Allium chemistry: Synthesis, natural occurrence, biological activity, and chemistry of Se-alk(en)ylselenocysteines and their γ-glutamyl derivatives and oxidation products

Article abstract of DOI:10.1021/jf001097b

Syntheses are reported for γ-glutamyl Se-methylselenocysteine (8a), selenolanthionine (16), Se-1-propenylselenocysteine (6d), Se-2-methyl-2-propenyl-L-selenocysteine (6e), and Se-2-propynyl-L-selenocysteine (6f). Oxidation of 8a and Se-methylselenocysteine (6a) gives methaneseleninic acid (24), characterized by X-ray crystallography, and dimethyl diselenide (25). Oxidation of Se-2-propenyl-L-selenocysteine (6c) gives allyl alcohol and 3-seleninoalanine (22). Compound 22 is also formed on oxidation of 16 and selenocystine (4). Oxidation of 6d gives 2-[(E,Z)-1-propenylseleno]-propanal (36). These oxidations occur by way of selenoxides, detected by chromatographic and spectroscopic methods. The natural occurrence of many of the Se-alk(en)ylselenocysteines and their γ-glutamyl derivatives and oxidation products is discussed. Three homologues of the potent cancer chemoprevention agents 6a and 6c, namely 6d-f, were evaluated for effects on cell growth, induction of apoptosis, and Dna-damaging activity using two murine mammary epithelial cell lines. Although each compound displays a unique profile of activity, none of these compounds (6d-f) is likely to exceed the chemopreventive efficacy of selenocysteine Se-conjugates 6a and 6c.

Full text of DOI:10.1021/jf001097b

458
J. Agric. Food Chem. 2001, 49, 458−470
Alliu m Ch em istr y: Syn th esis, Na tu r a l Occu r r en ce, Biologica l
Activity, a n d Ch em istr y of Se-Alk (en )ylselen ocystein es a n d Th eir
γ-Glu ta m yl Der iva tives a n d Oxid a tion P r od u cts
Eric Block,*,† Marc Birringer,†,§ Weiqin J iang,‡ Tsukasa Nakahodo,† Henry J . Thompson,‡
Paul J . Toscano,† Horst Uzar,§ Xing Zhang,† and Zongjian Zhu‡
Department of Chemistry, State University of NewYorksAlbany, Albany, New York 12222; Department of
Chemistry, Universita¨t-Gesamthochschule-Siegen, Siegen, Germany; and Center for Nutrition in the
Prevention of Disease, AMC Cancer Research Center, Denver, Colorado 80214
Syntheses are reported for γ-glutamyl Se-methylselenocysteine (8a ), selenolanthionine (16), Se-1-
propenylselenocysteine (6d ), Se-2-methyl-2-propenyl-L-selenocysteine (6e), and Se-2-propynyl-L-
selenocysteine (6f). Oxidation of 8a and Se-methylselenocysteine (6a ) gives methaneseleninic acid
(24), characterized by X-ray crystallography, and dimethyl diselenide (25). Oxidation of Se-2-
propenyl-L-selenocysteine (6c) gives allyl alcohol and 3-seleninoalanine (22). Compound 22 is also
formed on oxidation of 16 and selenocystine (4). Oxidation of 6d gives 2-[(E,Z)-1-propenylseleno]-
propanal (36). These oxidations occur by way of selenoxides, detected by chromatographic and
spectroscopic methods. The natural occurrence of many of the Se-alk(en)ylselenocysteines and their
γ-glutamyl derivatives and oxidation products is discussed. Three homologues of the potent cancer
chemoprevention agents 6a and 6c, namely 6d -f, were evaluated for effects on cell growth, induction
of apoptosis, and DNA-damaging activity using two murine mammary epithelial cell lines. Although
each compound displays a unique profile of activity, none of these compounds (6d -f) is likely to
exceed the chemopreventive efficacy of selenocysteine Se-conjugates 6a and 6c.
Keyw or d s: γ-Glutamyl Se-methylselenocysteine; selenoxides; Allium species; selenocysteines
INTRODUCTION
may also be present in these plants. Thus, from analysis
of the extracts of 75Se-treated onions (Allium cepa),
evidence was obtained for the possible presence of
selenocystine (4), selenomethionine (5), Se-â-carboxy-
Genus Allium plants contain S-alk(en)ylcysteine S-
oxides [2a -d , RS(O)CH2CH(NH2)COOH; R ) Me, n-Pr,
CH2dCHCH2, and MeCHdCH], which originate from
γ-glutamyl S-alk(en)ylcysteine storage compounds (1)
via S-alk(en)ylcysteines (Scheme 1). Crushing these
Sch em e 1
propylselenocysteine (6g), Se-methylselenocysteine Se-
oxide (7a ), Se-1-propenylselenocysteine Se-oxide (7d ),
and γ-glutamyl Se-1-propenylselenocysteine (8b). The
presence of 4 and 5 was confirmed using authentic
compounds (3). In an effort to confirm the identity of
the other species, Spåre and Virtanen synthesized Se-
methyl-, Se-propyl-, and Se-2-propenylselenocysteine
(6a -c, respectively) but were unsuccessful in their
attempts to oxidize 6a -c to the corresponding Se-oxides
7a -c.
plants releases sulfur-containing flavorants 3, which are
formed through the action of alliinase (â-lyase) enzymes
on 2 (1, 2). In 1964 Spåre and Virtanen (3) first
suggested that analogous types of selenium compounds
* To whom correspondence should be addressed [phone (518)
442-4459; fax (518) 442-3462; e-mail eb801@csc.albany.edu].
† SUNY-Albany.
§ Universita¨t-Siegen.
‡ AMC Cancer Center.
10.1021/jf001097b CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/20/2000

Chemistry and Biological Activity of Selenoamino Acids and Their Se Oxides
J. Agric. Food Chem., Vol. 49, No. 1, 2001 459
EXPERIMENTAL PROCEDURES
Since the seminal studies by Spåre and Virtanen (3),
much progress has been made in Allium organosele-
nium chemistry and, more broadly, in understanding
the natural products chemistry of selenium and its
importance to human health (4). Interest in identifying
Allium organoselenium compounds has been heightened
by the discovery that Se-enriched garlic (Allium sati-
vum) is very effective in mammary cancer chemopre-
vention in a well-characterized experimental model
system (5-10) and that this capacity appears to be
generalizable in that Se-enriched broccoli (Brassica
oleracea) is similarly effective (11). Of particular interest
is the evidence that Se-enriched Allium plants are much
more active in cancer prevention than their unenriched
Allium counterparts and that synthetic Se-2-propenyl-
selenocysteine (6c) shows stronger chemopreventive
effects than Se-methylselenocysteine (6a ). However, in
vitro 6c (but not 6a ) damaged DNA; neither compound
damaged DNA in vivo. Against this background several
questions emerged in the selenium cancer chemopre-
vention field: (1) to what extent does the spectrum of
Alllium organselenium compounds parallel that of
sulfur-containing compounds in this genus; (2) does
oxidation of Se-2-propenylselenocysteine (6c) create
transitory chemopreventive metabolites responsible for
the uniquely stronger chemopreventive effects of 6c
compared to those of Se-methylselenocysteine (6a ); and
(3) are there compounds related to 6c which can be
prepared that retain the superior chemopreventive
effects without damaging DNA?
The work reported in this paper is a logical extension
of our previous investigations in this area wherein the
organoselenium compounds in Allium extracts (and
related plants including broccoli and Se-enriched yeast)
were identified using high-performance liquid chroma-
tography with inductively coupled plasma-mass spec-
trometric (HPLC-ICP-MS) and electrospray mass spec-
trometric (HPLC-ESI-MS) detection (12-17). In this
previous work the major selenium compound in both Se-
enriched and unenriched garlic was identified as
γ-glutamyl Se-methylselenocysteine (8a ) along with
lesser amounts of Se-methylselenocysteine (6a ), sele-
nocystine (4), and selenomethionine (5), among other
compounds (16). In the first part of this paper, details
of chemical syntheses of organoselenium compounds
necessary for the further development of the field are
provided, including 8a , Se-(E,Z)-1-propenylselenocys-
teine (6d ), derivatives of which are claimed to be present
in Allium species (3), and Se-2-methyl-2-propenylsele-
nocysteine (6e) and Se-2-propynylselenocysteine (6f),
which were synthesized to compare their biological
activity with that of Se-2-propenylselenocysteine (6c).
On the basis of what is known about the occurrence of
Allium sulfur-containing compounds, the second part
of the paper addresses a strategic issue, namely, whether
efforts to exploit Allium organoselenium chemistry for
cancer prevention should be focused on already identi-
fied Se-alk(en)ylcysteine storage compounds or on their
Se-oxides. The formation and novel chemistry of Se-
oxides of the Se-alk(en)yl storage compounds will be
described. Evidence is presented that natural levels of
Se-alk(en)ylcysteines Se-oxides in Allium species must
be very low. The third part of the paper provides an
initial assessment of the biological activities of com-
pounds 6d -f, which are compared with the known
activities of 6a -c.
NMR spectra were obtained with Bruker AC200 or AMX
400 NMR spectrometers (200 or 400 MHz) or a Varian Gemini
spectrometer (300 MHz), with TMS or sodium 3-(trimethylsil-
yl)propanesulfonate as internal standard in CDCl3 or aqueous
solutions, respectively, and MeSeMe (Strem Chemicals, New-
buryport, MA) or MeSeSeMe (Aldrich, Milwaukee, WI) in glass
capillary tubes as 77Se standards. Hewlett-Packard HP 5988
or 5898 mass spectrometers were used for GC-MS and LC-
MS, a Bruker-Hewlett-Packard Esquire-LC mass spectrometer
(Bruker-Franzen Analytik GmbH, Bremen, Germany) was
used for ESI-MS (by M. Kotrebai), and a Finnigan TSQ 7000
(Finnigan MAT, San J ose, CA) equipped with an APCI
interface was used for APCI-MS (by Dr. Elizabeth Calvey).
Mass spectra are given for the most abundant 80Se isotope only.
The following were purchased from commercial sources: Dul-
becco’s modified Eagle’s medium and F-12 medium, Triton
X-100, glutaraldehyde, crystal violet (all from Sigma, St. Louis,
MO); adult bovine serum (Gemini Bioproducts, Calabasas, CA);
insulin and epidermal growth factor (Intergen, Purchase, NY);
gentamicin reagent solution and agarose (Gibco BRL, Grand
Island, NY); oligreen (Molecular Probes, Eugene, OR); and
L-selenocystine, L-selenomethionine, and â-chloro-L-alanine
(Aldrich, Milwaukee, WI). Compounds 6a -c were prepared
as described elsewhere (10, 16 18). Most reactions were carried
out under dry N2 or argon. Diethyl ether (“ether”) and THF
were distilled under N2 from sodium-benzophenone ketyl, CH2-
Cl2 was distilled from calcium hydride, MeOH was distilled
from Mg, and ethyl acetate was distilled before use. Analytical
TLC was performed on precoated silica gel plates (Art. No.
5715, Merck) with a 254 nm fluorescent indicator. HPLC was
performed on Rainin HPX and HPXL systems on 5 µm C-18
or normal phase silica columns with UV detection at 254 nm.
With the C-18 column, a 7:3 H2O/CH3CN mixture was typically
employed. A model 7942T Chromatotron (Harrison Research,
Palo Alto, CA) was also used with silica gel from Merck (Art.
No. 7749).
Ben zyl (Z)-1-P r op en yl Selen id e [(Z)-10]. Magnesium
turnings (0.67 g, 28 mmol) were placed in a 100 mL three-
neck round-bottom flask equipped with reflux condenser,
addition funnel, and spin bar. A small amount of (Z)-1-
bromopropene (3.4 g, 28 mmol) in dry THF (25 mL) was added
with stirring to the magnesium to initiate the vigorous
reaction. Subsequent addition was at a rate sufficient to
maintain gentle reflux. The yellow solution was stirred for 0.5
h and cooled to 0 °C, and dibenzyl diselenide (4.9 g, 14 mmol)
in THF (20 mL) was added dropwise. The mixture was warmed
to 20 °C and poured into 10% HCl solution, and the organic
layer was washed with 5% NaOH, saturated NaHCO3, and
brine, dried (Na2SO4), and purified using a Chromatotron to
give (Z)-10 (2.55 g, 43%) as a yellow liquid: 1H NMR (CDCl3)
δ 7.22 (m, 5 H), 6.14 (dq, J ) 9.0, 1.4 Hz, 1 H), 5.86 (m, 1 H),
3.82 (s, 2 H), 1.59 (dd, J ) 6.6, 1.4 Hz, 3 H); 13C NMR (CDCl3)
δ 139.1, 128.8, 128.5, 128.3, 126.8, 29.4, 16.7; 77Se NMR
(CDCl3, MeSeSeMe) δ 282; EI-GC-MS, m/z 212 (M+, 11%), 91
(100%).
Ben zyl (E)-1-P r op en yl Selen id e [(E)-10]. In a similar
manner using (E)-1-bromopropene, a 3:1 mixture of (E)-/(Z)-
10 was prepared in 43% yield. Compound (E,Z)-10 showed the
following: 1H NMR (CDCl3) δ 7.27 (m, 5 H), 6.20 (dq, J ) 15.4,
1.4 Hz, 1 H), 5.91 (m, 1 H), 3.87 (s, 2 H), 1.74 (dd, J ) 6.5, 1.5
Hz, 3 H); 13C NMR (CDCl3) δ 132.9, 128.8, 128.4, 126.7, 116.6,
29.8, 16.8; 77Se NMR (CDCl3, MeSeSeMe) δ 326; EI-GC-MS,
m/z 212 (M+, 11%), 91 (100%). Distillation of another batch
containing 1:2.3 (E)-/(Z)-10 gave a yellow oil, bp 139 °C (16
mbar). Anal. Calcd for C10H12Se: C, 56.87; H, 5.72. Found: C,
56.99; H, 5.71.
Bis[(Z)-1-p r op en yl] Diselen id e [(Z,Z)-13]. Lithium (0.14
g, 20 mmol) was added to liquid ammonia (200 mL) at -78 °C
under argon. After the lithium had dissolved, (Z)-10 (1.67 g,
7.9 mmol) in THF (40 mL) was added dropwise with stirring.
The reaction mixture was stirred at -65 °C for 1.5 h, warmed
to room temperature, and concentrated in vacuo. At 0 °C, water
(100 mL) and ether (150 mL) were added followed by an

