Isolation and identification of a new cysteine sulfoxide and volatile sulfur compounds from Allium subgenus Melanocrommyum

Article abstract of DOI:10.1021/jf902294c

A new cysteine sulfoxide containing a pyridyl residue was identified in bulbs of Allium L. species belonging to the subgenus Melanocrommyum section Megaloprason. One of these species, Allium stipitatum, is widely used as a crop plant and in folk medicine in Central Asia. The new cysteine sulfoxide was identified as L-(+)-S-(2-pyridyl)-cysteine sulfoxide. Aside from this cysteine sulfoxide, several pyridyl compounds could be identified, which were formed out of cysteine sulfoxides by the action of alliinase. Found cysteine sulfoxides and their metabolites are chemically unstable; thus, the analysis is rather difficult. By combining high-performance liquid chromatography (HPLC), HPLC tandem mass spectrometry, NMR, IR, and photometric methods, full structure elucidation of the cysteine sulfoxide was possible. Alliinase reaction products were mainly determined by various MS techniques. The achieved results give new insights in the chemistry of Allium crop plants and are probably useful for chemotaxonomical classification of the subgenus Melanocrommyum.

Full text of DOI:10.1021/jf902294c

520 J. Agric. Food Chem. 2010, 58, 520–526
DOI:10.1021/jf902294c
Isolation and Identification of a New Cysteine Sulfoxide and
Volatile Sulfur Compounds from Allium Subgenus
Melanocrommyum
†
,
JAN
KUSTERER, ANJA
V
OGT
AND
M
ICHAEL
KEUSGEN
*
€
€
Institut fur Pharmazeutische Chemie der Philipps-Universitat Marburg, Marbacher Weg 6-10,
D-35037 Marburg, Germany. † Current address: Department of Chemistry, University of
Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada
A new cysteine sulfoxide containing a pyridyl residue was identified in bulbs of Allium L. species
belonging to the subgenus Melanocrommyum section Megaloprason. One of these species, Allium
stipitatum, is widely used as a crop plant and in folk medicine in Central Asia. The new cysteine
sulfoxide was identified as
L
-(þ)-S-(2-pyridyl)-cysteine sulfoxide. Aside from this cysteine
sulfoxide, several pyridyl compounds could be identified, which were formed out of cysteine
sulfoxides by the action of alliinase. Found cysteine sulfoxides and their metabolites are chemically
unstable; thus, the analysis is rather difficult. By combining high-performance liquid chromatography
(HPLC), HPLC tandem mass spectrometry, NMR, IR, and photometric methods, full structure
elucidation of the cysteine sulfoxide was possible. Alliinase reaction products were mainly deter-
mined by various MS techniques. The achieved results give new insights in the chemistry of
Allium crop plants and are probably useful for chemotaxonomical classification of the subgenus
Melanocrommyum.
KEYWORDS: Allium stipitatum; Allium altissimum; Melanocrommyum; HPLC; L-(þ)-S-(2-pyridyl)-
cysteine sulfoxide; volatile sulfur compounds
INTRODUCTION
and Uzbekistan. Some of these species are known as ornamental
plants in the western hemisphere, the so-called “drumstick
onions”. Genetic analysis displayed a high similarity of most of
these ornamental plants. These species show a high polymorph-
ism. By using genetic analysis, these plants could be related to the
subgenus Melanocrommyum (16).
The genus Allium L. has a large diversity, and more than 700
species worldwide are described until now. Nearly all of them
occur in the semiarid regions of Europe, North America, North
Africa, and Asia. The most common Allium species are garlic
(Allium sativum L.) and onion (Allium cepa L.). These two species
havebeen used asfoods and spicessince ancient times. Aside from
their importance for nutrition, they have known pharmaceutical
values. Remarkable are the antidiabetic and antithrombotic
effects of A. sativum L. (1, 2). The described effects are cor-
related to several sulfur compounds (3, 4). Further important
substances found in Allium species are flavonoids and steroid
saponins. Steroid saponins are common in species of the families
Liliaceae and Alliaceae (11). The most common types of steroid
saponins are furostanol and spirostanol derivatives, which can be
found in an amount of 0.3-1.1% related to the dry weight in
A. sativum L. (12). Flavonoids like quercetin were observed in the
whole genus Allium. In red bulbs of A. cepa, the concentration of
flavonoids can be up to 2.1% related to the dry weight (13).
Allium species of the subgenus Melanocrommyum exhibit a
great variability (>200 species are known until now), and the
center of distribution is in Middle and in Southwest Asia. Coun-
tries with a high diversity of this subgenus are Iran, Tajikistan,
In Middle Asia, Allium species of the subgenus Melanocrommyum
have a large range of usage. The characteristic smell and taste make
the bulbs and leaves of these plants favored vegetables of the native
populations. In several cases, the amounts of cysteine sulfoxides and
their metabolites are rather high, so that the plants are used as spicy
vegetables or are even not edible. Allium stipitatum Regel is a very
common edible Allium species in Central Asia and is intensively used
by local populations as a spicy vegetable and medicinal plant.
In Iran, the plant is named “Musir”, and in countries of the former
Soviet Union, it is known as “Anzur”. Beside A. stipitatum, several
species of the subgenus Melanocrommyum are used in folk medicine.
As an example, leaves and bulbs of Allium severtzovioides R.M.
Fritsch are applied against stomach and duodenum diseases (5).
Allium motor Kamelin et Levichev leaves are served as a tonic
soup. Allium komarowii Lipsky is used as an anabolic for horses.
Beside these extraordinary uses, it is applied against anemia and
bad blood circulation. Allium suworowii Regel is used against early
forms of bronchitis and tuberculosis. Studies peformed in our
working group showed a very high antioxidative effect of these
species (7).
