Precursors and formation of pyrithione and other pyridyl-containing sulfur compounds in drumstick onion, Allium stipitatum

Article abstract of DOI:10.1021/jf200704n

Two novel, structurally unusual cysteine derivatives were isolated from the bulbs of Allium stipitatum (Allium subg. Melanocrommyum) and shown to be S-(2-pyridyl)cysteine N-oxide and S-(2-pyridyl)glutathione N-oxide. The former compound is the first example of a naturally occurring alliinase substrate that contains an N-oxide functionality instead of the S-oxide group. In addition, S-methylcysteine S-oxide (methiin) and S-(methylthiomethyl)cysteine 4-oxide (marasmin) were found in the bulbs. Presented data suggest that the previously reported identification of S-(2-pyridyl)cysteine S-oxide was most likely erroneous. The alliinase-mediated formation of pyridyl-containing compounds following disruption of A. stipitatum bulbs was studied by a combination of HPLC-MS, HPLC-PDA, DART-MS, and NMR techniques. It was found that no pyridyl-containing thiosulfinates are present in homogenized bulbs in detectable quantities. Instead, various pyridine N-oxide derivatives are formed, including N-hydroxypyridine-2(1H)-thione (pyrithione), 2-(methyldithio)pyridine N-oxide, 2-[(methylthio)methyldithio]pyridine N-oxide, di(2-pyridyl) disulfide N-oxide, and di(2-pyridyl) disulfide N,N′-dioxide. This represents the first report of pyrithione formation as a natural product.

Full text of DOI:10.1021/jf200704n

ARTICLE
pubs.acs.org/JAFC
Precursors and Formation of Pyrithione and Other Pyridyl-Containing
Sulfur Compounds in Drumstick Onion, Allium stipitatum
,†
†
‡
#
#
§
ꢀ
Roman Kubec,* Petra Krejꢀcovꢁa, Petr Simek, Lukꢁaꢀs Vꢁaclavík, Jana Hajꢀslovꢁa, and Jan Schraml
†
ꢀ
Department of Applied Chemistry, University of South Bohemia, Braniꢀsovskꢁa 31, 370 05 Ceskꢁe Budꢀejovice, Czech Republic
‡
ꢀ
Laboratory of Analytical Biochemistry, Biology Centre of the ASCR, v.v.i., Braniꢀsovskꢁa 31, 370 05, Ceskꢁe Budꢀejovice, Czech Republic
#Department of Food Chemistry and Analysis, Institute of Chemical Technology, Technickꢁa 5, 166 28 Prague 6, Czech Republic
§Institute of Chemical Process Fundamentals of the ASCR, v.v.i., Rozvojovꢁa 135, 165 02 Prague 6, Czech Republic
S Supporting Information
b
ABSTRACT: Two novel, structurally unusual cysteine derivatives were isolated from the bulbs of Allium stipitatum (Allium subg.
Melanocrommyum) and shown to be S-(2-pyridyl)cysteine N-oxide and S-(2-pyridyl)glutathione N-oxide. The former compound is
the first example of a naturally occurring alliinase substrate that contains an N-oxide functionality instead of the S-oxide group. In
addition, S-methylcysteine S-oxide (methiin) and S-(methylthiomethyl)cysteine 4-oxide (marasmin) were found in the bulbs.
Presented data suggest that the previously reported identification of S-(2-pyridyl)cysteine S-oxide was most likely erroneous. The
alliinase-mediated formation of pyridyl-containing compounds following disruption of A. stipitatum bulbs was studied by a combination of
HPLC-MS, HPLC-PDA, DART-MS, and NMR techniques. It was found that no pyridyl-containing thiosulfinates are present in
homogenized bulbs in detectable quantities. Instead, various pyridine N-oxide derivatives are formed, including N-hydroxypyridine-
2(1H)-thione (pyrithione), 2-(methyldithio)pyridine N-oxide, 2-[(methylthio)methyldithio]pyridine N-oxide, di(2-pyridyl) disulfide
N-oxide, and di(2-pyridyl) disulfide N,N0-dioxide. This represents the first report of pyrithione formation as a natural product.
KEYWORDS: Allium stipitatum, S-(2-pyridyl)cysteine N-oxide, marasmin, sulfenic acid, DART-MS, pyrithione
’ INTRODUCTION
thanks to the recent study of O’Donnell et al.,4 who isolated three
sulfur-containing pyridine N-oxides from the bulbs of this plant
(Figure 1). The identity of these structurally unusual compounds
was rigorously confirmed by spectroscopic methods and by synthe-
sis of authentic samples. On the other hand, Kusterer et al.5
reported detection of several 2-pyridyl sulfur compounds in homo-
genized A. stipitatum bulbs, none of which reportedly contained
the N-oxide moiety (Figure 1). However, the identification of
compounds described in the latter study was mainly based on
LC-MS/MS data without comparison with authentic standards.
This study was aimed at resolving the puzzling controversy
between the aforementioned reports of O’Donnell et al.4 and Kusterer
et al.5 In this paper, we describe the isolation and full spectral
characterization of two structurally unique heterocyclic S-substituted
cysteine derivatives present in intact bulbs of A. stipitatum. Further-
more, alliinase-mediated formation of pyridyl-containing compounds
following tissue disruption was studied in detail by a combination
of HPLC-MS, HPLC-PDA, DART-MS, and NMR techniques.
The subgenus Melanocrommyum (Webb & Berthel.) Rouy
belongs to the largest groups within the genus Allium (Alliaceae).
It comprises about 160 mostly perennial species native to arid
regions of the Mediterranean, the Near and Middle East, north-
western China, Pakistan, and Central Asia. Many Melanocrom-
myum species are popular ornamentals thanks to very attractive
inflorescences (e.g., Allium macleanii J. G. Baker, Allium giganteum
Regel, Allium christophii Trautv., and Allium stipitatum Regel),
whereas some are important food crops cultivated in Central Asia
(e.g., A. stipitatum Regel and Allium suworovii Regel).1,2
A. stipitatum produces sturdy stalks 80ꢀ100 cm tall, each
bearing a showy, fireworks-like cluster of star-shaped flowers
(see the Supporting Information), hence its common name
drumstick onion (this common name is usually used for the
whole group of Melanocrommyum species producing drumstick-
like inflorescences). The flowers are typically purple, although
white varieties are also known. This species is native to the dry
steppes of eastern Afghanistan, Pakistan, Pamir, and the Tien-Shan
Mountains. In these regions, it is frequently collected in the wild or
cultivated under the local name “anzur” and used as a condiment
(typically as pickled bulbs). It should be noted, however, that the
same local name is also used for several other closely related
Melanocrommyum species, for example, Allium rosenbachianum or
A. suworovii.2,3
’ MATERIALS AND METHODS
General Methods. 1H, 13C, and 15N NMR spectra were recorded
on a Varian INOVA 500 MHz spectrometer (Varian, Palo Alto, CA).