460 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Block et al.
aqueous solution (40 mL) of KI3 [from I2 (1.2 g, 7.5 mmol) and
KI (8 g, 48 mmol)]. The ether layer was separated, the aqueous
layer was extracted with ether (2 × 200 mL), and the combined
organic layers were washed with sodium thiosulfate solution
(3 × 100 mL) and NH4Cl solution, dried (MgSO4), filtered, and
concentrated in vacuo. Chromatography (Chromatotron) gave
(Z,Z)-13 (0.69 g, 72%) as a yellow liquid: 1H NMR (CDCl3) δ
6.66 (dq, J ) 8.9, 1.4 Hz, 2 H), 5.92 (m, 2 H), 1.79 (dd, J ) 6.9,
1.4 Hz, 6 H); 13C NMR (CDCl3) δ 130.8, 123.3, 16.3; 77Se NMR
(CDCl3, MeSeSeMe) δ 356; EI-GC-MS, m/z 242 (M+, 81%), 41
(100%).
Bis(2-m eth yl-2-p r op yl) Diselen id e (14). 2-Bromo-2-meth-
ylpropane (27.4 g, 0.2 mol) and Et2O (100 mL) were added to
magnesium turnings (4.86 g, 0.2 mol). After completion of the
reaction, selenium (14.2 g, 0.18 mol) was added gradually over
a period of 30 min at such a rate as to maintain gentle
refluxing without heating. Stirring was continued for an
additional 30 min. At 0 °C, NH4Cl solution (100 mL) was
added. An aqueous solution (40 mL) of KI3 (prepared as
described for 13) was added slowly. The organic layer was
separated, and the aqueous solution was extracted with ether
(2 × 200 mL). The combined organic layers were washed [10%
sodium thiosulfate (3 × 10 mL) and NH4Cl solution], dried
(MgSO4), filtered, and concentrated. Distillation gave 14, a
yellow liquid (11.0 g, 45% yield), bp 100 °C (11 Torr) (Lit. 53-
56 °C, 0.15 Torr; 19).
2-Meth yl-2-p r op yl (E,Z)-1-P r op en yl Selen id e (15). In a
similar manner the Grignard reagent prepared from (Z)-1-
bromopropene (5.6 g, 46 mmol) was reacted with 14 (6.0 g, 22
mmol). After workup and concentration, the residue was
distilled under vacuum using a Vigreux column to give (E,Z)-
15 (3.1 g, 40%), bp 53-55 °C (11 Torr): 1H NMR (CDCl3) δ
5.8-6.5 (m, 2 H), 1.68 (d, J ) 5.4 Hz), 1.66 (d, J ) 3.3 Hz; 3
H total), 1.44, 1.43 (s, 9 H total); 13C NMR (CDCl3) δ 129.4,
127.6, 120.7, 118.8 (CHd), 41.4, 41.3 (CMe3), 32.3, 32.2 (CH3-
CHd), 16.9, 16.6 [(CH3)3C]; EI-GC-MS, m/z 178 (M+, 48%), 121
(62%), 93 (38%), 57 (100%).
Se-(E,Z)-1-P r op en yl-L-selen ocystein e (6d ). Lithium (7.5
mg, 1.07 mmol) was added to liquid ammonia (20 mL) at -78
°C in a 50 mL three-neck round-bottom flask. After stirring
for 10 min, (E,Z)-10 (200 mg, 0.94 mmol) in THF (2 mL) was
added dropwise. The reaction mixture was stirred at -70 °C
for 1 h, and then â-chloro-L-alanine (12, 0.137 g, 1 mmol) was
added as a solid all at once. After 20 min, the ammonia was
evaporated and water was added. Compound 6d (138 mg, 65%,
1:1.8 E:Z) was obtained as a mixture of isomers after RP-
HPLC. For (Z)-6d : 1H NMR (D2O; ref. H2O δ 4.63) δ 6.12 (dq,
J ) 15.3, 1.2 Hz, 1 H), 5.88-5.97 (m, 1 H), 3.82 (dd, J ) 7.5,
4.2 Hz, 1 H), 2.86-3.13 (m, 2 H), 1.60 (dd, J ) 6.3, 1.5 Hz, 3
H); 13C NMR (D2O, acetone-d6 internal ref) δ 172.7, 136.4,
114.4, 54.5, 26.0, 19.2; 77Se NMR (D2O, MeSeSeMe) δ 154; EI-
MS, m/z 209 (M+, 12%), 43 (100%). For (E)-6d : 1H NMR (D2O)
δ 6.14 (dq, J ) 8.8, 1.4 Hz, 1 H), 5.91-6.04 (m, 1 H), 3.84 (dd,
J ) 6.6, 4.4 Hz, 1 H), 2.86-3.13 (m, 2 H), 1.54 (dd, J ) 6.9,
1.5 Hz, 3 H); 13C NMR (D2O, acetone-d6) δ 172.7, 131.0, 118.8,
54.7, 26.0, 16.2; 77Se NMR (D2O, MeSeSeMe) δ 199; EI-MS
same as (Z)-6d . A 1:2.3 E:Z mixture of crystalline light yellow
6d had mp 164 °C (dec). Anal. Calcd for C6H11NO2Se: C, 34.63;
H, 5.33. Found: C, 34.58; H, 5.39.
Se-2-P r op yn yl-L-selen ocystein e (6f). As in the synthesis
of 6e, using 3-chloropropyne (0.15 mL, 2.07 mmol), 6f (19 mg,
30%) was prepared as a colorless solid: 1H NMR (300 MHz,
D2O) δ 4.14 (dd, J ) 7.0, 4.6 Hz, 1 H, CRH), 3.42 (d, J ) 2.8
Hz, 2 H, tCCH2), 3.40 (dd, J ) 12.7, 4.6 Hz, 1 H, CâH2), 3.31
(dd, J ) 14.1, 7.1 Hz, 2 H, CâH2), 2.80 (t, J ) 2.6 Hz, 1 H,
HCt); 13C NMR (D2O, acetone-d6) δ 172.8, 81.5, 72.9, 54.2,
24.3, 7.8; 77Se NMR (D2O, MeSeSeMe) δ 210.
Se-Met h yl-L-m et h ylselen ocyst ein a t e H yd r och lor id e
(17). Compound 6a (1.41 g, 7.74 mmol) was suspended in dry
methanol (100 mL) in a 250 mL three-neck flask, fitted with
gas inlet and reflux condenser. Dry HCl gas was bubbled into
the suspension until the exothermic reaction subsided. The
solution was evaporated in vacuo. The oily residue was
extracted with CH2Cl2, and the extracts were dried (Na2SO4).
After evaporation, 17 crystallized (1.80 g, 100%): 1H NMR
(CDCl3) δ 8.8 (br s, 3 H), 4.53 (br s, 1 H), 3.86 (s, 3 H), 3.27 (br
s, 2 H), 2.12 (s, 3 H); 13C NMR (CDCl3) δ 168.7, 53.5, 53.2,
24.1, 5.9.
L-γ-Glu t a m yl-Se-m et h yl-L-selen ocyst ein e (8a ). Com-
pounds 18 (6.33 g, 12.9 mmol) (20) and 17 (3.00 g, 12.9 mmol)
were dissolved in CH2Cl2 (180 mL) and cooled to -10 °C. A
solution of dicyclohexylcarbodiimide (3.20 g, 15.5 mmol) in
CH2Cl2 (20 mL) was added, and the mixture was stirred for
20 h, filtered, and then concentrated in vacuo. The residue
was dissolved in EtOAc (200 mL) and filtered again. The
filtrate was washed with 1 N HCl (3 × 10 mL) and brine (2 ×
20 mL) and dried (Na2SO4). The mixture was concentrated to
give an oil, which was chromatographed (silica gel; CHCl3:
MeOH 20:1). The eluate was evaporated to an oil (19; Scheme
6), which was directly used for the next step. The oil was sus-
pended in H2O (20 mL), heated to reflux and then treated with
acetic acid (20 mL). Refluxing was continued for 5 min, the
solution was cooled to room temperature, and H2O (40 mL)
was added, whereupon triphenylcarbinol precipitated and was
removed by filtration. Concentration of the residue gave a
solid, which was dissolved in a mixture of water and MeOH
(1:1) and cooled to 15 °C. The pH was continuously adjusted
to 12 with aqueous 1 N LiOH over a period of 3 h. The solution
was then acidified with 2 N HCl to pH 6 and evaporated below
40 °C to a volume of 15 mL. The solution was applied to an
Amberlite IR45 column (OH- form, 1.5 × 30 cm). After the
column had been sequentially washed with water, 0.25 N Na-
OH, and water, the product was eluted with water. The eluate
was evaporated to 2 mL, and dry ethanol was added to pre-
cipitate the peptide. The white solid 8a was collected and dried
under reduced pressure (476 mg, 12%; two-step overall yield),
mp 159-161 °C: 1H NMR (300 MHz, D2O) δ 4.22 (dd, J )
4.5, 8.4 Hz, 1 H), 3.59 (t, J ) 6.2, 1 H), 2.85 (dd, J ) 4.5, 13.0
Hz, 1 H), 2.69 (dd, J ) 8.5, 17 Hz, 1 H), 2.33 (t, J ) 7.6, 2 H),
1.99 (m, 2 H), 1.85 (s, 3 H); 13C NMR (75.5 MHz, D2O) δ 182.3,
179.9, 177.7 (CdO), 60.l, 57.4, 34.9, 32.1, 29.7, 6.89 (Me); 77Se
NMR (D2O, MeSeSeMe) δ 53; HPLC-ESI-MS, m/z 313 (MH+);
APCI-MS, m/z 313 (MH+, 100%), 130, (23%); MS-MS of m/z )
313 gave m/z 295 (26%), 224 (4%), 184 (58%), 167 (100%).
Bis(r-am in opr opion ic acid)selen ide (16, Selen olan th io-
n in e). Selenocystine (4), 200 mg, 0.6 mmol) was suspended
in EtOH (5 mL) under argon atmosphere. Solid NaBH4 (100
mg, 3 mmol) was added, and the solution was refluxed for 5
min until the solution was clear and colorless. â-Chloro-L-
alanine (150 mg, 1.2 mmol) was added, and after 3 min, a
precipitate was formed. The solution was refluxed for an addi-
tional 10 min and cooled to room temperature. The precipitate
was filtered and dried. A solution of the precipitate in water
(3 mL) was applied to an Amberlite column (OH- form) and
eluted as described for 8a . After concentration in vacuo, 16
was precipitated with ethanol as a colorless solid (190 mg,
68%), mp 225-226 °C (dec): 1H NMR (300 MHz, D2O) δ 2.90
Se-2-Meth yl-2-p r op en yl-L-selen ocystein e (6e). Seleno-
cystine (125 mg, 0.375 mmol) was dissolved in 1 M HCl (2 mL).
The yellow solution was flushed with argon, and NaBH4 was
added until the solution turned colorless. A solution of NaOH
was added to give pH 7 as monitored by a glass electrode.
3-Chloro-2-methylpropene (0.15 mL, 1.52 mmol) was added,
and the mixture was stirred at room temperature overnight.
The mixture was then concentrated in vacuo to ∼1 mL, and
the residue was subjected to reverse phase (RP) HPLC (7:3
H2O/CH3CN) to give 6e (64 mg, 48%) as a colorless solid: 1H
NMR (D2O) δ 4.77 (m, 2 H), 3.79 (dd, J ) 6.7, 4.7 Hz, 1 H),
3.15 (d, J ) 3 Hz, 1 H), 2.88 (dd, J ) 13.8, 5.1 Hz, 1 H), 2.82
(dd, J ) 13.8, 6.9 Hz, 1 H), 1.69 (s, 3 H); 13C NMR (D2O,
acetone-d6) δ 173.2, 142.5, 114.0, 54.2, 31.6, 23.3, 21.3; 77Se
NMR (D2O, MeSeSeMe) δ 133; EI-MS, m/z 223 (M+, 0.4%),
135 (15%), 109 (13%), 67 (14%), 55 (89%), 44 (100%).
3
3
(d, J ) 5.5 Hz, 2 H, CH2), 3.50 (t, J ) 5.5 Hz, 1 H, CH); 13C
NMR (75 MHz, D2O) δ 22.6 (Câ), 48.6 (CR), 173.3 (COO); 77Se
NMR (D2O, MeSeMe) δ 82; HPLC-ESI-MS, m/z 257 (MH+),
279 (M + Na+), 168 [HO2C(NH2)CHCH2Se+]. Anal. Calcd for
C6H12N2O4Se: C, 28.25; H, 4.74. Found: C, 27.91; H, 4.40.
2-[(E,Z)-1-P r op en ylselen o]p r op a n a l (36) fr om Oxid a -
tion of 6d . A stirred solution of 6d (80 mg, 0.39 mmol) in

