Determination of the Absolute Configurations and Sensory Properties of the Enantiomers of a Homologous Series (C6-C10) of 2-Mercapto-4-alkanones

Article abstract of DOI:10.1021/acs.jafc.8b06599

The enantiomers of a homologous series (C6-C10) of 2-mercapto-4-alkanones were obtained by lipase-catalyzed kinetic resolution of the corresponding racemic 2-acetylthio-4-alkanones. Their configurations were assigned via vibrational circular dichroism and ¹H NMR anisotropy based methods. Odor thresholds and odor qualities were determined by capillary gas chromatography-olfactometry using chiral stationary phases. There were minima of the odor thresholds for the chain lengths C7 and C8. Except for chain length C8, the enantiomers of the other homologues showed similar odor thresholds. The odor qualities ranged from pungent (C5) to mushroom (C9 and C10) and were similar to those known for the corresponding 1-alken-3-ones with one less C atom. In contrast to their positional isomers (4-mercapto-2-alkanones), the investigated 2-mercapto-4-alkanones do not meet the requirements of a "tropical olfactophore" (i.e., compounds possessing a 1,3-oxygen-sulfur functionality and specific arrangements of the substituents).

Full text of DOI:10.1021/acs.jafc.8b06599

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Chemistry and Biology of Aroma and Taste
Determination of the Absolute Configurations and
the Sensory Properties of the Enantiomers of a
Homologous Series (C6–C10) of 2-Mercapto-4-alkanones
Christiane Kiske, Anja Devenie Riegel, Ronja Hopf, Anna Kvindt, Iulia Poplacean, Tohru
Taniguchi, Mahadeva M. M. Swamy, Kenji Monde, Wolfgang Eisenreich, and Karl-Heinz Engel
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06599 • Publication Date (Web): 03 Jan 2019
Downloaded from http://pubs.acs.org on January 3, 2019
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Page 1 of 41
Journal of Agricultural and Food Chemistry
Determination of the Absolute Configurations and the Sensory
Properties of the Enantiomers of a Homologous Series (C6–C10) of
2-Mercapto-4-alkanones
Christiane Kiske†, Anja Devenie Riegel†, Ronja Hopf†, Anna Kvindt†, Iulia
Poplacean†, Tohru Taniguchi‡, Mahadeva M. M. Swamy ‡, Kenji Monde‡, Wolfgang
Eisenreich§ and Karl-Heinz Engel†*
† Technische Universität München, Lehrstuhl für Allgemeine
Lebensmitteltechnologie, Maximus-von-Imhof-Forum 2, D-85354 Freising-
Weihenstephan, Germany
‡ Frontier Research Center for Advanced Material and Life Science, Faculty of
Advanced Life Science, Hokkaido University, Kita 21 Nishi 11, Sapporo 001-0021,
Japan
§ Technische Universität München, Lehrstuhl für Biochemie, Lichtenbergstraße 4, D-
85747 Garching, Germany
CORRESPONDING AUTHOR FOOTNOTE.
* Author to whom correspondence should be addressed [Tel.: +49-(0)8161-71-4250.
Fax: +49-(0)8161-71-4259. E-mail: k.h.engel@wzw.tum.de]
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Abstract
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The enantiomers of a homologous series (C6-C10) of 2-mercapto-4-alkanones were
obtained by lipase-catalyzed kinetic resolution of the corresponding racemic 2-
acetylthio-4-alkanones. Their configurations were assigned via vibrational circular
dichroism and 1H-NMR anisotropy-based methods. Odor thresholds and odor qualities
were determined by capillary gas chromatography/olfactometry using chiral stationary
phases. There were minima of the odor thresholds for the chain lengths C7/C8. Except
for chain length C8, the enantiomers of the other homologs showed similar odor
thresholds. The odor qualities ranged from pungent (C5) to mushroom (C9/10) and
were similar to those known for the corresponding 1-alken-3-ones with one C-atom
less. In contrast to their positional isomers (4-mercapto-2-alkanones), the investigated
2-mercapto-4-alkanones do not meet the requirements of a “tropical olfactophore”, i.e.
compounds possessing a 1,3-oxygen-sulfur functionality and specific arrangements of
the substituents.
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Keywords
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2-mercapto-4-alkanones; absolute configuration; VCD; H-NMR spectroscopy; odor
threshold; odor quality
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Introduction
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Sulfur-containing volatiles play prominent roles for the aroma of many foods.1-3 Among
these important odorants, polyfunctional thiols are of particular interest, owing to their
pronounced odor qualities and their low odor thresholds.4,5 Recent systematic studies
on structure-odor correlations in homologous series of mercaptoalkanols confirmed the
potencies and the attractive odor qualities of this class of sulfur-containing aroma
compounds.6
β-Mercaptoalkanones and β-mercaptoalkanols are representatives of polyfunctional
thiols which have been reported to occur in cooked red bell pepper7 and in aged
Cheddar cheese.8,9 Wakabayashi et al.10 investigated the stereoisomers of a
homologues series (chain lengths C5-C10) of 4-mercapto-2-alkanones and the
corresponding 4-acetylthio-2-alkanones. The enantiomers have been separated via
capillary gas chromatography (GC) using chiral stationary phases and the absolute
1
configurations have been assigned using the H-NMR anisotropy method. Odor
thresholds and odor qualities of the enantiomers have also been determined.11 For the
homologous series (C5-C10) of 4-mercapto-2-alkanols, GC separations of the
stereoisomers as well as assignments of the configurations and assessments of odor
qualities have been achieved.12,13
Cooked red bell pepper has been reported to contain not only 4-mercapto-2-heptanone
and 4-mercapto-2-heptanol but also the positional isomers 2-mercapto-4-heptanone
and 2-mercapto-4-heptanol.7 For 2-mercapto-4-heptanone, the separation of the
enantiomers and the assignment of the configurations have been described,14 and the
distribution of the enantiomers in bell peppers has been determined.15
The objective of this study was to extend the analytical knowledge elaborated for 2-
mercapto-4-heptanone to a homologous series (C6-C10) of 2-mercapto-4-alkanones.
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The absolute configurations of the enantiomers should be assigned and their GC-order
of elution should be determined. By comparison of the odor thresholds and odor
qualities of the enantiomers with data available for the homologous series of 4-
mercapto-2-alkanones, further insight into structure-odor relationships for the class of
β-mercaptoalkanones should be gained.