*To whom correspondence should be addressed. Tel: þ49(0)6421/
2825808. Fax:þ49(0)6421/2826652. E-mail: keusgen@staff.uni-marburg.
de.
pubs.acs.org/JAFC
Published on Web 11/18/2009
© 2009 American Chemical Society

Article
A number of species of the subgenus Melanocrommyum
develop a very characteristic coloration. By cutting an onion
and damaging the cells, an orange to red dye appears after some
J. Agric. Food Chem., Vol. 58, No. 1, 2010 521
device. The HPLC system used for quantitative analysis was a Merck
Hitachi 7000 series HPLC system. A VP 250/4 Nucleodur 100-5 C18 EC
€
column (Macherey Nagel, Duren, Germany) was used for all HPLC
measurements. The column oven was set to 30 °C. Calibration of the
minutes. This red dye is a metabolite of
L
-(þ)-S-(3-pyrrolyl)-
system was done with an OPA derivatized
matographic conditions were as follows:
L
-(þ)-alliin standard. Chro-
cysteine sulfoxide, a newly discovered cysteine sulfoxide (6). This
substance can be found in nearly all species of the subgenus
Melanocrommyum but mostly in low concentrations. How-
ever, some species of the subgenus Melanocrommyum, section
Megaloprason, do not contain this pyrrolyl-cysteine sulfoxide.
Especially bulbs of A. stipitatum Regel and A. altissimum Regel
showed no coloration after cutting (8). In the bulbs of
A. stipitatum Regel and A. suworowii Regel, new steroid saponins
of the spirostan series were isolated and identified (14,15), but no
reports about unusual cysteine sulfoxides were found. However,
the alliinase activity of A. stipitatum Regel was found to be similar
to other Allium species. The pH optimum (pH 7.5) and the
temperature optimum (38 °C) were typical values (17).
A
phosphate buffer pH
6.5/ acetonitrile gradient with a constant flow rate of 1 mL was used (A,
phosphate buffer, pH 6.5; B, acetonitrile): 78% A for 20 min; 78-75% A
over 29 min; 75% A for 1 min; 75-71% A over 4 min; 71% A for 1 min;
71-68% A over 8 min; 68-63% A over 2 min; 63% A for 10 min; and
78% A for 10 min; UV detection at 334 nm.
For qualitative HPLC-electrospray ionization (ESI)-MS, measure-
ments were performed on a Shimadzu LC 20 HPLC system containing
an autosampler, a high-pressure mixing pump, a column oven, and an UV
detector in combination with a QTrap 2000 equipped with a TurboIon-
spray ion source (Applied Biosystems/MDS Sciex, Toronto, Canada).
ESI-MS operating conditions for the qualitative analysis of the extracts
were as follows: positive ionization mode; scan range, 30-500 amu; source
temperature, 200 °C; ion spray voltage, 5.5 kV; curtain gas, 10; decluster-
ing potential, 110 V; entrance potential, 11 V; and flow rate, 0.25 mL/min
(HPLC separation). The LC conditions were as follows: a 50 μM
ammonium acetate buffer, pH 6.5/acetonitrile gradient with a constant
flow rate of 0.25 mL was used (A, ammonium acetate buffer, pH 6.5; B,
acetonitrile): gradient for quantitative analysis was also used for qualita-
tive HPLC/MS experiments; UV detection at 334 nm. A 250/2 Nucleodur
100-5 C18 EC column (Macherey Nagel) was used for all experiments.
Plant Processing for MS Fragmentation Experiments of Com-
pound 1. A bulb of A. stipitatum Regel (2.1 g fresh weight) was cleaned,
sliced, and placed in 10 mL of methanol to inhibit the enzymatic activity.
After 20 min of heating under reflux, the extract was filtered, the solvent
was evaporated under reduced pressure, and the residue was rediluted in
1 mL of methanol. This solution was filtered through a 0.2 μm cellulose
acetate filter and injected directly into the MS device.
Beside these findings, O’Donnell et al. suggested three new
volatile compounds in samples of A. stipitatum, which showed
antimicrobial activity (9). These compounds were identified as
N-oxides, also described for basidomycetes (10). Extracts showed
an activity against Mycobacterium tuberculosis. An explanation
for the formation of these three new compounds is an alliinase-
catalyzed reaction of an unknown cysteine sulfoxide. This hypo-
thetical sulfoxide should contain a pyridine ring. According to
O’Donnell et al., this pyridine ring should be substituted with a
cysteine sulfoxide at position 2. The formation of these volatile
compounds by an alliinase reaction insists that the main meta-
bolites are sulfoxides, not pyridine-N-oxides. The aim of the now
described work is the identification and structure elucidation of
this hypothetic cysteine sulfoxide as well as investigation of direct
metabolites of the alliinase reaction.
Plant Processing for Analysis of Volatile Sulfur Compounds.
Several bulbs of A. stipitatum Regel (53.3 g fresh weight) were cleaned and
sliced. To enable the alliinase reaction, the material was placed in 400 mL
of water and shaken for 30 min. Afterward, the water phase was extracted
with three portions of 40 mL of ethyl acetate p.a. The combined ethyl
actetate layers were evaporated at 30 °C under reduced pressure until
dryness and rediluted in 5 mL of methanol. Samples were cooled down to
3 °C and analyzed immediately.
Plant Processing for Structure Elucidation of Nonvolatile Com-
pounds. Bulbs of A. stipitatum Regel (105 g fresh weight) were cleaned,
sliced, and instantly placed in methanol to inhibit enzymatic activity. After
20 min of heating under reflux, the pieces were homogenized and extracted
under reflux for further 20 min. The extract was filtered, and the solvent
was removed under reduced pressure. The obtained residue was resolvedin
30 mL of OPA reagent and 300 μL of t-butylthiol. After an incubation time
of 30 min, 500 μL of a methanolic iodacetamide dilution (1 mM) was
added. The solution was filtered through a 0.45 μm cellulose acetate filter
after 3 min of incubation. The solvent was removed under reduced
pressure, and the residue was stored at -20 °C for further processing.