Chemistry and Biological Activity of Selenoamino Acids and Their Se Oxides
J. Agric. Food Chem., Vol. 49, No. 1, 2001 461
sodium phosphate buffer (pH 8, 1 mL) was cooled to 10 °C,
and H2O2 (30%, 0.04 mL, 44 mg, 0.39 mmol) was added
dropwise via syringe, leading to a vigorous reaction. The
mixture was stirred for 10 min, whereupon a yellow oil
separated. The organic layer was extracted with ether (3 × 2
mL). The combined extracts were washed with brine, dried
(Na2SO4), and purified by chromatography to give 36 (48 mg,
69%) as a yellow liquid, which was an inseparable E/Z
mixture: 1H NMR (CDCl3) δ 9.35, 9.36 (two d, 1 H, CHdO),
6.2-6.0 (m, 2H, CHdC), 3.57, 3.50 [two q, J ) 7 Hz, 1 H,
MeCH(SeR)CHO], 1.79, 1.75 (two d, J ) 6.4, 3H), 1.53 (t, J )
7 Hz, 3 H); 13C NMR (CDCl3) δ 192.8, 192.7 (HCdO), 139.0,
132.3, 115.8, 112.2 (CHdC′H), 44.4, 44.0 [CH3CH(SeR)CHO],
19.9, 16.9 (CH3CHd), 13.5, 13.3 [CH3CH(SeR)CHO]; 77Se NMR
(CDCl3, MeSeMe) δ 240; EI-GC-MS, m/z 178 (M+, 14%), 149
(M+ - CHO, 16%), 121 [M+ - MeCH(CHO), 14%], 107 (CH2-
CHSe, 27%), 93 (HCSe, 64), 67 (100%); IR (νmax) 2859, 1702
cm-1. Oxidation of 13 and 15 was done in a similar manner.
The compound (1 mmol) was dissolved in THF (2 mL) and
cooled, and an equimolar amount of H2O2 (30%) was added
via syringe. After 10 min, the solvent was evaporated and the
residue was dissolved in ether, washed, dried, and purified
by chromatography as described above. In both cases 36 was
the major product, isolated in >50% yield.
Gen er a l Oxid a tion Con d ition s (Oxid a tion in a n NMR
Tu be). The selenoamino acid or γ-glutamylpeptide (50-80 mg)
was dissolved in deuterated sodium phosphate buffer (pD 8, 1
mL) and cooled to 10 °C, and a crystal of internal standard
[sodium 3-(trimethylsilyl)propanesulfonate] was added. Equimo-
lar H2O2 (30%) was added dropwise via syringe. NMR spectra
were taken and, in case of gas evolution, the mixture was
shaken. When an organic layer separated, it was extracted by
CDCl3 and NMR spectra were taken. Some reactions were
monitored by HPLC (15 cm C8 or C18 RP column, 0.1% TFA
+ 2% MeOH).
described for 6a ]. Compounds 24 and 25 were the final
products after 6 days.
Oxid a tion of Se-2-P r op en yl-L-selen ocystein e (6c). Oxi-
dation with H2O2 at pH 8 led to a reaction that was 95%
complete after 1.5 h. The final products were allyl alcohol [δ
1
6.0 (m, 1 H), 5.29 (dd, 1H), 5.19 (dd, 1H), 4.12 (ddt, 2 H); H
NMR spectrum and GC-MS identical to those of authentic
material], selenate (by ICP-MS), and a third compound,
1
identified as 3-seleninoalanine (22), showing H NMR δ 4.32
(dd, J ) 10.7, 6.2 Hz; 1 H; CH), δ 3.03 (dd, J ) 14.3, 6.2 Hz),
and 2.88 (dd, J ) 14.3, 10.7 Hz; CH2, 2 H); 13C NMR δ 53.1
(CH2; 77Se-satellites with J ) 75 Hz), 54.9 (CH), 180.1 ppm
(COOH). On oxidation at pD 5 the same rapid exothermic
reaction could be observed. Besides allyl alcohol, a second
product had the same NMR patterns as seen at pH 8, but
slightly shifted chemical shifts [1H NMR δ 4.32 for CH (dd, J
) 10.7, 6.2 Hz), 3.48 (dd, J ) 14.3, 10.7 Hz), and 3.28 (dd, J )
14.3, 6.2 Hz) for CH2; 13C NMR δ 52.5 (Câ), 55.9(CR) and 175.2
(COOH)]. No elimination products (ammonium pyruvate) were
observed. (The above NMR data were obtained in Siegen at
200 MHz for 1H and 50 MHz for 13C.) After 2 h, HPLC-ESI-
MS of selenium products showed one Se-containing peak with
+
m/z 155 (100%), 137, and 109, possibly AllSe(OH)2 from 7c,
and a second Se-containing peak with m/z 226, 208, and 168.
On oxidation the HPLC retention time changes from 12 min
(6c) to 3.85 min (7c).
Oxid a tion of Selen ola n th ion in e (16). Oxidation was
achieved using excess H2O2 (30%) under neutral conditions.
The 77Se NMR spectrum (D2O, MeSeMe) changes from a single
peak at 82 ppm to double peaks at 858 and 865 ppm to a
product with a single peak at 1195 ppm. The 1H NMR
spectrum (300 MHz) showed δ 4.33 for CH (dd, J ) 10.0, 6.1
Hz), 3.38 (dd, J ) 12.5, 9.1 Hz), and 3.18 (dd, J ) 12.5, 7.1
Hz) for CH2; 13C NMR spectrum (75 MHz) δ 50 (Câ), 53 (CR),
and 172 (COOH). After 1.5 h, ammonium pyruvate could be
detected. The final product is identical to 3-seleninoalanine
Oxid a tion of Se-Meth yl-L-selen ocystein e (6a ). With
2-
1
(22) from 6c. By ICP-MS SeO4 was also found.
H2O2 at pD 8 the H NMR spectrum of selenoxide 7a shows a
peak at δ 2.80, which at high resolution is resolved into two
singlets at δ 2.797 and 2.803, shifted from the δ 2.05 peak for
6a . Two signals also appear in the 77Se NMR spectrum at δ
853 and 854 ppm, shifted from the signal for 6a at δ 38. In
the 1H NMR spectrum a singlet at δ 2.45 appeared after 17
min, which could be a hydrate. The oxidation was complete in
∼24 h. After 5 days, the final products were methaneseleninic
acid (24; δ 2.79, s) and dimethyl diselenide (25, δ 2.61, s) in a
2:1 ratio and ammonium pyruvate (δ 2.19, s, 3H; 7.15, t, 4 H).
Identities of products were confirmed by spiking with authen-
tic materials and, in some cases by ESI-MS [e.g., m/z 129 for
MeSe(OH)2+]. With H2O2 at pD 1 a fast exothermic reaction
occurs (in the first 2 min the δ 2.13 1H NMR singlet was
replaced by one at δ 3.28), which was finished after 1 h. By
HPLC, the retention time of the major peak changes from 3.4
min for 6a to 2.0 min for 7a ; after 1 min, no signal of the
starting material was found. The final products are 24, 25 (2:1
ratio), and ammonium pyruvate.
Oxid a tion of Selen ocystin e (4). Oxidation was achieved
using excess H2O2 (30%) in D2O/DCl (1 M). The 1H NMR
spectrum (300 MHz) showed δ 4.32 for CH (dd, J ) 10.6, 6.1
Hz), 3.48 (dd, J ) 12.2, 10.7 Hz), and 3.26 (dd, J ) 12.2, 6.2
Hz) for CH2; 13C NMR spectrum (75 MHz) δ 50 (Câ), 54 (CR),
and 172 (COOH). Oxidation at pH 7 gave a product with 77Se
1208 ppm (in D2O), shifted from the position of 4 at 290 ppm
(DCl/D2O). This final product is identical to 3-seleninoalanine
(22) from 6c [note that selenoamino acid 77Se NMR shifts are
known to vary with pH (21)].
Oxid a tion of 6b, (E)- a n d (Z)-6d , 6e, a n d 6f. On oxidation,
HPLC retention times changed from 21 min for 6b to 4.8 min
for 7b, from 15 and 17 min for (E)- and (Z)-6d to 3.8-4 min
for (E,Z)-7d , from 38 min for 6e to 2 min for 7e, and from 5
min for 6f to 1.9 min for 7f. Treatment of 7b,d -f with sodium
thiosulfate shortly after oxidation regenerated 6b,d -f. By LC-
ESI-MS, oxidation of 6b gave a product showing m/z 157 (n-
PrSeO2H2+).
Oxid a tion of Selen om eth ion in e (5). Oxidation was car-
ried out in D2O with an equimolar amount of H2O2. By HPLC
(0.1% HFBA), the retention time of the major peak changes
from 12.95 min for 5 to 2.61 min for selenomethionine Se-oxide
Cell Cu ltu r e. The mouse mammary hyperplastic epithelial
cell lines TM2H and TM12 were obtained from the laboratory
of Daniel Medina (22). Cells were grown at 37 °C in a
humidified incubator containing 5% CO2 in Dulbecco’s modified
Eagle’s medium and F-12 medium (1:1 DMEM/F-12) contain-
ing 2% adult bovine serum, 10 µg/mL insulin, 5 ng/mL
epidermal growth factor, and 5 µg/mL gentamicin. TM12 cells
have an intact functional (wild-type) p53 gene. This gene is
part of the genetic circuitry that participates in cellular
responses to genotoxicity. Thus, it was anticipated that this
cell line would be very responsive to genotoxic effects of the
compounds investigated. The TM2H cell line is biologically
similar to the TM12 cell line in terms of its origin: both are
derived from a mouse mammary epithelial hyperplasias, but
TM2H cells lack p53 activity; functionally, this eliminates a
component of the cellular response to genotoxic insult. By
1
(20). The oxidized product gives two H NMR signals (δ 3.01
and 3.05, shifted from δ 2.02 in 5), which correspond to the
methyl protons of the two diastereomers 20. The 77Se-NMR
spectrum of 20 showed signals at δ 838 and 845 ppm, whereas
HPLC-ESI-MS showed the presence of the selenoxide hydrate
at m/z 232 [MeSe(OH)2CH2CH2CH(NH3)CO2H+].
Oxid a tion of L-γ-Glu ta m yl-Se-m eth yl-L-selen ocystein e
(8a ). Oxidation with H2O2 at pH 7 occurs rapidly, and two 1H
NMR signals for the methyl protons were found at δ 2.74 and
2.76. After 2 h, a new signal, presumed to be a hydrate, could
be seen rising under the γ-protons at δ 2.4 and was the major
signal after 3 days but was gone after 6 days. After 3 days,
elimination was complete [the 1H NMR spectrum showed
olefinic protons at δ 5.69 and 5.89 and two singlets for
methaneseleninic acid (24) and dimethyl diselenide (25), as
using both cell lines, the opportunity to detect
a wider
spectrum of biological responses to a particular compound is
enhanced.

