Analysis and Sensory Evaluation of the Stereoisomers of a Homologous Series (C5-C10) of 4-Mercapto-2-alkanols

Article abstract of DOI:10.1021/acs.jafc.7b03543

A homologous series of 4-mercapto-2-alkanols (C5-C10) was used to investigate the impact of the stereochemistry on the sensory properties of a class of naturally occurring polyfunctional thiols having a 1,3-oxygen-sulfur functionality. Stereoisomers were obtained via syntheses of racemic mixtures and subsequent lipase-catalyzed kinetic resolutions. Analytical separations of the stereoisomers were achieved by capillary gas chromatography (GC) using chiral stationary phases. The absolute configurations were assigned via NMR analysis. Sensory evaluations by means of GC/olfactometry revealed odor threshold minima for the medium-chain homologues (C7-C9) of the 4-mercapto-2-alkanol stereoisomers. Except for the C5 homologue, the lowest odor thresholds were determined for the (2R,4R)-configured stereoisomers. The variability in odor qualities was mainly determined by the chain length. None of the 4-mercapto-2-alkanol stereoisomers showed consistent odor qualities for all homologues.

Full text of DOI:10.1021/acs.jafc.7b03543

Subscriber access provided by Purdue University Libraries
Article
Analysis and Sensory Evaluation of the Stereoisomers of
a Homologous Series (C5–C10) of 4-Mercapto-2-alkanols
Svenja Nörenberg, Christiane Kiske, Bastian Reichardt, Verena Andelfinger,
Anne Pfeiffer, Franziska Schmidts, Wolfgang Eisenreich, and Karl-Heinz Engel
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03543 • Publication Date (Web): 17 Sep 2017
Downloaded from http://pubs.acs.org on September 27, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 40
Journal of Agricultural and Food Chemistry
Analysis and Sensory Evaluation of the Stereoisomers of a
Homologous Series (C5ꢀC10) of 4ꢀMercaptoꢀ2ꢀalkanols
†
†
†
†
Svenja Nörenberg , Christiane Kiske , Bastian Reichardt , Verena Andelfinger , Anne
†
†
‡
†
Pfeiffer , Franziska Schmidts , Wolfgang Eisenreich and KarlꢀHeinz Engel *
†
Technische Universität München, Lehrstuhl für Allgemeine Lebensmitteltechnologie,
MaximusꢀvonꢀImhofꢀForum 2, Dꢀ85354 FreisingꢀWeihenstephan, Germany
‡
Technische Universität München, Lehrstuhl für Biochemie, Lichtenbergstraße 4, Dꢀ
85747 Garching, Germany
CORRESPONDING AUTHOR FOOTNOTE.
*
Author to whom correspondence should be addressed (Tel.: +49ꢀ(0)8161ꢀ71ꢀ4250.
Fax: +49ꢀ(0)8161ꢀ71ꢀ4259. Eꢀmail: k.h.engel@wzw.tum.de)
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 40
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Abstract
A homologous series of 4ꢀmercaptoꢀ2ꢀalkanols (C5ꢀC10) was used to investigate the
impact of the stereochemistry on the sensory properties of a class of naturally
occurring polyfunctional thiols having a 1,3ꢀoxygenꢀsulfur functionality. Stereoisomers
were obtained via syntheses of racemic mixtures and subsequent lipaseꢀcatalyzed
kinetic resolutions. Analytical separations of the stereoisomers were achieved by
capillary gas chromatography (GC) using chiral stationary phases. The absolute
configurations were assigned via NMR analysis. Sensory evaluations by means of
GC/Olfactometry revealed odor threshold minima for the mediumꢀchain homologs
(C7ꢀC9) of the 4ꢀmercaptoꢀ2ꢀalkanol stereoisomers. Except for the C5 homolog, the
lowest odor thresholds were determined for the (2R,4R)ꢀconfigured stereoisomers.
The variability in odor qualities was mainly determined by the chain length. None of
the 4ꢀmercaptoꢀ2ꢀalkanol stereoisomers showed consistent odor qualities for all
homologs.
1
1
1
1
1
1
1
1
1
Keywords
4ꢀMercaptoꢀ2ꢀalkanols; polyfunctional thiols; absolute configuration; odor threshold;
odor quality
2
ACS Paragon Plus Environment

Page 3 of 40
Journal of Agricultural and Food Chemistry
1
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
Introduction
Polyfunctional thiols are known as one of the classes of sulfurꢀcontaining volatiles
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
1ꢀ6
playing important roles for the aroma of various foods. They are characterized by
4,7
low odor thresholds and pronounced odor qualities. Fruity, tropical and vegetable
odor notes have been particularly associated with numerous polyfunctional thiols
7,8
possessing a 1,3ꢀoxygenꢀsulfur function.
Compounds fulfilling this essential
structural feature of the soꢀcalled ‘tropical olfactophore’ are for example the fruity
smelling 3ꢀmercaptoꢀ1ꢀpentanol, 3ꢀmercaptoꢀ4ꢀmethylꢀ1ꢀpentanol and its acetate
9
10
identified in hop and beer as well as the brothꢀlike, sweaty and leekꢀlike smelling 3ꢀ
11,12
mercaptoꢀ2ꢀmethylꢀ1ꢀpentanol isolated from raw onions.
As known for other chiral
aroma compounds, the stereochemistry also has been shown to play a role in the
13
olfactory perception of polyfunctional sulfurꢀcontaining volatiles. For example,
different odor qualities have been described for the esters of 3ꢀ(methylthio)ꢀ1ꢀhexanol
14,15
identified in yellow passion fruits.
Enantiodifferentiation in terms of odor intensity
has been recognized for 3ꢀmercaptoꢀ2ꢀmethylꢀ1ꢀpentanol; the odor thresholds of the
12
stereoisomers differed by a factor up to 1000.
The importance of the stereochemistry for the sensory properties has also been
demonstrated for a homologous series (chain lengths C5ꢀC10) of 4ꢀmercaptoꢀ2ꢀ
16ꢀ18
19ꢀ21
alkanones
, a class of naturally occurring βꢀmercaptoketones.
For the
corresponding 4ꢀmercaptoꢀ2ꢀalkanols, it was shown that the odor thresholds depend
on the chain length and that there are differences in the sensory properties of the
22,23
diastereomeric pairs.
However, data demonstrating differences between
24,25
enantiomers have been limited to the respective C7ꢀhomolog
identified in cooked
21
red bell pepper . The analysis of 4ꢀmercaptoꢀ2ꢀheptanol and its acetyl derivatives
via GC/O demonstrated that the odor properties of the stereoisomers were not only
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 40
4
4
4
4
4
4
5
5
5
4
5
6
7
8
9
0
1
2
affected by acetylation but also by the configurations of the two asymmetric
24,25
centers.
The aim of the present investigation was to evaluate the sensory properties of the
stereoisomers of the complete homologous series (C5ꢀC10) of 4ꢀmercaptoꢀ2ꢀ
alkanols. To this end, the first part of the study was devoted to the synthesis of the
stereoisomers, their separation via GC using chiral stationary phases and the
assignment of their absolute configurations. Subsequently, odor qualities and odor
thresholds of the stereoisomers were determined via GC/O.
5
5
3
4
Materials and Methods
Chemicals. 3ꢀHeptenꢀ2ꢀone and (S)ꢀ(+)ꢀ2ꢀmethoxyꢀ2ꢀ(1ꢀnaphthyl)propionic acid ((S)ꢀ
MαNP) were obtained from TCI Europe (Zwijndrecht, Belgium). 3ꢀOctenꢀ2ꢀone was
provided by Alfa Aesar (Karlsruhe, Germany) and 3ꢀdecenꢀ2ꢀone by SAFC (Buchs,
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Switzerland). 3ꢀPentenꢀ2ꢀone, 3ꢀnonenꢀ2ꢀone, (E)ꢀ2ꢀdecenal (≥ 95%), thioacetic acid,
lithium aluminum hydride, lipases from porcine pancreas (Type II, PPL, lot numbers:
020M1589V and SLBL 2143V) and Candida antarctica (B lipase, adsorbed on a
macroporous acrylic resin, CALꢀB, lot number: SLBG4222V), deuterochloroform
(CDCl ), 4ꢀ(dimethylamino)pyridine (DMAP), N,N’ꢀdicyclohexylcarbodiimide (DCC)
3
®
and Celite 503 were purchased from SigmaꢀAldrich (Steinheim, Germany). 3ꢀ
Hexenꢀ2ꢀone was synthesized by Knoevenagel reaction of 3ꢀoxobutanoic acid and
18
propanal as previously reported.
Syntheses. A homologous series (chain lengths C5–C10) of 4ꢀacetylthioꢀ2ꢀ
alkanones 7ꢀ12 were synthesized by Michaelꢀtype addition of thioacetic acid to the
17,18
corresponding 3ꢀalkenꢀ2ꢀones according to previously described procedures.
4ꢀ
Mercaptoꢀ2ꢀalkanols 1ꢀ6 were prepared by adding the synthesized 4ꢀacetylthioꢀ2ꢀ
alkanones (6.63 mmol for 1, 5.74 mmol for 2, 10.60 mmol for 3, 3.11 mmol for 4,
4
ACS Paragon Plus Environment

