Volatile organic sulfur-containing constituents in Poncirus trifoliata (L) Raf. (Rutaceae)

Article abstract of DOI:10.1021/jf063453h

During our screening of plant materials to find new natural fragrance and flavor ingredients, we discovered two series of 3-sulfanylalkyl alkanoates in a peel extract of fruits of wild-growing Poncirus trifoliata (L) Raf. (Rutaceae), a species closely related to Citrus. The two series belong to alkanoates of 3-methyl-3-sulfanylbutan-1-ol and 3-sulfanylhexan-1-ol, respectively, and thus are members of a family of natural molecules having in common a 1,3-positioned O,S moiety. The alkanoate residues comprise all even-numbered saturated fatty acids from C2 (acetate) to C18 (octadecanoate). Among the 20 sulfur-containing compounds identified, 14 are described for the first time as naturally occurring in a botanical species. Several cysteine-S-conjugates were synthesized as hypothetical precursors of the new volatile sulfur-containing constituents, where after S-(3-hydroxy-1,1-dimethylpropyl)-L-cysteine, S-[3-(acetyloxy)-1,1- dimethylpropyl]-L-cysteine, and S-[1-(2-hydroxyethyl)butyl]-L-cysteine were identified in the fruit peel. No cysteine-S-conjugates were detected in the fruit juice.

Full text of DOI:10.1021/jf063453h

J. Agric. Food Chem. 2007, 55, 4511−4517 4511
Volatile Organic Sulfur-Containing Constituents in
Poncirus trifoliata (L.) Raf. (Rutaceae)
CHRISTIAN STARKENMANN,* YVAN NICLASS, AND SINA ESCHER
Firmenich SA, Corporate R&D Division, P.O. Box 239, CH-1211 Geneva 8, Switzerland
During our screening of plant materials to find new natural fragrance and flavor ingredients, we
discovered two series of 3-sulfanylalkyl alkanoates in a peel extract of fruits of wild-growing Poncirus
trifoliata (L.) Raf. (Rutaceae), a species closely related to Citrus. The two series belong to alkanoates
of 3-methyl-3-sulfanylbutan-1-ol and 3-sulfanylhexan-1-ol, respectively, and thus are members of a
family of natural molecules having in common a 1,3-positioned O,S moiety. The alkanoate residues
comprise all even-numbered saturated fatty acids from C2 (acetate) to C18 (octadecanoate). Among
the 20 sulfur-containing compounds identified, 14 are described for the first time as naturally occurring
in a botanical species. Several cysteine-S-conjugates were synthesized as hypothetical precursors
of the new volatile sulfur-containing constituents, where after S-(3-hydroxy-1,1-dimethylpropyl)-L-
cysteine, S-[3-(acetyloxy)-1,1-dimethylpropyl]-L-cysteine, and S-[1-(2-hydroxyethyl)butyl]-L-cysteine
were identified in the fruit peel. No cysteine-S-conjugates were detected in the fruit juice.
KEYWORDS: Poncirus trifoliata (L.) Raf.; 3-methyl-3-sulfanylbutan-1-ol; 3-methyl-3-sulfanylhexan-1-ol;
3-methyl-3-sulfanylbutyl alkanoates; 3-sulfanylhexyl alkanoates; cysteine-S-conjugates; natural occur-
rence; fragrance and flavor chemical
INTRODUCTION
Flash Chromatography (FC). Silica gel 60, 35-70 µm (sodium
dodecyl sulfate), was used for normal-phase FC, and LiChroprep RP-
18, 40-63 µm (Merck, catalog no. 1.1390.0250), was used for reversed-
phase FC.
Poncirus trifoliata (L.) Raf. (Rutaceae; previous classification
Citrus trifolata L.) is native of China and closely related to
Citrus species. It is a wild-growing, up to 2 m high shrub armed
with big spines, and it survives temperatures as low as -20
°C. Many commercially important Citrus varieties are grown
on Poncirus rootstocks, which provide resistance to the cold
and raise the quality of the citrus fruit. The strong odor of P.
trifoliata fruits reminds many of jasmine flowers and over ripe,
rotten, sulfury mangos and is very different from the fresh odor
of a lemon or an orange fruit. The essential oil of P. trifoliata
peel is composed of monoterpene hydrocarbons such as
â-pinene, myrcene, limonene, camphene, γ-terpinene, and
p-cymene, as well as oxygenated compounds and sesquiterpenes.
However, the published data (1, 2) do not give enough
information to understand the very peculiar odor tonalities. This
prompted us to isolate and analyze the volatile sulfur-containing
components of P. trifoliata fruits. In addition to the chemical
and organoleptic analyses, this study discusses possible precur-
sors of the new volatile sulfur-containing compounds identified.
Gas Chromatography/Electron Impact-Mass Spectrometry (GC/
EI-MS). An Agilent-GC-6890 system connected to an Agilent-MSD-
5973 quadrupole mass spectrometer was operated at an electron energy
of ca. 70 eV. Helium was the carrier gas at a constant flow rate of 0.7
mL/min. The spectra are listed as follows: fragment ions m/z (relative
intensity). Separations were performed on fused-silica capillary columns,
either coated with SPB-1 (apolar; 0.25 mm i.d. × 30 m; 0.25 µm d.f.;
Supelco) or coated with Supelcowax10 (polar; SPWax; 0.25 mm i.d.
× 30 m; 0.25 µm d.f.; Supelco). The standard oven program was the
following: 50 °C for 5 min, then 50-240 °C at 5°/min, and hold at
240 °C. Retention indices (I) were calculated by linear extrapolation
from the retention times (tR; in min) of the analytes and the two closest
alkanes eluting just before and just after the analyte. Injection modes
were adapted accordingly to the quality of the sample; synthetic
compounds were injected with a 50 to 1 split ratio, and natural extracts
were injected in splitless mode.