Immediately before LC separation, frozen samples were rediluted in 5 mL
of ammonium acetate buffer (50 μM).
MATERIALS AND METHODS
Chemicals. Used chemicals were purchased either from Fluka
(Deisenhofen, Germany) or from Sigma (Munich, Germany). Millipore-
grade water was used for all experiments.
Derivatization Reagent [ortho-Phthaldialdehyde (OPA) Re-
agent]. Under magnetic stirring, 140 mg of OPA was solubilized in
5 mL of methanol. Afterward, 200 μL of t-butylthiol was added. Then,
50 mL of borate buffer, pH 9.5, was slowly mixed with the OPA solution.
The reagent was stored for at least 12 h under light protectionbefore usage.
Plant Material. Plant material (A. stipitatum Regel) for structure
elucidation was obtained from the living plant collection at IPK
Gatersleben, Germany. The plant material for the quantitative analysis
was collected in various regions of Middle Asia. A. stipitatum Regel
samples 1086/2, 1090/2, and 1240 Z1 were collected in Iran. A. stipitatum
samples 4238/1 and 4238/2 were collected in Turkmenistan. The bulbs of
Allium altissimum Regel listed as samples 4206/1 and 4206/2 were collected
in Uzbekistan. Voucher species were planted in the living Allium collection
at IPK Gatersleben. Taxonomic determination of used plant material was
done by Dr. R. M. Fritsch of the same institute.
HPLC Separation of OPA-Derivatized Cysteine Sulfoxides.
A
Waters HPLC system (600 E System controller and a Waters 991 PAD)
was usedfor separation. LC separations were performed using a VP250/21
Nucleodur 100-5 C18 EC column (Macherey Nagel). The column oven
was set to 30 °C. An acetonitrile/50μM ammonium acetate buffer, pH 6.5,
gradient with a constant flow rate of 10 mL/min was used (A, acetonitrile;
B, ammonium acetate buffer): 78% B for 25 min; 78-75% B over 25 min;
UV detection at 334 nm. The OPA-derivatized cysteine sulfoxide eluted at
35 min. The solvent of the collected fractions of 1a was evaporated under
reduced pressure at 30 °C. To remove traces of ammonium acetate by sub-
limation, the fractions were further dried under high vacuum for several
hours. Afterward, the fractions were stored in the freezer at -20 °C until
further analysis.
Plant Processing and Instrumental Setup for Qualitative and
Quantitative Analysis of Cysteine Sulfoxides. Bulbs were cleaned and
sliced into pieces of 200-800 mg weight. Pieces were instantly placed in
20 mL of methanol to inhibit enzymatic activity and heated under reflux.
After 10 min, the pieces were homogenized, and extraction was continued
with a mixture of methanol/water (VT 1:1) by heating under reflux.
Afterward, the extract was filtered, and the solvent was carefully evapo-
rated under reduced pressure. The obtained completely dried extracts were
storedin thefreezer at -20 °C. Formeasurements, extracts were solubilized
in 4.93 mL of OPA reagent and 50 μL of 2-methylpropanethiol. After a
derivatization for 30 min in darkness, 20 μL of iodacetamide was added.
For quantitative measurements, 200 μL of samples was diluted in
800 μL of water. For qualitative measurements, 500 μL of samples was
diluted in 500 μL of water. The diluted samples were injected either into the
high-performance liquid chromatography (HPLC) or the HPLC MS/MS
HPLC Separation of the Volatile Sulfur Compounds. The Waters
HPLC system already described above was used. LC separations were
performed using a VP 250/21 Nucleodur 100-5 C18 EC column (Macherey

522 J. Agric. Food Chem., Vol. 58, No. 1, 2010
Kusterer et al.
Nagel). A methanol/water gradient with a constant flow rate of 10 mL/min
was used (A, methanol; B, water): 90% B for 2 min; 90-10% B over
15 min; UV detection with a photodiode array (PDA) over a range of
200-400 nm. The solvent of the collected fractions containing 3 and 6 was
removed under reduced pressure at 30 °C. Afterward, the fractions were
stored in the freezer at -20 °C.
HPLC-MS Analysis of Volatile Sulfur Compounds. HPLC-ESI-
MS measurements were performed on a Shimadzu LC 20 HPLC system
containing an autosampler, a high-pressure mixing pump, a column oven,
and an UV detector in combination with a QTrap 2000 equipped with a
TurboIonspray ion source (Applied Biosystems/MDS Sciex, Toronto,
Canada). The LC ESI-MS operating conditions for the analysis of 3-7
were as follows (positive ionization mode): scan range, 30-500 amu;
source temperature, 200 °C; ion spray voltage, 5.5 kV; curtain gas,
10; declustering potential, 110 V; entrance potential, 11 V; flow rate,
0.2 mL/min (HPLC separation). Chromatographic conditions were as
follows: a methanol/water gradient with a constant flow rate of 0.2 mL was
used (A, methanol; B, water): 90% B for 2 min; 90-10% B over 13 min;
10-5% B over 15 min; and 5% B for 15 min. A 250/2 Nucleodur 100-5
C18 EC column (Macherey Nagel) was used for the separation.
The ESI-MS/MS operating conditions for the fragmentation of 1 and
3-7 were as follows: positive or negative ionization mode; scan range,
30-1000 amu; source temperature deactivated; ion spray voltage, 5.5 kV;
curtain gas, 10; declustering potential, 110 V; entrance potential, 10 V;
collision energy, 22-52; collision cell entrance potential, 25.42 V; collision
cell exit potential, 3 V; and flow rate, 20 μL/min (direct injection).