Page 5 of 40
Journal of Agricultural and Food Chemistry
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6.93 mmol for 5 and 12.93 mmol for 6) dissolved in 20 mL of dry tetrahydrofuran
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
(THF) slowly to a suspension of lithium aluminum hydride (LiAlH : 29.84 mmol for 1,
4
25.83 mmol for 2, 47.70 mmol for 3, 14.01 mmol for 4, 31.19 mmol for 5 and
58.19 mmol for 6) in 50 mL of dry THF under argon atmosphere at 0 °C. The reaction
mixture was stirred at RT overnight. After being cooled to 0 °C, distilled water was
carefully added and the aqueous phase was adjusted to pH 2 using hydrochloric acid
(5%) and extracted three times with dichloromethane. The organic phase was dried
with anhydrous sodium sulfate and evaporated under reduced pressure to give the
following crude products: 4ꢀmercaptoꢀ2ꢀpentanol, 1: 0.41 g (3.41 mmol, mol yield:
51%, purity (GC): 76%, diastereomeric ratio, dr (%) = 37:63, purity after column
2
chromatography on silica gel (nꢀhexane/Et O, 3:2, v/v): 96% (by GC)). 4ꢀMercaptoꢀ2ꢀ
hexanol, 2: 0.83 g (6.17 mmol, mol yield: 108%, purity: 84% by GC, dr (%) = 42:58).
4ꢀMercaptoꢀ2ꢀheptanol, 3: 1.54 g (10.40 mmol, mol yield: 97%, purity: 96% by GC,
dr (%) = 39:61). 4ꢀMercaptoꢀ2ꢀoctanol, 4: 0.49 g (3.02 mmol, mol yield: 97%, purity:
89% by GC, dr (%) = 47:53, purity after column chromatography on silica gel (nꢀ
2
hexane/Et O, 5:3, v/v): 96% (by GC)). 4ꢀMercaptoꢀ2ꢀnonanol, 5: 1.25 g (7.09 mmol,
mol yield: 102%, purity: 92% by GC, dr (%) = 45:55). 4ꢀMercaptoꢀ2ꢀdecanol, 6: 1.77 g
(9.27 mmol, mol yield: 72%, purity: 91% by GC, dr (%) = 43:57). Chromatographic,
mass spectrometric and NMR data were in agreement with those previously
7,21,22,24,26
reported.
Enzymeꢀcatalyzed kinetic resolution of racemic 4ꢀacetylthioꢀ2ꢀalkanones. In
17,18
analogy to the method described by Wakabayashi et al.
, 5 mmol of synthesized
4ꢀacetylthioꢀ2ꢀalkanone (0.80 g of 7, 0.87 g of 8, 0.94 g of 9, 1.01 g of 10, 1.08 g of
11 and 1.15 g of 12, respectively) were dissolved in 50 mL of potassium phosphate
buffer (50 mM, pH 7.4). After adding 1.0 g of the enzyme preparation (CALꢀB resin
for 7 and PPL for 8ꢀ12), the mixture was stirred at RT for a defined time (4 h for 7, 6 h
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 40
9
9
9
9
6
7
8
9
for 8, 7.5 h for 9, 1 h for 10 and 11, and 2 h for 12). The enzyme was filtered off and
the aqueous phase was extracted four times with 50 mL Et O. The organic phase
2
was dried with anhydrous sodium sulfate and the solvent was removed under
reduced pressure using an aspirator at 40 °C. An aliquot of the reaction mixture
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
2
(1 ꢁL/mL in Et O) was subjected to GC analysis using heptakis(2,3ꢀdiꢀOꢀmethylꢀ6ꢀOꢀ
tertꢀbutyl dimethylsilyl)ꢀβꢀcyclodextrin as chiral stationary phase. The following
conversion rates of substrates (56% for 7, 75% for 8, 79% for 9, 43% for 10, 38% for
11 and 63% for 12) as well as the enantiomeric ratios (er) of products (4ꢀmercaptoꢀ2ꢀ
alkanones (C5ꢀC10, 13ꢀ18), er (%) = 12:88 for 13, 66:34 for 14, 38:62 for 15, 82:18
for 16, 86:14 for 17 and 72:28 for 18) and of the remaining substrates (4ꢀacetylthioꢀ2ꢀ
alkanones, er (%) = 99.6:0.4 for 7, 97:3 for 8, 98:2 for 9, 74:26 for 10 and 72:28 for
27
11) were calculated.
The separation of the reaction mixture was carried out by column chromatography on
silica gel using a mixture of nꢀhexane and Et O (3:1 for 7 and 13, 4:1 for 8 and 14 as
well as for 9 and 15, 5:1 for 10 and 16 as well as for 11 and 17, and 6:1 for 12 and
2
24,25
18, v/v). In analogy to the method described for the C7 homolog
, the separated 4ꢀ
acetylthioꢀ2ꢀalkanones of 10 and 11 were used as starting substances to synthesize
the (4R)ꢀconfigured diastereomers of 4ꢀmercaptoꢀ2ꢀoctanol 4 and 4ꢀmercaptoꢀ2ꢀ
nonanol 5 employing the same procedure as described above for the synthesis of 4ꢀ
mercaptoꢀ2ꢀalkanols. The reduction of the remaining substrate of the C5 homolog 7
resulted in the formation of the (4S)ꢀconfigured diastereomers of 4ꢀmercaptoꢀ2ꢀ
pentanol 1 since CALꢀB resin was used for the enzymeꢀcatalyzed hydrolysis instead
28
of PPL. In case of the C6 and C10 homolog, the obtained products 4ꢀmercaptoꢀ2ꢀ
hexanone 14 and 4ꢀmercaptoꢀ2ꢀdecanone 18 were reduced to the (4S)ꢀconfigured
4
diastereomers of the corresponding mercaptoalkanols 2 and 6 using LiAlH .
6
ACS Paragon Plus Environment