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MATERIALS AND METHODS
Chemicals. 4-Hexen-3-one, 2,2-dimethyl-1,3-dioxan-4,6-dione (Meldrum’s acid),
hexanoyl chloride, heptanoyl chloride, oxalyl chloride, 4-(dimethylamino)pyridine
(DMAP), N,N’-dicyclohexylcarbodiimide (DCC), (R)-(-)-2-methoxy-2-phenylacetic acid
((R)-MPA), lipase from Candida antarctica (B lipase, adsorbed on a macroporous
acrylic resin, CAL-B), (E)-2-decenal, and deuterochloroform (CDCl3) were purchased
from Sigma-Aldrich (Steinheim, Germany). Thioacetic acid was obtained from Merck
Schuchardt OHG (Hohenbrunn, Germany). 2-Octen-4-one, (S)-(+)-2-methoxy-2-(1-
naphthyl)propionic acid ((S)-MαNP), and (R)-(-)-2-phenylpropionic acid ((R)-HTA)
were purchased from TCI Europe (Zwijndrecht, Belgium) and silica gel (NormaSil60,
40-63 µm) from VWR Chemicals (Leuven, Belgium). CDCl3 used for VCD
measurements was purchased from Cambridge Isotope Laboratories (Tewksbury,
MA).
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Syntheses. 2-Nonen-4-one and 2-decen-4-one. The starting materials methyl 3-
oxooctanoate and methyl 3-oxononanoate were synthesized according to Oikawa et
al.16, starting with 69.4 mmol Meldrum’s acid in 70 mL dichloromethane (CH2Cl2). After
addition of 138.8 mmol (2.0 equiv.) pyridine and 76.3 mmol (1.1 equiv.) hexanoyl
chloride (for C9) or heptanoyl chloride (for C10), the reaction mixture was stirred for
1 h under ice cooling and for another hour at room temperature (RT) under argon.
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Subsequently, it was washed with diluted (10%) HCl (3x20 mL) and H2O (2x20 mL),
dried over anhydrous sodium sulfate, and the solvent was removed using a rotary
evaporator. The crude product was mixed with 10 mL of methanol and stirred for 2 h
under reflux, followed by evaporation of the solvent and column chromatography on
silica gel with a mixture of n-hexane/Et2O (6+1, v/v). The 3-oxoacid esters were directly
used for the alkenone synthesis.7,14 The alkenones were purified by distillation under
vacuum: 2-Nonen-4-one: boiling point (bp): 78-80 °C (11 mbar), yield (y): 56.0%, purity
by GC: (p) 84.7%; linear retention indices (LRI) determined by GC: 1485 (Rtx®-WAX),
1101 (DB-1); 2-decen-4-one: bp: 83-85 °C (5 mbar), y: 18.9%, p: 76.2%, LRI: 1578,
1203.
2-Acetylthio-4-alkanones. 6 and 8-10 (Figure 1) were synthesized by Michael-type
addition of thioacetic acid (1.1 equiv.) to the respective alkenones.10,11 2-Acetylthio-4-
hexanone 6: yield (y): 107.1%, purity by GC (p): 92.4%, LRI: 1822 (Rtx®-WAX), 1225
(DB-1); 2-acetylthio-4-octanone 8: y: 97.1%, p: 96.0%, LRI: 1989, 1411; 2-acetylthio-
4-nonanone 9: y: 115.1%, p: 88.0%, LRI: 2093, 1511, and 2-acetylthio-4-decanone 10:
y: 116.6%, p: 89.1%, LRI: 2198, 1613.
2-Mercapto-4-alkanones. 1 and 3-5 (Figure 1) were obtained by treatment of the 2-
acetylthio-4-alkanones with methanol/sulfuric acid.14 Crude products were purified by
column chromatography on silica gel by elution with a mixture of n-hexane/Et2O (4+1,
v/v) for 1, (7+1, v/v) for 3, and (8+1, v/v) for 4 and 5; the obtained fractions were
checked by TLC. 2-Mercapto-4-hexanone 1: yield (y): 46.7%, purity by GC (p): 92.1%,
LRI: 1471 (Rtx®-WAX), 982 (DB-1); 2-mercapto-4-octanone 3: y: 54.2%, p: 95.7%, LRI:
1649, 1176; 2-mercapto-4-nonanone 4: y: 89.8%, p: 73.0%, LRI: 1755, 1279; 2-
mercapto-4-decanone 5: y: 41.8%, p: 93.1%, LRI: 1861, 1383.
Mass spectrometric and NMR data of the synthesized compounds are given in the
Supporting Information.
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Lipase-catalyzed kinetic resolutions. The 2-acetylthio-4-alkanones were mixed with
50 mM potassium phosphate buffer (pH 7.4), CAL-B was added, and the mixture was
stirred magnetically with a Teflon stir bar at RT. After defined reaction times, the
mixture was filtered and extracted with Et2O (3x15 mL). The combined organic layers
were dried with anhydrous sodium sulfate, filtered, and the solvent was removed under
reduced pressure. The formed (R)-2-mercapto-4-alkanones were separated from the
remaining substrates via column chromatography on silica gel by elution with a mixture
of n-hexane/Et2O (4+1, v/v) for 1, (7+1, v/v) for 3, and (8+1, v/v) for 4 and 5; the
obtained fractions were checked by TLC (ALUGRAM® SIL G/UV254, Macherey-
Nagel, Germany), visualization was achieved by spraying with 10% sulfuric acid and
subsequent heating until dryness. The remaining (S)-acetylthioalkanone substrates
were transesterified with 10 mL acidified methanol (pH 1-2) under reflux for 24 h. The
reaction mixture was cooled to RT, diluted with 30 mL of Et2O, and washed with water
(3x10 mL). After drying with anhydrous sodium sulfate, the solvent was removed under
reduced pressure to yield the mercaptoalkanone enantiomers. The employed amounts
of substrate and enzyme, reaction times, conversion rates, enantiomeric excesses,
purities, and yields are presented in Table 1.
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Formation of diastereoisomers with chiral auxiliaries. (R)-hydratropic acid (HTA)
thioesters. The thioesters were synthesized as previously described14 using (R)-HTA
(1.09 mmol), 0.72 mL oxalyl chloride, and 0.36 mmol of the respective 2-mercapto-4-
alkanone enantiomer (reaction time 72 h). (R)-HTA thioester 11 of (R)-1: 84.3 mg, yield
(y): 88.9%; 12 of (S)-1: 64.2 mg, y: 66.7%; 13 of (R)-3: 89.1 mg, y: 86.1%; 14 of (S)-3:
55.5 mg, y: 52.8%; 15 of (R)-4: 101.8 mg, y: 91.7%; 16 of (S)-4: 74.5 mg, y: 66.7%; 17
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of (R)-5: 92.1 mg, y: 80.6%; 18 of (S)-5: 56.8 mg, y: 50.0%. H-NMR data are
presented in Table 2.