Structure Elucidation. NMR experiments were performed on a
JEOL ECA-500 spectrometer. By the aid of standard correlation experi-
ments [correlated spectroscopy (COSY), heteronuclear multiple quantum
coherence (HMQC), and heteronuclear multiple bond coherence
(HMBC)] and nuclear Overhauser effect spectroscopy (NOESY) experi-
ments, structure elucidation of compounds 1a and 3 was performed. IR
measurements were performed on Bruker Alpha-P Fourier transforma-
tion infrared (FT-IR) (Ettlingen, Germany). The HR-ESI experiment of
1a was carried out on a Finnigan LTQ-FT hybrid mass-spectrometer. HR-
ESI experiments of the compounds 3, 4, 6, and 7 were performed with a
Micromass Autospec (Manchester, England). The optical rotation of 1a
was measured on a Chemdata Jasco DIP-370 Digital Photometer
(Zimmern, Germany) at 589 nm and 20 °C. The sample was dissolved in
methanol.
Figure 1. Structure of the newly described
sulfoxide (1) and its OPA derivative (1a), which was used for structure
elucidation.
L
-(þ)-S-(2-pyridyl)-cysteine
Table 1. NMR Data of the Pyridyl Residue of Compound 1a and 3
compound 1a (acetonitrile-d3)
[ppm]
compound 3 (methanol-d4)
[ppm]
no.
1H
13C
no.
1H
13C
2
3
151.7
126.8
2,20
153.3
129.2
7.43 (d, 1,
J = 8.0 Hz)
7.29 (dt, 1,
3,30 7.70 (dd, 2,
J = 1.7, 8.3 Hz)
4,40 7.54 (dt, 2,
J = 1.2, 8.3 Hz)
5,50 7.40 (dt, 2,
J = 1.7, 6.6 Hz)
6,60 8.41 (dt, 2,
J = 1.2, 6.6 Hz)
4
5
6
122.7
121.0
138.7
123.5
122.3
139.1
J = 7.7, 8.0 Hz)
7.07 (dt, 1,
J = 6.3, 7.5 Hz,)
8.13 (dd, 1,
J = 6.3 Hz)
801 (w), 760 (s), 703 (m). UV (λmax, MeOH): 238, 264, 310. ESI MS/MS,
m/z (relative intensity): 78 (100), 79 (11), 98 (16), 111 (5), 126 (29), 127 (18),
142 (18), 253 (26). HR-ESI-MS, 253.0130; molecular formula
C10H9N2O2S2. MS calculated (M þ Hþ): 253.0105. Compound 4 UV
(λmax, MeOH): 238, 264, 310. ESI MS/MS, m/z (relative intensity): 66.9
(16), 78 (100), 79 (7), 98 (18), 111 (57), 126 (37), 127 (18), 237 (57). HR-
ESI-MS, 237.0140; molecular formula C10H9N2OS2. MS calculated (Mþ
Hþ): 237.0156. Sodium adduct C10H9N2OS2Na: HR-ESI-MS, 259.0025;
MS calculated, 258.9976. Compound 5 UV (λmax, MeOH): 238, 282. ESI
MS/MS, m/z (relative intensity): 67 (8), 79 (3) 111 (100), 187 (8), 221 (49).
Compound 6 FT-IR [νmax cm-1, intensity given as strong (s), medium (m)
or weak (w)]: 3102 (w), 2921 (m), 1737 (m), 1679 (m), 1557 (w), 1509 (w),
1466 (s), 1422 (s), 1372 (w), 1316 (w), 1263 (w), 1245 (w), 1221 (m), 1206
(m), 1137 (w), 1080 (w), 1040 (s), 836 (m), 800 (w), 761 (m), 703 (w). UV
(λmax, MeOH): 238, 260, 310. ESI MS/MS, m/z (relative intensity): 78 (89),
98 (17), 111 (20), 114 (40), 125 (38), 126 (54), 127 (43), 173 (14), 174 (100).
Sodium adduct C6H7NOS2Na: HR-ESI-MS, 195.9873; MS calculated,
195.9867. Compound 7 UV (λmax, MeOH): 238, 260, 310. ESI MS/MS,
m/z (relative intensity): 45 (10), 61 (23), 63 (26), 78 (9), 93 (100), 109 (11),
111 (11), 112 (12), 126 (4), 142 (3) 174 (5), 220 (5). Sodium adduct
C7H9NOS3Na: HR-ESI-MS, 241.9749; MS calculated, 241.9744.
Analytical Data of the Identified Compounds. Compound 1
(Figure 1): ESI MS/MS, m/z (relative intensity %) 70 (13), 78 (5), 88
(44), 110 (8), 112 (8), 124 (4), 128 (100), 152 (3), 159 (4), 169 (6), 198 (14),
215 (31).