Page 7 of 40
Journal of Agricultural and Food Chemistry
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
Preparation of (S)ꢀMαNP thioesters of (R)ꢀ and (S)ꢀ4ꢀmercaptoꢀ2ꢀoctanone. (R)ꢀ
4ꢀmercaptoꢀ2ꢀoctanone, (R)ꢀ16. In accordance with the previously described
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
17
enzymeꢀcatalyzed kinetic resolution , 37.5 mmol of racemic 4ꢀacetylthioꢀ2ꢀoctanone
10 were dissolved in potassium phosphate buffer and 7.5 g of PPL were added. After
®
stirring for 3 h at RT, the enzyme was filtered off using Celite and the aqueous
2
phase was extracted with Et O (4x25 mL). The organic phase was dried over
anhydrous sodium sulfate and the solvent was removed under reduced pressure.
(R)ꢀ4ꢀacetylthioꢀ2ꢀoctanone 10 was obtained after column chromatography on silica
gel using a mixture of nꢀhexane and Et
2
O (7:1, v/v): mol yield: 1.7%, purity (GC):
97.8%, er (%) = 91:9. (R)ꢀ4ꢀmercaptoꢀ2ꢀoctanone (R)ꢀ16 was obtained via
28
transesterification starting with 0.5 mmol of (R)ꢀ10 followed by purification (column
chromatography on silica gel using a mixture of nꢀhexane and Et O (7:1, v/v)): mol
2
yield: 0.7%, purity (GC): 97.5%, er (%) = 76:14.
(S)ꢀ4ꢀmercaptoꢀ2ꢀoctanone, (S)ꢀ16. For (S)ꢀ16, 12.5 mmol of racemic 4ꢀacetylthioꢀ2ꢀ
octanone 10 were dissolved in potassium phosphate buffer and 5 g of PPL were
®
added. After stirring for 1 h at RT, the enzyme was filtered off using Celite and the
aqueous phase was extracted with Et O (4x25 mL). The organic phase was dried
2
over anhydrous sodium sulfate and the solvent was removed under reduced
pressure. (S)ꢀ16 was obtained after column chromatography on silica gel using a
2
mixture of nꢀhexane and Et O (7:1, v/v): mol yield: 6.4%, purity (GC): 97.1%, er (%) =
88:12.
(S)ꢀMαNP thioesters of (R)ꢀ and (S)ꢀ16. The diastereomers were prepared according
28
to Kiske et al. and separated by semiꢀpreparative HPLC using 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 50ꢀ5 column
(CS Chromatography, Langerwehe, Germany). Isocratic elution was performed at
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 40
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
30 °C with a mixture of nꢀhexane/isopropanol 96:4 (v/v) as the eluent and a flow rate
of 2 mL/min. (S)ꢀMαNP thioesters of (R)ꢀ16: 10.0 mg, 31.0%; (S)ꢀMαNP thioesters of
(S)ꢀ16: 5.5 mg, 17.2%.
Capillary Gas Chromatography (GC-FID). The column used was
a
30 m x 0.25 mm i.d., 0.5 ꢁm; DBꢀWax column (J&W Scientific, Agilent Technologies,
Waldbronn, Germany, column 1) installed into an HP5890 A gas chromatograph
(HewlettꢀPackard, Heilbronn, Germany) equipped with a split/splitless injector
(215 °C, split ratio of 1:7) and an FID (350 °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 135 kPa.
A 30 m x 0.25 mm i.d.,1.0 ꢁm; DBꢀ1 column (J&W Scientific, Agilent Technologies,
column 2) was installed into a CE 5160 instrument (Carlo Erba Instruments, Hofheim,
Germany) equipped with a split/splitless injector (200 °C, split ratio of 1:10) and an
FID (260 °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 74 kPa. Linear retention
29
8
indices (LRI) were determined according to van den Dool and Kratz , using C ꢀC40
nꢀalkane standard solutions (SigmaꢀAldrich).
Enantioselective analyses of 4ꢀacetylthioꢀ2ꢀalkanones 7ꢀ12 and 4ꢀmercaptoꢀ2ꢀ
alkanones 13ꢀ18 were performed on a 30 m x 0.25 mm i.d., 0.25 ꢁm; CycloSilꢀB
column (J&W Scientific, Agilent Technologies, column 3) installed into an HP5890
Series II gas chromatograph (HewlettꢀPackard) equipped with a split/splitless injector
(200 °C, split ratio of 1:5) and an FID (350 °C); temperature program: from 75 °C
(0 min hold) to 180 °C (5 min hold) at 1 °C/min; carrier gas: hydrogen at a constant
pressure of 176 kPa.
GC separation of the stereoisomers of 1ꢀ6 was performed on two inꢀhouse prepared
30 m x 0.25 mm i.d. fused silica capillary columns coated with 50% heptakis(2,3ꢀdiꢀ
8
ACS Paragon Plus Environment

Page 9 of 40
Journal of Agricultural and Food Chemistry
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
73
Oꢀacetylꢀ6ꢀOꢀtertꢀbutyl dimethylsilyl)ꢀβꢀcyclodextrin (column 4) and 28% heptakis(2,3ꢀ
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
diꢀOꢀmethoxymethylꢀ6ꢀOꢀtertꢀbutyl
dimethylsilyl)ꢀβꢀcyclodextrin
in
OV1701ꢀvi
(column 5), respectively. The syntheses of the cyclodextrins and the column
preparations were carried out as previously described.30,31 Columns 4 and 5 were
installed into a CE 5160 instrument (Carlo Erba Instruments) equipped with a
split/splitless injector (200 °C, split ratio of 1:10) and an FID (260 °C); hydrogen at
constant pressure of 75 kPa and 110 kPa, respectively, was used as carrier gas. A
TM
30 m x 0.25 mm i.d., 0.25 ꢁm; Inert Cap Chiramix column (GL Science, Tokyo,
Japan, column 6) installed into an HP5890 Series II gas chromatograph (Hewlettꢀ
Packard) equipped with a split/splitless injector (230 °C, split ratio of 1:30) and an
FID (250 °C); carrier gas: hydrogen at a constant pressure of 110 kPa was also used
to separate the stereoisomers of 1ꢀ6.
Multidimensional Gas Chromatography (MDGC). The instrumentation consisted of
two coupled GC 8000 (Carlo Erba Instruments). A Moving Column Stream Switching
device (MCSS) and a 1 m x 0.25 mm i.d. deactivated fused silica transfer capillary
were used to transfer the diastereomers of 1ꢀ6 from the preꢀcolumn (GC 1) onto the
main column (GC 2). A 60 m x 0.32 mm i.d., 0.25 ꢁm; DBꢀWax column (J&W
Scientific, Agilent Technologies) was installed into GC 1, equipped with a
split/splitless injector (215 °C, split ratio of 1:5) and an FID (230 °C); temperature
program: from 40 °C (5 min hold) to 240 °C (25 min hold) at 4 °C/min; carrier gas:
hydrogen at a constant pressure of 165 kPa. The columns 4ꢀ6 were installed as main
columns in GC 2 equipped with an FID (230 °C); outlet pressure was 98 kPa. Data
were processed via ChromꢀCard software (Thermo Fisher Scientific, Dreieich,
Germany).
Capillary Gas ChromatographyꢀOlfactometry (GC/O). Sensory analyses of the
stereoisomers of 1ꢀ6 were performed on columns 4ꢀ6. Columns 4 and 5 were
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 40
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
installed into an HP5890 A Series II gas chromatograph (HewlettꢀPackard) equipped
with a coldꢀonꢀcolumn injector (40 °C), a heated sniffing port (200 °C) and an FID
(250 °C); carrier gas: hydrogen at a constant pressure of 75 kPa. Column 6 was
installed into a Fractovap 4200 (Carlo Erba Instruments), equipped with a
split/splitless injector (220 °C, split ratio of 1:10), a sniffing port (230 °C) and an FID
(230 °C); carrier gas: hydrogen at a constant pressure of 98 kPa; makeꢀup gas:
nitrogen at 50 kPa. For both GC/O systems, the effluent was split 1:1 via a pressꢀfit
Yꢀsplitter and 30 cm x 0.25 mm i.d. deactivated fused silica capillaries (BGB Analytik
AG, Rheinfelden, Germany) among sniffing port and FID.
The sensory analyses of the stereoisomers of 1ꢀ6 were performed by three panelists
(females; 20ꢀ30 years old). Panelist 1 and 2 had no prior experience with GC/O
assessments, whereas panelist 3 had extensive training for more than three years.
Panelist 1 determined the odor properties of the stereoisomers of 1, 3a/a', 4, 5a/a'
and 6 using column 4. The sensory evaluation of the synꢀenantiomeric pair of 3b/b'
was performed using octakis(2,3ꢀdiꢀOꢀnꢀbutyrylꢀ6ꢀOꢀtertꢀbutyl dimethylsilyl)ꢀγꢀCD as
24,25
chiral stationary phase.
Panelist 2 used column 6 for the determination of odor
thresholds and odor qualities of the stereoisomers of 1, 2, and 4ꢀ6. Panelist 3
determined the odor properties of the stereoisomers of 2 and 3 using column 4 and
24,25
5.
Odor thresholds in air were determined following the procedure described by
Ullrich and Grosch using (E)ꢀ2ꢀdecenal with the reported odor threshold of 2.7 ng/L in
32,33
air as internal standard.
Known amounts of the internal standard and of the 4ꢀ
mercaptoꢀ2ꢀalkanols were dissolved in Et O and diluted stepwise by a factor of 1:2
2
(v/v). Stock solutions were freshly prepared for each panelist. 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
24
the odor was consistently perceived in three consecutive GC/Oꢀruns. Flavor dilution
1
0
ACS Paragon Plus Environment