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(S)- and (R)-MPA thioesters. The derivatization was performed according to Porto et
al.17 using 0.17 mmol (1.0 equiv.) of the (R)-mercaptoalkanone, 0.21 mmol (1.2 equiv.)
of (R)- or (S)-MPA, 0.21 mmol (1.2 equiv.) of DCC, and DMAP (5 mg) in 1 mL of dry
CH2Cl2. The reaction mixtures were stirred for 2 h under RT. After work up, purification
was achieved by semi-preparative HPLC. (R)-MPA thioester 19 of (R)-1: 7.0 mg, yield
(y): 14.4%; (S)-MPA thioester 20 of (R)-1: 14.5 mg, y: 29.8%; (R)-MPA thioester 21 of
(R)-3: 7.0 mg, y: 13.2%; (S)-MPA thioester 22 of (R)-3: 14.5 mg, y: 27.1%; (R)-MPA
thioester 23 of (R)-4: 7.7 mg, y: 13.8%; (S)-MPA thioester 24 of (R)-4: 13.9 mg, y:
24.8%; (R)-MPA thioester 25 of (R)-5: 20.8 mg, y: 35.6%; (S)-MPA thioester 26 of (R)-
5: 14.3 mg, y: 24.5%.
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(S)-MαNP thioesters. A solution of DCC (2.0 equiv.) and DMAP (1.0 equiv.) in 1 mL
dry CH2Cl2 was added to a solution of (S)-MαNP (1.0 equiv.) and the respective
enantiomerically enriched mercaptoalkanone in 1 mL dry CH2Cl2. The mixture was
stirred at RT for 20 h, filtered through a syringe filter (0.45 µm), and dried under a N2-
stream. The residue was separated by semi-preparative HPLC. (S)-MαNP thioester 27
of (R)-1: 3.6 mg, yield (y): 23.1%; 28 of (S)-1: 4.6 mg, y: 23.3%; 29 of (R)-3: 4.8 mg, y:
29.5%; 30 of (S)-3: 5.5 mg, y: 27.9%; 31 of (R)-4: 7.7 mg, y: 40.4%; 32 of (S)-4: 8.0 mg,
y: 43.4%; 33 of (R)-5: 8.4 mg, y: 31.4%; 34 of (S)-5: 7.8 mg, y: 29.6%. 1H-NMR data
are presented in Table 3.
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High Performance Liquid Chromatography (HPLC). Semi-preparative separations
of the diastereoisomers of (R)-HTA, (S)- and (R)-MPA, and (S)-MαNP thioesters of
mercaptoalkanones were carried out on a Dionex HPLC system (UltiMate 3000 series,
Dionex, Germering, Germany) equipped with a 3100 wavelength detector set at
254 nm using a 250 x 8 mm i.d. Nucleosil 50-5 column (CS Chromatography,
Langerwehe, Germany). Isocratic elution was performed at 30 °C with the following
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eluents and flow rates: for HTA-thioesters 11-16 and MPA-thioesters 19 and 20:
Hex/EtOAc (90/10, v/v) with 3 mL/min; for HTA-thioesters 17 and 18: Hex/EtOAc
(85/15, v/v) with 3 mL/min; for MPA-thioesters 21 and 22: Hex/EtOAc 92/8 with
3.5 mL/min; for MPA-thioesters 23 and 24: Hex/EtOAc 95/5 with 3.5 mL/min; for MPA-
thioesters 25 and 26: Hex/EtOAc 96/4 with 4.0 mL/min, for MαNP-thioesters 27 and
28: Hex/EtOAc 10/1 with 4.0 mL/min; for MαNP-thioesters 29 and 30: Hex/EtOAc 20/1
with 4.0 mL/min; for MαNP-thioesters 31-34: Hex/EtOAc 25/1 with 4.0 mL/min.
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NMR Spectroscopy. CDCl3 was used as solvent. 1H-NMR and 13C-NMR spectra were
recorded at 500 MHz and 126 MHz, respectively, with Avance 500 spectrometers
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(Bruker, Billerica, MA, USA). H-detected experiments were done with an inverse
1H/13C probehead. Direct 13C-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, and HMBC). Data processing was typically done with the
MestreNova software (Mestrelab Research, Santiago de Compostela, Spain).
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VCD Spectroscopy. VCD and IR spectra were measured on a JASCO FVS-6000
spectrometer (JASCO International Co. Ltd., Tokyo, Japan) for 3000 and 16 scans,
respectively. All spectra were recorded in CDCl3 (c = 0.6 M) using a 50-mm BaF2 cell
at a resolution of 4 cm-1 at ambient temperature. All VCD spectra were corrected by
using those measured for the enantiomers of each sample, and all IR spectra were
corrected using a solvent spectrum obtained under the identical experimental
conditions. The resultant VCD and IR spectra are presented in Δε and ε (both in M-1
cm-1) units, respectively.
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Computation. Molecular Mechanics Force Field (MMFF) MonteCarlo search was
performed on SPARTAN’10 software18 and density functional theory (DFT) calculation
was carried out on Gaussian 09 package.19 All calculations were conducted without
considering solvent effects. Theoretical calculations of VCD and IR spectra started with
a preliminary MMFF search using the arbitrarily selected (R)-enantiomer of 2-
mercapto-4-hexanone 1. The obtained conformers within 16 kJ/mol from the most
stable were further optimized using the DFT/B3LYP/6-311+G(2df,2p) level of theory.
VCD and IR spectra of the resultant 8 stable conformers within a 1.8 kcal/mol window
were calculated at the DFT/B3LYP/6-311+G(2df,2p) level and simulated with
Lorentzian lineshapes of 6 cm-1 width. The calculated frequencies ν were scaled with
a factor of 0.98. Final spectra were obtained based on the Boltzmann population
average of the spectra of each conformer.
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GC Analyses. Capillary Gas Chromatography (GC-FID). The column used was a 30 m
x 0.25 mm i.d.; 0.5 µm film thickness (df) Rtx®-Wax (Restek, Bad Homburg, Germany)
installed into a HP5890 A gas chromatograph (Hewlett-Packard INC, Waldbronn,
Germany) equipped with a split/splitless injector (215 °C, split ratio of 1:10) and an FID
(300 °C); temperature program: from 40 °C (5 min hold) to 240 °C (30 min hold) at
4 °C/min; carrier gas: hydrogen at a constant pressure of 150 kPa.