Chemical shifts of 1 and 2 were referenced externally to the signal
Despite the fact that many Melanocrommyum species are
frequently consumed and widely used in folk medicine,3 only
little attention has been paid to the research of sulfur compounds
present in these plants. A. stipitatum attracted our attention
Received: February 19, 2011
Revised:
April 18, 2011
Accepted: April 22, 2011
Published: April 22, 2011
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2011 American Chemical Society
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Journal of Agricultural and Food Chemistry
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disproportionating into 16 and di(2-pyridyl) disulfide (all attempts to
purify it by preparative C-8 HPLC or silica gel chromatography failed).
2-[(Methylthiomethyl)dithio]pyridine N-oxide (11) was prepared by
oxidation of equimolar amounts of (methylthio)methanethiol and
2-sulfanylpyridine N-oxide by hydrogen peroxide in acetone and subse-
quent purification by preparative HPLC chromatography. 2-[(Methyl-
thiomethyl)dithio]pyridine was prepared analogously to the synthesis of
11, starting from (methylthio)methanethiol and pyridine-2-thiol.
Isolation of Crude CꢀS Lyases (Alliinases). The procedure
described in ref 13 was followed for the isolation of crude CꢀS lyases
from the bulbs of A. stipitatum and onion. The purity and specific activity
of the obtained preparations were not examined in detail.
GC-MS and HPLC Analysis. S-Substituted cysteines present in the
bulbs of A. stipitatum were analyzed by the GC-MS and HPLC methods
of Kubec and Dadꢁakovꢁa.9 The presence of the 18 various cysteine
derivatives listed in ref 6 was monitored.
Isolation of Compounds 1 and 2. Bulbs of A. stipitatum (337 g)
were cut in quarters and finely homogenized in 1 L of MeOH/H2O/HCl
(90:9:1, v/v/v), and the slurry was allowed to gently boil under reflux for
5 min and filtered through a layer of cotton wool. The extraction was
repeated with another 1 L portion of MeOH/H2O/HCl (90:9:1, v/v/v).
The extracts were combined and concentrated to approximately 150 mL
by vacuum evaporation (<35 °C). After centrifugation, the precipitate
was discarded, and the supernatant was adjusted to pH 2.5 by 5 M KOH
and applied onto a cation-exchange column (22 ꢁ 3 cm; Amberlite IR-
120, Hþ form, 16ꢀ45 mesh). After the column had been washed with
H2O (100 mL), the amino acid-containing fraction was eluted with
0.5 M NH4OH. The ninhydrin-positive fractions were collected, their
pH was adjusted to 5.5ꢀ6.0, and then they were freeze-dried. The
residue obtained was redissolved in 20 mL of 50 mM KH2PO4 buffer
(pH 5.5) and subjected to preparative C-8 HPLC, with 50 mM KH2PO4
(pH 5.5, solvent A) and acetonitrile (solvent B) as the mobile phase.
The gradient was as follows: A/B 97:3 (0 min), 97:3 (in 5 min), 40:60
(in 10 min), 40:60 (in 15 min), and 97:3 (in 22 min), with a flow rate of
18 mL minꢀ1. The fractions eluting at 7.2 and 11.3 min were collected,
pooled, and freeze-dried. The residues obtained were extracted with
2ꢁ 150 mL of MeOH and filtered, and the combined extracts were carefully
evaporated (<30 °C) to yield 1 (569 mg) and 2 (50 mg), respectively.
Extraction of Enzymatically Formed Compounds. Bulbs of
A. stipitatum (94.4 g) were cut in quarters and finely homogenized in
150 mL of H2O using a blender. The homogenate was allowed to stand
at room temperature for 30 min. Diethyl ether (200 mL) was added to
the homogenate, and the resulting slurry was filtered through a layer of
cotton wool. The layers were separated by centrifugation, and the
aqueous layers was extracted with another 100 mL portion of diethyl
ether. The combined ether portions were dried over MgSO4 and
concentrated to dryness by vacuum evaporation (<23 °C). The residue
obtained was redissolved in 1 mL of CH3CN, filtered through a syringe-
tip PTFE filter (0.45 μm), and analyzed by C-8 HPLC with H2O
(solvent A) and acetonitrile (solvent B) as the mobile phase. The
gradient was as follows: A/B 90:10 (0 min), 85:15 (in 10 min), 10:90
(in 40 min), 10:90 (in 50 min), and 90:10 (in 55 min), with a flow rate of
Figure 1. Structures of pyridyl-containing compounds previously re-
ported to occur in Allium stipitatum.
1
of DSS. H chemical shifts of compounds measured in CDCl3 were
referenced to the signal of HMDSS (δ 0.04) and 13C shifts to the central
line of the solvent signal (δ 76.99). 15N NMR chemical shifts were
referenced externally to the signal of nitromethane (δ 0.00) in the same
deuterated solvent. 1H chemical shifts of 15 measured by LC-NMR were
referenced to the signal of CH3CN (δ 2.00). Specific rotation values
were recordedby means of an Autopol IV polarimeter (Rudolph Research
Analytical, Hackettstown, NJ). Melting points (uncorrected) were deter-
mined using a Stuart SMP 10 apparatus. HPLC separations were
performed on a Dynamax SD-210 binary pump system (Varian), employ-
ing a Varian PDA 335 detector and a C-8 column (Varian Microsorb-MV
100 Å, 250 ꢁ 4.6 mm, 5 μm). Alternatively, a preparative C-8 column
(Varian Dynamax-100 Å, 250 ꢁ 21.4 mm, 8 μm) was used. LC-NMR,
GC-MS, IR, ESI-TOF-HRMS, and DART-MS experiments were con-
ducted using the instrumentation and settings as described in ref 5.
Plant Material. The bulbs of A. stipitatum ‘Glory of Pamir’ were
obtained from Dr. Leonid Bondarenko (Lithuanian Rare Bulb Garden,
Vilnius, Lithuania) in August 2009. The bulbs were originally collected
in Hodja-obi-Garm (Hissar Mountains, Pamir, Tajikistan). For a picture
of the plant see the Supporting Information. Voucher specimens are still
cultivated in the Alliaceae species collection at University of South
Bohemia and can be accessed upon request.
Chemicals. 2-Sulfanylpyridine N-oxide/N-hydroxypyridine-2(1H)-
thione (5/8), pyridine-2-thiol, S-methyl methanethiosulfonate, L-cy-
steine, and glutathione were purchased from Acros Organics (Geel,
Belgium). Chloromethyl methyl sulfide and 2-bromopyridine N-oxide
hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO), and
acetonitrile was supplied by LabScan (Dublin, Ireland). Other chemicals
were purchased from Spolana (Neratovice, Czech Republic).