Page 11 of 40
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
25
factors (FD) of the internal standard and of the target compounds were obtained by
34
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
aroma extract dilution analysis (AEDA). The odor impressions obtained during the
AEDA were collected and those with injection volumes corresponding to
approximately 1.5 ng for each stereoisomer at the sniffing port were used as
descriptors of the odor qualities.
Capillary Gas Chromatography ꢀ Mass Spectrometry (GCꢀMS).
A 30 m x 0.25 mm i.d., 0.5 ꢁm; DBꢀWaxetr fused silica capillary column (J&W
TOP
Scientific, Agilent Technologies) installed into a GC 8000
gas chromatograph (CE
Instruments, Hindley Green, United Kingdom) directly coupled to
a
TOP
Fisons MD8000
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 and 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 ꢀ 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).
1
13
NMR Spectroscopy. H NMR and C NMR spectra were recorded at 500 MHz and
126 MHz, respectively with Avance 500 spectrometers (Bruker, Billerica, MA, USA).
1
1
13
13
Hꢀdetected experiments were done with an inverse H/ C probehead, direct Cꢀ
13
31 29
19
1
measurements were performed with a QNP C/ P/ Si/ F/ H cryoprobe. The
experiments were done in full automation using standard parameter sets of the
13
TOPSPIN 3.0 software package (Bruker). C NMR spectra were recorded in protonꢀ
decoupled mode. The compounds were dissolved in deuterated chloroform.
The spectra were recorded at 27 °C. All signals were assigned by protonꢀproton and
protonꢀcarbon correlation experiments (COSY, HSQC and HMBC). Data processing
1
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 40
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
was typically done with the MestreNova software (Mestrelab Research, Santiago de
Compostela, Spain).
Optical Rotations. Optical rotations were measured on a PolartronicꢀE polarimeter
(Schmidt & Haensch, Berlin, Germany) fitted with a measuring cell (path length 1 dm)
and a sodium lamp (wavelength 589 nm). Samples were diluted in ethanol and the
measurements were performed at a temperature of 21 °C. (S)ꢀ16: [α]
D
+17.0,
concentration (c): 2.07 g/100 mL, GC purity (p): 97.1%, enantiomeric excess (ee):
D D
75.9%; (R)ꢀ10: [α] +15.6, c: 0.96, p: 97.8, ee: 82.8; (R)ꢀ16: [α] ꢀ16.9, c: 1.16, p:
97.5, ee: 71.5.
Results and Discussion. GCꢀseparation of the stereoisomers of 4ꢀmercaptoꢀ2ꢀ
alkanols. 4ꢀMercaptoꢀ2ꢀalkanols 1ꢀ6 (Table 1) were synthesized by Michaelꢀtype
addition of thioacetic acid to the corresponding 3ꢀalkenꢀ2ꢀones and subsequent
reduction of the obtained 4ꢀacetylthioꢀ2ꢀalkanones 7ꢀ12 with lithium aluminum
16ꢀ18,21,24
hydride.
The GC separations of the diastereomeric pairs of the homologous
series (chain lengths C5ꢀC10) are shown in Figure 1A. For separation of the four
stereoisomers of each homolog several chiral stationary phases were tested. Figure
1B shows the separation obtained by using heptakis(2,3ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyl
dimethylsilyl)ꢀβꢀcyclodextrin as chiral stationary phase. The use of this cyclodextrin
derivative was suitable for the separation of the four stereoisomers, except for chain
length C6. For this homolog 2, only an incomplete separation could be achieved
(Figure 1C), and there were coelutions with stereoisomers of 1. Therefore,
heptakis(2,3ꢀdiꢀOꢀmethoxymethylꢀ6ꢀOꢀtertꢀbutyl dimethylsilyl)ꢀβꢀcyclodextrin was
employed; the use of this chiral stationary phase enabled the separation of all four
stereoisomers of 2 (Figure 1D).
1
2
ACS Paragon Plus Environment

Page 13 of 40
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
76
Determination of the absolute configurations and order of elution of the stereoisomers
of 4ꢀmercaptoꢀ2ꢀalkanols. The absolute configurations and the order of elution of the
stereoisomers of 4ꢀmercaptoꢀ2ꢀalkanols with chain lengths C5 1, C6 2, and C8ꢀC10
4ꢀ6 were determined according to the principles previously reported for the
assignment of the GCꢀelution order of the stereoisomers of the C7 homolog 4ꢀ
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
24,25
mercaptoꢀ2ꢀheptanol 3.
This involved (i) a comparison of the NMR data of the LCꢀ
separated diastereomers of 3 with those reported for antiꢀ(2S,4S)ꢀ4ꢀmercaptoꢀ2ꢀ
heptanol and (ii) the formation of enantioꢀenriched diastereomers of 3 via lipaseꢀ
catalyzed kinetic resolution of 4ꢀacetylthioꢀ2ꢀheptanone 9 and subsequent reduction
24ꢀ26
of the remaining substrate.
Recently, the configurations of 4ꢀmercaptoꢀ2ꢀ
pentanone 13 and 4ꢀmercaptoꢀ2ꢀheptanone 15 have been reꢀinvestigated by
1
vibrational circular dichroism (VCD) and H NMR analyses of (R)ꢀhydratropic acid
2
8
thioesters and 2ꢀmethoxyꢀ2ꢀphenylacetic acid thioesters.
The absolute
configurations of the enantiomers of 13 and 15 determined with these approaches
1
were not in agreement with those deduced via the H NMR anisotropy method using
16ꢀ18
(S)ꢀ2ꢀmethoxyꢀ2ꢀ(1ꢀnaphthyl)propionic acid (MαNP) as chiral reagent.
As a
consequence, the published assignments of the absolute configurations of the
stereoisomers of 3 had to be revised since their assignments were based on the
absolute configuration of 4ꢀmercaptoꢀ2ꢀheptanone 15 determined by Wakabayashi et
17,24,25
al.
As a result, the order of elution of the diastereomers of 3 were assigned as
antiꢀ before synꢀ on a DBꢀWax column (Figure 1A) and as (2S,4S)ꢀ3a before
(2R,4R)ꢀ3a' and (2R,4S)ꢀ3b before (2S,4R)ꢀ3b' for the stereoisomers using
heptakis(2,3ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyl
dimethylsilyl)ꢀβꢀcyclodextrin
as
chiral
stationary phase (Figure 1B).
1
The analysis of the diastereomeric mixture of 3 via H NMR spectroscopy enabled
the determination of the diastereomeric ratios (Hꢀ2 and Hꢀ4) at the stereogenic
1
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 40
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
centers (Figure 2A). As outlined in Table 2, the ratios measured via integral analysis
of the appropriate pairs of protons (syn/anti) were nearly identical to the ratio of antiꢀ
and synꢀdiastereomers determined by GC analysis (Figure 1A). Based on this result,
the comparison of diastereomeric ratios of synthesized 4ꢀmercaptoꢀ2ꢀalkanols
1
obtained via capillary gas chromatography and H NMR spectroscopy was used to
assign the antiꢀ/synꢀconfigurations for the complete homologous series. For the
synthesized mercaptoalkanols (chain lengths C5 1, C6 2, and C8ꢀC10 4ꢀ6) there
were also good agreements between the GC and the NMR data, as shown in Table
2. The diastereomeric ratios determined at the Hꢀ2 and Hꢀ4 positions of the
mercaptoalkanols with the shortest (C5, 1) and the longest chain lengths (C10, 6) are
exemplarily shown in Figure 2B and 2C. As a result, the order of elution of the
diastereomers of the investigated 4ꢀmercaptoꢀ2ꢀalkanols was consistently assigned
as anti before syn (Figure 1A).
The final step was the assignment of the order of elution of the two antiꢀconfigured
[(2S,4S) and (2R,4R)] and the two synꢀconfigured [(2S,4R) and (2R,4S)]
enantiomers. To this end, a procedure based on enzymeꢀcatalyzed kinetic resolution
was used to obtain enantiomerically enriched thiols. The used approach is
exemplarily shown for 4ꢀmercaptoꢀ2ꢀpentanol 1 in Figure 3. The first step was the
CALꢀBꢀmediated hydrolysis of the thioester bond of racemic 4ꢀacetylthioꢀ2ꢀpentanone
7 which resulted in the formation of the (R)ꢀconfigured thiol 13 as product and (4S)ꢀ
configured 7 as remaining substrate (Figure 3, step A). The absolute configurations
28
were assigned according to Kiske et al. GC analysis of the enantiomers of 7 and 13
was performed using heptakis(2,3ꢀdiꢀOꢀmethylꢀ6ꢀOꢀtertꢀbutyl dimethylsilyl)ꢀβꢀ
cyclodextrin as chiral stationary phase (Figure 3, step A). In the next step, the
reaction mixture was separated by column chromatography and the nearly
enantiomerically pure 4ꢀacetylthioꢀ2ꢀpentanone (S)ꢀ7 was subjected to reduction with
1
4
ACS Paragon Plus Environment