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A 30 m x 0.25 mm i.d.; 1.0 µm df DB-1 column (J&W Scientific, Waldbronn, Germany)
was installed into a 6890N instrument (Agilent Technologies, Waldbronn, Germany)
equipped with a split/splitless injector (230 °C, split ratio of 1:10) and an FID (300 °C);
temperature program: from 60 °C (5 min hold) to 250 °C (5 min hold) at 5 °C/min;
carrier gas: hydrogen at a constant pressure of 72 kPa.
Linear retention indices (LRI) were determined according to van den Dool and Kratz20
using C8-C40 n-alkane standard solutions (Sigma Aldrich).
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Enantioselective analysis. For the enantioselective analysis of 2-mercapto-4-
alkanones and 2-acetylthio-4-alkanones a 25 m x 0.25 mm i.d., 0.25 µm df MEGA-DEX
DET-Beta column, diethyl tert-butylsilyl-β-cyclodextrin (Mega s.n.c., Legnano, Italy),
was used in a 6890N chromatograph (Agilent Technologies) equipped with a
split/splitless injector (230 °C, split ratio of 1:10) and an FID (300 °C); carrier gas:
hydrogen at a constant pressure of 75 kPa.
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Capillary Gas Chromatography/Olfactometry (GC/O). Sensory analyses were
performed on a Trace GC Ultra (Thermo Fisher Scientific, Dreieich, Germany)
equipped with a cold-on-column injector (35 °C), a heated sniffing port (200 °C) and
an FID (250 °C); carrier gas: hydrogen at a constant pressure of 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, Rheinfelden, Germany) among sniffing port and FID. For
compounds 1-10, a 25 m x 0.25 mm i.d., 0.25 µm df MEGA-DEX DET-Beta column,
diethyl tert-butylsilyl-β-cyclodextrin (Mega s.n.c.), and for 4-mercapto-2-pentanone and
4-acetylthio-2-pentanone, a 30 m x 0.25 mm i.d.; 0.25 µm df CycloSil-B column, 30 %
heptakis(2,3-di-O-methyl-6-O-tert-butyl dimethylsilyl)-β-cyclodextrin in DB-1701 (J&W
Scientific), were installed. The temperature programs used are given in the Supporting
Information.
The sensory analyses of the mercapto- and acetylthio-enantiomers were performed by
three panelists (females, 22-37 years old). The odor thresholds of the mercapto-
enantiomers were additionally evaluated by a fourth panelist (panelist 4, female, 21
years old). Panelists 1 and 4 had no prior experience with GC/O assessments,
whereas panelist 2 and 3 were experienced. Odor thresholds in air were determined
according to the procedure described by Ullrich and Grosch using (E)-2-decenal with
the reported odor threshold of 2.7 ng/L in air as internal standard.21,22 Known amounts
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of the internal standard, of 2-mercapto-4-alkanones, and of 2-acetylthio-4-alkanones
were dissolved in Et2O and diluted stepwise (1+1, (v/v)). The aliquots were analyzed
by GC/O until no odor was perceivable. The panelists considered a concentration level
only as odor threshold if it was the lowest dilution step at which the odor was
consistently perceived in three consecutive GC/O-runs.13 Flavor dilution factors (FD)
of the internal standard and of the target compounds were obtained by aroma extract
dilution analysis (AEDA).23 The odor qualities were determined at one dilution step
above the odor threshold. Threshold values were determined in duplicate analysis by
panelists 2 (except C5) and 4; mean values were calculated.
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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 8000TOP gas chromatograph (CE Instruments, Hindley Green, United Kingdom)
and directly coupled to a Fisons MD8000TOP mass spectrometer (Fisons Instruments,
Manchester, UK) was used for compound identifications. The temperature was
programmed from 40 °C (5 min hold) to 240 °C (25 min hold) at 4 °C/min. A
split/splitless injector (220 °C, split ratio 1:50) was used, the carrier gas was helium at
a constant inlet pressure of 75 kPa. The mass spectra in the electron impact mode (EI)
were measured at 70 eV in a scan range from m/z 30 to 250. The source temperature
was 200 °C and the interface temperature 240 °C. Data acquisition was done via
Xcalibur software, version 1.4 (Thermo Fisher Scientific).
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Determination of Optical Rotations. A Polartronic-E polarimeter (Schmidt &
Haensch, Berlin, Germany) fitted with a measuring cell (path length 1 dm) and a
sodium lamp (wavelength 589 nm) was used. Samples were diluted in ethanol and
measurements were performed at 24 °C. (S)-1: [α]D +59.4, concentration (c):
3.25 g/100 mL, GC purity (p): 98.3%, enantiomeric excess (ee): 84.4%; (R)-1: [α]D -
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55.3, c: 2.18, p: 96.8, ee: 92.9; (S)-3: [α]D +55.9, c: 1.96, p: 97.3, ee: 88.6; (R)-3: [α]D -
25.0, c: 0.86, p: 90.7, ee: 92.9; (S)-4: [α]D +50.6, c: 1.49, p: 97.8, ee: 94.2; (R)-4: [α]D -
47.1, c: 1.46, p: 95.6, ee: 91.7; (S)-5: [α]D +45.0, c: 1.48, p: 96.7, ee: 93.1; (R)-5: [α]D -
42.1, c: 1.23, p: 84.3, ee: 94.6; (S)-6: [α]D -11.6, c: 2.22, p: 97.0, ee: 99.6; (S)-8: [α]D -
16.5, c: 1.52, p: 96.7, ee: 99.6; (S)-9: [α]D -18.4, c: 1.07, p: 91.2, ee: 96.4; (S)-10: [α]D
-18.4, c: 1.44, p: 95.0, ee: 97.9;
RESULTS AND DISCUSSION.
In analogy to the procedure previously described for 2-mercapto-4-heptanone 2, 2-
mercapto-4-alkanones with carbon chain lengths C6 to C10 (1 and 3-5, Figure 1) were
synthesized by Michael-type addition of thioacetic acid to the respective alkenones and
subsequent treatment of the formed 2-acetylthio-4-alkanones (6 and 8-10, Figure 1)
with methanol/sulfuric acid.7,14 GC separations of the enantiomers of the homologous
series of 2-mercapto-4-alkanones (Figure 2A) and 2-acetylthio-4-alkanones (Figure
2B) were achieved using diethyl tert-butylsilyl-β-cyclodextrin as chiral stationary phase.