462 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Block et al.
An a lysis of Cell Gr ow th In h ibition . The overall effect
of 6d -f on cell number homeostasis was determined by
evaluating the number of adherent cells accumulating over
different periods of time. This approach provides an estimate
of the net effect of a compound on cell proliferation and cell
death. The assay used crystal violet staining of adherent cells
as an endpoint (23). Briefly, cells (1000 cells/well) were seeded
in flat-bottom 96-well plates in 100 µL of culture medium.
Twenty-four hours after initial seeding, cells were allowed to
continue growing in either the same medium (no Se supple-
ment) or that medium supplemented with 6d -f at various
concentrations of Se. After 24 h of incubation, the medium was
removed, and the cells were fixed for 15 min with 100 µL of
1% glutaraldehyde in phosphate-buffered saline (PBS) per
well. The fixative was removed and replaced with 150 µL of
PBS per well, and the plates were stored under PBS at 4 °C.
At the end of the experiment, all plates were stained simul-
taneously with 0.02% aqueous crystal violet solution (100 µL/
well) for 30 min. Excess dye was removed by rinsing the plates
with distilled water, and excess water was blotted out. The
stain bound by the cells was redissolved in 70% ethanol (180
µL/well) while the microplates were shaken for 2 h on a
Titertek shaker (Titertek Instruments Inc., Huntsville, AL).
Absorbance was measured at 590 nm using a Thermomax
Microplate Reader (Molecular Devices, Sunnyvale, CA).
Com et Assa y. We adapted without modification the previ-
ously reported single-cell gel electrophoresis assay as a method
for assessing DNA damage (24). The assay, more commonly
known as the comet assay, is a rapid and sensitive method
for measuring DNA strand breaks at the level of individual
cells. Quantification of DNA damage was performed as recom-
mended (25).
oxidation of 6d described below. These latter studies
also required 2-methyl-2-propyl (Z)-1-propenyl selenide
(15), prepared from bis(2-methyl-2-propyl) diselenide
(14; Scheme 3), synthesized according to an improved
Sch em e 3
procedure. Compounds 6e and 6f were prepared by al-
kylation of the sodium salt of selenocysteine [from boro-
hydride reduction of selenocystine (4)] with 3-chloro-2-
methylpropene or 3-chloropropyne, respectively (Scheme
1
4). The H and 13C NMR spectra of 6f were similar to
Sch em e 4
those of S-2-propynyl-L-cystine (E. Block and T. Naka-
hodo, unpublished data; 29). Reaction of the sodium salt
of selenocysteine with â-chloro-L-alanine (12) gave sele-
nolanthionine (16, Scheme 5), used in connection with
Ap op tosis Cou n tin g. Effects of selenium compounds on
apoptosis of cultured cells were determined morphologically
by fluorescent microscopy after labeling with acridine orange
and ethidium bromide as reported (26).
Sch em e 5
Sta tistica l An a lyses. Differences in cellular responses to
selenium compounds were evaluated by factorial analysis of
variance (ANOVA). The factors investigated were dose of
compound and treatment duration. Post hoc comparisons
among treatment conditions were made using the Bonferroni
multiple-range test (27).
studies of the oxidation of 6c described below. γ-Glutamyl
Se-methylselenocysteine (8a ) was prepared by condens-
ing Se-methyl-L-methylselenocysteinate hydrochloride
(17) with triethylammonium-N-(trityl)-L-γ-glutamate
(18) (20) in the presence of dicyclohexylcarbodiimide
(DCC) and then deprotecting condensation product 19
(Scheme 6). The 1H NMR spectrum of 8a was very
RESULTS AND DISCUSSION
Syn th esis. Se-Methylselenocysteine (6a ), Se-propyl-
L-selenocysteine (6b), and Se-2-propenyl-L-selenocys-
teine (6c) were synthesized according to known proce-
dures [6a ,b (10, 28)], which were also used in the
synthesis of Se-(E,Z)-1-propenylselenocysteine (6d ), Se-
2-methyl-2-propenyl-L-selenocysteine (6e), and Se-2-
propynyl-L-selenocysteine (6f). Thus, 6d was prepared
by alkylation of â-chloro-L-alanine (12) with lithium
(E,Z)-1-propeneselenolate (11), from Li-NH3 reduction
of benzyl (E,Z)-1-propenyl selenide [10; Scheme 2; only
(Z)-isomers shown]. Oxidation of 11 gave bis(1-propenyl)
diselenide 13, used in connection with studies of the
Sch em e 6
Sch em e 2
similar to that of L-γ-glutamyl-S-methyl-L-cysteine (1,
R ) Me), run under identical conditions, and to the
published 1H and 13C NMR spectra of related L-γ-
glutamyl-S-alk(en)yl-L-cysteines (30).
Oxid a tion Stu d ies. Background. Selenomethionine
(5) is said to give a stable selenoxide 20 or its hydrate
21 (31, 32), readily reducible back to 5 with thiosulfate
(33) and by other means (34, 35). Under the same
conditions, selenocysteine derivatives afforded only de-
hydroalanines, by elimination (31). Selenomethionine