Compound 1a (Figure 1): 13C NMR (125.77 MHz, acetonitrile-d3): δ
30.3 (C22, C23, C24), 35.5 (C8), 49.7 (C21), 59.3 (C9), 110.6 (C19), 115.2
(C12), 120.0 (C17), 120.4 (C14), 120.9 (C15), 121.0 (C5), 122.1 (C16), 122.7
(C4), 124.5 (C13), 126.8 (C3), 130.6 (C18), 138.7 (C6), 151.7 (C2), 172.3
(C10). 1H NMR (500.16 MHz, acetonitrile-d3): δ 1.14 (s, 9, H22, H22,
H22, H23, H23, H23, H24, H24, H24), 3.56 (dd, 1, J = 14.03, 10.02 Hz,
H8), 3.89 (dd, 1, J=14.03, 4.58 Hz, H8), 5.84 (dd, 1, J=10.02, 4.58 Hz,
H9), 6.88 (dt, 1, J = 0.86, 8.31 Hz, H15), 6.96 (dt, 1, J = 0.86, 8.31 Hz,
H16), 7.07 (dt, 1, J=6.30, 7.45 Hz, H5), 7.29 (dt, 1, J=7.73, 8.02 Hz, H4),
7.43 (d, 1, J=8.02 Hz, H3), 7.5 (d, 1, J= 8.59 Hz, H14), 7.58 (dd, 1 J=
8.59, 0.86 Hz, H17), 7.67 (s, 1, H12), 8.13 (dd, 1, J=6.30 Hz, H6). FT-IR
[νmax cm-1, intensity given as strong (s), medium (m) or weak (w)]: 3187
(m), 3013 (m), 2858 (m), 1694 (m), 1544 (m), 1402 (s), 1366 (s), 1269 (m),
1046 (w), 1015 (m), 923 (w), 883 (w), 833 (w), 748 (w), 658 (s), 621 (s), 491
(m), 448 (s). UV (λmax, MeOH): 233, 320, 335. HR-ESI-MS, 403.1144;
molecular formula C20H23S2O3N2; MS calculated (M þ Hþ), 403.1145.
Sodium adduct C20H22S2O3N2Na: HR-ESI-MS, 425.0964; MS calculated,
425.0964; specific optical rotation [RD20], -9.2 mL g-1 dm-1 (589 nm, in
methanol).
RESULTS AND DISCUSSION
Isolation and Structure Elucidation of the Precursor
L
-(þ)-S-(2-
Pyridyl)-cysteine Sulfoxide. To elucidate the structure of the
proposed alliinase precursor 1, the OPA derivatization method
for amino acids was used. This strategy was chosen because direct
isolation of 1 failed due to large impurities caused by various
sugars. Derivatization with OPA and a mercapto-alkyl (OPA
reagent) allows isolation of a nearly pure fraction of compound
1a. MS data of 1 and 1a emphasized a substance with two
nitrogen atoms and, for 1a, a mass difference in accordance with
an OPA derivatization reaction (1, 214 g/mol; 1a, 402 g/mol; mass
difference, 188 amu). Furthermore, a molecular formula of
C20H23S2O3N2 was determined by high-resolution mass spectro-
metry (HR-MS) (1a).
Analytical Data of Isolated Volatile Sulfur Compounds. Com-
pound 3: 1H NMR (500.16 MHz, methanol-d4): δ 7.40 (dt, 2, J=1.72, 6.59
Hz, H5, H50), 7.54 (dt, 2, J=1.15, 8.31 Hz, H4, H40), 7.70 (dd, 2, J=1.72,
8.31 Hz, H3, H30), 8.41 (dd, 2, J = 1.15, 6.59 Hz, H6, H60). 13C NMR
(125.77 MHz, acetonitrile-d3) are given in the Table 1. FT-IR [νmax cm-1
,
Further structure elucidation was performed mainly by various
NMR techniques. First, correlation experiments (HMQC,
HMBC, and COSY) were used. The OPA moiety of 1a consists
intensity given as strong (s), medium (m) or weak (w)]: 3103 (w), 2932 (m),
2861 (w), 1635 (w), 1590 (m), 1556 (w), 1519 (m), 1465 (s), 1422 (s), 1266
(m), 1249 (w), 1221 (m), 1206 (m), 1139 (m), 1083 (m), 1035 (m), 838 (s),

Article
J. Agric. Food Chem., Vol. 58, No. 1, 2010 523
Figure 2. Amounts of cysteine sulfoxides in various samples of A. stipitatum Regel and A. altissimum Regel.
of an isoindole system and a t-butylthiol. These substructures
exhibited characteristic signals in NMR experiments. The HMBC
spectrum showed a strong correlation between the t-butyl alkyl
protons (H22, H23, H24; δ 1.14) and the quaternary carbon atom
(C21; δ 49.7). The isoindole substructure was elucidated by all
three correlation experiments. COSY experiments showed specific
signals for the isoindole ring. Significant here are the correlations
between the protons H14 (δ 7.5), H15 (δ 6.88), H16 (δ 6.96), and
H17 (δ 7.58). The HMBC correlations between the quaternary
carbon atoms C13 (δ 124.5), C18 (δ 130.6), and C19 (δ 110.6) with
the protons of the isoindole ring H12 (δ 7.58), H14, H15, H16, and
H17 proved the expected substructure of the OPA moiety.
The comparison of IR measurements of (-)- and (þ)-cysteine
sulfoxides underlined that compound 1a is a (þ)-isomer. The
absorption band of the sulfoxide of compound 1a shows the same
profile as IR measurement of (þ)-cysteine sulfoxides (20).
A rather strong band at 1015 cm-1 supports the (þ)-configura-
tion, whereas the (-)-sulfoxide shows a band at 1000 cm-1
.
The decreased optical rotation of compound 1a in comparison
to the optical rotations of (þ)-cysteine sulfoxides is caused by the
OPA substituent at C9. The published specific optical rotation of
L
-(þ)-alliin is given as [RD20] þ62.8 mL g-1 dm-1 (21). The
20
specific optical rotation of the derivatized precursor 1a at [RD
]
-9.2 mL g-1 dm-1 (589 nm, in methanol) is due to the larger
substituent at C9.
The cysteine structural part was identified by comparison of
the NMR data with those of already published cysteine sulf-
In MS/MS experiments, compound 1 showed several charac-
teristic fragments leading to the assumed structure. The most
important fragment is 78 m/z in positive ionization mode, which
indicates the pyridyl moiety. The fragment of 88 m/z in positive
ionization mode is typical for cysteine derivatives. The results
oxides, like
L
-(þ)-alliin (20). The signals of the protons H8 (δ 3.56,
1
δ 3.89) matched the expected shifts in H NMR. COSY and
HMBC correlation of the protons H8 with the proton at the
stereogenic center C9 (H9, δ 5.84) was obvious. The carbonic acid
C10 (δ 172.3) was identified by HMBC experiments. A strong
3J coupling of H9 and a 4J coupling of the two hydrogens of C8
were obtained.
obtained underline the hypothesis that compound 1 is a
cysteine sulfoxide.