Page 15 of 40
Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
28
LiAlH
4
to form the stereoisomers of 4ꢀmercaptoꢀ2ꢀpentanol 1 with the corresponding
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
excess of the (4S)ꢀconfigured diastereomers (Figure 3, step B). GC analysis using
heptakis(2,3ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyl dimethylsilyl)ꢀβꢀcyclodextrin as chiral
stationary phase demonstrated that the (4S)ꢀconfigured diastereomers coeluted with
the first peaks of the pairs of stereoisomers obtained for the synthesized 4ꢀmercaptoꢀ
2ꢀpentanol 1. The assignment of the antiꢀ and synꢀdiastereomers achieved in the first
step in combination with the assignment of the enantiomeric pairs to their
corresponding diastereomers via MDGC enabled the determination of the absolute
configurations and GC orders of elution of the four stereoisomers of 4ꢀmercaptoꢀ2ꢀ
pentanol (Figure 3; step C) as (2S,4S)ꢀ1a before (2R,4R)ꢀ1a' and (2R,4S)ꢀ1b before
(2S,4R)ꢀ1b'.
An analogous procedure was applied to assign the order of elution of the
stereoisomers of 4ꢀmercaptoꢀ2ꢀoctanol 4. To this end, the (4S)ꢀ and (4R)ꢀconfigured
enantiomers of 16 were prepared via kinetic resolution using PPL in accordance with
28
the previously described procedure.
The obtained enantiomerically enriched
mercaptoalkanones were reacted with (S)ꢀMαNP (Figure 4), purified with semiꢀ
preparative HPLC, and the diastereomers were analyzed by NMR spectroscopy
1
(Table 3). Previous studies had demonstrated consistent H NMR anisotropy effects
for the complete homologous series of (S)ꢀMαNP thioesters of 4ꢀmercaptoꢀ2ꢀ
17,18
alkanones of chain lengths C5 to C10.
Therefore, it was assumed that the
1
recently revised sector rule for secondary thiols, verified via VCD and H NMR
analysis of diastereomeric thioesters formed with other auxiliary reagents for 4ꢀ
28
mercaptoꢀ2ꢀalkanones of chain lengths C5 and C7, can be applied. The ∆δ values
of Hꢀ1 and Hꢀ3 are positive (0.06 and 0.05, respectively) and are placed on the left
side whereas the ∆δ values for Hꢀ5 – Hꢀ8 are negative (ꢀ0.02, ꢀ0.03, ꢀ0.03 and ꢀ0.04,
respectively) and are placed on the right side. This results in (R)ꢀconfiguration at the
1
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 40
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
Cꢀ4 position of the first eluting diastereomer and thus corresponds to the (S,R)
diastereomeric MαNP thioester of 16 which elutes before the (S,S) diastereomer.
Based on this result, PPLꢀmediated hydrolysis of racemic 4ꢀacetylthioꢀ2ꢀoctanone
racꢀ10 resulted in the formation of the (S)ꢀconfigured thiol 16 as product and (4R)ꢀ
configured 10 as remaining substrate. After column chromatography, (4R)ꢀconfigured
4
10 was subjected to reduction with LiAlH to form the (4R)ꢀconfigured diastereomers
of 4. Comparing the order of elution of the (4R)ꢀconfigured diastereomers of 4 to
those of the enantiomeric pairs of antiꢀ and synꢀconfigured 4 via MDGC resulted in an
order of elution of (2S,4S)ꢀ4a before (2R,4R)ꢀ4a' and (2R,4S)ꢀ4b before (2S,4R)ꢀ4b'
using heptakis(2,3ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyl dimethylsilyl)ꢀβꢀcyclodextrin as chiral
stationary phase (Figure 1B).
In analogy, the absolute configurations of the stereoisomers of the remaining
homologs 2, 5 and 6 were assigned via PPLꢀmediated hydrolyses of 4ꢀacetylthioꢀ2ꢀ
hexanone 8, 4ꢀacetylthioꢀ2ꢀnonanone 11 and 4ꢀacetylthioꢀ2ꢀdecanone 12. The
formed (S)ꢀconfigured thiols (14, 17 and 18) and the remaining (R)ꢀconfigured
substrates (8, 11 and 12) were analyzed using heptakis(2,3ꢀdiꢀOꢀmethylꢀ6ꢀOꢀtertꢀ
16ꢀ18,28
butyl dimethylsilyl)ꢀβꢀcyclodextrin as chiral stationary phase.
chromatography, (4R)ꢀconfigured 11 as well as (4S)ꢀconfigured 14 and 18 were
subjected to reduction with LiAlH to form the corresponding enantiomerically
After column
4
enriched diastereomers of 2, 5 and 6. GC analyses of (4R)ꢀconfigured 5, (4S)ꢀ
configured 6 and the respective racemic reference substances resulted in orders of
elution of (2S,4S)ꢀa before (2R,4R)ꢀa' and (2R,4S)ꢀb before (2S,4R)ꢀb' using
heptakis(2,3ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyl
dimethylsilyl)ꢀβꢀcyclodextrin
as
chiral
stationary phase (Figure 1B). The same order of elution was assigned for the
stereoisomers of the C6ꢀhomolog 2 separated on heptakis(2,3ꢀdiꢀOꢀmethoxymethylꢀ
6ꢀOꢀtertꢀbutyl dimethylsilyl)ꢀβꢀcyclodextrin as stationary phase (Figure 1D).
1
6
ACS Paragon Plus Environment