Chemistry and Biological Activity of Selenoamino Acids and Their Se Oxides
J. Agric. Food Chem., Vol. 49, No. 1, 2001 463
spectrum of selenoxide 7a shows a peak at δ 2.80, which
at high resolution is resolved into two singlets at δ 2.797
and 2.803 replacing the δ 2.05 peak for 6a ; similarly,
the 77Se NMR spectrum showed two singlets at δ 853
and 854 replacing the δ 38 peak for 6a . These observa-
tions are consistent with the conversion of 6a to a pair
of diastereomeric selenoxides 7a . After 17 min, a 1H
NMR singlet at δ 2.45 appeared, which could be a
hydrate. The oxidation was complete in 24 h. The final
products were 2:1 methaneseleninic acid (24)/dimethyl
diselenide (25) and ammonium pyruvate, as confirmed
by NMR by spiking with authentic samples and by
HPLC-ESI-MS (44) (Scheme 7). The results were similar
Se-oxide (20) and Se-methylselenocysteine Se-oxide (7a )
are reported to be present in marine phytoplankton (36,
37), clover (38), and cabbage (39). Several papers (37,
40-43) propose the possible natural occurrence of
3-seleninoalanine [22, HO2SeCH2CH(NH2)COOH; “sele-
nocysteine seleninic acid”] and/or 3-selenonoalanine [23,
HO3SeCH2CH(NH2)COOH; “selenocysteic acid”] but
provide no spectroscopic proof or authentic samples.
In collaboration with Professor Peter Uden and co-
workers, oxidation of selenoamino standards 6a -f with
excess H2O2 was investigated using HPLC-ICP-MS (17).
For each selenoamino acid oxidized a new selenium-
containing peak was produced and the original sele-
noamino acid peak disappeared. The selenoamino acid
oxidation products eluted at much shorter retention
times than their precursors on the reverse phase (RP)
column, consistent with the increased polarity expected
for selenoxides. Oxidation of a mixture of (E/ Z)-Se-1-
propenyl selenocysteine (6d ) led to a pair of new peaks,
indicating that geometrical isomerism persisted un-
changed in the oxidation products. Treatment of solu-
tions of the oxidized selenoamino acids with thiosulfate
restored the original selenoamino acids if the thiosulfate
treatment was performed shortly after oxidation, before
selenoxide decomposition took place.
Sch em e 7
at pD 1, but the reactions were much faster. In a similar
manner, oxidation of 8a rapidly led to elimination
products 24-26 via selenoxide 9 (Scheme 8). Colorless
Sch em e 8
Oxidation of Selenomethionine (5). As monitored by
RP-HPLC, addition of an equivalent of H2O2 to 5 led to
an immediate change in retention time from 7.3 to 2.2
1
min. With H2O2 at pD 8 in D2O, the H NMR spectrum
showed two singlets at δ 3.05 and 3.01 replacing the δ
2.02 peak for 5; similarly, the 77Se NMR spectrum
showed two singlets at δ 845 and 838 replacing the δ
75 (D2O, pD 4) peak for 5. These observations are
consistent with the conversion of 5 to a pair of diaster-
eomeric selenoxides 20. Analysis of a solution of oxidized
5 by HPLC-ESI-MS showed the presence of the sele-
noxide hydrate (21) at m/z 232.
Oxidation of Se-Methyl-L-selenocysteine (6a ) and L-γ-
Glutamyl-Se-methyl-L-selenocysteine (8a ). As monitored
by RP-HPLC, addition of an equivalent of H2O2 to 6a
led to an immediate change in retention time from 3.4
to 2.0 min. With H2O2 at pD 8 in D2O, the 1H NMR
crystals of 24, obtained by slow evaporation of a
saturated aqueous solution at 0-5 °C, were character-
ized by X-ray crystallography. The configuration about
the selenium atom is pyramidal with Se-C ) 1.925(8)
Å, Se-O ) 1.672(7) Å, Se-OH ) 1.756(7) Å, OSeO )
103.0(3)°, HO-Se-C ) 93.5(3)°, and OSeC ) 101.4-
(3)°. Hydrogen bonds link the molecules together in
spirals along the c axis (Figure 1). The structure is
isomorphous to that of methanesulfinic acid (45). Al-
though structures of aliphatic seleninic acids have not
F igu r e 1. X-ray crystal structure of methaneseleninic acid (24) showing intermolecular hydrogen bonding.

464 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Block et al.
Ta ble 1. NMR Da ta for Selected Selen iu m -Con ta in in g Am in o Acid s a n d Rela ted Mod el System sa
compd
4b
ECH2
CH
CO2H
77Se
295
3.34 (dd, 7.7, 14.1)
3.48 (4.6, 14.1) [26.6]
3.20 (t, 7.2) [24.1]
SeCH3: 2.13 [5.7]
2.90 (5.5) [22.6]
2.00 [6.0]
4.34 (dd, 4.6, 7.7) [53.0]
[170.2]
6a c
4.47 (t, 7.2) [53.1]
3.50 (5.5) [48.6]
[170.5]
[173.3]
38
16
82
0
Me2Se
Me2SeO
22d
3.05
812e
1195
3.28 (6.2, 14.3)
3.48 (10.7, 14.3) [52.5 (75)]
2.79 [39.2]
4.32 (6.2, 10.7) [55.9]
4.50 (3.5, 9.0) [45.8]
[175.2]
24
25
A
1216e
275e
2.61
2.82 (9.0, 14.0)
2.99 (3.5, 14.0) [54.2]
a
E
) S or Se; 1H NMR shifts, ppm (coupling constants in hertz)[13C NMR shifts, ppm (coupling constants in hertz)]; A )
HO2SCH2CH(NH3+)CO2H; 4, selenocystine; 6a , Se-methylselenocysteine; 16, selenolanthionine; 24, methaneseleninic acid; 25, dimethyl
diselenide; 22, 3-seleninoalanine. Stocking et al. (52). c M. Birringer, Ph.D. Thesis, Universita¨t-Siegen, 1997; solvent is DMF-d7. From
b
d
6c, at pH (pD) 5. e Paulmier, C. Selenium Reagents and Intermediates in Organic Synthesis; Pergamon Press: Oxford, U.K., 1986.
been previously reported, the crystal structures of both
benzeneseleninic acid and p-chlorobenzeneseleninic acid
are known (46, 47), and the crystal structure at 2.0-Å
resolution of the seleninic acid from selenosubtilisin has
been published (48).
Oxidation of Se-2-Propenyl-L-selenocysteine (6c) and
Selenolanthionine (16). Oxidation of 6c with an equiva-
lent of H2O2 at pD 8 in D2O led to a reaction that was
95% complete after 1.5 h as judged by NMR, giving allyl
alcohol (proven by 1H NMR and GC-MS) and a selenium-
containing compound 22. The latter compound is also
formed on oxidation of selenolanthionine (16). On the
basis of the spectroscopic data summarized in Table 1
and mechanistic grounds (Scheme 9), 22 is identified
L-selenocysteine Se-oxide (7c) (49) followed by hydrolysis
of the resultant selenenic ester 27 to 3-selenenoalanine
(28). The latter compound then undergoes further
reactions. The 13C NMR spectrum of 22 indicated two
aliphatic carbons (CH, δ 54.9; and CH2, 53.1) and a
COOH carbon at 180.1 ppm. The CH2 group, which
shows 77Se-satellites, is deshielded compared to model
compounds such as selenocystine (4) (δ 26.6) but is
similar to the chemical shift of the CH2 group in
3-sulfinoalanine [HO2SCH2CH(NH3+)CO2H; 54.2]. The
1H NMR spectrum of 22 is consistent with a compound
of type X-SeCHaHbCH(NH3+)CO2-. The results of
oxidation at pD 5 were similar. Using RP-HPLC-ICP-
MS and RP-HPLC-ESI-MS within 2 h of addition of
H2O2, as well as 24 h later, the formation of a short-
lived, polar selenium-containing oxidation product, most
likely 7c or 27, was indicated by a peak of m/z 226, and
a second, more persistent, polar selenium-containing
compound was indicated by a peak with the highest
mass at m/z 185 (44).
Sch em e 9
In an effort to identify the unknown selenium-
containing product, we examined the oxidations of
selenolanthionine (16) and selenocystine (4). We antici-
pated that the selenoxide 29 of 16 would decompose to
pyruvate and 28 (Scheme 9). According to the literature
(37), oxidation of 4 gives 3-seleninoalanine (22) or
3-selenonoalanine (23). Oxidation of 16 with H2O2 gave
ammonium pyruvate after 1.5 h. As monitored by RP-
HPLC-ICP-MS, addition of an equivalent of H2O2 to 16
led to immediate formation of a polar selenium-contain-
ing compound with a retention time similar to that of
the more persistent m/z 185 product from oxidation of
6c (44). Initially this peak had a (presumed) MH+ ion
at m/z 218, corresponding to selenocysteine perseleninic
acid (30). After 24 h, this peak was replaced with a peak
of retention time and a mass spectral pattern (m/z 185,
154, 131, and 111) identical to that of the more
persistent m/z 185 product from oxidation of 6c (44).
Although the identical, persistent m/z 185 peaks from
oxidation of 6c and 16 might be selenenic acid 28
(perhaps stabilized by intramolecular hydrogen bond-
ing), it is more likely that the m/z 185 peak is a fragment
ion derived from a heavier parent, for example, loss of
HO• from selenocysteine seleninic acid, because under
the ESI-MS conditions used, compounds 6a , 6c, and 6d
show no parent ions but only fragment ions. Further-
more, in the mass spectrum of MeSeO2H the fragment
resulting from loss of HO• is 58% as abundant as the
MH+ ion.
as 3-seleninoalanine (selenocysteine seleninic acid). We
postulate that allyl alcohol and 22 are produced by a
facile [2,3]-sigmatropic rearrangement of Se-2-propenyl-