L
-(þ)-
Qualitative and Quantitative Analysis of A. stipitatum and A.
altissimum Samples. All investigated samples of A. stipitatum and
A. altissimum contained the new cysteine sulfoxide 1 and the
already described methiin 2, which could be clearly identified by
HPLC-MS/MS and authentic standards (20). These compounds
were also analyzed as their corresponding OPA derivatives. This
method allows sufficient separation of cysteine sulfoxides from
various amino acids, which are also present in all Allium samples.
The amount of the new cysteine sulfoxide 1, related to the fresh
weight of bulbs, was found to be between 0.13 and 0.44%.
Methiin was present in amounts of 0.03-0.86% (Figure 2). The
examined bulbs contained 1 in amounts comparable to methiin.
This suggests that numerous mixed thiosulfinates can be expected
by the reaction of alliinase with these two cysteine sulfoxides
(compare Figure 3).
High variations in the amounts of 1 and 2 can have several
reasons: (i) Material was collected at various places in nature;
(ii) the time of harvest was not the same for all bulbs; and (iii) it
can be assumed that A. stipitatum and related species are
taxonomically not sufficiently described. The species A. stipitatum
can probably be divided in a number of subspecies. The area
of distribution is strongly disrupted by large desert areas and
mountain ranges higher as 5000 m. Leaves can show strong and
dense hairs but are sometimes also nearly hairless.
Finally, the sulfoxide moiety and its substitution had to be
identified. No further aliphatic protons and carbons were ob-
served in the NMR spectra. All data strongly supported a pyridyl
substituent. 1H NMR measurement showed four proton signals
with a shift between 7.0 and 8.2 ppm. These signals showed
couplings in COSY and HMBC. The couplings correlated in a
clear order; it could be deduced that these protons are in positions
3 (H3 δ 7.43), 4 (H4 δ 7.29), 5 (H5 δ 7.07), and 6 (H6 δ 8.13) of the
pyridyl substituent. Therefore, the sulfoxide group must beplaced
at position 2 of the pyridine ring.
Until now, mainly (þ)-sulfoxides were found in nature. Relat-
ing to 1H NMR spectra of synthetic (-)- and (þ)-cysteine
sulfoxides (20), compound 1a, respectively, compound 1, is the
sulfur (þ)-isomer. For (þ)-cysteine sulfoxides, the two protons at
C8 show a difference in chemical shifts between 0.2 and 0.3 ppm.
The signals of these protons nearly merge to one single signal in
the case of (-)-cysteine sulfoxides. For instance, the chemical
shifts of the two protons at this carbon atom are 3.21 and 3.45
ppm for (þ)-alliin (20), but 3.36 and 3.43 ppm for (-)-alliin. The
precursor 1a showed a difference of 0.33 ppm between the
chemical shifts of the two protons at C8 suporting a (þ)-
sulfoxide. The slightly larger shift in comparison to literature
data can be explained by the isoindole ring, which is located at C9.

524 J. Agric. Food Chem., Vol. 58, No. 1, 2010
Kusterer et al.
Figure 5. Main fragments of pyridyl compounds found in MS/MS experi-
ments (positive ionization mode).
as MS fragmentation of 5 and aldrithiol-2 were found to be
identical.
The HPLC-MS/MS measurements showed correlations be-
tween the retention times and the polarity of the proposed
structures. Peaks related to compounds 3-5 showed MS signals
correlated to retention times as follows (RP column): 3, m/z 253
[MþH]þ, 16.4 min; 4, m/z 237 [MþH]þ, 22.4 min; and 5, m/z 221
[MþH]þ, 27.5 min. This change in retention time correlates with
the amount of oxygen in the different compounds (3, two
oxygens; 4, one oxygen; and 5, no oxygen). Compound 6 eluted
at 19.2 min, and compound 7 eluted at 23.12 min.
MS/MS experiments of compounds 3, 4, 6, and 7 gave a m/z
126 [M]þ fragment, which indicates the pyridyl sulfoxide moiety,
and a m/z 78 [M]þ fragment, which is characteristic for the pyridyl
moiety (Figure 5). The fragment at m/z 111 [M þ H]þ for
compounds 3-7 emphasizes the presence of a pyridyl disulfide
substructure. This fragment might be formed by a homolytic
cleavage of the disulfide bridge. The presence of m/z 126 [M]þ
suggests that compounds 6 and 7 were products of 1 and 2 with a
thiosulfinate moiety, which is in full accordance with the alliinase
reaction theory. Due to the fragment m/z 126, this oxygen must be
located at the sulfur next to the pyridyl substituent. Furthermore,
the intensity of m/z 126 [M]þ is much higher as the intensity of m/z
111 [M þ H]þ.
Figure 3. Alliinase-catalyzed reaction starting with
cysteine sulfoxide (1) and methiin (2) as precursors.
L
-(þ)-S-(2-pyridyl)-
For compound 3, a strong signal at m/z 142 [M]þ was found
resulting from a homolytic cleavage between the two sulfoxy
functionalities. To prove the suggested structure of 3, compound
3a was synthesized according to Barton and Ramesh (22) and
subjected to MS/MS fragmentation experiment. The fragmenta-
tion pattern was found to be completely different from 3.
Compounds 3 and 6 were separated by HPLC in a semipre-
parative scale and examined by FT-IR measurements. Com-
pound 6 showed the typical absorption bands of a pyridyl
moiety at 702, 762, 836, 1206, 1220, 1249, 1266, 1422, and 1466
cm-1. Beside these pyridyl bands, the typical band of the expected
Figure 4. Structures of volatile compounds found after alliinase reaction.