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Determination of the Absolute Configurations. Enantiomerically enriched 2-mercapto-
4-alkanones were obtained by kinetic resolutions of the respective racemic 2-
acetylthio-4-alkanones using the lipase CAL-B as biocatalyst. The employed
procedure is exemplarily shown for 2-mercapto-4-octanone 3 in Figure 3. Yields and
optical purities of the enantiomers of 2-mercapto-4-alkanones obtained by this
approach are summarized in Table 1.
The absolute configurations and the orders of elution of the enantiomers of 2-
mercapto-4-alkanones and 2-acetylthio-4-alkanones with chain lengths C6 and C8-
C10 were determined based on the procedures previously applied to 2-mercapto-4-
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heptanone, i.e. VCD spectroscopy as well as 1H-NMR analyses of diastereoisomeric
thioesters using different chiral auxiliaries.14
As a first step, the obtained enantiomers were subjected to VCD experiments. A
preliminary MMFF conformational search and subsequent DFT/B3LYP/6-
311+G(2df,2p) optimization were conducted for the arbitrarily chosen (R)-enantiomer
of 2-mercapto-4-hexanone 1, resulting in 8 stable conformers within 1.8 kcal/mol of the
most stable one. VCD and IR spectra were calculated for these conformers at the
DFT/B3LYP/6-311+G(2df,2p) level of theory. The final VCD and IR spectra were
obtained based on the Boltzmann population average of the spectrum of each
conformer (Figure 4A and B). Experimental VCD and IR spectra of 1-E1 were obtained
as CDCl3 solutions, at concentrations of 0.60 M. The peak positions and the signs of
each signal of the observed VCD spectrum showed excellent agreement with those
calculated for (R)-1. Therefore, this VCD result concludes the absolute configuration
of 1-E1 as (R).
Assuming that the elongation of the carbon chain length from C6 to C10 did not affect
their conformations, the VCD and IR spectra of 3-E1, 4-E1 and 5-E1 (also measured
in CDCl3 solutions, at the same concentration as 1) were superimposed on those
calculated for 1-E1. The observed VCD similarities strongly suggested the absolute
configurations of 3-E1, 4-E1, and 5-E1 as (R).
Using the method described by Helmchen and Schmierer24, 2-mercapto-4-alkanone
enantiomers obtained via lipase-catalyzed kinetic resolution were reacted with (R)-
hydratropic acid chloride. As examples, 1H-NMR spectra of (R)-HTA thioesters 11 and
12 of (R)-1 and (S)-1, respectively, are shown in Figures 5 A and B. The protons of the
methyl group (C-6) as well as both methylene bridges (C-3 and C-5) of (R)-HTA
thioester 11 showed a relative upfield shift compared to the protons of (R)-HTA
thioester 12. In contrast, the protons of the other terminal methyl group (C-1) of (R)-
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HTA thioester 11 underwent a downfield shift compared to the equivalent protons of
(R)-HTA thioester 12. The described shifting effects can be attributed to a cisoid
arrangement of the acid’s phenyl ring in the depicted constellations of (R)-HTA
thioester 11 and 12. Therefore, the (R)-HTA thioester 11 was confirmed as (R,R)
whereas the (R)-HTA thioester 12 was confirmed as (R,S). In Table 2, 1H-NMR data
of (R)-HTA thioesters 11-18 of the enantiomers of 1 and 3-5 are shown.
As second chiral derivatizing reagent, 2-methoxy-2-phenylacetic acid (MPA) was
applied. Porto et al.17,25 have previously shown its suitability for the assignment of the
absolute configurations of chiral thiols. The ΔδRS signs determined for the (R)- and (S)-
MPA thioesters obtained after derivatization of the (R)-enantiomers of 1 and 3-5 were
consistent within the homologous series as shown in Figure 6. According to the model
developed by Porto et al.25, the spatial arrangement of the L1/L2 side chains confirmed
the (R)-configuration of the 2-mercapto-4-alkanones.
Finally, (S)-MαNP was used as chiral auxiliary. The (R)- and (S)-enantiomers obtained
after lipase-catalyzed kinetic resolution were reacted with (S)-MαNP, purified by semi-
1
preparative HPLC, and analyzed by H-NMR spectroscopy (Table 3). Applying the
revised sector rule for β-mercaptoalkanones and thiols14 to the (S)-MαNP thioester of
1, the Δδ value of H-1 is negative (-0.01) and is placed on the right side whereas the
Δδ values for H-3, H-5, and H-6 are positive (0.04, 0.04, and 0.03, respectively) and
are placed on the left side. This resulted in (R)-configuration at the C-2 position of the
first eluting compound (corresponding to LC-peak I, 27, Table 3). The same results
were found for the (S)-MαNP thioesters of 3-5.
Based on the consistent results obtained by VCD and NMR analyses, the GC-order of
elution of the enantiomers of 2-mercapto-4-alkanones (1 and 3-5) as well as of 2-
acetylthio-4-alkanones (6 and 8-10) on the employed chiral stationary phase could be
assigned as (S) before (R), (Figure 2A and B).
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Determination of Odor Thresholds. Odor thresholds of the enantiomers of 2-mercapto-
4-alkanones (1-5) and 2-acetylthio-4-alkanones (6-10) were determined via GC/O
using the method described by Ullrich and Grosch21. Table 4 shows the results
obtained for the two homologous series from C6 to C10 as well as for the C5-homologs
4-mercapto-2-pentanone and 4-acetylthio-2-pentanone. The mercaptoalkanones were
assessed by four panelists, the acetylthioalkanones by three. Regarding their
variability, panelist 4 showed a high sensitivity for the C5 and C7 homologs, e.g. factor
49 for (S)-4-mercapto-2-pentanone compared to panelist 3 or factor 13 for (S)- and
(R)-2-mercapto-4-heptanone 2 compared to panelist 1. However, for the other
compounds and panelists the individual odor thresholds either were the same or
differed only by factors between 2 and approximately 4. This corresponds to one or
two dilution steps in the course of the AEDA and thus indicates the reproducibility of
the sensory assessments.