Chemistry and Biological Activity of Selenoamino Acids and Their Se Oxides
J. Agric. Food Chem., Vol. 49, No. 1, 2001 465
It is known that selenenic acids can be oxidized to
seleninic acids when generated in the presence of H2O2
(50) in addition to undergoing disproportionation to
seleninic acids. During the course of the oxidation of 16,
the 77Se NMR spectrum underwent a change from a
single peak in the starting material at δ 82 to a pair of
peaks at δ 858 and 865 (presumably selenoxide 29) to
a final product showing a peak at δ 1195, which is
within the range of chemical shifts reported for seleninic
acids (δ 1240-1175) (21) but distinct from the chemical
shifts for selenenic acids (δ ∼1143-1066) (51, 53 54)
and selenonic acids (δ 1022) (55). In particular, the
observed shift of δ 1195 is quite close to the values of δ
1188 and 1190 reported for the 3-seleninoalanine com-
ponent of oxidized selenosubtilisin. Efforts to observe
the selenenic acid corresponding to selenosubtilisin were
unsuccessful (21). We also find that oxidation of sele-
nocystine (4) with H2O2 gives 22.
Oxidation of Se-(E,Z)-1-Propenyl-L-selenocysteine (6d ).
In view of the ready formation of methaneselenenic acid,
MeSeOH, on oxidation of Se-methylselenocysteine (6a ),
it was of interest to determine if a similar process would
occur with 6d giving 1-propeneselenenic acid (31; Scheme
10), because the latter might then rearrange to give the
unknown propaneselenal Se-oxide (32), the selenium
analogue of the onion lachrymatory factor (56). Oxida-
tion of 6d at pH 8 with H2O2 afforded in 69% yield a
yellow oil identified by spectroscopic means (see Ex-
perimental Procedures) as 2-[(E,Z)-1-propenylseleno]-
propanal (36). We propose that Se-(E,Z)-1-propenyl-L-
selenocysteine Se-oxide (7d ) undergoes elimination,
giving 1-propeneselenenic acid (31), which is in equi-
librium with the corresponding selenenic anhydride (33)
(53, 57). Compound 33 adds to itself to give intermediate
35, which on hydrolysis gives 36. Alternatively, addition
of 31 to 33 followed by hydrolysis of adduct 34 could
also afford 36. Similar processes have been reported by
others (58). In support of this mechanism, oxidation of
13 (which should afford 33) and 15 (which should yield
31) both gave 36, each in >50% yield.
Na tu r a l Occu r r en ce. Apart from the work of Spåre
and Virtanen (3) on the possible natural occurrence of
γ-glutamyl selenoamino acids discussed above, γ-glutamyl
Se-methylselenocysteine (8a ) has been detected in As-
tralagus bisulcatus (59) and in low levels in Melilotus
indica L. (an Se-tolerant grassland legume species) (60).
We have reported on the detection of 8a and γ-glutamyl
selenomethionine in garlic, onion, ramp, selenized yeast,
and Astragulus praleongus and the detection of seleno-
lanthionine (16) in selenized yeast by HPLC-ICP-MS
and HPLC-ESI-MS, calibrating our analyses with
samples of 8a and 16 prepared as described in the
present paper (13-16). Because little change was seen
following treatment of garlic and onion extracts with
thiosulfate, known to readily reduce selenoxides [includ-
ing those derived from Se-alk(en)yl selenocysteines and
selenomethionine], we conclude that selenoxides can at
most be present in Allium species at levels below the
detection limits of ICP-MS. These conclusions are also
supported by X-ray absorption spectroscopy (XAS) stud-
ies of the forms of selenium in normal and Se-enriched
garlic (I. Pickering and E. Block, unpublished studies,
Stanford Synchrotron). The same can be said about the
presence in Allium species of Se-alk(en)yl selenocys-
teines 6b-d . The thermal instability found by us for
Se-alk(en)yl selenocysteine selenoxides also makes their
natural occurrence improbable, other than as transient
intermediates in redox systems or as artifacts formed
by air oxidation during analysis (61).
Biologica l Activity. Background. As noted above,
Ip and Lisk showed that selenium-enriched garlic had
anticancer activity significantly higher than that of
unenriched garlic (6, 7). Synthetic Se-propylselenocys-
teine (6b) and Se-2-propenylselenocysteine (6c) were
selected for evaluation of their anticancer activity
because they are analogues of Se-methylselenocysteine
(6a ), which is found in high abundance in Se-enriched
garlic. Compound 6c showed particular promise: at 2
ppm of Se in the diet it caused an 86% inhibition in a
chemically induced mammary tumor model in rats (10).
This magnitude of cancer protection was significantly
greater than that observed with equivalent doses of
either 6a (5) or 6b. Differences in the anticancer activity
of allyl and propyl organosulfur compounds have been
noted; it has been suggested that the presence of a
double bond in the allyl moiety may have a unique effect
(62).
Sch em e 10
It is well recognized that the biological activity of
selenium is an expression of selenium in a variety of
chemical forms and not that of the element per se (63).
We have used defined cell culture models to characterize
the responses to different chemical forms of selenium
(64, 65). Cell culture models work well for this purpose
because the specificity of the assay endpoints can be
assessed without the complications of systemic metabo-
lism. In recent work, the effects of 6b and 6c have been
compared in cell culture (66). Compound 6c inhibited
cell growth and induced apoptosis in mammary epithe-
lial cell lines that have either wild-type or mutant p53
activity. Compound 6b was without effect. However, 6c
also induced a loss of DNA integrity measured as
alkaline labile breaks, an activity that could limit its
usefulness if this effect were manifest in vivo at doses
of 6c that inhibit the development of cancer (10). As
noted in Zhu et al. (66), and consistent with evidence
recently reported by Rooseboom et al. (67), selenocys-
teine Se-conjugates are likely to undergo selenoxidation