Substance 3a was synthesized according to Barton and Ramesh (22) to
perform structure elucidation of 3.
Analysis of Volatile Sulfur Compounds. According to the
alliinase activity of A. stipitatum (17), cysteine sulfoxides should
first react to thiosulfinates as primary volatile compounds.
However, the analysis of the formed metabolites was crucial,
because obtained yields were rather low. To exclude further
reaction of formed thiosulfinates, the extract was processed and
analyzed rapidly after alliinase reaction. Under these conditions,
the formation of volatile compounds should follow the pathway
described in Figure 3.
Identified compounds are given in Figure 4. The pure fractions
containing 3 and 6 were stored at -20 °C to minimize side
reactions. Structure elucidation was performed by 1H NMR, FT-
IR, HPLC MS/MS, and MS/MS fragmentation. According to
the alliinase reaction depicted in Figure 3, compounds 4 and 6
were expected. Beside these substances, three related volatile
compounds (3, 5, and 7) were identified by HPLC-MS/MS.
HR-ESI-MS data of 3, 4, 6, and 7 were gathered and showed
good correlation with the proposed structures. Typical MS/MS
fragments found by MS/MS fragmentation are given in Figure 5.
Compound 5 is known as “aldrithiol-2”, which can be obtained
from Sigma-Aldrich (Munich, Germany). Retention time as well
thiosulfinate was observed with a strong intensity at 1040 cm-1
.
Compound 3 also showed the typical pyridyl bands. This sub-
stance was redissolved in methanol-d4 and further analyzed by 1H
NMR techniques. The four expected proton signals of the two
identical pyridyl moiety occurred at δ 7.70 (H3, H30), 7.54 (H4,
H40), 7.40 (H5, H50), and 8.41 (H6, H60). This suggests that 3 is a
disulfoxide.
As alternative structure, compound 3a is thinkable. The syn-
thetic reference material of 3a was also subjected to NMR
1
experiments showing eight different proton signals in H NMR
(between δ 7.4 and 8.7 instead of four proton signals for 3). H2
COSY measurements of 3a showed the expected correlation of the
proton signals. Additionally, there were differences in the IR
spectrum between 3 and 3a. The found 1H NMR between 1a and 3
is rather similar, strongly supporting the supposed structure of 3.
In MS/MS experiments, the main fragments of compound 3
were m/z 142 [M]þ, m/z 126 [M]þ, and m/z 78 [M]þ. The m/z 142
[M]þ fragment is formed due to an intramolecular shift of the
oxygen during the fragmentation. However, the structure of 3
does not fit with the “classical” alliinase theory (disulfoxide

Article
J. Agric. Food Chem., Vol. 58, No. 1, 2010 525
instead of a thiosulfinate). In previous experiments of our group,
it was found that alliinase isolated from Melanocrommyum
species has a different substrate specificity as alliinase from
A. sativum and A. cepa (8). As a remarkable feature of the
Melanocrommyum alliinase, an oxidase side activity was found.
Therefore, the formation of the second sulfoxide group of 3
instead of a thiosulfinate can be a direct effect of alliinase activity.
However, this oxidase activity needs further investigation.
Special attention was paid to the question whether the found
oxygen atoms are attached to the sulfur atom or to the nitrogen of
the pyridine ring. The alliinase pathway only allows attachment
to the sulfur. According to O’Donnell et al. (9), one antimicrobial
component of A. stipitatum is a 2,20-dithio-bis-pyridine-N-oxide.
The ESI-MS measurements of this substance showed a mass
signal of m/z 220 [M]þ (10). This fragment had a relative intensity
of 20% and is formed by splitting of the two pyridine N-O
bonds. This fragment did not occur in the MS/MS measurements
of compound 3. Furthermore, in the MS spectrum of 2,20-dithio-
bis-pyridine-N-oxide, a fragment at m/z 110 [M]þ showed a high
relative intensity of 84% (10). This is formed by the reduction of
the nitrogens and a homolytic cleavage of the disulfide bond. In
contrast, the main fragments of compound 3 were m/z 142 [M]þ,
m/z 126 [M]þ, and m/z 78 [M]þ.
The total area under the curve of the intensities of the UV signals
of compounds 3-7 was set to 100%. Compound 7 showed the
largest relative amount (39.2%). The other compounds had
amounts as follows: 3 (24.4%), 4 (4.6%), 5 (0.8%), and 6
(30.5%). Expected volatile compounds consisting of two meth-
yl-sulfur moieties could not be safely determined by LC-MS
experiments, probably due to low concentrations. It can be
assumed that alliinase products of methiin nearly completely
react to compounds 6 and 7, which are the leading substances of
the volatile fraction.
Conclusions. A new class of cysteine sulfoxides was isolated
from A. stipitatum, which is also present in closely related species.
This probably allows chemo-taxonomical classification of this
section inside the subgenus Melanocrommyum. Still, further
samples have to be analyzed. Because of high variations in the
amount of cysteine sulfoxides, it can be expected that this genus
has to be divided into different subgenera.
A. stipitatum is widely used as a spicy vegetable and as a
traditional medicine. However, no information is available about
possible toxicity of the pyridine derivatives. The observed strong
antibiotic effects are an interesting observation, but it can be
assumed that the sulfur compounds do also show further bioac-
tivities. This is of importance, because bulbs of A. stipitatum are
eaten by several million people.
Conversion of compound 4 into a dipyridine-di-N-oxide is
rather unlikely as a direct product of alliinase activity. The
absence of a N-oxide is further supported by comparison of
NMR data. The 1H and 13C NMR data of compound 1a
(precursor) and compound 3 (product) are also very similar
(Table 1). However, formation of a pyridine-N-oxide would result
in significant shifts in the proton NMR spectra of 3, especially at
position 3 and 6 of the pyridine ring. A migration of the oxygen
atom from sulfur to nitrogen is also theoretically possible.