Chiral GC/Atomic Emission Detector (AED). An Agilent-GC-
6890 system was coupled to an AED (Jass, Germany). The chiral
column was a fused-silica capillary column DiMePeBeta-1701 (0.25
mm i.d. × 10 m; 0.25 µm d.f.; Mega, from Brechbu¨hler, Switzerland).
The oven temperature was programmed from 50 to 200 °C at 1 °C/
min. The enantiomeric ratios were measured at the S-specific emission
detection wavelength 181 nm. The elution order tR (R) < tR (S) of the
enantiomers (R)/(S)-2 and (R)/(S)-4 was confirmed by coinjections of
authentic reference samples (3).
EXPERIMENTAL PROCEDURES
General. Commercially available reagents and solvents of adequate
quality were used without further purification. Reactions were per-
formed in standard glassware. Yields were not optimized. Organic
extracts were washed to neutrality with aqueous H2SO4 solution and/
or NaHCO3 and NaCl solutions, dried (Na2SO4), and evaporated.
GC/MS-Olfaction (GC/MS-O). The GC part of a GC/EI-MS
system as described above was equipped with an effluent splitter and
a heated sniffing port. The apolar fused-silica capillary column was
* To whom correspondence should be addressed. Tel: +41 22 780 34
77. Fax: +41 22 780 33 34. E-mail: christian.starkenmann@firmenich.com.
10.1021/jf063453h CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/08/2007

4512 J. Agric. Food Chem., Vol. 55, No. 11, 2007
Starkenmann et al.
coated with SPB-1 (0.53 mm i.d. × 30 m; 1.0 µm d.f.; Supelco). The
polar column was a SPWax (0.53 mm i.d. × 30 m; 1.0 µm d.f.;
Supelco).
Preparation of Extracts for the Analysis of Cys-S-Conjugates
in the Fruit Peel. The peel material, which was recovered from the
above juice preparation (300 g), was homogenized in a kitchen aid in
the presence of ice, and S-[1-(2-hydroxyethyl)-1-methylbutyl]-L-cysteine
(26) (6) (1 mg) was added as an internal standard. The slurry was
centrifuged (1 h, 4000 rpm/min, 10 °C). The solid was removed, and
the liquid was lyophilized to give 20 g. This material was rediluted
with water and filtered on SiO2-RP18 as described above. The internal
standard 26 was in the fraction (2.3 g) eluted with water/ethanol 7:3.
Further purification of this fraction was performed on an ion exchange
column (10 g, Dowex 50WX8, Aldrich, catalog no. 21751-4). The
elution was performed with an aqueous 0.3-1.5 M ammonia gradient.
The internal standard 26 as well as 21, 22, and 24 were detected in the
fraction eluted with 1.2 M ammonia (20 mg) (HPLC/ESI+-MS).
Quantification of 21 and 24, as well as the determination of the recovery
factor of 26, was measured in this fraction.
HPLC/MS. An Agilent 1100 LC-MS system equipped with a
B1312A binary pump was used. The separations were performed on a
Zorbax SB-C18 column (rapid resolution HT; 2.1 mm i.d. × 50 mm;
1.8 µm particle size; Agilent, catalog no. 822700-902). The elution
solvents were CH3CN and water containing 0.02% formic acid. The
gradient profile was started at 5% CH3CN and held for 0.5 min and
then increased to 100% CH3CN in 5 min. The flow rate was 0.3 mL/
min. The retention times (tR) are expressed in minutes. The mass
spectrometer was a Thermo Finnigan LCQ ion-trap spectrometer, with
an electrospray ion source operated in positive mode (ESI+). The spray
voltage was 4.5 kV. The capillary temperature was heated at 250 °C.
The sheath gas was nitrogen at a flow rate of 70 (Finnigan arbitrary
units). The auxiliary gas was also nitrogen at a flow rate of 5 (Finnigan
arbitrary units). Analyses were performed in selected ion monitoring
(SIM) mode according to [M + 1]+ of expected cysteine-S-conjugates.
To achieve high sensitivity and selectivity, quantitative data were
recorded as follows: The [M + 1]+ ion of each compound was
fragmented in the ion trap by applying 30% normalized collision energy.
The most intense fragment was extracted, and the corresponding peak
was integrated using Xcalibur software. Compound 26 was fragmented
at 236, and ion 122 was extracted. Compound 21: fragmentation at
208, extraction of ion 122. Compound 22: fragmentation at 250,
extraction of ion 233. Compound 24: fragmentation at 222, extraction
of ion 205.
Quantification of the Cys-S-Conjugates. The calibration curve was
established by replicate injections of 5 µL of standard solutions
containing 21, 22, 24, and 26, each at 1, 5, 10, 20, and 50 µg/mL
(HPLC/ESI+-MS). The precursors were then quantified in the purified
peel extract. The recovery of the internal standard 26 was only 0.033
( 0.004 mg instead of 1 mg added to 300 g of peel.
General Procedure for the Synthesis of 3-Sulfanylalkyl Esters
as Reference Compounds. The 3-methyl-3-sulfanylbutan-1-ol (1)
(Firmenich, catalog no. 958055) or 3-sulfanylhexan-1-ol (2) (Alfa
Aesus, catalog no. 821965) (10 mmol) was diluted in CH2Cl2 (10 mL).
The desired acid chloride (10 mmol; diluted in 2 mL of CH2Cl2) was
added dropwise. After stirring at 22 °C for 18 h, the reaction mixture
was concentrated under partial vacuum to remove CH2Cl2. The crude
product was submitted to FC on SiO2 (250 g). The elution was done
with cyclohexane/diethyl ether 95:5. The reaction yield after purification
is given below for each preparation. The purified esters were analyzed
by 1H NMR, 13C NMR, and GC/MS. The retention indices were
calculated after injection on SPB-1 and SPWax GC columns.