Reference Compounds. (R)-S-(2-Pyridyl)cysteine N-oxide (1) and
S-(2-pyridyl)glutathione N-oxide (2) were prepared by the reaction of
2-bromopyridine N-oxide with L-cysteine or glutathione as described in the
Supporting Information. Various S-alk(en)yl-L-cysteine S-oxides were synthe-
sized or isolated as described elsewhere.6ꢀ11 2-(Methyldithio)pyridine
and 2-(methyldithio)pyridine N-oxide (10) were prepared as described
in ref 4. Di(2-pyridyl) disulfide N-oxide (15) and di(2-pyridyl) disulfide
N,N0-dioxide (16) were synthesized according to ref 12. However,
di(2-pyridyl) disulfide N-oxide (15) appeared to be quite labile, readily
0.9 mL minꢀ1
.
Model Experiments. Aliquots (1 mL) of stock solutions of 1, 3
and 4 (25 mM in 50 mM KH2PO4 buffer, pH 6.5) were placed in 10 mL
glass vials and mixed with 0.5 mL of a crude alliinase solution (10 mg/
1 mL). The model mixtures were incubated with stirring at 23 °C for
30 min (in some experiments only 2 min) and extracted with 3 mL
of CH2Cl2, and the organic portion was stripped off using argon. The
residues obtained were redissolved in acetonitrile (300 μL), filtered
through a syringe-tip PTFE filter (0.45 μm), and analyzed by C-8 HPLC
with H2O (solvent A) and acetonitrile (solvent B) as the mobile phase,
emloying the same solvent gradient as described above. For DART-MS
experiments, analogous model mixtures were prepared, incubated for
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1 min, and analyzed without filtration or extraction. When alliinase-
mediated cleavage of 1 was studied by 1H NMR, 10 mg of 1 in 0.3 mL of
D2O was mixed with 0.4 mL of a crude alliinase solution (10 mg/1 mL)
directly in an NMR tube. After shimming, the 1H NMR spectrum was
recorded.
C10H8N2NaOS2, 258.9970 [M þ Na]þ; found, 258.9971 (for NMR,
ESI-MS, and UV spectra see the Supporting Information).
Di(2-pyridyl) disulfide N,N0-dioxide (16): colorless solid; mp 215ꢀ218 °C;
UV (PDA, rel int) 236 nm (1.00), 262 nm (sh, 0.47), 302 nm (sh, 0.15);
1H NMR (CDCl3, 500 MHz) δ 7.19 (ddd, 2H, J = 7.8, 6.4, and 1.8 Hz,
H-5/H-50), 7.28 (ddd, 2H, J = 8.2, 7.8, and 1.1 Hz, H-4/H-40), 7.60
(ddd, 2H, J = 8.2, 1.8, and 0.5 Hz, H-3/H-30), 8.30 (ddd, 2H, J = 6.5, 1.1,
and 0.5 Hz, H-6/H-60); 13C NMR (CDCl3, 125 MHz) δ 122.9 (C-3/
C-30), 123.5 (C-5/C-50), 127.5 (C-4/C-40), 139.6 (C-6/C-60), 150.2
(C-2/C-20); 15N NMR (CDCl3, 50.7 MHz) δ ꢀ96.0; IR (KBr) 1462,
1419, 1250, 1227, 837, 764 cmꢀ1. ESI-TOF HRMS: calcd for
C10H9N2O2S2, 253.0100 [M þ H]þ; found, 253.0100; calcd for
C10H8N2NaO2S2, 274.9919 [M þ Na]þ; found, 274.9920 (for NMR,
IR, ESI-MS, and UV spectra see the Supporting Information).
Analytical Data of the Identified Compounds. (R)-S-(2-
Pyridyl)cysteine N-oxide (2-PyCNO, 1): colorless solid; mp 179ꢀ181 °C;
[R]2D2 þ29.4° (H2O); UV (PDA, rel int) 237 nm (1.00), 262 nm (sh, 0.33),
304 nm (0.11); 1H NMR(D2O, 500 MHz) δ 3.38 (dd, 1H, J = 14.7 and 8.5
Hz, H-3a), 3.62 (dd, 1H, J = 14.7 and 3.9 Hz, H-3b), 3.95 (dd, 1H, J = 8.5
and 3.9 Hz, H-2), 7.27 (ddd, 1H, J = 7.5, 6.5, and 1.5 Hz, H-50), 7.52 (ddd,
1H, J = 8.0, 1.5, and 1.1 Hz, H-30), 7.58 (ddd, 1H, J = 8.0, 7.5, and 0.6 Hz,
H-40), 8.22 (ddd, 1H, J = 6.5, 1.1, and 0.6 Hz, H-60); 13C NMR (D2O, 125
MHz) δ 31.8 (C-3), 53.3 (C-2), 122.6 (C-50), 123.6 (C-30), 131.7 (C-40),
139.5 (C-60), 150.6 (C-20), 173.1 (C-1); 15N NMR (D2O, 50.7 MHz) δ
ꢀ342.8 (NH2), ꢀ115.5 (NdO); IR (KBr) 3090ꢀ2540, 1628, 1574, 1361,
1219, 833, 764 cmꢀ1. ESI-TOF HRMS: calcd for C8H11N2O3S, 215.0485
[M þ H]þ; found, 215.0485 (for NMR, IR, ESI-MS, and UV spectra see
the Supporting Information).
(SC2,RC7)-S-(2-Pyridyl)glutathione N-oxide (2): colorless hygroscopic
solid; [R]2D2 ꢀ6.5° (H2O); UV (PDA, rel int) 238 nm (1.00), 261 nm
(sh, 0.39), 307 nm (0.15); 1H NMR (D2O, 500 MHz) δ 1.89 (dd, 2H,
J = 14.5 and 7.5 Hz, H-3), 2.33 (dt, 2H, J = 7.5 and 3.0 Hz, H-4), 3.30
(dd, 1H, J = 14.3 and 8.8 Hz, H-12a), 3.45 (t, 1H, J = 5.9 Hz, H-2), 3.54
(dd, 1H, J = 14.3 and 5.1 Hz, H-12b), 3.62 (q, 2H, J = 17.2 Hz, H-10),
4.68 (dd, 1H, J = 9.0 and 4.9 Hz, H-7), 7.25 (ddd, 1H, J = 7.6, 6.5, and
1.6 Hz, H-50), 7.52 (ddd, 1H, J = 8.4, 1.6, and 0.4 Hz, H-30), 7.56 (ddd,
1H, J = 8.4, 7.6, and 1.3 Hz, H-40), 8.20 (ddd, 1H, J = 6.5, 1.3, and 0.4 Hz,
H-60); 13C NMR (D2O, 125 MHz) δ 27.7 (C-3), 31.6 (C-12), 31.8
(C-4), 43.6 (C-10), 52.0 (C-7), 54.7 (C-2), 122.3 (C-50), 123.4 (C-30),
131.5 (C-40), 139.4 (C-60), 151.3 (C-20), 171.2 (C-8), 175.5 (C-5),
176.4 (C-11), 176.7 (C-1); IR (KBr) 3530ꢀ2650, 1658, 1608, 1419,
1222, 837 cmꢀ1. ESI-TOF HRMS: calcd for C15H21N4O7S, 401.1125
[M þ H]þ; found, 401.1127; calcd for C15H19N4O7S, 399.0980
[M ꢀ H]ꢀ; found, 399.0981 (for NMR, IR, ESI-MS, and UV spectra
see the Supporting Information).