Page 17 of 40
Journal of Agricultural and Food Chemistry
®
3
3
3
3
3
3
3
3
3
3
80
An interesting phenomenon was observed when screening Chiramix , a column
81
82
83
84
85
86
87
88
89
coated with a mixture of the two chiral stationary phases heptakis(2,6ꢀdiꢀOꢀmethylꢀ3ꢀ
Oꢀpentyl)ꢀβꢀcyclodextrin
and
octakis(2,6ꢀdiꢀOꢀmethylꢀ3ꢀOꢀtrifluoroacetyl)ꢀγꢀ
35
cyclodextrin as alternative chiral stationary phase (Figure 5). There were changes
in the order of elution of the stereoisomers of 1ꢀ6 depending on the chain lengths.
For the longꢀchain homologs 4ꢀ6 the antiꢀconfigured (2S,4S)ꢀa and (2R,4R)ꢀa'
stereoisomers consistently eluted before the corresponding synꢀconfigured (2S,4R)ꢀ
b' and (2R,4S)ꢀb stereoisomers. The changed order of elution of the four
stereoisomers of the shortꢀchain homologs 1 and 2 as well as the coelution of the
(4R)ꢀconfigured diastereomers of the C7 homolog 3a' and 3b' appears to be due to
an increasing shift of the (2R,4R)ꢀconfigured stereoisomers 1ꢀ3a' to later retention
times with decreasing chain lengths.
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
Determination of odor thresholds. The odor thresholds of the stereoisomers of 1ꢀ6
were determined via GC/O (Table 4). In total three panelists participated; except for
5b and 5b', each stereoisomer was evaluated by two assessors. Regarding the
variability of the panelists, there were a few cases in which high differences between
odor thresholds were observed, i.e. for 4a' (factor: 60) and 5a' (factor: 30) between
panelists 1 and 2 as well as for 2b' (factor: 30), 2a (factor: 22) and 2b (factor: 7)
between panelists 2 and 3. However, for most of the stereoisomers the individual
odor thresholds were either the same or differed up to a maximum of factors 3 to 5,
corresponding to approximately two dilution steps in the course of the AEDA. Figure
6 illustrates the odor threshold curves for the four stereoisomers of 4ꢀmercaptoꢀ2ꢀ
alkanols 1ꢀ6 based on the geometric means calculated from the assessments by the
respective panelists (except for 5b and 5b', for which only single sensory evaluations
were available).
1
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 40
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
The data set revealed two effects: (i) For all stereoisomers odor threshold minima
were observed for the mediumꢀchain homologs (C7ꢀC9). This is in good agreement
with the recently reported odor thresholds for the racemic mixtures of a homologous
22
series of 4ꢀmercaptoꢀ2ꢀalkanols. The individual curves for the diastereomers
[(2R,4S), (2S,4R)] and [(2S,4S), (2R,4R)] also fit very well to those reported for the
23
respective diastereomeric mixtures. This good agreement with sensory data
22,23
generated in a different laboratory and by trained panelists
supports the reliability
of the data obtained in the present study, despite the limited number of (trained)
panelists and the observed individual differences. Threshold minima for mediumꢀ
chain representatives have been observed within homologous series of various
22
mercaptoalkanols and for the enantiomers of the homologous series of 4ꢀmercaptoꢀ
2ꢀalkanones (minima at carbon chain lengths C8) and 4ꢀacetylthioꢀ2ꢀalkanones
1
8
(minima at carbon chain lengths C7/C8). (ii) Except for C5, the lowest odor
thresholds were determined for the (2R,4R)ꢀconfigured stereoisomers. The visual
differences seen in Figure 6 were verified by calculating the ratios of the geometrical
means of the thresholds determined for the (2R,4R)ꢀconfigured stereoisomers and
those of the second most intensive smelling stereoisomers. Particularly for the
homologs with chain lengths C8 and C9 these ratios were pronounced
(approximately 18).
Using the example of 4ꢀmercaptoꢀ2ꢀheptanol and its acetyl derivatives, it has been
shown that the odor thresholds were nearly independent from Sꢀ and/or Oꢀacetylation
24,28
if the structural prerequisite of (2R)ꢀconfiguration was fulfilled.
A high sensory
potency was also observed for the (4R)ꢀconfigured isomers of shortꢀ and mediumꢀ
chain homologs (C5ꢀC8) of 4ꢀmercaptoꢀ2ꢀalkanones (C5ꢀC10) taking into account the
18,28
reꢀinvestigated absolute configurations.
These data indicate that the (R)ꢀ
configurations might play key roles for the thresholds of these ßꢀmercaptoalcohols
1
8
ACS Paragon Plus Environment

Page 19 of 40
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
31
and ßꢀmercaptoketones. However, Sꢀacetylation of 4ꢀmercaptoꢀ2ꢀalkanones (C5ꢀ
C10) reduced the odor intensity, and the odor thresholds of the enantiomers were
highly impacted by the chain length.
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
Determination of odor qualities. Odor qualities of the stereoisomers of 1ꢀ6 were also
determined by panelists 1ꢀ3 via GC/O (Table 5). It is known that odor qualities may
show pronounced concentrationꢀdependent changes. Nevertheless, for the purpose
of comparison the sensory assessments were performed at constant amounts of
1.5 ng at the sniffing port for each stereoisomer. Considering the variability of the
dominant odor impressions (printed in bold type), it is obvious that none of the
stereoisomers showed consistent odor qualities for all homologs. For example, the
odor qualities of the (2R,4R)ꢀconfigured stereoisomers (1ꢀ6a') ranged from onion to
plasticꢀsolventꢀlike odor notes. The comparison of the odor descriptions for the
different chain lengths showed that for the C5 homolog 1 similar odor qualities such
as onion (1a and 1a') or sweat (1b and 1b') were detected for the enantiomeric pairs.
Different odor notes such as fruity (2a and 3a), onion (2a', 3a' and 3b), savory (2b
and 3b'), and green/herbꢀlike (2b') were obtained for the isomers of the chain lengths
C6 and C7. The odor descriptions of the stereoisomers of the chain lengths C8 to
C10 changed towards unpleasant chemical notes such as burnt, plastic, solvent, or
pungent. These data demonstrate that the chain length is the main factor determining
the variability in odor qualities of stereoisomers of 4ꢀmercaptoꢀ2ꢀalkanols. A similar
effect was reported for the odor properties of the diastereomers of 4ꢀmercaptoꢀ2ꢀ
22,23
alkanols (C5ꢀC10).
GC/O analysis resulted in onion and meaty notes for the C5
homologs, a fruityꢀlike odor reminiscent of grapefruit dominated the odor impressions
of the C6ꢀC9 homologs, and further elongation led to fatty and burnt odor notes. No
significant differences in the odor qualities have been described for the
diastereomeric pairs of the 4ꢀmercaptoꢀ2ꢀalkanols (C5ꢀC10), except for the C6
1
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 40
2
2,23
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
homolog, having either a more meaty or fruity odor note.
The odor qualities of the
enantiomers of the corresponding 4ꢀmercaptoꢀ2ꢀalkanones (C5ꢀC10) are highly
16,18
impacted by the stereochemistry.
GC/O analyses revealed fruity odor notes for
the (R)ꢀenantiomers and more unpleasant notes such as catty and sulfury for the (S)ꢀ
enantiomers, taking into account the results of the reinvestigation of the absolute
16,18,28
configurations of βꢀmercaptoalkanones.
As expected for compounds
possessing a 1,3ꢀoxygenꢀsulfur functionality, tropical, fruity, and vegetable odor notes
were obtained for stereoisomers of the investigated 4ꢀmercaptoꢀ2ꢀalkanols (Table 5).
However, specific notes such as meaty, savory, sweaty as well as chemical notes
were additionally perceived by the panelists (Table 5) and have also been reported in
7,21
literature to describe the odor qualities of 4ꢀmercaptoꢀ2ꢀalkanols.
In conclusion, the GC separation and the assignment of the absolute configurations
of the stereoisomers for the homologous series of 4ꢀmercaptoꢀ2ꢀalkanols with chain
lengths from C5 to C10 have been achieved. GC/O analyses revealed that the odor
thresholds of the stereoisomers of 4ꢀmercaptoꢀ2ꢀalkanols were highly impacted by
the stereochemistry as the lowest odor thresholds were determined for the (2R,4R)ꢀ
configured stereoisomers, except for C5. In contrast, the odor qualities were mainly
influenced by the chain length. The data provide another example for the impact of
both chain length and stereochemistry on the sensory properties of members of
homologous series of aroma compounds.
4
4
4
4
4
74
75
76
77
78
ASSOCIATED CONTENT
4
4
4
4
79
80
81
82
Supporting information: Experimental details of the syntheses of 4ꢀacetylthioꢀ2ꢀ
alkanones (C5ꢀC10). MS and NMR data of the synthesized 4ꢀmercaptoꢀ2ꢀalkanols
(C5ꢀC10). Temperature programs used for the GC separations of the 4ꢀmercaptoꢀ2ꢀ
alkanol stereoisomers. GC linear retention indices of the diastereomers of 4ꢀ
2
0
ACS Paragon Plus Environment