3-Methyl-3-sulfanylbutyl Acetate (3). Yield: 1.45 g (89.5%). ISPB-1
1H and 13C NMR Spectra. The NMR spectra were recorded on a
Bruker-Avance-500 spectrometer at 500.13 and 125.76 MHz, respec-
tively. If not stated otherwise, the solvent was CDCl3. δ Values are in
ppm downfield from (CH3)4Si () 0 ppm). The cysteine-S-conjugates
were measured in D2O with sodium 3-(trimethylsilyl)tetradeuterio-
propionate as an internal standard. The assignments by correlation
spectroscopy, heteronuclear single quantum correlation, and hetero-
nuclear multiple quantum coherence experiments were performed with
standard Bruker software (XWINNMR 3.1).
1
1080; ISPWax 1566. H NMR: 4.29 (t, J ) 7.1 Hz, 2H), 1.98 (s, 3H),
1.90 (t, J ) 7.1 Hz, 2H), 1.69 (br, SH), 1.42 (s, 6H). 13C NMR: 180.0
(s), 62.0 (t), 44.4 (t), 42.9 (s), 33.1 (2q), 21.1(q). MS: 162 (2, M+),
102 (40), 87 (12), 75 (15), 69 (100), 59 (9), 43 (60), 41 (35).
3-Methyl-3-sulfanylbutyl Butanoate (5). Yield: 1.65 g (86.8%). ISPB-1
1266; ISPWax 1725. 1H NMR: 4.28 (t, J ) 7.1 Hz, 2H), 2.29 (t, J ) 7.1
Hz, 2H), 1.93 (t, J ) 7.1 Hz, 2H), 1.70 (br, SH), 1.59-1.68 (m, 2H),
1.42 (s, 6H), 0.95 (t, J ) 7.1 Hz, 3H). 13C NMR: 173.5 (s), 61.8 (t),
44.4 (t), 42.9 (s), 36.3 (t), 33.1 (2q), 18.4 (t), 13.7 (q). MS: 190
(2, M+), 102 (40), 87 (12), 75 (10), 71 (48), 69 (100), 59 (9), 43 (35),
41 (35).
Organic Extract of P. trifoliata (L.) Raf. Fruit Peel. In October
2005, 80 fruits of P. trifoliata (L.) Raf. were collected in the botanical
garden of Geneva, Switzerland (registry CJBG 19801106). The outer
peel layer, which corresponded mostly to the flavedo, was removed
with a kitchen rasp, and the material (300 g) was macerated in CH2Cl2
(500 mL) at 4 °C for 16 h. The solvent extract was decanted, dried
(Na2SO4), and concentrated to 67.9 g in a Vigreux apparatus at 40 °C.
This extract was used for further analysis. The same operation was
repeated with 40 fruits coming from a private garden in the Geneva
area. The analytical results were comparable.
3-Methyl-3-sulfanylbutyl Hexanoate (7). Yield: 1.7 g (78.0%). ISPB-1
1468; ISPWax 1920. 1H NMR: 4.28 (t, J ) 7.1 Hz, 2H), 2.29 (t, J ) 7.1
Hz, 2H), 1.94 (t, J ) 7.1 Hz, 2H), 1.75 (br, SH), 1.58-1.67 (m, 2H),
1.42 (s, 6H), 1.24-1.35 (m, 4H), 0.90 (t, J ) 7.1 Hz, 3H). 13C NMR:
173.8 (s), 61.8 (t), 44.4 (t), 42.9 (s), 34.4 (t), 33.1 (2q), 31.3 (t), 24.6
(t), 22.3 (t), 13.9 (q). MS: 218 (2, M+), 102 (45), 99 (32), 87 (12), 71
(22), 69 (100), 59 (9), 55 (12), 43 (28), 41 (31).
Isolation of the Fraction Containing Compounds with Free SH
Groups (“Thiol” Fraction). To a 10 g aliquot of the organic extract
was added octane-1-thiol (0.01 mg) as an internal standard, and the
spiked extract was submitted to affinity chromatography on Affi-Gel
501 (4, 5). The thiol fraction was carefully concentrated in a Vigreux
apparatus to 0.1 mL and immediately submitted to GC/EI-MS analyses
on SPB-1 and SPWax columns (injection volume, 1 µL; closed split).
The natural thiol compounds of interest were confirmed by comparing
their MS and retention indices (I) with those of authentic reference
compounds on both polar and apolar columns. Quantitative data are
only an approximation based on the comparison with the GC peak area
of the internal standard.
Preparation of Extracts for the Analysis of Cys-S-Conjugates
in the Fruit Juice. To a juice (160 g), which had been prepared from
20 fruits using a kitchen juicer, was added S-[1-(2-hydroxyethyl)-1-
methylbutyl]-L-cysteine (26) (6) (1 mg) as an internal standard and
lyophilized to give 21 g. This solid (1 g) was diluted in 0.02% aqueous
formic acid (10 mL), centrifuged, and filtered on an Acrodisk (0.45
µm pore size). A 5 µL aliquot of the filtrate was injected to HPLC/
ESI+-MS. Only 26 was detected. The remaining 20 g was rediluted in
water (100 mL) and filtered on SiO2-RP18 (column 7 cm i.d. × 5 cm;
conditioned with ethanol and then rinsed with 10 volumes of water).
Elution was performed with five 200 mL portions (water, water/ethanol
9:1, 8:2, 7:3, and 1:1, respectively). Except for 26, present in the fraction
7:3, no other Cys-S-conjugates were detected.