2-(Methyldithio)pyridine N-oxide (10): colorless solid; mp 107ꢀ110 °C;
UV (PDA, rel int) 237 nm (1.00), 264 nm (sh, 0.41), 303 nm (sh, 0.12); 1H
NMR (CDCl3, 500 MHz) δ 2.45 (s, 3H, CH3), 7.15 (ddd, 1H, J = 7.6, 6.4,
and 1.8 Hz, H-50), 7.38 (ddd, 1H, J = 8.3, 7.6, and 1.2 Hz, H-40), 7.87 (ddd,
1H, J = 8.3, 1.8, and 0.5 Hz, H-30), 8.25 (ddd, 1H, J = 6.4, 1.2, and 0.5 Hz,
H-60); 13C NMR (CDCl3, 125 MHz) δ 21.7 (CH3), 121.3 (C-30), 121.6
(C-50), 126.2 (C-40), 138.7 (C-60), 151.7 (C-20); 15N NMR (CDCl3, 50.7
MHz) δ ꢀ97.6; IR (KBr) 1466, 1423, 1246, 1142, 833, 752 cmꢀ1. ESI-TOF
HRMS: calcd for C6H8NOS2, 174.0042 [M þ H]þ; found, 174.0041 (for
NMR, IR, ESI-MS, and UV spectra see the Supporting Information).
2-[(Methylthio)methyldithio]pyridine N-oxide (11): yellow oil; UV
(PDA, rel int) 239 nm (1.00), 265 nm (sh, 0.51), 304 nm (sh, 0.14); 1H
NMR (CDCl3, 500 MHz) δ 2.31 (s, 3H, CH3), 3.88 (s, 2H, CH2), 7.15
(ddd, 1H, J = 7.6, 6.4, and 1.8 Hz, H-50), 7.36 (ddd, 1H, J = 8.6, 7.6, and
1.2 Hz, H-40), 7.90 (dd, 1H, J = 8.3 and 1.8 Hz, H-30), 8.24 (ddd, 1H, J =
6.4, 1.2, and 0.5 Hz, H-60); 13C NMR (CDCl3, 125 MHz) δ 15.8 (CH3),
43.7 (CH2), 121.6 (C-30), 121.8 (C-50), 126.0 (C-40), 138.6 (C-60),
151.6 (C-20); 15N NMR (CDCl3, 50.7 MHz) δ ꢀ97.5; IR (KBr) 1462,
1423, 1261, 836, 760 cmꢀ1. ESI-TOF HRMS: calcd for C7H10NOS3,
219.9919 [M þ H]þ; found, 219.9920; calcd for C7H9NNaOS3,
241.9739 [M þ Na]þ; found, 241.9738 (for NMR, IR, ESI-MS, and
UV spectra see the Supporting Information).
’ RESULTS AND DISCUSSION
An amino acid/oligopeptide-containing fraction from the
bulbs of A. stipitatum was obtained by extraction with acidified
aqueous methanol and subsequent treatment by cation-exchange
chromatography. C-8 HPLC analysis of the fraction revealed the
presence of several compounds exhibiting significant absorp-
tion in the region of 230ꢀ260 nm. Two of these compounds
were subsequently isolated by preparative C-8 HPLC and fully
characterized by spectroscopic methods.
13C NMR data of the major isolated compound (1) indicated
the presence of five magnetically different aromatic carbons,
together with two sp3-hybridized carbon atoms and one car-
boxylic group. Further NMR experiments (including COSY,
HETCOR, and HMBC) revealed the presence of two isolated
structural subunits: (i) a C-monosubstituted pyridyl moiety and
(ii) a ꢀCH2CH(X)COOH chain. Unambiguous evidence that
the pyridyl moiety was substituted at position 2 was obtained by a
detailed analysis of the 1H spin system and from 1H NMR spectra
measured with selective proton decoupling. These spectra
showed that the four aromatic hydrogens formed a coupling
pathway chain with each of the two internal hydrogens being
vicinally coupled (J ∼ 7 Hz) to two other hydrogens. Those at δ
8.22 and 7.52 terminated the chain as they were coupled to only
one neighboring hydrogen (see the Supporting Information).
The ESI-TOF HRMS exhibited [M þ H]þ of 215.0485 (calcd
for C8H11N2O3S, 215.0485), indicating that the compound was
an oxide of S-(2-pyridyl)cysteine. The most difficult task in the
structure elucidation of 1 was to determine the site of oxidation.
Two possible isomeric structures were taken into consideration,
namely, S-(2-pyridyl)cysteine S-oxide and S-(2-pyridyl)cysteine
N-oxide (Figure 2). The abundant and widespread occurrence of
various S-substituted cysteine S-oxides in alliaceous plants initi-
ally favored the S-oxide structure. However, the 13C and 15N
NMR shifts together with the IR spectrum of 1 suggested that
the compound was rather the N-oxide. The most indicative of
correct assignment was the 13C NMR shift of the carbon C-3. It
has been observed that 13C NMR shifts of C-3 carbons in various
S-substituted cysteine S-oxides vary only slightly between δ 50
and 55, whereas those in S-substituted cysteines appear in the
region of δ 31ꢀ35 (in D2O). It is noteworthy that the S-bound
substituent has only a marginal effect on the shift (see the
Supporting Information). The signal of C-3 in 1 was found at
δ 31.8, indicating that the sulfur was not oxidized. This conclu-
sion was further supported by the fact that 1 was not cleaved
by onion alliinase, which is known to act only upon S-oxide
derivatives of L-cysteine14 [e.g., S-(2-pyrrolyl)cysteine S-oxide
was readily cleaved by the same crude onion alliinase preparation6].
Di(2-pyridyl) disulfide N-oxide (15): UV (PDA, rel int) 236 nm
(1.00), 266 nm (0.80), 308 nm (sh, 0.18); LC-1H NMR (D2O/CH3CN,
500 MHz) δ 7.31 (t, 1H, J = 5.8 Hz, H-5), 7.39 (t, 1H, J = 6.5 Hz, H-50),
7.56ꢀ7.62 (m, 2H, H-4/H-40), 7.76ꢀ7.82 (m, 2H, H-3/H-30), 8.32
(d, 1H, J = 6.2 Hz, H-6), 8.40 (s, 1H, H-60). ESI-TOF HRMS: calcd for
C10H9N2OS2, 237.0151 [M þ H]þ; found, 237.0150; calcd for
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is structurally markedly distinct from all derivatives previously
isolated from alliaceous plants in that it contains the N-oxide
moiety instead of the S-oxide group.
ESI-TOF HRMS data of the second compound (2) isolated from
the extract corresponded to the molecular formula of C15H20N4O7S.