Page 21 of 40
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
83
mercaptoꢀ2ꢀalkanols on DBꢀWax and DBꢀ1. Separation factors α and resolutions R
s
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
of the enantiomeric pairs of 4ꢀmercaptoꢀ2ꢀalkanols. Geometric means of the
individual odor thresholds of the stereoisomers of 4ꢀmercaptoꢀ2ꢀalkanols determined
by the panelists via GC/O.
This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49ꢀ(0)8161ꢀ71ꢀ4250. Fax: +49ꢀ(0)8161ꢀ71ꢀ4259. Eꢀmail:
k.h.engel@wzw.tum.de.
Notes
The authors declare no competing financial interest.
Acknowledgement
We thank Christine Schwarz for recording the NMR spectra.
References
(1) Boelens, M. H.; van Gemert, L. J. Volatile characterꢀimpact sulfur
compounds and their sensory properties. Perfum. Flavor. 1993, 18, 29ꢀ39.
(2) Mussinan, C. J.; Keelan, M. E. Sulfur compounds in foods. In Sulfur
compounds in foods; Mussinan, C. J.; Keelan, M. E., Eds.; ACS Symposium
Series 564; American Chemical Society: Washington DC, 1994, pp. 1ꢀ6.
(3) Blank, I. Sensory relevance of volatile organic sulfur compounds in food. In
Heteroatomic aroma compounds; Reineccius, G. A.; Reineccius, T. A., Eds.; ACS
Symposium Series 826; American Chemical Society: Washington DC, 2002, pp.
25ꢀ53.
2
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 40
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
(4) Vermeulen, C.; Pellaud, J.; Gijs, L.; Collin, S. Combinatorial synthesis and
sensorial properties of polyfunctional thiols. J. Agric. Food Chem. 2001, 49, 5445ꢀ
5449.
(5) Vermeulen, C.; Gijs, L.; Collin, S. Sensorial contribution and formation
pathways of thiols in foods: a review. Food Rev. Int. 2005, 21, 69ꢀ137.
(6) Vermeulen, C.; Collin, S. Combinatorial synthesis and screening of novel
odorants such as polyfunctional thiols. Comb. Chem. High Throughput Screen.
2006, 9, 583ꢀ590.
(7) Vermeulen, C.; GuyotꢀDeclerck, C.; Collin, S. Combinatorial synthesis and
sensorial properties of mercapto primary alcohols and analogues. J. Agric. Food
Chem. 2003, 51, 3623ꢀ3628.
(8) 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, UK, 2002, pp. 202ꢀ226.
(9) Takoi, K.; Degueil, M.; Shinkaruk, S.; Thibon, C.; Maeda, K.; Ito, K.;
Bennetau, B.; Dubourdieu, D.; Tominaga, T. Identification and characteristics of
new volatile thiols derived from the hop (Humulus lupulus L.) cultivar Nelson
Sauvin. J. Agric. Food Chem. 2009, 57, 2493ꢀ2502.
(10) Gros, J.; Peeters, F.; Collin, S. Occurrence of odorant polyfunctional thiols
in beers hopped with different cultivars. First evidence of an Sꢀcysteine conjugate
in hop (Humulus lupulus L.). J. Agric. Food Chem. 2012, 60, 7805ꢀ7816.
(11) Widder, S.; Sabater Lüntzel, C.; Dittner, T.; Pickenhagen, W. 3ꢀMercaptoꢀ
2ꢀmethylpentanꢀ1ꢀol, a new powerful aroma compound. J. Agric. Food Chem.
2000, 48, 418ꢀ423.
2
2
ACS Paragon Plus Environment

Page 23 of 40
Journal of Agricultural and Food Chemistry
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
33
(12) Sabater Lüntzel, C.; Widder, S.; Vössing, T.; Pickenhagen, W.
Enantioselective syntheses and sensory properties of the 3ꢀmercaptoꢀ2ꢀ
methylpentanols. J. Agric. Food Chem. 2000, 48, 424ꢀ427.
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
(13) Engel, K.ꢀH.; Takeoka, G. Importance of chirality to flavor compounds;
Engel, K.ꢀH.; Takeoka, G., Eds.; ACS Symposium Series 1212; American
Chemical Society: Washington, DC, 2015.
(14) Engel, K.ꢀH.; Tressl, R. Identification of new sulfurꢀcontaining volatiles in
yellow passion fruits (Passiflora edulis f. flavicarpa). J. Agric. Food Chem. 1991,
39, 2249ꢀ2252.
(15) Weber, B.; Haag, H.ꢀP.; Mosandl, A. Stereoisomere Aromastoffe. LIX. 3ꢀ
Mercaptohexylꢀ und 3ꢀMethylthiohexylalkanoate – Struktur und Eigenschaften der
Enantiomeren. Z. Lebensm. Unters. Forsch. 1992, 195, 426ꢀ428.
(16) Wakabayashi, M.; Wakabayashi, H.; Nörenberg, S.; Reichardt, B.;
Kubota, K.; Engel, K.ꢀH. Analytical and sensory characterization of chiral 4ꢀ
mercaptoꢀ2ꢀalkanones, 4ꢀacetylthioꢀ2ꢀalkanones, and the corresponding alcohols.
In Recent advances in the analysis of food and flavors; Toth, S.; Mussinan, C.,
Eds.; ACS Symposium Series 1098; American Chemical Society: Washington,
DC, 2012, pp. 27ꢀ39.
(17) Wakabayashi, M.; Wakabayashi, H.; Eisenreich, W.; Morimitsu, Y.;
Kubota, K.; Engel, K.ꢀH. Determination of the absolute configurations of 4ꢀ
1
mercaptoꢀ2ꢀalkanones using the H NMR anisotropy method and enzymeꢀ
catalyzed kinetic resolution of the corresponding 4ꢀacetylthioꢀ2ꢀalkanones. Eur.
Food Res. Technol. 2011, 232, 753ꢀ760.
(18) Wakabayashi, M.; Wakabayashi, H.; Nörenberg, S.; Kubota, K.; Engel, K.ꢀ
H. Comparison of odor thresholds and odor qualities of the enantiomers of 4ꢀ
2
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 40
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
mercaptoꢀ2ꢀalkanones and 4ꢀacetylthioꢀ2ꢀalkanones. Flavour Fragrance J. 2015,
30, 171ꢀ178.
(19) Kleinhenz, J. K.; Kuo, C. J.; Harper, W. J. Evaluation of polyfunctional
thiol compounds in aged Cheddar cheese: identification. Milchwissenschaft 2006,
61, 300ꢀ304.
(20) Kleinhenz, J. K.; Kuo, C. J.; Harper, W. J. Evaluation of polyfunctional
thiol compounds in aged Cheddar cheese: estimated concentrations.
Milchwissenschaft 2007, 62, 181ꢀ183.
(21) Naef, R.; Velluz, A.; Jaquier, A. New volatile sulfurꢀcontaining constituents
in a simultaneous distillationꢀextraction extract of red bell peppers (Capsicum
annuum). J. Agric. Food Chem. 2008, 56, 517ꢀ527.
(22) Polster, J.; Schieberle, P. Structureꢀodor correlations in homologous
series of mercaptoalkanols. J. Agric. Food Chem. 2017, 65, 4329ꢀ4340.
(23) Polster, J. Dissertation, Technische Universität München: Freising,
Germany, 2012.
(24) Nörenberg, S.; Reichardt, B.; Andelfinger, V.; Eisenreich, W.; Engel, K.ꢀH.
Influence of the stereochemistry on the sensory properties of 4ꢀmercaptoꢀ2ꢀ
heptanol and its acetylꢀderivatives. J. Agric. Food Chem. 2013, 61, 2062ꢀ2069.
(25) Engel, K.ꢀH.; Nörenberg, S.; Reichardt, B.; Andelfinger, V. Impact of the
stereochemistry on the sensory properties of βꢀmercaptoalkanꢀ2ꢀol derivatives. In
Current topics in flavor chemistry & biology, Proceedings of the 10th Wartburg
Aroma Symposium, Eisenach; Hofmann, T.; Krautwurst, D.; Schieberle, P., Eds.;
Deutsche Forschungsanstalt für Lebensmittelchemie: Freising, Germany, 2014,
pp. 221ꢀ226.
(26) Ozeki, M.; Nishide, K.; Teraoka, F.; Node, M. Diastereoꢀ and
enantioselective synthesis of antiꢀ1,3ꢀmercapto alcohols from α,βꢀunsaturated
2
4
ACS Paragon Plus Environment