3-Methyl-3-sulfanylbutyl Octanoate (9). Yield: 2.35 g (95.5%). ISPB-1
1678; ISPWax 2130. 1H NMR: 4.28 (t, J ) 7.1 Hz, 2H), 2.29 (t, J ) 7.1
Hz, 2H), 1.94 (t, J ) 7.1 Hz, 2H), 1.76 (br, SH), 1.57-1.66 (m, 2H),
1.43 (s, 6H), 1.22-1.36 (m, 8H), 0.88 (t, J ) 7.1 Hz, 3H). 13C NMR:
173.8 (s), 61.8 (t), 44.4 (t), 42.9 (s), 34.4 (t), 33.1 (2q), 31.6 (t), 29.1
(t), 28.9 (t), 24.9 (t), 22.5 (t), 14.0 (q). MS: 246 (2, M+), 127 (28),
102 (58), 87 (12), 71 (8), 69 (100), 57 (40), 55 (15), 43 (15), 41 (32).
3-Methyl-3-sulfanylbutyl Decanoate (11). Yield: 2.45 g (89.4%).
1
ISPB-1 1876; ISPWax 2342. H NMR: 4.28 (t, J ) 7.1 Hz, 2H), 2.29 (t,
J ) 7.1 Hz, 2H), 1.94 (t, J ) 7.1 Hz, 2H), 1.76 (br, SH), 1.57-1.66
(m, 2H), 1.43 (s, 6H), 1.22-1.36 (m, 12H), 0.88 (t, J ) 7.1, 3H). 13
C
NMR: 173.8 (s), 61.8 (t), 44.4 (t), 42.9 (s), 34.4 (t), 33.1 (2q), 31.9
(t), 29.4 (t), 29.3 (2t), 29.1 (t), 24.9 (t), 22.7 (t), 14.1 (q). MS: 274 (2,
M+), 155 (22), 102 (68), 87 (12), 71 (18), 69 (100), 68 (21), 57 (17),
55 (19), 43 (20), 41 (31).
3-Methyl-3-sulfanylbutyl Dodecanoate (13). Yield: 2.0 g (66.2%).
1
ISPB-1 2082; ISPWax 2557. H NMR: 4.28 (t, J ) 7.1 Hz, 2H), 2.29
(t, J ) 7.1 Hz, 2H), 1.94 (t, J ) 7.1 Hz, 2H), 1.76 (br, SH), 1.57-1.66

Constituents in Poncirus trifoliata (L.) Raf. (Rutaceae)
J. Agric. Food Chem., Vol. 55, No. 11, 2007 4513
(m, 2H), 1.43 (s, 6H), 1.22-1.36 (m, 16H), 0.88 (t, J ) 7.1 Hz, 3H).
13C NMR: 173.8 (s), 61.8 (t), 44.4 (t), 42.9 (s), 34.4 (t), 33.1 (2q),
31.9 (t), 29.6-29.2 (6t), 25.0 (t), 22.7 (t), 14.1 (q). MS: 183 (18), 102
(82), 87 (12), 69 (100), 68 (30), 57 (22), 55 (19), 43 (22), 41 (31).
3-Methyl-3-sulfanylbutyl Tetradecanoate (15). Yield: 3.1 g (94.0%).
(s), 62.0 (t), 41.2 (t), 37.9 (t), 37.3 (d), 34.3 (t), 31.9 (t), 29.1-29.7
(12t), 25.0 (t), 22.6 (t), 20.1 (t), 14.1 (q), 13.7 (q). MS: 285 (6), 267
(10), 116 (100), 101 (15), 88 (45), 87 (20), 83 (38), 71 (12), 57 (16),
55 (22), 43 (18), 41 (11).
General Procedure for the Synthesis of Cys-S-Conjugates as
Reference Compounds. Preparation of S-(3-Hydroxy-1,1-dimethyl-
propyl)-L-cysteine (21). N-Boc-cysteine (2 g, 9 mmol) was added to
3-methylbut-2-enal (1.21 g, 14.4 mmol) in CH3CN (3 mL) in the
presence of Cs2CO3 (1.5 g, 4.6 mmol). The mixture was stirred at 22
°C for 16 h. NaBH4 (304 mg, 8 mmol) was added portion wise. After
1 h, the reaction was diluted with water, acidified to pH 1 with 2 M
HCl, and extracted with ethyl acetate. The water phase was lyophilized
and treated with 6 mL of concentrated HCl for 10 min. Water (20 mL)
was added, and the mixture was loaded on a Dowex 50WX8 column
(40 g, H+ form). The resin was washed to neutrality with water (200
mL) and then eluted with an aqueous 0.3-2.1 M ammonia gradient.
Compound 21 was obtained in 40% yield (750 mg). HPLC/ESI+-MS:
1
ISPB-1 2287; ISPWax 2770. H NMR: 4.28 (t, J ) 7.1 Hz, 2H), 2.29 (t,
J ) 7.1 Hz, 2H), 1.94 (t, J ) 7.1 Hz, 2H), 1.76 (br, SH), 1.57-1.66
(m, 2H), 1.43 (s, 6H), 1.22-1.36 (m, 20H), 0.89 (t, J ) 7.1 Hz, 3H).
13C NMR: 173.8 (s), 61.8 (t), 44.4 (t), 42.9 (s), 34.4 (t), 33.1 (2q),
31.9 (t), 29.7-29.2 (8t), 25.0 (t), 22.7 (t), 14.1 (q). MS: 211 (18), 102
(92), 87 (15), 69 (100), 68 (30), 57 (22), 55 (20), 43 (22), 41 (34).
3-Methyl-3-sulfanylbutyl Hexadecanoate (17). Yield: 3.4 g (95.0%).
