes, Werner; Mevissen, Lutz. Model reactions on roast aroma formation. VI.
Volatile reaction products from the reaction of phenylalanine with glucose during
cooking and roasting. Z. Lebensm. Unters. Forsch. 1988, 187, 209-14.
18. Lu, G.; Yu, T.-H.; Ho, C.-T. Generation of Flavor Compounds by the Reaction of
2-Deoxyglucose with Selected Amino Acids. J. Agric. Food Chem. 1997, 45, 233-
236.
19. Froehlich, O.; Duque, C.; Schreier, P. Volatile constituents of curuba (Passiflora
mollissima) fruit. J. Agric. Food Chem. 1989, 37, 421-425
20. Werkhoff, P.; Güntert, M.; Krammer, G.; Sommer, H.; Kaulen, J. Vacuum
Headspace Method in Aroma Research:ꢀ Flavor Chemistry of Yellow Passion
Fruits. J. Agric. Food Chem. 1998, 46, 1076-1093
21. Jordán M.J.; Goodner; K.L.; Shaw, P.E. Characterization of the Aromatic Profile
in Aqueous Essence and Fruit Juice of Yellow Passion Fruit (Passiflora edulis
Sims F. Flavicarpa degner) by GC−MS and GC/O. J. Agric. Food Chem. 2002,
50, 1523−1528
22. Cannon, R.J., Agyemang, D.;, Curto, N.L.;, Yusuf, A.; Chen, M.Z.; Janczuk, A.J.
In-depth analysis of Ciflorette strawberries (Fragaria x ananassa ‘Ciflorette’)
23
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568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
by multidimensional gas chromatography and gas chromatography-olfactometry.
Flavour Fragr. J. 2015, 30, 302–319.
23. van den Dool, H.; Kratz, P. D. A Generalization of the Retention Index System
Including Linear Temperature Programmed Gas-Liquid Partition
Chromatography. J. Chromatogr. 1963, 11, 463–471.
24. Dia, R.-E.; Belaqziz, R.; Romane, A.; Antoniotti, S.; Duñach, E.. Flavouring and
odorant thiols from renewable natural resources by InIII-catalysed
hydrothioacetylation and lipase-catalysed solvolysis. Tetrahedron Lett. 2010, 51
2164–2167.
25. Rana, N.K.; Unhale, R.; Singh, V.K. Enantioselective sulfa-Michael addition of
thioacids to α,β-unsaturated ketones with bifunctional organocatalyst.
Tetrahedron Lett. 2012, 53, 2121–2124
26. Chen, C.S.; Fujimoto, Y.; Gordailas, G.;, Sih, C.J. Quantitative analyses of
biochemical kinetic resolutions of enantiomers. J. Am. Chem. Soc. 1982, 104,
7294–7299.
27. Schmarr, H. G. Beiträge zur On-line LC-GC Kopplung und modifizierte
Cyclodextrine als chirale stationäre Phasen in der Kapillar GC. Dissertation,
Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany, 1992.
28. Ullrich, F.; Grosch, W. Identification of the most intense volatile flavour
compounds formed during autoxidation of linoleic acid. Z. Lebensm. Unters.
Forsch. 1987, 184, 277–282.
29. Boelens, M. H.; van Gemert, L. J. Physicochemical parameters related to
organoleptic properties of flavor components. In Developments in Food Flavours;
Birch, G. G., Lindley, M. G., Eds.; Elsevier Applied Science: London, U.K., 1986;
pp 23–49.
30. Grosch, W. Detection of potent odorants in foods by aroma extract dilution
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593
594
595
596
597
598
599
600
analysis. Trends Food Sci. Technol. 1993, 4, 68–73.
31. Gawronski,J.; Gawronska, K.; Wynberg, H. Alkaloid-catalysed asymmetric
addition of thiocarboxylic S-acids to cyclohex-2-en-1-ones. Absolute configuration
of the adducts by O,S-dibenzoate Cotton effect. J. Chem. Soc., Chem. Commun.,
1981, 307-308
32. Polster, J.; Schieberle, P. Structure−Odor Correlations in Homologous Series of
Mercaptoalkanols. J. Agric. Food Chem. 2017, 65, 4329-4340
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Figure Captions
Figure 1. Structures of (A) 3-mercaptocycloalkanones 1-3 and the respective 3-
acetylthiocycloalkanones 4-6; (B) 3-mercaptocycloalkanols 7-8 and the respective 3-
acetylthiocycloalkanols 10-12.
Figure 2. Capillary gas chromatographic separation of the enantiomers of (A) 3-
mercaptocyclopentanone
mercaptocyclohexanone
1
and
3-acetylthiocyclopentanone
4,
(B)
3-
2
and 3-acetylthiocyclohexanone 5, and (C) 3-
mercaptocycloheptanone 3 and 3-acetylthiocycloheptanone 6 on octakis(2,3-di-O-
butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin. The peak numbers correspond to
those in Table 1. For conditions, see Supporting Information.