A graphic presentation of the geometric means of the odor thresholds depending on
the chain lengths is given in Figure 7 (solid lines). The data illustrate that there were
minima of the odor thresholds for the chain lengths C7/C8 for both 2-mercapto-4-
alkanones (Figure 7A) and 2-acetylthio-4-alkanones (Figure 7B). The graphs for the
enantiomers were quite similar; significantly lower odor thresholds were only observed
for the (R)-enantiomers of the C8-homologs. This is confirmed by a comparison of the
geometric means for the enantiomers shown in Table 4. Except for 2-mercapto-4-
octanone (factor 3) and 2-acetylthio-4-octanone (factor 7), the geometric means of the
odor thresholds calculated for the enantiomers were either the same or differed only
by factors up to 2 (Table 4).
Figures 7A and B (dotted lines) also show the odor thresholds previously reported for
the homologous series of 4-mercapto-2-alkanones and 4-acetylthio-2-alkanones.11
The range of odor thresholds (0.1-2 ng/L) previously described for 4-mercapto-2-
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pentanone is in the same order of magnitude as those determined in this study (0.1-
4.9 ng/L). The C8 and C9 homologs of 4-mercapto-2-alkanones showed lower odor
threshold values than the respective 2-mercapto-4-alkanones; another striking
difference was the high odor threshold determined for (S)-4-mercapto-2-hexanone.
The odor thresholds of 4-acetylthio-2-pentanone (7-44 ng/L) determined by the
panelists in this study were significantly lower than those previously reported (70-200
ng/L).11 The odor thresholds of (R)-2-acetylthio-4-octanone and the 2-acetylthio-4-
decanone enantiomers were significantly lower than those of the respective 4-
acetylthio-2-alkanone positional isomers. Except for these differences, the shapes of
the curves depending on the chain lengths showed similar minima and maxima and
were comparable for the positional isomers. The significant differences in odor
thresholds between the mercaptoalkanones and the respective acetylthioalkanones
were similarly expressed for the homologous series of both positional isomers.
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Determination of Odor Qualities. Odor qualities of the enantiomers of 2-mercapto-4-
alkanones and 2-acetylthio-4-alkanones were determined via GC/O at one dilution step
above the odor threshold (Tables 5 and 6). It is striking that the fruity and sulfury-catty
notes reported for the (R)- and (S)-enantiomers of 4-mercapto-2-alkanones,
respectively, were not observed for 2-mercapto-4-alkanones. As summarized in Table
5, their odor qualities changed, depending on the chain length, from pungent, plastic
(C6/C7) and roasty (C8) to earthy and mushroom (C9 and C10). No consistent
differences between the (R)- and (S)-enantiomers were observed.
Structural requirements for a “tropical olfactophore” have been proposed by Rowe26
and were extended to the respective acetyl compounds by Robert et al.27 (Figure 8A).
In contrast to this model, the 2-mercapto-4-alkanones and 2-acetylthio-4-alkanones
(chain lengths C6–C10) possess alkyl groups (from ethyl to hexyl) rather than
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hydrogen, a methyl group, a ring system, or an ether moiety as the substituent R4. This
may explain the lack of tropical, fruity notes. Among the β-mercaptoalkanones
investigated in this study, only the C5 homolog 4-mercapto-2-pentanone meets the
structural requirements suggested by Rowe;26 this is in line with the descriptions “fruity”
by two of the panelists (Table 5).
In contrast, within both homologous series the C9 and C10 homologs exhibited
pronounced earthy and mushroom odor notes. Figure 8B shows the structural similarity
between 2-mercapto-4-nonanone 4 and the known mushroom odorant 1-octen-3-one.
The lengths of the alkyl chains (corresponding to the substituent R4 in Figure 8A) are
identical; the β-mercapto group in 2-mercapto-4-nonanone 4 seems to play the role of
the terminal double bond in 1-octen-3-one. This is strongly supported by the fact that
the odor qualities determined for the other 2-mercapto-4-alkanones 1-3 and 5 are also
in very good agreement with odor qualities described for the respective homologous
series of 1-alken-3-ones.28 The odor thresholds of the homologous 1-alken-3-ones28
are significantly lower than those determined for the 2-mercapto-4-alkanones in this
study. However, the odor descriptions reported for 1-alken-3-ones in the literature, e.g.
pungent and plastic for 1-penten-3-one and 1-hexen-3-one28-31, vegetable-like for 1-
hepten-3-one28, and mushroom for 1-nonen-3-one28-34, nearly perfectly match those
determined in this study for the corresponding 2-mercapto-4-alkanones with one C-
atom more. Preliminary sensory assessments of a homologous series of 2-mercapto-
4-alkanols indicated that this phenomenon might also apply to the corresponding
alcohols (data not shown). This observation is in agreement with the role of (R)-1-
octen-3-ol as another key mushroom aroma compound35,36 and is supported by the
description of mushroom-like odors also for 1-mercapto-3-octanol and 1-mercapto-3-
nonanol.6
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The 2-acetylthio-4-alkanones showed mainly vegetable notes for the (R)-enantiomers
(C6-C8). Within the homologous series, the predominating notes also changed to
earthy and mushroom for 2-acetylthio-4-nonanone and 2-acetylthio-4-decanone,
indicating that the phenomenon is also valid for the acetyl-compounds.
The flavor of 2-mercapto-4-heptanone 2 tasted in NaCl and sugar solutions has been
described as grapefruit, sesame, earthy, and rocket.7 The main descriptions
associated with this homolog in the course of the GC/O evaluation were vegetables,
onion, and pungent. However, it is noteworthy that one of the panelists mentioned bell
pepper as a descriptor for both enantiomers. In cooked bell pepper, (S)-2-mercapto-4-
heptanone has been determined as the predominating enantiomer.15 A determination
of the odor threshold in water and a calculation of the odor activity value would be
required to assess its actual contribution to the aroma of bell peppers.
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In conclusion, the sensory assessments showed that in contrast to the data reported
for the homologous series of 3-mercapto-2-methylalkanols and 1-mercapto-2-methyl-
3-alkanols6, the exchange of the positions of the functional groups (SH and carbonyl)
in β-mercaptoalkanones did not cause significant changes in the GC-odor thresholds.
However, there is a significant impact of the positions of the functional groups on the
odor qualities.
Acknowledgement
We thank Christine Schwarz for recording the NMR spectra.