The 13C NMR spectrum showed the presence of 15 different carbon
atoms, including 5 aromatic and 4 CdO carbons. Additional NMR
experiments (COSY, HETCOR, HMBC, and HSQC) revealed
the presence of several isolated structural subunits, namely, an
N-oxy-2-pyridyl moiety and ꢀC(O)CH2CH2CH(X)COOH
ꢀCH2CH(X)COꢀ ꢀCH2(X)COOH chains. These spectral
data indicated the compound to be an S-substituted glutathione
derivative, S-(2-pyridyl)glutathione N-oxide (Figure 2). Indeed,
this tripeptide was subsequently prepared by synthesis and found
to have spectral properties (NMR, MS, and UV) identical to those
of 2. Analogous S-substituted glutathione derivatives (bearing
2-carboxypropyl or methyl moieties) occur in onion, garlic, and
other alliaceous species and are thought to be intermediates in the
biogenesis of S-substituted cysteine S-oxides.15 Thus, it seems to
be reasonable to assume that 2 is a biochemical precursor of 1.
GC-MS and HPLC analysis of the amino acid fraction isolated
from A. stipitatum bulbs also revealed the presence of two other
S-substituted cysteine derivatives, namely, (SS,RC)-S-methylcys-
teine S-oxide (methiin, MCSO, 3) and (RS,RC)-S-(methylthio-
methyl)cysteine 4-oxide (marasmin, MTMCSO, 4). The identity
and absolute configurations of 3 and 4 were confirmed by com-
parison with authentic standards. The relative ratio of 1/3/4
was found to be 51:17:32 in the bulbs of A. stipitatum we
analyzed. Whereas methiin occurs abundantly in all allia-
ceous species,6,8,9,11,16 the presence of marasmin has not thus
far been reported in any genus Allium plant. This amino acid is
the key odor precursor of society garlic (Tulbaghia violacea
Harv.), an alliaceous plant belonging to the taxonomically closely
related genus Tulbaghia, which comprises species native to South
Africa.10
Figure 2. Structures of pyridyl-containing amino compounds isolated
from Allium stipitatum in this study (1 and 2) and the compound (10)
reportedly isolated by Kusterer et al.5
Furthermore, the IR spectrum contained no significant absorp-
tion band in the region of 980ꢀ1040 cmꢀ1 (ꢀSdO). Fool-
proof evidence about the identity of 1 was obtained by its
comparison with an authentic sample of (R)-S-(2-pyridyl)-
cysteine N-oxide prepared by synthesis. The compound iso-
lated from the bulbs was found to be identical in all respects
(NMR, MS, UV, optical rotation) with the standard. Therefore,
the structure of 1 could be unambiguously assigned as (R)-S-(2-
pyridyl)cysteine N-oxide (2-PyCNO) (Figure 2).
Kusterer et al.5 recently reported the isolation of an OPA-
derivatized amino acid from A. stipitatum that they claimed to be
S-(2-pyridyl)cysteine S-oxide (10). However, we did not detect
any other compound with Mr 214 except for 1 in the amino acid
fraction by HPLC-MS. Careful evaluation of the NMR data given
by Kusterer et al. for the compound they isolated revealed a
very close similarity of its 1H and 13C NMR shifts to those of 1.
In particular, the 13C NMR shift of carbon C-3 in the OPA
derivative was δ 35.5 (in CD3CN), clearly indicating that the
compound was not an S-oxide. Thus, we believe that the com-
pound isolated by Kusterer et al. was in fact the OPA derivative of
S-(2-pyridyl)cysteine N-oxide (1). A great deal of attention was
also paid to ruling out the possibility that 1 was formed from the
putative 10 during isolation. Therefore, a bulb of the plant was
homogenized in cold methanol, and the extract obtained was
directly analyzed by HPLC (without any exposure to elevated
temperature and pHextremes). Indeed, 1 was the onlycomponent
with Mr 214 present in the extract, clearly confirming that this
amino acid is not an artifact formed from 10 during isolation.
Herein, we report the isolation and full spectral characteriza-
tion of pure 1 for the first time. It is noteworthy that 2-PyCNO
(1) is only the third naturally occurring example of an aromatic
S-substituted cysteine derivative. The other two compounds are
S-benzylcysteine S-oxide (petiveriin) from the Amazonian plant
Petiveria alliacea L. (Phytolaccaceae) and S-(2-pyrrolyl)cysteine
S-oxide from giant onion (A. giganteum Regel) and some other
subgenus Melanocrommyum species.6,7 However, 2-PyCNO (1)
The alliinase-mediated conversion of the three cysteine deri-
vatives concomitantly present in the intact bulbs (1, 3, and 4)
was studied by a combination of HPLC-MS, HPLC-PDA,
1
DART-MS, and H NMR techniques. A diethyl ether extract
of homogenized bulbs of A. stipitatum was prepared and im-
mediately analyzed by HPLC-PDA. The chromatogram obtained
was surprisingly very simple, revealing the presence of only
a few compounds, with two of them accounting for 74% of the
total peak area (at 210 nm; see the Supporting Information).
HPLC-MS analysis of the extract indicated that the two most
abundant components exhibited Mr of 173 and 219. These
compounds were subsequently shown to be identical in all
respects (chromatographic behavior, UV and MS spectra) with
authentic samples of 2-(methyldithio)pyridine N-oxide (10)
and 2-[(methylthiomethyl)dithio]pyridine N-oxide (11), re-
spectively. In addition, N-hydroxypyridine-2(1H)-thione (8),
di(2-pyridyl) disulfide N-oxide (15), and di(2-pyridyl) disulfide
N,N0-dioxide (16) were identified as minor components in the
extract by HPLC-MS and by comparison with standards. It should
be mentioned that compounds 8, 10, 11, 15, and 16 appeared to
be quite photolabile, which rendered the use of preparative HPLC
for their isolation impossible (unless the effluent was diverted
before the PDA detector cell). Also during freeze-drying or
purification by silica gel column chromatography, most samples
decomposed, giving rise to bluish degradation products.
It is noteworthy that no other compounds having a molecular
weight of Mr 173, 219, 236, or 252 (corresponding to the presumable
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Journal of Agricultural and Food Chemistry
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Table 1. Allium stipitatum NI-DART and PI-DART Measurements
ionization
NI-DART
compound
species
[CH3OS]ꢀ
[C2H5OS2]ꢀ
[C5H4NOS]ꢀ
calcd
found
rel abundance (%)
6
62.9910
108.9787
126.0019
62.9898
108.9776
126.0008
0.49
0.56
100
7
5/8
PI-DART
9
[C2H6OS2 þ H]þ
[C3H8OS3 þ H]þ
[C6H7ONS2 þ H]þ
[C4H10OS4 þ H]þ
[C7H9NOS3 þ H]þ
[C10H8N2OS2 þ H]þ
110.9933
156.9810
174.0042
202.9687
219.9919
237.0151
110.9934
156.9810
174.0042
202.9688
219.9920
237.0150
0.24
nda
100
nd
13/14
10
12
11
7.31
0.38
15
a Not detected after puncturing the bulb (abundant in a model mixture consisting of 3, 4, and crude alliinase).