Page 25 of 40
Journal of Agricultural and Food Chemistry
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
84
ketones via tandem Michael additionꢀMPV reduction. Tetrahedron: Asymmetry
2004, 15, 895–907.
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
(27) Ghanem A.; AboulꢀEnein, H. Y. Application of lipases in kinetic resolution
of racemates. Chirality 2005, 17, 1ꢀ15.
(28) Kiske, C.; Nörenberg, S.; Ecker, M.; Ma, X.; Taniguchi, T.; Monde, K.;
Eisenreich, W.; Engel, K.ꢀH. Reinvestigation of the absolute configurations of
1
chiral βꢀmercaptoalkanones using vibrational circular dichroism and H NMR
analysis. J. Agric. Food Chem. 2016, 64, 8563ꢀ8571.
(29) van den Dool, H.; Kratz, P. D. A generalization of the retention index
system including linear temperature programmed gasꢀliquid partition
chromatography. J. Chromatogr. 1963, 11, 463ꢀ471.
(30) Dietrich, A.; Maas, B.; Karl, V.; Kreis, P.; Lehmann, D.; Weber, B.;
Mosandl, A. Stereoisomeric flavor compounds. Part LV: stereodifferentiation of
some chiral volatiles on heptakis(2,3ꢀdiꢀOꢀacetylꢀ6ꢀOꢀtertꢀbutyldimethylsilyl)ꢀβꢀ
cyclodextrin. J. High. Resol. Chromatogr. 1992, 15, 176ꢀ179.
(31) Takahisa, E.; Engel, K.ꢀH.
2,3ꢀDiꢀOꢀmethoxymethylꢀ6ꢀOꢀtertꢀ
butyldimethylsilylꢀβꢀcyclodextrin, useful stationary phase for gas
a
chromatographic separation of enantiomers. J. Chromatogr. A 2005, 1076, 148ꢀ
154.
(32) Ullrich, F.; Grosch, W. Identification of the most intense volatile flavour
compounds formed during autoxidation of linoleic acid. Z. Lebensm. Unters.
Forsch. 1987, 184, 277ꢀ282.
(33) Boelens, M. H.; van Gemert, L. J. Physicochemical parameters related to
organoleptic properties of flavour components. In Developments in food flavours;
Birch, G. G.; Lindley, M. G., Eds.; Elsevier applied science: London, U.K., 1986;
pp 23ꢀ49.
2
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 40
6
6
6
10
11
12
(34) Grosch, W. Detection of potent odorants in foods by aroma extract
dilution analysis. Trends Food Sci. Technol. 1993, 4, 68ꢀ73.
(35) Tamogami, S.; Awano, K.; Amaike, M.; Takagi, Y.; Kitahara, T.
6
6
6
13
14
15
Development of an efficient GLC system with a mixed chiral stationary phase and
its application to the separation of optical isomers. Flavour Fragrance J. 2001,16,
349ꢀ352.
2
6
ACS Paragon Plus Environment

Page 27 of 40
Journal of Agricultural and Food Chemistry
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
16
Figure Captions
Figure 1.
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
(A) Capillary gas chromatographic separation of the diastereomers of 4ꢀmercaptoꢀ2ꢀ
alkanols 1ꢀ6 on a DBꢀWax column; (B) separation of the stereoisomers of 4ꢀ
mercaptoꢀ2ꢀalkanols 1, 3ꢀ6 on heptakis(2,3ꢀdiꢀOꢀacetylꢀ6ꢀOꢀTBDMS)ꢀβꢀCD; (C)
separation of the stereoisomers of 4ꢀmercaptoꢀ2ꢀhexanol 2 on heptakis(2,3ꢀdiꢀOꢀ
acetylꢀ6ꢀOꢀTBDMS)ꢀβꢀCD; (D) separation of the stereoisomers of 4ꢀmercaptoꢀ2ꢀ
hexanol 2 on heptakis(2,3ꢀdiꢀOꢀmethoxymethylꢀ6ꢀOꢀTBDMS)ꢀβꢀCD.
Figure 2.
1
H NMR data of synthesized (A) 4ꢀmercaptoꢀ2ꢀheptanol 3, (B) 4ꢀmercaptoꢀ2ꢀpentanol
1, and (C) 4ꢀmercaptoꢀ2ꢀdecanol 6.
Figure 3.
Approach employed to assign the GC order of elution of the stereoisomers of 4ꢀ
mercaptoꢀ2ꢀalkanols, shown for 4ꢀmercaptoꢀ2ꢀpentanol 1 as example.
Figure 4.
6
6
6
34
35
36
Structures of synthesized diastereomeric (S)ꢀMαNP thioesters of (R)ꢀ and (S)ꢀ4ꢀ
mercaptoꢀ2ꢀoctanone 16.
6
6
6
6
6
37
38
39
40
41
Figure 5.
®
GC separation of the stereoisomers of 4ꢀmercaptoꢀ2ꢀalkanols 1ꢀ6 on Chiramix .
2
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 40
6
6
6
6
42
43
44
45
Figure 6.
Geometric means of the odor thresholds of the stereoisomers of 4ꢀmercaptoꢀ2ꢀ
alkanols (except for (2R,4S) and (2S,4R) of C9, for which only single sensory
evaluations were available).
2
8
ACS Paragon Plus Environment

Page 29 of 40
Journal of Agricultural and Food Chemistry
Table 1. Structures of the Stereoisomers of 4ꢀMercaptoꢀ2ꢀalkanols 1ꢀ6 with Chain
Lengths from C5 to C10
4
ꢀmercaptoꢀ2ꢀalkanols
no.
chain
R
structure
length
anti
syn
1
2
C5
C6
ꢀCH3
SH OH
SH OH
ꢀCH ꢀCH3
2
R
R
3
4
5
6
C7
C8
ꢀ(CH ) ꢀCH
2
2
3
3
3
3
a: (2S,4S)
b: (2R,4S)
ꢀ(CH ) ꢀCH
2
3
SH OH
R
a': (2R,4R)
SH OH
C9
ꢀ(CH ) ꢀCH
2 4
R
C10
ꢀ(CH ) ꢀCH
b': (2S,4R)
2
5
ACS Paragon Plus Environment