Supporting Information
MS and NMR data of 2-acetylthio-4-alkanones and 2-mercapto-4-alkanones (C6-C10)
as well as 2-nonen-4-one and 2-decen-4-one. 1H-NMR data and Δδ values of (R)- and
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(S)-MPA thioesters of (R)-(-)-2-mercapto-4-hexanone 1. 1H-NMR data and Δδ values
of (R)- and (S)-MPA thioesters of (R)-(-)-2-mercapto-4-octanone 3. 1H-NMR data and
Δδ values of (R)- and (S)-MPA thioesters of (R)-(-)-2-mercapto-4-nonanone 4. 1H-NMR
data and Δδ values of (R)- and (S)-MPA thioesters of (R)-(-)-2-mercapto-4-decanone
5. 13C-NMR data of synthesized diastereoisomeric derivatives. Temperature programs
used for the separations of 2-acetylthio-4-alkanones and 2-mercapto-4-alkanones (C6-
C10) as well as 4-acetylthio-2-pentanone and 4-mercapto-2-pentanone.
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assignment of their absolute configuration by 1H NMR. Org. Lett. 2007, 9, 5015-5018.
(26) Rowe, D. J. High impact aroma chemicals. In Advances in Flavours and
Fragrances–From the Sensation to the Synthesis, Swift, K. A. D., Ed. Royal Society of
Chemistry: Cambridge, UK, 2002; pp 202-226.
(27) Robert, F.; Heritier, 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.
(28) Lorber, K.; Schieberle, P.; Buettner, A. Influence of the Chemical Structure on
Odor Qualities and Odor Thresholds in Homologous Series of Alka-1,5-dien-3-ones,
Alk-1-en-3-ones, Alka-1,5-dien-3-ols, and Alk-1-en-3-ols. J. Agric. Food Chem. 2014,
62, 1025-1031.
(29) Buettner, A.; Schieberle, P. Characterization of the Most Odor-Active Volatiles
in Fresh, Hand-Squeezed Juice of Grapefruit (Citrus paradisi Macfayden). J. Agric.
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(30) Song, H.; Cadwallader, K. R. Aroma components of American country ham. J.
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528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
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Raspberry Aroma Using a Retronasal Aroma Simulator and Gas Chromatography
Olfactometry. J. Agric. Food Chem. 1996, 44, 3919-3925.
(33) Schnermann, P.; Schieberle, P. Evaluation of Key Odorants in Milk Chocolate
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(34) Cullere, L.; Fernandez de Simon, B.; Cadahia, E.; Ferreira, V.; Hernandez-Orte,
P.; Cacho, J. Characterization by gas chromatography-olfactometry of the most odor-
active compounds in extracts prepared from acacia, chestnut, cherry, ash and oak
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Figure Captions
Figure 1.
545
546
Structures of investigated 2-mercapto-4-alkanones 1-5 and the respective 2-acetylthio-
4-alkanones 6-10.
547
548
549
550
Figure 2.
Capillary gas chromatographic separation of the enantiomers of (A) 2-mercapto-4-
alkanones 1-5 and (B) 2-acetylthio-4-alkanones 6-10.
551
552
553
Figure 3.
Preparation of the 2-mercapto-4-octanone enantiomers 3-E1 and 3-E2 via kinetic
resolution of 2-acetylthio-4-octanone 8, catalyzed by CAL-B.
554
555
556
Figure 4.
Comparison of (A) IR and (B) VCD spectra calculated for (R)-2-mercapto-4-hexanone
(R)-1 and observed for 1-E1, 3-E1, 4-E1, and 5-E1.
557
558
559
Figure 5.
1H-NMR spectra of (R)-hydratropic acid thioesters (A) (R,R)-11 of (R)-1 and (B) (R,S)-
12 of (S)-1.
560
561
562
563
Figure 6.
Structures of (R)- and (S)-MPA thioesters of (R)-1, (R)-3, (R)-4, and (R)-5 with δ values
(ppm) and ΔδRS values (ppm). L1 (front side) and L2 (rear side) correspond to the side
chains at the asymmetric centers of the mercaptoalkanone moieties.
564
565
566
Figure 7.
Geometric means of the odor thresholds of (A) 2-mercapto-4-alkanones: (R)-
enantiomer (●), (S)-enantiomer (●) and 4-mercapto-2-alkanones11: (R)-enantiomer
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567
568
(Δ), (S)-enantiomer (Δ) and (B) 2-acetylthio-4-alkanones: (R)-enantiomer (●), (S)-
enantiomer (●) 4-acetylthio-2-alkanones11: (R)-enantiomer (Δ), (S)-enantiomer (Δ).
569
570
571
572
573
Figure 8.
(A) Structural requirements for a ‘tropical olfactophore’ as proposed by Rowe26 and
extended by Robert et al.27 (A: H, SCH3, ring; B: H, CH3, acyl, absent if carbonyl; R1/R2:
H, alkyl; R3: H, alkyl, ring; R4: H, CH3, ring, OR; R5: H, absent if carbonyl. (B) Structures
of 2-mercapto-4-nonanone 4 and 1-octen-3-one.