thiosulfinates and vic-disulfoxide reported by Kusterer et al.) were
detected in the extract by HPLC-MS, despite our specifically
searching for them. Even when various model mixtures consisting
of 1, 3, and 4 were injected shortly (within 2 min) after mixing
with crude alliinase from A. stipitatum, no such compounds were
detected.
and di(2-pyridyl) disulfide N,N0-dioxide (16), are probably
formed by secondary transformations of 5 and 8, as documented
by numerous studies (Figure 4).12,19ꢀ21 The assumption that 15
and 16 are not primary products of the alliinase-mediated cleavage
of 2-PyCNO (1) is also supported by the facts that only low
amounts of these two compounds were found in the ether extract
by HPLC and the formation of 16 was not observed by DART-MS
immediately after tissue disruption. To our surprise, no evidence
was obtained about the expected presence of the three marasmin-
derived thiosulfinates 12ꢀ14, presumably formed by condensa-
tion of 6 and 7 (Figure 3). The presence of these thiosulfinates was
not detected by HPLC-MS in the ether extract or by DART-MS
upon punctureof thebulb. These compounds werefoundonly in a
model mixture consisting of 3, 4, and crude alliinase in the absence
of 1 (Table 1). We therefore assume that, in the bulbs we analyzed,
sulfenic acid 7 had been completely trapped by 5 before it could
form the thiosulfinates.
Our results clearly show that no pyridyl-containing thiosulfi-
nates are formed in detectable quantities in crushed A. stipitatum
tissue. These findings are in major conflict with the study of
Kusterer et al.,5 who reported detection of several 2-pyridyl
thiosulfinates and one S,S0-dioxide derivative (Figure 1). How-
ever, they deduced structures of these compounds mainly from
HPLC-MS/MS data (without comparison with authentic samples),
assuming the precursor to be S-(2-pyridyl)cysteine S-oxide (10).
The only compound they characterized more rigorously (by NMR
and IR) was claimed to be di(2-pyridyl) disulfide S,S0-dioxide.
However, the presence of this compound would be highly surpris-
ing, as vic-disulfoxides are known to be extremely reactive,
nonisolatable intermediates of only a fleeting existence at room
temperature.22,23 The 1H NMR data given by Kusterer et al.5 for
this compound were carefully evaluated and found to be nearly
identical to those of di(2-pyridyl) disulfide N,N0-dioxide (16).
Further evidence that the compounds described in ref 5 were in
fact the corresponding N-oxides was obtained from their UV
spectra. We observed that 2-pyridyl-containing disulfides ex-
hibit considerably different UV spectra from those of their
N-oxide counterparts. UV spectra of the former compounds
contain an intense absorption band around 282 nm, whereas
pyridine N-oxides do not show significant absorption in this
region (see the Supporting Information). On the other hand,
the latter compounds typically exhibit a maximum at 236ꢀ239 nm,
with two considerably less intense shoulders around 262 and
304 nm (in H2O/CH3CN). All compounds detected by Kusterer
et al. exhibited UV absorption at 238, 260ꢀ264, and 310 nm (in
CH3OH), clearly suggesting they were pyridine N-oxides rather
To get more detailed insight into the formation pathways of
8ꢀ16, direct analysis in real time mass spectrometry (DART-
MS) was also employed. This exceptionally mild desorption
ionization technique allows one to observe the formation of
compounds of only a fleeting existence without the necessity for
any sample pretreatment, simply by momentarily holding the
tested material in the DART gas stream.6,11,17 A bulb of A. stipitatum
was punctured by a sampling capillary, which was immediately
(within 2ꢀ3 s after tissue disruption) inserted in the DART source
ionization region. As shown in Table 1, the predominant signal
detected by negative ionization (NI) DART-HRMS corresponded
to a compound(s) with the molecular formula of C5H5NOS. It can
be assumed that this compound was 2-sulfanylpyridine N-oxide (5)
arising by alliinase-mediated cleavage of 2-PyCNO (1). Further-
more, signals corresponding to the two sulfenic acids 6 and 7
(formed from 3 and 4, respectively) were also detected by NI-
DART-HRMS. Positive ionization (PI) DART-HRMS experiments
confirmed the abundant presence of 10 and 11, along with minor
amounts of 9 and 15 (see the Supporting Information). Similar
results were also obtained by analyzing various model mixtures
consisting of 1, 3, 4, and crude alliinase from A. stipitatum. The
alliinase-mediated cleavage of 2-PyCNO (1) was also studied by
NMR. 2-PyCNO was mixed with crude alliinase directly in an NMR
1
tube, and the H NMR spectrum was recorded. 2-PyCNO was
completely consumed within 3 min, and the only signals detected in
the region of δ 6ꢀ9 were those belonging to N-hydroxypyridine-
2(1H)-thione (8) (see the Supporting Information).
On the basis of the data obtained, it could be proposed that
compounds 5ꢀ16 are formed as outlined in Figures 3 and 4.
Upon tissue disruption, 2-PyCNO (1) is cleaved by alliinase to
yield 2-sulfanylpyridine N-oxide (5), which in turn condenses
with sulfenic acids 6 and 7, giving rise to 10 and 11, respectively.
Indeed, when 5 was added into model mixtures consisting of
crude alliinase and either 3 or 4, the formation of 10 and 11,
respectively, was observed. Compound 5 can also spontaneously
rearrange into tautomeric and more stable N-hydroxypyridine-
2(1H)-thione (8). It has been reported that, in aqueous media,
the thione form (8) predominates over the N-oxide (5), with
the molar ratio of 8/5 being approximately 54.18,19 The other
pyridine N-oxides detected, di(2-pyridyl) disulfide N-oxide (15)
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Journal of Agricultural and Food Chemistry
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Figure 3. Alliinase-mediated formation of volatile compounds in Allium stipitatum (the compounds marked by / were detected only in some model
mixtures).
Figure 4. Proposed formation of pyridyl-containing compounds from pyrithione (adapted with permission from ref 20).
than thiosulfinates. Finally, if thiosulfinates had been formed,
both regiomers differing in the position of the oxygen atom
[i.e., RS(O)SR0 and RSS(O)R0] should have been observed.
However, no such pairs of compounds were reported in ref 5.
Compounds 10,11,and16 were already isolated from A. stipitatum
by O’Donnell et al.,4 and 16 was also found in a basidiomycetous
mushroom of the genus Cortinarius native to New Zealand.24
These three compounds were shown to possess potent inhibitory
activity against several multidrug-resistant strains of microorgan-
isms including Staphylococcus aureus.4 Of particular interest is the
formation of N-hydroxypyridine-2(1H)-thione (pyrithione, 8)
upon crushing of the bulbs of A. stipitatum. To the best of our
knowledge, this is the first report of pyrithione formation from
a naturally occurring precursor (i.e., 2-PyCNO, 1). This com-
pound, a distant analogue of aspergillic acid, is known to exhibit
significant antimicrobial properties25 and is widely used (under
the comercial name Omadine) as an antifouling agent in various
paints, coatings, polymers, and other materials. Pyrithione and its
zinc salt are also the active components of antidandruff shampoos
and other cosmetics.26 Thus, A. stipitatum is equipped with a very
efficient and powerful weaponry to defend itself against attacks
of various predators (insect, ruminants, microorganisms etc.).