However, the dissociation enthalpy of the N-O bond of a
pyridine-N-oxide is given as 301.7 ( 2.8 kJ mol-1 at 298.15
K (18). The dissociation enthalpy of sulfoxides is listed as 420 kJ
mol-1 at 298.15K (19). Under stricttemperature and time control
as applied in the here-described experiments, an intramolecular
shift of the oxygen is very unlikely. Furthermore, the proposed
structure of 3 was also supported by the radiolysis of 2-thiopyri-
dine derivatives (23). Sulfoxides instead of N-oxides were ob-
served as reaction products.
Only some of the pyridine compounds of A. stipitatum could be
elucidated. Analogously to A. sativum and A. cepa, it can be
expected that different postharvest treatments of the crude bulb
material will lead to different patterns of pyridine sulfur com-
pounds. The proposed enzymatic cleavage of the
L
-(þ)-S-(2-
pyridyl)-cysteine sulfoxide leads to thiosulfinates like 4 and 6.
The alliinase theory only allows thiosulfinates as primary enzy-
matic products, also mixed thiosulfinates. The presence of 3, 5,
and 7 is somewhat surprising and can be explained by secondary
oxidation or reduction reactions. As described previously, oxida-
tion reactions can be also directly catalyzed byMelanocrommyum-
alliinase. Volatile compounds with a R-SO-SO-R structure are
already described in literature (3). Normally, these compounds are
intermediate products. The relatively high amount in the samples
of our investigation is a surprising observation. Compound 7 is
probably caused by fast reactions of two molecules of methylsul-
fenic acid with the 2-pyridinesulfenic acid (see Figure 3). The
primarily formed thiosulfinates are highly reactive and are prob-
ably subject to secondary and tertiary modifications. It is unclear
if further enzymatic steps are involved in this procedure. Never-
theless, the results strongly emphasize that N-oxides were not
formed during or directly after alliinase reaction. The already
described N-oxides are probably caused by further fermentation
processes. Because of the complicated taxonomy of A. stipitatum,
it can also be assumed that investigations described in ref 9 were
performed with a different species.
In the MS/MS measurements of compound 5, a different
fragmentation pattern in comparison to 3, 4, 6, and 7 was
observed. The high amount of the fragment at m/z 111 [M þ
H]þ and the absence of fragment m/z 78 [MþH]þ emphasize the
presence of a disulfide bond instead of a thiosulfinate or a
disulfoxide functionality. The found fragmentation pattern was
identical with this reference material (aldrithiol-2).
The main fragments of compound 7 were m/z 174 [M þ H]þ
and m/z 93 [M]þ. The m/z 174 [MþH]þ fragment is identical with
the positively charged compound 6. The fragment of m/z 93
showed the highest intensity and is formed by the cleavage of the
disulfide bond. These findings strongly support that compound 7
is homologous to 6 (one additional CH2-S group).
UV spectra were obtained for all compounds during the HPLC
separations by a PDA detector. The spectra of 3 and 4 showed
three UV maxima at 238, 264, and 310 nm. The maximum at 310
nm had a lower intensity for compound 4. Compound 5 only
showed two UV maxima at 238 and 282 nm. The missing of the
UV minor maxima at 310 indicates the absence of the sulfoxide
functionality. The UV spectra of compounds 6 and 7 showed
similarities to the spectra of compounds 3 and 4. Three maxima at
238, 260, and 310 nm were found.
ABBREVIATIONS USED
OPA, o-phthaldialdehyde; COSY, correlated spectroscopy;
HMQC, heteronuclear multiple quantum coherence; HMBC,
heteronuclear multiple bond coherence; NOESY, nuclear Over-
hauser effect spectroscopy; MS, mass spectrometry; ESI, electro-
spray ionization; HR-MS, high-resolution mass spectrometry;
HPLC, high-performance liquid chromatography; FT-IR, Four-
ier transformation infrared; PDA, photodiode array.
SAFETY
The smell and toxicity of the 2-methylpropanethiol (t-butyl-
thiol) insist that the preparation of the OPA derivatization
reagent be performed under a fume hood.
The relative amounts of the volatile sulfur compounds were
estimated by the area under the curve of the UV signals at 254 nm.

526 J. Agric. Food Chem., Vol. 58, No. 1, 2010
Kusterer et al.
ACKNOWLEDGMENT
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Thanks are due to Professor Dr. M. Abbasi, Plant Pests and
Diseases Research Institute (Tehran, Iran), for organization of
field trips in 2006 and 2007. We thank Dr. R. M. Fritsch, IPK
Gatersleben, for the identification of collected bulbs and provid-
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Supporting Information Available: FT-IR of compounds 1a,
3, 3a, and 6; MS/MS of compounds 3 and 3a; MS of compound 5;
and 1H NMR of compounds 3 and 3a. This material is available
free of charge via the Internet at http://pubs.acs.org.
(14) Vollerner, Y. S.; Kravets, S. D.; Shashkov, A. S.; Gorovits, M. B.;
Abubakirov, N. K. Steroids of the spirostan and furostan series
from plants of the genus Allium. XXIV. Structure of anzurogenin
A from Allium suvorowii and Allium stipitatum. Khimiya Prirodnykh
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Abubakirov, N. K. Steroids of the spirostan and furostan series
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Received for review July 13, 2009. Revised manuscript received
October 17, 2009. Accepted October 18, 2009. This research was
partially supported by the VolkswagenStiftung, Hannover, as part of
the program “Zwischen Europa und Orient—Mittelasien/Kaukasus im
Fokus der Wissenschaft”.