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Table 1. Preparation of 2-Mercapto-4-alkanone Enantiomers via Lipase-catalyzed Hydrolysis of the Respective 2-Acetylthio-4-
alkanones
starting
compound
obtained
enantiomer optical rotation
configurationb,
amountc
[g]
lipase
CAL-B [g]
volume of
buffer [mL]
reaction
time [h]
conversion
rated [%]
eee [%] purityd [%]
yieldf [%]
a
1-E1
1-E2
3-E1
3-E2
4-E1
4-E2
5-E1
5-E2
(R)-(-)
(S)-(+)
(R)-(-)
(S)-(+)
(R)-(-)
(S)-(+)
(R)-(-)
(S)-(+)
0.866
0.689
1.947
1.36
1.00
0.8
50
40
0.5
2
33
50
33
46
62
46
42
48
92.9
84.4
92.9
88.6
91.7
94.2
94.6
93.1
96.8
98.3
98
11.0
12.6
11.9
5.3
6
8
9
2.21
1.61
1.21
2.51
2.52
1.31
110
95
0.5
2
97.3
95.6
97.8
97.1
96.7
1.029
2.130
2.132
1.068
60
1
23.3
12.4
22.4
6.4
150
150
70
4
1
10
6
a Numbering refers to the enantiomers obtained either as direct hydrolysis product (E1) or via the remaining substrate (E2)
b Configurations determined via VCD and 1H-NMR analysis of HTA-, MPA-, and (S)-MαNP-thioesters; for optical rotations see Materials and Methods
c Used amount of the starting compound
d Determined via GC (DBWAX)
e Enantiomeric excess, determined via GC (MEGA-DEX DET-Beta)
f Molar yields
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Table 2. 1H-NMR Data of (R)-HTA Thioesters of the Enantiomers of 2-mercapto-4-hexanone 1, 2-mercapto-4-octanone 3, 2-mercapto-
4-nonanone 4, and 2-mercapto-4-decanone 5
2-mercapto-4-hexanone 1
2-mercapto-4-octanone 3
(R)-HTA thioester
2-mercapto-4-nonanone 4
2-mercapto-4-decanone 5
1-E1
1-E2
3-E1
3-E2
4-E1
4-E2
5-E1
5-E2
Δδ
Δδ
Δδ
Δδ
H
(R,R)-11
(R,S)-12
(R,R)-13
(R,S)-14
(R,R)-15
(R,S)-16
(R,R)-17
(R,S)-18
1
2
1.22 (d, 7.0)
3.81 (m)
1.18 (d, 7.0)
3.80 (m)
-0.04
-0.01
1.22 (s)
3.80 (m)
1.18 (s)
3.80 (m)
-0.04
0
1.22 (d, 7.0)
3.80 (m)
1.18 (d, 7.0)
3.80 (m)
-0.04
0
1.22 (d, 6.9)
3.81 (m)
1.18 (d,7.0)
3.80 (m)
-0.04
-0.01
3
3'
5
2.62
2.68
0.06
0.05
0.08
0.05
2.62
(dd, 16.7, 5.0)
2.68
(dd, 16.7, 5.3)
0.06
0.05
0.05
2.62
(dd, 16.7, 4.9)
2.68
(dd, 16.7, 5.3)
0.06
0.05
0.06
2.62
2.68
0.06
0.05
0.05
(dd, 16.7, 5.0) (dd, 16.7, 5.4)
(dd, 16.7, 4.9) (dd, 16.7, 5.4)
2.47 2.52
(dd, 16.6, 8.3) (dd, 16.7, 8.1)
2.46
(dd, 16.7, 3.3)
2.51
(dd, 16.7, 8.0)
2.46
(dd, 16.8, 8.3)
2.51
(dd, 16.7, 8.1)
2.46 2.51
(dd, 16.8, 8.3) (dd, 16.7, 8.1)
2.26 (m)
2.32 (q, 7.4)
2.24
(td, 7.3, 3.6)
2.29
(dd, 7.9, 7.0)
2.23
(td, 7.3, 3.7)
2.29 (m)
2.24
(td, 7.3, 3.7)
2.29 (m)
6
7
0.91 (t, 7.3)
0.96 (t, 7.3)
1.41 (m)
1.46 (m)
1.22 (m)
0.05
0.04
0.02
1.43 (m)
1.14 (m)
1.46 (m)
1.15 (m)
0.03
0.01
0.03
0.01
1.42 (m)
1.17 (m)
1.47 (m)
1.20 (m)
0.05
0.03
0.03
0.03
0.01
1.18 (t, 7.3)
0.80 (t, 7.3)
8
0.82 (t, 7.3)
1.19 (m)
1.22 (m)
1.17 (m)
1.20 (m)
9
0.80 (t, 7.2)
0.81 (t, 7.1)
1.17 (m)
1.20 (m)
10
0.80 (t, 7.1)
0.81 (t, 7.0)
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Table 3. 1H-NMR Data of (S)-MαNP Thioesters of the Enantiomers of 2-mercapto-4-hexanone 1, 2-mercapto-4-octanone 3, 2-
mercapto-4-nonanone 4, and 2-mercapto-4-decanone 5
2-mercapto-4-hexanone 1
2-mercapto-4-octanone 3
2-mercapto-4-nonanone 4
2-mercapto-4-decanone 5
(S)-MαNP thioester
LC Peak Ia
LC Peak IIa
LC Peak Ia
LC Peak IIa
LC Peak Ib
LC Peak IIb
LC Peak Ib
LC Peak IIb
Δδ
Δδ
Δδ
Δδ
H
(S,R)-27
(S,S)-28
(S,R)-29
(S,S)-30
(S,R)-31
(S,S)-32
(S,R)-33
(S,S)-34
1
2
3
1.24 (d, 7.0)
3.80 (m)
2.64
1.23 (d, 7.9) -0.01
1.23 (d, 7.0)
3.80 (m)
2.64
1.22 (d, 7.0)
3.79 (m)
2.68
-0.01
-0.01
0.04
1.23 (d, 6.9)
3.79 (m)
2.64
1.22 (d, 7.0) -0.01
1.23 (d, 7.0)
3.79 (m)
1.22 (d, 6.9) -0.01
3.81 (m)
2.68
-0.01
0.04
3.80 (m)
2.68
0.1
3.80 (m)
0.01
0.04
0.04
2.64
2.68
(dd, 16.6, 5.0) (dd, 16.5, 5.6)
2.51 2.50
(dd, 16.5, 8.5) (dd, 15.6, 8.1)
(dd, 18.5, 5.1) (dd, 16.6, 5.5)
2.49 2.49
(dd, 16.6, 8.5) (dd, 16.5, 8.2)
(dd, 16.6, 4.8) (dd, 16.6, 5.2)
2.49 2.49
(dd, 16.6, 8.6) (dd, 16.7, 8.2)
(dd, 16.6, 4.9)
(dd, 16.6, 5.5)
3'
-0.01
0
0
2.49
(dd, 16.6, 8.6)
2.49
(dd, 16.6, 8.2)
0
5
6
2.27 (m)
2.31 (m)
0.04
0.03
2.24
(td, 7.4, 4.3)
1.41 (m)
2.29
(td, 7.2, 1.8)
1.44 (m)
0.05
0.03
0.01
0.03
2.23 (m)
1.42 (m)
2.28 (m)
1.46 (m)
0.05
0.04
0.01
0.01
0.03
2.23 (td, 7.5, 4.2)
1.41 (m)
2.29 (m)
1.45 (m)
1.18 (m)
1.18 (m)
1.18 (m)
0.80 (t, 6.8)
0.06
0.04
0.03
0.03
0.03
0.02
0.91 (t, 7.3)
0.94 (t, 7.3)
7
1.18 (m)
1.19 (m)
1.16 (m)
1.17 (m)
1.15 (m)
8
0.78 (t, 7.3)
0.81 (t, 7.4)
1.16 (m)
1.17 (m)
1.15 (m)
9
0.77 (t, 7.2)
0.80 (t, 7.1)
1.15 (m)
10
0.78 (t, 6.9)
a Eluent: hexane/ethyl acetate 20/1 (v/v)
b Eluent: hexane/ethyl acetate 25/1 (v/v)
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