466 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Block et al.
Ta ble 2. Effects of 6d -f on TM2H a n d TM12 Cell Gr ow th a
% untreated control
TM2H
TM12
48 h
selenium (µM)
24 h
48 h
72 h
24 h
72 h
6d
25
50
100
6e
25
50
100
6f
25
50
99 ( 5.4ab
91 ( 3.8b
81 ( 1.8b
92 ( 2.1ab
86 ( 2.3b
67 ( 1.8c
72 ( 3.8b
65 ( 1.0b
33 ( 0.8c
95 ( 3.2ab
87 ( 3.0b
84 ( 2.4b
87 ( 2.0b
76 ( 0.9c
66 ( 1.2d
78 ( 2.4b
61 ( 4.0c
33 ( 0.8d
93 ( 3.4ab
84 ( 4.4bc
78 ( 3.4bc
91 ( 3.3a
80 ( 2.4b
64 ( 1.3c
85 ( 1.8b
70 ( 3.8c
54 ( 1.5d
98 ( 2.6ab
90 ( 1.4ab
88 ( 3.5b
98 ( 1.8ab
92 ( 1.6bc
87 ( 1.1c
99 ( 1.9a
97 ( 2.8a
95 ( 1.8a
98 ( 3.9a
95 ( 2.5a
93 ( 2.6a
91 ( 2.3ab
78 ( 11bc
66 ( 6.6c
88 ( 1.4b
73 ( 1.3c
48 ( 1.9d
97 ( 2.1a
85 ( 2.0b
80 ( 3.3b
91 ( 1.8b
77 ( 3.0c
59 ( 1.0d
84 ( 3.0b
53 ( 0.8c
32 ( 0.9d
100
a
Effect of treatment with 6d -f on cell growth inhibition. All experiments were repeated three times. In each experiment, eight replicates
of each dose of each compound were analyzed. The results of a representative experiment are presented. Data are expressed as percent
of untreated control, mean ( SE (n ) 8). Cell number was estimated as an absorbance value of crystal violet stained cells, λ ) 590 nm.
The absolute absorbance of TM2H cells in the untreated control was 0.081 ( 0.004, 0.170 ( 0.02, and 0.248 ( 0.02 at 24, 48, and 72 h,
respectively; the absolute absorbance of TM12 cells in the untreated control was 0.072 ( 0.005, 0.153 ( 0.03, and 0.265 ( 0.02 at 24, 48,
and 72 h, respectively. For each compound and treatment duration, values with different superscripts were statistically different, p <
0.05.
Ta ble 3. Effects of 24 h of Exp osu r e to 6d -f on Ra te of Ap op tosis in TM2H a n d TM12 Cellsa
apoptotic cells/100 cells counted
6d
6e
6f
Se (µM)
TM2H
TM12
TM2H
TM12
TM2H
TM12
0
12.5
25
1.9 ( 0.4a
5.5 ( 1.2ab
8.0 ( 2.4ab
9.8 ( 2.1b
8.9 ( 1.6a
15.2 ( 2.1a
17.5 ( 2.1b
19.2 ( 2.3b
1.9 ( 0.2a
3.9 ( 0.4b
4.0 ( 0.5b
4.2 ( 0.5b
8.9 ( 1.6a
11.8 ( 1.3a
10.7 ( 2.1a
9.7 ( 1.4a
1.9 ( 0.3ab
1.5 ( 0.5a
1.9 ( 0.4ab
3.0 ( 0.4b
8.9 ( 1.6a
12.6 ( 2.3a
13.9 ( 2.3a
27.3 ( 2.2b
50
a
Effect of treatment with 6d -f on apoptosis. The data are expressed as a percent of cells counted that were apoptotic, mean ( SE (n
) 6). Two hundred cells were counted for each apoptosis assay. In an experiment, each treatment was evaluated in duplicate. The effects
of each treatment were studied in three independent experiments. The results of all three experiments were combined for the purpose of
statistical analysis. For each compound and treatment duration, values with different superscripts were statistically different, p < 0.05.
followed by a syn-elimination, a reaction mechanism
that proceeds at least to some extent under physiological
conditions despite the presence within the cell of glu-
tathione, which is known to be able to rapidly reduce
selenoxides (34). In this regard, it is interesting to note
that 24 (MeSeO2H), the disproportionation product of
methaneselenenic acid (MeSeOH) released from 7a and
9, shows significant anticancer activity (68, 69). In the
case of 6c, oxidative metabolism could ultimately give
rise to allyl alcohol, which could be converted to acrolein,
a metabolite that has been reported to have DNA-
damaging activity. Compounds 6e and 6f would be
predicted to form related products, for example, methyl-
allyl and propargyl alcohols, respectively, if they were
similarly metabolized. Therefore, we have used previ-
ously developed cell culture models and endpoints (66)
to study the activities of these three synthetic homo-
logues of 6c, namely, Se-(E,Z)-1-propenylselenocysteine
(6d ), Se-2-methyl-2-propenylselenocysteine (6e), and Se-
2-propynylselenocysteine (6f). Our goal was to identify
a homologue of 6c that had comparable activities in
inhibiting cell growth and inducing apoptosis without
affecting DNA integrity.
apoptosis, which can be induced by several different
sequences of cellular events, is recognized as a powerful
effector of cancer inhibitory activity and has been
reported to be induced by many selenium compounds,
some of which induce DNA damage (63, 65). Because
some selenium compounds induce DNA damage whereas
others do not, we also measured DNA integrity using
the comet assay. Selenium compounds that inhibit cell
growth and induce apoptosis in the absence of DNA
damage in these cell lines have generally been found to
be well tolerated and to exert cancer inhibitory activity
in vivo (65).
In both TM2H and TM12 cells, Se-(E,Z)-1-propenyl-
selenocysteine (6d ), used as a mixture of isomers,
inhibited cell growth in a dose- and time-dependent
manner (Table 2) and also induced apoptosis (Table 3).
Levels of DNA damage were increased by treatment of
either cell line with 6d (Table 4). Although the addition
of a methyl group in the 2-position of the allyl moiety
eliminated DNA-damaging activity, the presence of a
double bond conjugated to the selenium atom is activat-
ing. The levels of DNA damage observed in the TM2H
cell line, which cannot eliminate damaged cells by a p53-
mediated growth arrest mechanism, were the highest
that we have observed with any selenoamino acid tested
to date. Consistent with this observation was an in-
crease in levels of apoptosis, which can be a cellular
response to genotoxic events. Such effects are likely to
account for the progressive increase in the degree of cell
growth inhibition by 6d with increasing treatment
duration. Because of the DNA-damaging activity of 6d ,
Results. Compounds 6d -f were evaluated for their
effects on cell growth, induction of apoptosis, and DNA
integrity in two cell lines that differ in their p53
activity: TM12, which has wild-type p53 activity, and
TM2H, which lacks p53 activity due to a mutation in
that gene (66). As noted above, these cell lines differ in
the mechanisms by which they respond to genotoxic
stimuli and undergo apoptotic cell death. Cell death by

Chemistry and Biological Activity of Selenoamino Acids and Their Se Oxides
J. Agric. Food Chem., Vol. 49, No. 1, 2001 467
Ta ble 4. Effects of 6d -f on DNA In tegr ity Mea su r ed by th e Com et Assa y in TM2H a n d TM12 Cells for 24 h a
arbitrary units
6d
6e
6f
Se (µM)
TM2H
TM12
TM2H
TM12
TM2H
TM12
0
12.5
25
35 ( 2.4a
46 ( 2.2a
154 ( 11b
187 ( 11c
45 ( 5.3a
40 ( 11a
66 ( 11a
125 ( 15b
35 ( 2.4a
36 ( 3.1a
40 ( 4.7a
40 ( 4.4a
45 ( 5.3a
42 ( 4.5ac
22 ( 2.3b
30 ( 1.7bc
35 ( 2.4a
34 ( 2.3a
32 ( 3.0a
34 ( 3.6a
45 ( 5.3a
33 ( 4.0ab
21 ( 2.4bc
14 ( 2.5c
50
a
Effect of treatment with 6d -f on DNA integrity. The data are expressed as arbitrary units of alkaline labile DNA damage, mean (
SE (n ) 9). The score ranges from a minimum of 0 to a maximum of 400. Two hundred cells were counted for each comet assay. In an
experiment, each treatment was replicated three times. The effects of each treatment were studied in three independent experiments.
The results of all three experiments were combined for the purpose of statistical analysis. For each compound and treatment duration,
values with different superscripts were statistically different, p < 0.05.
Ta ble 5. Com p a r ison of in Vitr o Effects of Selen oa m in o Acid s 6a -f in TM2H a n d TM12 Cellsa
% of untreated control
cell growtha
apoptosisb
DNA integrityb
compd
TM2H
TM12
TM2H
TM12
TM2H
TM12
6a c,d
6be
6ce
6d
6e
6f
28 ( 2.9c
94 ( 1.1
31 ( 0.7
65 ( 1.0
70 ( 3.8
73 ( 1.3
47 ( 5.6c
89 ( 1.2
5 ( 0.4
61 ( 4.0
97 ( 2.8
53 ( 0.8
225 ( 30d
175 ( 25
225 ( 20
516 ( 216
221 ( 26
158 ( 21
176 ( 15d
141 ( 9
65 ( 8d
65 ( 3
168 ( 10
534 ( 31
114 ( 13
97 ( 10
95 ( 5d
98 ( 2
318 ( 9
158 ( 5
278 ( 33
67 ( 4
216 ( 26
109 ( 15
307 ( 24
31 ( 6
a
Comparison made with 50 µM Se of each compound following 72 h of exposure. Data are represented as percent of control, i.e., cells
b
not treated. Comparison made with 50 µM Se of each compound following 24 h of exposure. Data are represented as percent of control,
i.e., cells not treated. c Unpublished data. Ip et al. (68). e Zhu et al. (66).
d
modifications of the propenyl chain in this manner
appear to be of limited value relative to chemopreven-
tion because of the potential for genotoxicity.
on growth were consistently greater at each dose-time
point in TM12 versus TM2H cells. Interestingly, 6f had
a marginal effect on the induction of apoptosis in TM2H
cells, whereas a 3-fold increase was observed in TM12
cells. Compound 6f had no effect on DNA integrity in
TM2H, but treatment was associated with lower levels
of DNA strand breaks in TM12 cells. These data indicate
the substitution of a triple bond for the double bond in
the allyl moiety eliminates DNA-damaging activity
while retaining the ability to inhibit cell growth and
induce apoptosis. Although the effects of 6f on cell
growth were substantial after 72 h of treatment with
50 µM Se (73 and 53% of the cell number in TM2H and
TM12 untreated cultures, respectively), 6c induced
greater effects on cell growth under the same treatment
conditions (31 and 5% of the cell number in TM2H and
TM12 untreated cultures, respectively; see Table 5).
Table 5 summarizes the relative effects of the six
selenocysteine compounds tested in our cell culture
model system. Although each compound displays a
unique profile of activity, the compounds can be rank
ordered in terms of their probability of being well
tolerated in vivo and having cancer inhibitory activity
in animal models. The ranking is 6a > 6c . 6f > 6e >
6b > 6d . This ranking takes into account, in decreasing
order of importance, the degree of cell growth inhibition
by each compound, the magnitude of DNA-damaging
activity, and the consistency of apoptosis induction in
both cell lines studied. Thus, on the basis of our assay
results, it appears that the compounds evaluated in the
biological assays in this study are unlikely to exceed the
chemopreventive efficacy of other selenocysteine com-
pounds that have already been evaluated, that is, 6a
and 6c. This analysis is made with one significant
reservation. We have recently observed that the â-lyase
activity of cells in this model system may limit the rate
of release of the selenol moiety from the selenoamino
acid (H. Thompson, unpublished data). Moreover, it is
unclear how the Km and Vmax of the lyases will vary for
Se-2-Methyl-2-propenylselenocysteine (6e) inhibited
cell growth in the TM2H cell line in a dose- and time-
dependent manner; however, it had only a small effect
in the TM12 cell line (Table 2). Consistent with this
observation, 6e also induced apoptosis in the TM2H cell
line (a 2-fold increase), but not in TM12 cells (Table 3).
Whereas 6e was without effect on DNA integrity in
TM2H cells, lower levels of DNA damage were observed
with increasing concentrations of 6e in TM12 cells
(Table 4). These data indicate that the presence of a
methyl group in the 2-position of the allyl moiety of this
selenocysteine homologue reduces the level of DNA
damage in treated cells in comparison to Se-2-prope-
nylselenocysteine (6c) (see Table 5). The inhibition of
cell growth and induction of apoptosis by 6e was
generally greater than that achieved using Se-propylse-
lenocysteine (6b) but less than that when 6c was used
(Table 5). These data imply that the addition of a methyl
group to the double bond reduces DNA-damaging activ-
ity while retaining a modest level of cell growth inhibi-
tory activity and ability to induce apoptosis. These data
suggest that effects of 6e on cell number homeostasis
are independent of the DNA-damaging activity that has
previously been associated with the allyl moiety of 6c.
The fact that the effects of 6e were observed primarily
in TM2H cells indicates that mechanisms of apoptosis
induction were independent of p53. Whether or not the
effect on cell growth is mediated by influences on cell
cycle regulatory molecules merits consideration because
6c has recently been shown to dramatically reduce
levels of phosphorylated retinoblastoma protein (70).
Se-2-Propynylselenocysteine (6f) inhibited the growth
of both cell lines in a dose- and time-dependent manner
(Table 2). Overall, the effects of 6f on cell growth were
relatively small during the first 24 h of exposure but
then became marked at 48 and 72 h of exposure. Effects