Pyrithione was also shown to effectively inhibit the growth of
higher plants.27 It can be therefore assumed that the ability of
A. stipitatum to transform 2-PyCNO (1) into pyrithione and other
pyridine N-oxide derivatives represents an important ecological
advantage helping the plant to survive in the harsh environment.
Although no studies have been published regarding some
adverse effects related to the consumption of A. stipitatum,
potential health risks should be carefully considered. N-Hydro-
xypyridine-2(1H)-thione (pyrithione, 8), one of the compounds
formed from 2-PyCNO (1), is known to be very photolabile,
giving rise to the hydroxyl radical upon irradiation with UVꢀ
visible light (Figure 4).12,19ꢀ21 This reactive radical has been
shown to generate modifications of nucleic acid bases and to
cause mutations that can initiate carcinogenesis. It also plays an
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Journal of Agricultural and Food Chemistry
ARTICLE
important role in oxidative damage of cells leading to a number of
age-related diseases.28ꢀ30 Furthermore, pyrithione and products
of its transformations are suspected of being embryotoxic for fish
and other marine organisms and have also been shown to be
potentially cytotoxic and genotoxic.31ꢀ34 Thus, it cannot be ruled
out that regular consumption of A. stipitatum could have some
undesirable health consequences. Given the fact that A. stipitatum
and related species (e.g., A. rosenbachianum or A. suworovii) are
frequently consumed by large populations in Central Asia, it seems
to be highly pertinent to evaluate the biological properties of
2-PyCNO-derived compounds in more detail.
photodiode array; PI, positive ionization; PTFE, polytetrafluor-
ethene; 2-PyCNO, S-(2-pyridyl)cysteine N-oxide; SPE, solid phase
extraction; subg, subgenus; TOF, time-of-flight; UV, ultraviolet.
’ REFERENCES
(1) Fritsch, R. M.; Blattner, F. R.; Gurushidze, M. New classification
of Allium L. subg. Melanocrommyum (Webb & Berthel.) Rouy
(Alliaceae) based on molecular and morphological characters. Phyton
2010, 49, 145–220.
(2) Kamenetsky, R; Fritsch, R. M. Ornamental Alliums. In Allium
Crop Science: Recent Advances; Rabinowitch, H. D., Currah, L., Eds.;
CABI Publishing: New York, 2002; pp 459ꢀ491.
’ ASSOCIATED CONTENT
(3) Keusgen, M.; Fritsch, R. M.; Hisoriev, H.; Kurbonova, P. A.;
Khassanov, F. O. Wild Allium species (Alliaceae) used in folk medicine
of Tajikistan and Uzbekistan. J. Ethnobiol. Ethnomed. 2006, 2, 18.
(4) O’Donnell, G.; Poeschl, R.; Zimhony, O.; Gunaratnam, M.;
Moreira, J. B. C.; Neidle, S.; Evangelopoulos, D.; Bhakta, S.; Malkinson,
J. P.; Boshoff, H. I.; Lenaerts, A.; Gibbons, S. Bioactive pyridine-N-oxide
disulfides from Allium stipitatum. J. Nat. Prod. 2009, 72, 360–365.
(5) Kusterer, J.; Vogt, A.; Keusgen, M. Isolation and identification of
a new cysteine sulfoxide and volatile sulfur compounds from Allium
subgenus Melanocrommyum. J. Agric. Food Chem. 2010, 58, 520–526.
S
Supporting Information. Selected NMR, IR, UVꢀvis,
b
and ESI-MS spectra of compounds 1, 2, 10, 11, 15, and 16 (NMR
and IR spectra of 15 and 16 were recorded using synthetic
samples); NI-DART-HRMS and PI-DART-HRMS traces of
volatiles formed upon crushing a bulb of A. stipitatum; repre-
sentative HPLC-PDA chromatogram of an ether extract of
homogenized bulbs of A. stipitatum; 13C NMR data of various
S-substituted cysteine derivatives; picture of the Allium stipitatum
plant analyzed; and experimental details about the preparation of
compounds 1 and 2. This material is available free of charge via
the Internet at http://pubs.acs.org.
ꢀ
(6) Kuꢀcerovꢁa, P.; Kubec, R.; Simek, P.; Vꢁaclavík, L.; Schraml, J.
Allium discoloration: the precursor and formation of the red pigment in
giant onion (Allium giganteum Regel) and some other subgenus
Melanocrommyum species. J. Agric. Food Chem. 2011, 59, 1821–1828.
(7) Kubec, R.; Musah, R. A. Cysteine sulfoxide derivatives in
Petiveria alliacea. Phytochemistry 2001, 58, 981–985.
’ AUTHOR INFORMATION
(8) Kubec, R.; Svobodovꢁa, M.; Velíꢀsek, J. Distribution of S-alk-
(en)ylcysteine sulfoxides in some Allium species. Identification of a
new flavor precursor: S-ethylcysteine sulfoxide (ethiin). J. Agric. Food
Chem. 2000, 48, 428–433.
Corresponding Author
*Phone: þ420-38-7772664. Fax: þ420-38-5310405. E-mail:
kubecr@centrum.cz. or kubecr@yahoo.com
(9) Kubec, R.; Dadꢁakovꢁa, E. Chromatographic methods for deter-
mination of S-substituted cysteine derivatives ꢀ a comparative study.
J. Chromatogr., A 2009, 1216, 6957–6963.
(10) Kubec, R.; Velíꢀsek, J.; Musah, R. A. The amino acid precursors
and odor formation in society garlic (Tulbaghia violacea Harv.). Phy-
tochemistry 2002, 60, 21–25.
(11) Kubec, R.; Cody, R. B.; Dane, A. J.; Musah, R. A.; Schraml, J.;
Vattekkatte, A.; Block, E. Applications of direct analysis in real time-
mass spectrometry (DART-MS) in Allium chemistry. (Z)-Butanethial
S-oxide and 1-butenyl thiosulfinates and their S-(E)-1-butenylcysteine
S-oxide precursor from Allium siculum. J. Agric. Food Chem. 2010,
58, 1121–1128.
(12) Barton, D. H. R.; Chen, C.; Wall, G. M. Synthesis of disulfides
via sulfenylation of alkyl and aryldithiopyridine N-oxides. Tetrahedron
1991, 47, 6127–6138.
(13) Shen, C.; Parkin, K. L. In vitro biogeneration of pure thiosulfi-
nates and propanethial-S-oxide. J. Agric. Food Chem. 2000, 48, 6254–6260.
(14) Lancaster, J. E.; Shaw, M. L.; Joyce, M. D. P.; McCallum, J. A.;
McManus, M. T. A novel alliinase from onion roots. Biochemical
characterization and cDNA cloning. Plant Physiol. 2000, 122, 1269–1279.