1
ISPB-1 2492; ISPWax > 2800. H NMR: 4.28 (t, J ) 7.1 Hz, 2H), 2.29
(t, J ) 7.1 Hz, 2H), 1.94 (t, J ) 7.1 Hz, 2H), 1.76 (br, SH), 1.57-1.66
(m, 2H), 1.43 (s, 6H), 1.22-1.36 (m, 24H), 0.89 (t, J ) 7.1 Hz, 3H).
13C NMR: 173.8 (s), 61.8 (t), 44.4 (t), 42.9 (s), 34.4 (t), 33.1 (2q),
31.9 (t), 29.7-29.2 (10t), 25.0 (t), 22.7 (t), 14.1 (q). MS: 340 (2, M+
-18), 239 (18), 102 (100), 87 (15), 69 (95), 68 (24), 57 (22), 55 (20),
43 (22), 41 (29).
1
tR 1.17 min; [M + 1]+ 208. H NMR (D2O): 3.92 (dd, J ) 4.5 and 8
Hz, 1H), 3.77 (t, J ) 7 Hz, 2H), 3.14 (dd, J ) 4.5 and 14 Hz, 1H),
3.02 (dd, J ) 8 and 14 Hz, 1H), 1.85 (t, J ) 8 Hz, 2H), 1.44 (s, 6H).
13C NMR (D2O): 175.6 (s), 61.4 (t), 57.1 (d), 47.8 (s), 45.9 (t), 31.8
(t), 31.0 (2q).
3-Methyl-3-sulfanylbutyl Octadecanoate (19). Yield: 3.8 g (98.4%).
I
SPB-1 2696; ISPWax >2800. 1H NMR: 4.28 (t, J ) 7.1 Hz, 2H), 2.29 (t,
J ) 7.1 Hz, 2H), 1.94 (t, J ) 7.1 Hz, 2H), 1.76 (br, SH), 1.57-1.66
(m, 2H), 1.43 (s, 6H), 1.22-1.36 (m, 28H), 0.89 (t, J ) 7.1 Hz, 3H).
13C NMR: 173.8 (s), 61.8 (t), 44.4 (t), 42.9 (s), 34.4 (t), 33.1 (2q),
31.9 (t), 29.7-29.2 (12t), 25.0 (t), 22.7 (t), 14.1 (q). MS: 368 (2, M+
-18), 281 (8), 267 (12), 207 (18), 102 (100), 87 (15), 69 (78), 68
(12), 57 (18), 55 (17), 43 (18), 41 (25).
Preparation of S-[3-(Acetyloxy)-1,1-dimethylpropyl]-L-cysteine (22).
Compound N-Boc-21 (6.1 g, 20 mmol) was diluted in pyridine (20
mL) and treated with acetic anhydride (10 mL). After 4 h of stirring at
room temperature, toluene was added (100 mL), and the solvent was
distilled under vacuum. To the crude mixture, CH2Cl2 (20 mL) and
trifluoroacetic acid (TFA, 40 mL) were added. After 1 h of stirring at
22 °C, toluene (100 mL) was added for the azeotropic distillation. The
dried product was rediluted in water and submitted to FC on SiO2-
RP18 (130 g). The elution was performed by using 200 mL portions
of water and then water/ethanol mixtures containing 10-50% ethanol.
The solvent was distilled off. Compound 22 was obtained pure (730
mg, yield 15%). Note: During the N-Boc deprotection, the ester was
3-Sulfanylhexyl Octanoate (10). Yield: 2.4 g (93.8%). ISPB-1 1799;
1
ISPWax 2271. H NMR: 4.22-4.28 (m, 2H), 2.82-2.92 (m, 1H), 2.29
(t, J ) 7.1 Hz, 2H), 1.97-2.06 (m, 1H), 1.24-1.79 (m, 16H), 0.93 (t,
J ) 7.1 Hz, 2H), 0.89 (t, J ) 7.1 Hz, 2H). 13C NMR: 173.7 (s), 62.0
(t), 41.2 (t), 37.9 (t), 37.3 (d), 34.3 (t), 31.7 (t), 29.1 (t), 28.9 (t), 25.0
(t), 22.6 (t), 20.1 (t), 14.1 (q), 13.7 (q). MS: 242 (2, M+ -18), 145
(10), 127 (48), 116 (100), 101 (30), 88 (90), 87 (40), 83 (68), 73 (20),
57 (68), 55 (59), 43 (22), 41 (30).
1
partially cleaved. HPLC/ESI+-MS: tR 4.38 min; [M + 1]+ 250. H
NMR (D2O): 4.26 (t, J ) 7 Hz, 2H), 3.92 (dd, J ) 4 and 8 Hz, 1H),
3.13 (dd, J ) 4 and 14 Hz, 1H), 3.03 (dd, J ) 8 and 14 Hz, 1H), 2.08
(s, 3H), 1.94 (t, J ) 8 Hz, 2H), 1.44 (s, 6H). 13C NMR (D2O): 177.5
(s), 175.4 (s), 65.4 (t), 57.1 (d), 47.8 (s), 42.1 (t), 31.2 (t), 31.0 (2q),
23.5 (q).
3-Sulfanylhexyl Decanoate (12). Yield: 2.6 g (89.9%). ISPB-1 2003;
ISPWax 2480. H NMR: 4.22-4.28 (m, 2H), 2.82-2.92 (m, 1H), 2.29
1
(t, J ) 7.1 Hz, 3H), 1.97-2.06 (m, 1H), 1.24-1.79 (m, 20H), 0.93 (t,
J ) 7.1 Hz, 3H), 0.88 (t, J ) 7.1 Hz, 3H). 13C NMR: 173.7 (s), 62.0
(t), 41.2 (t), 37.9 (t), 37.3 (d), 34.3 (t), 31.9 (t), 29.1-29.4 (4t), 25.0
(t), 22.6 (t), 20.1 (t), 14.1 (q), 13.7 (q). MS: 288 (2, M+), 173 (8), 155
(25), 116 (100), 101 (20), 88 (75), 87 (35), 83 (68), 71 (20), 57 (18),
55 (45), 43 (28), 41 (23).