Figure 3. Capillary gas chromatographic separation of the stereoisomers of (A) 3-
mercaptocyclopentanol 7, 3-mercaptocyclohexanol 8, 3-acetylthiocyclopentanol 10 and
3-acetylthiocyclohexanol 11 on octakis(2,3-di-O-n-butyryl-6-O-tert-butyldimethylsilyl)-γ-
cyclodextrin, (B) 3-mercaptocycloheptanol 9 and 3-acetylthiocycloheptanol 12 on
heptakis(2,3-di-O-ethyl-6-tert-butylsilyl)-β-cyclodextrin). The peak numbers correspond
to those in Table 1. For conditions, see Supporting Information.
Figure 4. NOESY effects observed for the diastereoisomers of 3-acetylthiocyclohexanol
11 separated via normal phase HPLC. For NMR data, see Supporting Information.
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Figure 5. Determination of the order of elution of the stereoisomers of 3-
mercaptocyclohexanol 8 and 3-acetylthiocyclohexanol 11 on octakis(2,3-di-O-n-butyryl-
6-O-tert-butyldimethylsilyl)-γ-cyclodextrin by (A) preparation of enantiomerically
enriched 3-mercaptocyclohexanone 2 and 3-acetylthiocyclohexanone 5 via enzyme-
catalyzed kinetic resolution, (B) reduction to the corresponding enantiomerically
enriched 8 and 11, and (C) comparison of the retention times to those of the
stereoisomers of chemically synthesized 8 and 11. For chromatographic conditions, see
Supporting Information.
Figure 6. Comparison of the odor thresholds of (A) enantiomers of (I) 3-
mercaptocycloalkanones, (II) 4-mercapto-2-alkanones and (III) 2-mercapto-4-alkanones
and (B) enantiomers of (I) 3-acetylthiocycloalkanones, (II) 4-acetylthio-2-alkanones and
(III) 2-acetylthio-4-alkanones (C5-C7).
Figure 7. Stereoisomers of 3-mercaptocycloalkanols, 2-mercapto-4-alkanols and 4-
mercapto-2-alkanols showing the lowest odor thresholds (geometric means) determined
by GC/O for the respective chain length. The alkyl chains in the red dashed boxes
correspond to the substituent R4 in the model developed by Rowe.6
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Table 1. Configurations of the Investigated Stereoisomers of 3-Mercaptocycloalkanones
1-3, 3-Mercaptoalkanols 7-9, and the Respective 3-Acetylthio-Derivatives 4-6 and 10-12
(the Structures are Shown in the Supporting Information)
(S)
1a
2a
3a
(R)
1a´
2a´
3a´
1
2
3
3-mercaptocyclopentanone
3-mercaptocyclohexanone
3-mercaptocycloheptanone
4
5
6
3-acetylthiocyclopentanone
3-acetylthiocyclohexanone
3-acetylthiocycloheptanone
4a
5a
6a
4a´
5a´
6a´
cis
(1S,3R) (1R,3S)
trans
(1S,3S) (1R,3R)
7
8
9
3-mercaptocyclopentanol
3-mercaptocyclohexanol
3-mercaptocycloheptanol
7a
8a
9a
7a´
8a´
9a´
7b
8b
9b
7b´
8b´
9b´
10
11
12
3-acetylthiocyclopentanol
3-acetylthiocyclohexanol
3-acetylthiocycloheptanol
10a
11a
12a
10a´
11a´
12a´
10b
11b
12b
10b´
11b´
12b´
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Table 2. Enzyme-Catalyzed Kinetic Resolutions of 4-6
reaction
time (h)
4
conversion
preferred
enantiomer
(R)
substrate enzyme
porcine liver esterase
Ea
(%)
15
8
6
3
1.4
1.4
1.3
4
4
4
4
4
Rhizopus oryzae lipase
43
70
21
29
6
(R)
(R)
(R)
(R)
(R)
Candida antarctica lipase B
Candida cylindracea lipase
Pseudomonas cepacia lipase
porcine pancreas lipase
4
0.5
4
2
4
4
porcine liver esterase
42
31
51
6
41
19
25
10
6
3
1.2
1.2
(R)
(R)
(R)
(R)
(R)
(R)
Rhizopus oryzae lipase
Candida cylindracea lipase
porcine pancreas lipase
Candida antarctica lipase B
Pseudomonas cepacia lipase
5
6
4
2
4
2
porcine liver esterase
Candida antarctica lipase B
Candida cylindracea lipase
17
11
29
2
1.9
1.6
(R)
(R)
(R)
a E: enantioselectivity26
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