(15) Lancaster, J. E.; Shaw, M. L. γ-Glutamyl peptides in the
biosynthesis of S-alk(en)yl-L-cysteine sulphoxides (flavour precursors)
in Allium. Phytochemistry 1989, 28, 455–460.
Funding Sources
Financial support provided by the Grant Agency of South
Bohemia (GAJU 067/2010/Z and GAJU 028/2010/P) (R.K.
and P.K.), the Ministry of Education of the Czech Republic
(MSM6007665806 and MSM6046137305) (R.K. and J.H.), and
the Grant Agency of ASCR (IAA 400720706) (J.S.) is greatly
appreciated.
’ ACKNOWLEDGMENT
We thank Pavla Kruꢀzberskꢁa (Laboratory of Analytical Bio-
ꢀ
chemistry, Biology Centre, ASCR, Ceskꢁe Budꢀejovice), Petr
ꢀ
Zꢁaꢀcek (Institute of Organic Chemistry and Biochemistry, ASCR,
Prague), Martina Kluꢀcꢁakovꢁa (University of Technology, Brno),
and Jan Sꢁykora (Institute of Chemical Process Fundamentals,
ASCR, Prague) for their helpful technical assistance.
’ ABBREVIATIONS USED
COSY, correlation spectroscopy; DART, direct analysis in
real time; DSS, 4,4-dimethyl-4-silapentane-1-sulfonic acid; ESI,
electrospray ionization; GC-MS, gas chromatographyꢀmass
spectrometry; HETCOR, heteronuclear chemical shift correla-
tion; HMBC, heteronuclear multiple bond correlation; HMDSS,
hexamethyldisilane; HPLC, high-performance liquid chromatog-
raphy; HPLC-MS, high-performance liquid chromatographyꢀmass
spectrometry; HRMS, high-resolution mass spectrometry; HSQC,
heteronuclear single-quantum correlation; IR, infrared; MCSO,
S-methylcysteine S-oxide (methiin);MTMCSO, S-(methylthiome-
thyl)cysteine 4-oxide (marasmin); NI, negative ionization; NMR,
nuclear magnetic resonance; OPA, ortho-phthaldialdehyde; PDA,
(16) Fritsch, R. M.; Keusgen, M. Occurrence and taxonomic sig-
nificance of cysteine sulphoxides in the genus Allium L. (Alliaceae).
Phytochemistry 2006, 67, 1127–1135.
(17) Block, E.; Dane, A. J.; Thomas, S.; Cody, R. B. Applications of
direct analysis in real time mass spectrometry (DART-MS) in Allium
chemistry. 2-Propenesulfenic and 2-propenesulfinic acids, diallyl trisul-
fane S-oxide, and other reactive sulfur compounds from crushed garlic
and other Alliums. J. Agric. Food Chem. 2010, 58, 4617–4625.
(18) Jones, R. A.; Katritzky, A. R. N-Oxides and related compounds.
Part XVII. The tautomerism of mercapto- and acylamino-pyridine
l-oxides. J. Chem. Soc. 1960, 2937–2942.
5769
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Journal of Agricultural and Food Chemistry
ARTICLE
(19) Aveline, B. M.; Kochevar, I. E.; Redmond, R. W. Photochem-
istry of the nonspecific hydroxyl radical generator, N-hydroxypyridine-
2(1H)-thione. J. Am. Chem. Soc. 1996, 118, 10113–10123.
(20) Boivin, J.; Crꢁepon, E.; Zard, S. Z. Generation of hydroxyl
radicals from 1-hydroxypyridine-2(1H)-thione and their application to
organic synthesis. Bull. Soc. Chim. Fr. 1992, 129, 145–150.
(21) Lapinski, L.; Gerega, A.; Sobolowski, A. L.; Nowak, M. J.
Thioperoxy derivative generated by UV-induced transformation of
N-hydroxypyridine-2(1H)-thione isolated in low-temperature matrixes.
J. Phys. Chem. A 2008, 112, 238–248.
(22) Freeman, F. vic-Disulfoxides and OS-sulfenyl sulfinates. Chem.
Rev. 1984, 84, 117–135.
(23) Ishii, A. Syntheses and properties of vic-disulfoxides. J. Synth.
Org. Chem. Jpn. 2006, 64, 395–405.
(24) Nicholas, G. M.; Blunt, J. W.; Munro, M. H. G. Cortamidine
oxide, a novel disulfide metabolite from the New Zealand basidiomycete
(mushroom) Cortinarius species. J. Nat. Prod. 2001, 64, 341–344.
(25) Khattar, M. M.; Salt, W. G.; Stretton, R. J. The influence of
pyrithione on the growth of microorganisms. J. Appl. Bacteriol. 1988, 64,
265–272.
(26) Marks, R.; Pearse, A. D.; Walker, A. P. The effects of a shampoo
containing zinc pyrithione on the control of dandruff. Br. J. Dermatol.
1985, 112, 415–422.
(27) Norman, A. G. Growth repression of higher plants by 2-pyr-
idinethiol, 1-oxide. Plant Physiol. 1957, 32, 16–19.
(28) Adam, W.; Marquardt, S.; Saha-M€oller, C. R. Oxidative DNA
damage in the photolysis of N-hydroxy-2-pyridone, a specific hydroxyl-
radical source. Photochem. Photobiol. 1999, 70, 287–291.
(29) Epe, B.; Ballmaier, D.; Adam, W.; Grimm, G. N.; Saha-M€oller,
C. R. Photolysis of N-hydroxypyridinethiones: a new source of hydroxyl
radicals for the direct damage of cell-free and cellular DNA. Nucleic Acids
Res. 1999, 24, 1625–1631.
(30) Chaulk, S. G.; Pezacki, J. P.; MacMillan, A. M. Studies of RNA
cleavage by photolysis of N-hydroxypyridine-2(1H)-thione. A new
photochemical footprinting method. Biochemistry 2000, 39, 10448–10453.
(31) Goka, K. Embryotoxicity of zinc pyrithione, an antidandruff
chemical, in fish. Environ. Res. A 1999, 81, 81–83.
(32) M€oller, M.; Adam, W.; Saha-M€oller, C. R.; Stopper, H. Studies
on cytotoxic and genotoxic effects of N-hydroxypyridine-2-thione
(Omadine) in L5178Y mouse lymphoma cells. Toxicol. Lett. 2002, 136,
77–84.
(33) Doose, C. A.; Ranke, J.; Stock, F.; Bottin-Weber, U.; Jastorff, B.
Structureꢀactivity relationships of pyrithiones ꢀ IPC-81 toxicity tests
with the antifouling biocide zinc pyrithione and structural analogs. Green
Chem. 2004, 6, 259–266.
(34) Onduka, T.; Mochida, K.; Harino, H.; Ito, K.; Kakuno, A.; Fujii,
K. Toxicity of metal pyrithione photodegradation products to marine
organisms with indirect evidence for their presence in water. Arch.
Environ. Contam. Toxicol. 2010, 58, 991–997.
5770
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