Preparation of S-[3-(Butanoyloxy)-1,1-dimethylpropyl]-L-cysteine
(23). In the same way as described for 22, compound 23 (1227 mg,
1
43%) was obtained. HPLC/ESI+-MS: tR 5.30 min; [M + 1]+ 278. H
NMR (D2O): 4.28 (t, J ) 7 Hz, 2H), 4.01 (dd, J ) 4 and 8 Hz, 1H),
3.17 (dd, J ) 4 and 14 Hz, 1H), 3.06 (dd, J ) 8 and 14 Hz, 1H), 2.37
(t, J ) 7 Hz, 2H), 1.93-1.96 (m, 2H), 1.52-1.64 (m, 2H), 1.44 (s,
6H), 0.90 (t, J ) 7 Hz, 3H). 13C NMR (D2O): 180.0 (s), 174.8 (s),
65.2 (t), 56.7 (d), 48.0 (s), 42.2 (t), 38.9 (t), 31.0 (t + 2q), 20.9 (t),
15.8 (q).
3-Sulfanylhexyl Dodecanoate (14). Yield: 2.9 g (93.7%). ISPB-1 2208;
ISPWax 2696. H NMR: 4.22-4.28 (m, 2H), 2.82-2.92 (m, 1H), 2.29
1
(t, J ) 7.1 Hz, 3H), 1.97-2.06 (m, 1H), 1.24-1.79 (m, 24H), 0.93 (t,
J ) 7.1 Hz, 3H), 0.88 (t, J ) 7.1 Hz, 3H). 13C NMR: 173.7 (s), 62.0
(t), 41.2 (t), 37.9 (t), 37.3 (d), 34.3 (t), 31.9 (t), 29.1-29.6 (6t), 25.0
(t), 22.6 (t), 20.1 (t), 14.1 (q), 13.7 (q). MS: 201 (10), 183 (20), 116
(100), 101 (20), 88 (70), 87 (35), 83 (54), 71 (12), 57 (22), 55 (40), 43
(22), 41 (18).
Preparation of S-[1-(2-Hydroxyethyl)butyl]-L-cysteine (24). Com-
pound 24 was synthesized in the same way as was described for 22
but purified as a side product during of the N-Boc deprotection to
3-Sulfanylhexyl Tetradodecanoate (16). Yield: 2.6 g (75.9%). ISPB-1
1
prepare 25. HPLC/ESI+-MS: tR 5.27 min; [M + 1]+ 222. H NMR
1
2412; ISPWax >2800. H NMR: 4.22-4.28 (m, 2H), 2.82-2.92 (m,
(D2O): 3.98-4.02 (m, 1H), 3.67-3.77 (m, 2H), 3.00-3.18 (m, 2H),
2.82-2.93 (m, 1H), 1.82-1.91 (m, 1H), 1.70-1.78 (m, 1H), 1.56-
1.61 (m, 2H), 1.38-1.46 (m, 2H), 0.90 (t, J ) 7 Hz, 3H). 13C NMR
(D2O): 175.1 (s), 62.1 (t), 56.7 (d), 45.5 (d), 39.4 (t), 39.0 (t), 33.1 (t),
22.1 (t), 16.1 (q).
Preparation of S-{1-[2-(Acetyloxy)ethyl]butyl}-L-cysteine (25). Com-
pound 25 was prepared in the same way as was described for 22 (900
mg, yield 16%). HPLC/ESI+-MS: tR 6.74 and 6.77 min (two diaste-
reoisomers); [M + 1]+ 264. 1H NMR (D2O): 4.22-4.28 (m, 2H), 3.98-
4.02 (m, 1H), 3.00-3.18 (m, 2H), 2.88-2.93 (m, 1H), 2.10 (s, 3H),
1.97-2.02 (m, 1H), 1.80-1.90 (m, 1H), 1.56-1.61 (m, 2H), 1.39-
1.46 (m, 2H), 0.90 (t, J ) 7 Hz, 3H). 13C NMR (D2O): 177.6 (s),
175.4 (s), 65.9 (t), 56.9 (d), 45.5 (d), 39.2 (t), 35.3 (t), 33.4 (t), 23.4
(q), 22.1 (t), 16.0 (q).
1H), 2.29 (t, J ) 7.1 Hz, 2H), 1.97-2.06 (m, 1H), 1.24-1.79 (m, 28H),
0.93 (t, J ) 7.1 Hz, 3H), 0.88 (t, J ) 7.1 Hz, 3H). 13C NMR: 173.7
(s), 62.0 (t), 41.2 (t), 37.9 (t), 37.3 (d), 34.3 (t), 31.7 (t), 29.1-29.4
(8t), 25.0 (t), 22.6 (t), 20.1 (t), 14.1 (q), 13.7 (q). MS: 229 (8), 211
(15), 116 (100), 101 (18), 88 (60), 87 (30), 83 (49), 71 (12), 57 (22),
55 (33), 43 (22), 41 (17).
3-Sulfanylhexyl Hexadodecanoate (18). Yield: 3.4 g (91.3%). ISPB-1
1
2615; ISPWax >2800. H NMR: 4.22-4.28 (m, 2H), 2.82-2.92 (m,
1H), 2.29 (t, J ) 7.1 Hz, 2H), 1.97-2.06 (m, 1H), 1.24-1.79 (m, 32H),
0.93 (t, J ) 7.1 Hz, 3H), 0.88 (t, J ) 7.1 Hz, 3H). 13C NMR: 173.7
(s), 62.0 (t), 41.2 (t), 37.9 (t), 37.3 (d), 34.3 (t), 31.9 (t), 29.1-29.7
(10t), 25.0 (t), 22.6 (t), 20.1 (t), 14.1 (q), 13.7 (q). MS: 372 (0.5, M+),
258 (2), 239 (11), 116 (100), 101 (18), 88 (50), 87 (20), 83 (40), 71
(12), 57 (16), 55 (27), 43 (18), 41 (12).
3-Sulfanylhexyl Octadodecanoate (20). Yield: 3.5 g (87.5%). ISPB-1
RESULTS
1
2816; ISPWax >2800. H NMR: 4.22-4.28 (m, 2H), 2.82-2.92 (m,
Identification of Volatile Organic Sulfur-Containing Com-
pounds in P. trifoliata (L.) Raf. The fruits were collected on
1H), 2.29 (t, J ) 7.1 Hz, 2H), 1.97-2.06 (m, 1H), 1.24-1.79 (m, 36H),
0.93 (t, J ) 7.1 Hz, 3H), 0.88 (t, J ) 7.1 Hz, 3H). 13C NMR: 173.7

4514 J. Agric. Food Chem., Vol. 55, No. 11, 2007
Starkenmann et al.
Figure 1. (A) GC-TIC trace on apolar column of the crude extract. (B) Inverted GC-TIC trace of an equimolar mixture of synthetic thiols from 1 to 20.
Marks represent retention times where sulfury odors were smelled.
two different local shrubs. The crude peel extract was submitted
to GC/MS-O and GC/MS analyses. The odor profile was very
rich. It started with green notes produced by hexanal and (E)-
hex-2-en-1-ol, cheesy tonalities from butanoic acid, and fruity
notes from esters such as 3-methylbutyl and 2-methylbutyl
acetate, respectively. Between the calculated retention indices
I
SPB-1 900 and 1060 (Figure 1, zone 1), typical odors deriving
from terpenes such as R-pinene, sabinene, â-pinene, myrcene,
1,5-p-menthadiene, and limonene were observed and confirmed
by GC/MS and by retention indices on both polar and apolar
columns. After this zone and before a group of sequiterpenes
(Figure 1, zone 3), many very well-known odors corresponding
to linalool, citronellal, decanal, citronellol, indole, neryl acetate,
and geranyl acetate were perceived. Indole was detected at ISPB-1
1260 and ISPWax 2452. It is responsible for the jasmine type
smell of this Citrus-related species. We were mainly interested
in the strong tropical fruit and sulfury odors of the fruit that
were detected within these three zones at seven different
retention times (Figure 1). Unfortunately, the concentrations
were too low to record the mass spectra of these odorant
compounds suspected to contain a sulfur atom. For this reason,
the crude extract was submitted to affinity chromatography on
Affi-Gel 501 (4, 5), an efficient method to enrich compounds
with free SH groups. The thiol extract was analyzed by GC/
MS on apolar and polar columns. The retention indices I were
measured, and the mass spectra were compared with those of
authentic samples. The first thiol smelled sweaty, cheesy, roasted
pork skin, and had ISPB-1 942 and ISPWax 1663 with a typical
MS fragmentation pattern for 1. The next compound smelled
like mustard, rubbery, and tomato leafs. It had ISPB-1 1080 and
ISPWax 1716 and a mass spectrum corresponding to 3. We
reinjected the thiol extract in SIM mode monitoring the ions at
m/z 69 and 102 to screen for higher ester homologues. This
experiment allowed us to discover a whole series of 3-methyl-
3-sulfanylbutyl alkanoates from acetate 3 to octadecanoate 19
(Scheme 1).
Figure 2. Comparison of mass spectra from natural 19 with that of
synthetic 19.
1
was fully characterized by H and 13C NMR, MS (Figure 2),
as well as by ISPB-1 and ISPWax
.
We were still missing some organoleptically important
molecules that had strong tropical fruity, sulfury types of odors.
The compounds 4 and 6 were easily identified by GC/MS of
the Affi-Gel extract. The analysis was repeated in the SIM mode
monitoring the ions at m/z 116, 88, and 83. The ion m/z 116
results of the MacLafferty rearrangement involved the carbonyl
To confirm the hypothesis, the 3-methyl-3-sulfanylbutyl esters
3, 5, 7, 9, 11, 13, 15, 17, and 19 were prepared. Each compound

Constituents in Poncirus trifoliata (L.) Raf. (Rutaceae)
J. Agric. Food Chem., Vol. 55, No. 11, 2007 4515
Scheme 1. Compounds with a 1,3-Positioned O,S Moiety Identified in
P. trifoliata (L.) Raf.
* Identified for the first time in a plant source.
Scheme 2. Chemical Structures of Synthesized Precursors
Figure 3. Comparison of mass spectra from natural 20 with that of
synthetic 20.
group of the ester moiety. The ion at m/z 88, an even-numbered
mass, suggests a rearrangement rather than of a fragmentation.
Furthermore, the m/z 116 fragment loses SH to give the ion at
m/z 83. This selective MS analysis allowed us to bring out the
background of the GC trace the following 3-mercaptohexyl
alkanoates: 4, 6, 8, 10, 12, 14, 16, 18, and 20 (Figure 3).
On the basis of comparison with the GC-TIC peak area of
octane-1-thiol (internal standard), the concentrations in the peel
were estimated to be in the 50 ppb range for 1-4, in the 40
ppb range for 6, and in the 10 ppb range for 5-7. The higher
homologues were in the baseline of the TIC.
Preparation and Identification of Cysteine-S-Conjugates.
There is growing evidence that compounds with such 1,3-
positioned O,S moieties are formed in nature via an enzyme-
mediated addition of an S-nucleophile, for example, glutathione
or a metabolite thereof, to R,â-unsaturated carbonyl compounds.
The volatile thiols are released from such S-conjugates upon
the action of â-lyases (6-11). To screen P. trifoliata for such
potential precursors, several cysteine-S-conjugates were pre-
pared. N-Boc-cysteine was coupled to 3-methylbut-2-enal. The
crude product was directly reduced with NaBH4. The resulting
N-Boc-Cys-S-alkanol was deprotected in TFA and purified by
reversed phase FC. Compound 21 was then esterified with acetic
anhydride to give 22 or with butanoic anhydride to give 23.
The same procedure was repeated with (E)-hex-2-enal to prepare
24 and 25 (Scheme 2).
* Identified in P. trifoliata (L.) Raf.; IS, internal standard.
internal standard 26 (3.3 ppm) was added to the peel material
that remained from the juice preparation. After homogenization,
centrifugation, and filtration on reversed phase, the spiked peel
sample was analyzed by HPLC/ESI+-MS under instrumental
conditions that had been optimized with the authentic samples.
The Cys-S-conjugates 21, 22, and 24 were detected at the
corresponding retention times. The natural occurrence in P.
trifoliata peel has thus been proven. However, neither acetyloxy
derivative 23 nor butanoyloxy derivative 25 was detected. The
detection limits of the Cys-S-conjugates were 5 ng of 21 and
10-fold lower for the internal standard 26 due to the stronger
ionization of this latter compound. The low recovery factor of
1/30 for 26 can be explained by interactions like adsorptions
on the highly sticky and viscous peel matrix or by some
enzymatic residual activities degrading the Cys-S-conjugates.
Therefore, we are able to give only an indication of the amounts
of 21 and 24 present in the peel: 0.045 ( 0.004 and 0.011 (
0.04 mg/kg, respectively, without applying recovery factors. The
presence of 22 was confirmed, but the amount was too low to
give quantitative data.
DISCUSSION
The fruit juice was collected, and S-[1-(2-hydroxyethyl)-1-
methylbutyl]-L-cysteine (26) was added as an internal standard
(6). The lyophilized juice was analyzed by HPLC/ESI+-MS.
Except for the internal standard 26, no cysteine-S-conjugates
were detected. After the lyophilizate was desalted, 26 remained
again the only conjugate detected. In a further experiment, the
The presence of 18 sulfanylalkyl alkanoates in P. trifoliata,
a species closely related to Citrus, is quite remarkable, as if
nature had made use of combinatorial synthesis using the two
parent sulfanyl alcohols 1 and 2 or their respective precursors
as substrates for esterification.

4516 J. Agric. Food Chem., Vol. 55, No. 11, 2007
Starkenmann et al.
Only a few volatile sulfur-containing constituents have been
reported to occur in Citrus species. Undoubtedly, the most
famous among these compounds is p-menth-1-ene-8-thiol, a
powerful trace component of grapefruit juice (Citrus paradisi
Macfayden) (12-14), also identified in yuzu (Citrus junos Sieb.)
(15) and in late Valencia orange juice (16). The catty-smelling
4-methyl-4-sulfanylpentan-2-one has been detected in grapefruit
juice (13, 14) and in yuzu peel oil (17). The roles of 4-methyl-
4-sulfanylpentan-2-ol and methional were described in cold-
pressed grapefruit oil (18). Finally, S,S′-ethylidene dithioacetate
has been identified in the juice of blood oranges (19).
extraction of thiols (4, 5) but disulfides (R-S-S-R) or sulfides
(R-S-R) will not be extracted. This may explain why no structure
could be associated to some typical descriptors for sulfur
compounds like skunk odor (ISPB-1 810), rubbery (ISPB-1 854),
and cooked asparagus (ISPB-1 891).
ACKNOWLEDGMENT
We thank Francine Willcocks for suggesting the analysis of P.
trifoliata (L.) Raf. and Dr. Didier Roguet, curator of the botan-
ical garden of Geneva, Switzerland, for botanical certifications.
LITERATURE CITED
Among the 20 constituents with a free SH group identified
in P. trifoliata, only 1-4, 6, and 8 have been reported in natural
systems previously (for a recent review, see ref 20). The
3-sulfanylhexan-1-ol (2) and derivatives thereof are quite
common in nature [e.g., passion fruit (21, 22), Vitis Vinifera L.
var. Sauvignon blanc wine (23), clary sage (24), Ruta chalepen-
sis L. (17), and Persicaria odorata (5)]. The 3-methyl-3-
sulfanylbutan-1-ol (1) was reported to be present in Sauvignon
blanc wine (23) and in passion fruit (8). Acetate 3 was found
in coffee (25), in wine (26), and in passion fruit (8). The esters
5, 7, and 9-20 are new natural compounds.
The 3-sulfanylhexan-1-ol (2) and its esters have an asym-
metric carbon. Analysis of the Affi-Gel extract by chiral GC/
AED allowed determination of the ratio (R)-2/(S)-2 to be 2:3
whereas the ratio (R)-4/(S)-4 was 3:7. P. trifoliata L. is thus a
further natural organism in which such 3-sulfanylalkyl deriva-
tives are formed preferentially with the (S)-configuration [pas-
sion fruit (22, 27, 28), clary sage (24), R. chalepensis L. (17),
and human axillary sweat (29)].
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interesting organoleptic properties and may find use in a wide
range of flavor applications. Compounds 3, 5, 7, and 9 were
tasted at 1 ppm in plain water. The flavor profile of 3 had tomato
leaf, grapefruit, and mango tonalities. Compound 5 was less
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Constituents in Poncirus trifoliata (L.) Raf. (Rutaceae)
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