468 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Block et al.
(7) Ip, C.; Lisk, D. J . Efficacy of cancer prevention by high
selenium-garlic is primarily dependent on the action of
selenium. Carcinogenesis 1995, 16, 2649-2652.
(8) Ip, C.; Lisk, D. J . Modulation of phase I and phase II
xenobiotic metabolizing enzymes by selenium-enriched
garlic in rats. Nutr. Cancer 1997, 28, 184-188.
(9) Ip, C.; Lisk, D. J .; Stoewsand, G. S. Mammary cancer
prevention by regular garlic and selenium enriched
garlic. Nutr. Cancer 1992, 17, 279-286.
(10) Ip, C.; Zhu, Z.; Thompson, H. J .; Lisk, D.; Ganther, H.
E. Chemoprevention of mammary cancer with Se-
allylselenocysteine and other selenoamino acids in the
rat. Anticancer Res. 1999, 19, 2875-2880.
(11) Finley, J . W.; Davis, C. D.; Feng, Y. Selenium from high
selenium broccoli protects rats from colon cancer. J .
Nutr. 2000, 130, 2384-2389.
(12) Kotrebai, M.; Bird, S. M.; Tyson, J . F.; Block, E.; Uden,
P. C. Characterization of selenium species in biological
extracts by enhanced ion-pair liquid chromatography
with inductively coupled plasma- and electrospray ion-
ization-mass spectrometric detection. Spectrochim. Acta
B 1999, 54, 1573-1591.
(13) Kotrebai, M.; Birringer, M.; Tyson, J . F.; Block, E.; Uden,
P. C. Identification of the principal selenium compounds
in selenium-enriched natural sample extracts by ion-
pair liquid chromatography with inductively coupled
plasma- and electrospray ionization-mass spectrometric
detection. Anal. Commun. 1999, 36, 249-252.
(14) Kotrebai, M.; Birringer, M.; Tyson, J . F.; Block, E.; Uden,
P. C. Characterization of selenium compounds in sele-
nium-enriched and natural sample extracts using per-
fluorinated carboxylic acids as HPLC ion-pairing agents
with ICP- and electrospray ionization-mass spectromet-
ric detection. Analyst 2000, 125, 71-78.
each of the compounds evaluated. The above reservation
is significant because one mechanism for bioactivation
of selenocysteine Se-conjugates 6 involves â-elimination
of selenols from 6 by â-lyases (18, 63, 71, 72). Ap-
proaches are being developed to minimize this potential
limitation.
Finally, the biological activity of γ-glutamyl Se-
methylselenocysteine (8a ) has been found to be quite
similar to that of Se-methylselenocysteine (6a ); these
results will be presented elsewhere (73).
NOTE ADDED IN PROOF
Compound 8a has recently been identified in garlic
harvested in naturally seleniferous soil using size
exclusion chromatography in conjunction with ICP-MS
and ESI-MS; 8a accounts for 78% of the total selenium
present (74). It has also been reported that in bulbs from
Se-enriched ramps (Allium tricoccum), the predominant
form of selenium at all concentrations of selenium is 6a ,
with lower amounts of selenate, 8a , and selenocys-
tathionine (75).
SAFETY
Caution! Many of the organoselenium compounds
used in this work have very unpleasant smells and may
be toxic on ingestion or skin contact. All experiments
were carried out in a glovebox within a hood.
ABBREVIATIONS USED
(15) Kotrebai, M.; Tyson, J . F.; Block, E.; Uden, P. C. HPLC
of selenium compounds utilizing perfluorinated carbox-
ylic acid ion-pairing agents and inductively coupled
plasma- and electrospray ionization-mass spectrometric
detection, J . Chromatogr. A 2000, 866, 51-63.
(16) Ip, C.; Birringer, M.; Block, E.; Kotrebai, M.; Tyson, J .
F.; Uden, P. C.; Lisk, D. J . Chemical speciation influ-
ences comparative activity of selenium-enriched garlic
and yeast in mammary cancer prevention. J . Agric. Food
Chem. 2000, 48, 2062-2070.
(17) Uden, P. C.; Bird, S. M.; Kotrebai, M.; Nolibos, P.; Tyson,
J . F.; Block, E.; Denoyer, E. Analytical selenoamino acid
studies by chromatography with interfaced atomic mass
spectrometry and atomic emission spectral detection.
Fresenius’ J . Anal. Chem. 1998, 362, 447-456.
(18) Commandeur, J . N. M.; Andreadou, I.; Rooseboom, M.;
Out, M.; De Leur, L. J .; Groot, E.; Vermeulen, N. P. E.
Bioactivation of selenocysteine Se-conjugates by a highly
purified rat renal cysteine conjugate â-lyase/glutamine
transaminase K. J . Pharmacol. Exp. Ther. 2000, 294,
753-761.
HPLC, high-performance liquid chromatography; ICP,
inductively coupled plasma; MS, mass spectrometry;
ESI, electrospray ionization; GC, gas chromatography;
RP, reverse phase; PBS, phosphate-buffered saline.
ACKNOWLEDGMENT
We thank Dr. Elizabeth Calvey for APCI-MS data,
Professor Peter Uden and Dr. Mihaly Kotrebai for LC-
ESI-MS data, Professor Howard Ganther for helpful
comments, J in Chao for technical assistance, and Dr.
Martijn Rooseboom for sharing unpublished results.
Su p p or tin g In for m a tion Ava ila ble: Details of X-ray
structure determination of methaneseleninic acid (24). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(19) Anderson, J . A.; Odom, J . D.; Zozulin, A. J . Preparation
of unsymmetrical alkyl methyl and alkyl phenyl disele-
nides and determination of their selenium-77 chemical
shifts and 77Se-77Se spin-spin coupling constants.
Organometallics 1984, 3, 1458-1465.
(20) Amiard, G.; Heymes, R.; Velluz, L. New synthesis of
glutathione. Bull. Soc. Chim. Fr. 1956, 698-700.
(21) House, K. L.; Dunlap, R. B.; Odom, J . D.; Wu, Z.-P.;
Hilvert, D. Structural characterization of selenosubtili-
sin by 77Se NMR spectroscopy. J . Am. Chem. Soc. 1992,
114, 8573-8579.
(22) Medina, D.; Kittrell, F. S.; Liu, Y. J .; Schwartz, M.
Morphological and functional properties of TM preneo-
plastic mammary outgrowths. Cancer Res. 1993, 53,
663-667.
(23) Bernhardt, G.; Reile, H.; Birnbock, H.; Spruss, T.;
Schonenberger, H. Standardized kinetic microassay to
quantify differential chemosensitivity on the basis of
proliferative activity. J . Cancer Res. Clin. Oncol. 1992,
118, 35-43.
LITERATURE CITED
(1) Block, E. The organosulfur chemistry of the genus
Allium. Implications for organic sulfur chemistry. An-
gew. Chem., Int. Ed. Engl. 1992, 31, 1135-1178.
(2) Koch, H. P.; Lawson, L. D. Garlic. The Science and
Therapeutic Applications of Allium sativum L. and
Related Species; William and Wilkens: Baltimore, MD,
1996.
(3) Spåre, C.-G.; Virtanen, A. I. On the occurrence of free
selenium-containing amino acids in onion (Allium cepa).
Acta Chem. Scand. 1964, 18, 280-282.
(4) Rayman, M. P. The importance of selenium to human
health. Lancet 2000, 356, 233-241.
(5) Ip, C.; Ganther, H. E. Comparison of selenium and
sulfur analogs in cancer prevention. Carcinogenesis
1992, 13, 1167-1170.
(6) Ip, C.; Lisk, D. J . Enrichment of selenium in allium
vegetables for cancer prevention. Carcinogenesis 1994,
15, 1881-1885.

Chemistry and Biological Activity of Selenoamino Acids and Their Se Oxides
J. Agric. Food Chem., Vol. 49, No. 1, 2001 469
(24) Singh, N. P.; McCoy, M. T.; Tice, R. R.; Schneider, E. L.
A simple technique for quantitation of low levels of DNA
damage in individual cells. Exp. Cell Res. 1988, 175,
184-191.
(25) Collins, A.; Dusinska, M.; Franklin, M.; Somorovska, M.;
Petrovska, H.; Duthie, S.; Fillion, L.; Panayiotidi, M.;
Raslova, K.; Vaughan, N. Comet assay in human
biomonitoring studies: reliability, validation, and ap-
plications. Environ. Mol. Mutagen. 1997, 30, 139-146.
(26) Duke, R. C.; Cohen, J . J . Morphological and biochemical
assays of apoptosis. In Current Protocols in Immunology;
Coligan, J . E., Kruisbeak, A. M., Eds.; Wiley: New York,
1992; pp 3.17.1-3.17.16.
(27) Snedecor, G. J . W.; Cochran, W. G. Statistical Methods,
6th ed.; Iowa State University Press: Ames, IA, 1967;
pp 135-317.
(28) Andreadou, I.; Menge, W. M. P. B.; Commandeur, J . N.
M.; Worthington, E. A.; Vermeulen, N. P. E. Synthesis
of novel Se-substituted selenocysteine derivatives as
potential kidney selective prodrugs of biologically active
selenol compounds: evaluation of kinetics of â-elimina-
tion reactions in rat renal cytosol. J . Med Chem. 1996,
39, 2040-2046.
(29) Marrone, L.; Siemann, S.; Beecroft, M.; Viswanatha, T.
Specificity of lysine:N6-hydroxylase: a hypothesis for a
reactive substrate intermediate in the catalytic mech-
anism. Bioorg. Chem. 1996, 24, 401-416.
(30) Mu¨tsch-Eckner, M.; Meier, B.; Wright, A. D.; Sticher,
O. γ-Glutamyl peptides from Allium sativum bulbs.
Phytochemistry 1992, 31, 2389-2391.
(31) Walter, R.; Roy, J . Selenomethionine: a potential cata-
lytic antioxidant in biological systems. J . Org. Chem.
1971, 36, 2561-63.
(45) Seff, K.; Heidner, E. G.; Meyers, M.; Trueblood, K. N.
The crystal and molecular structure of methanesulfinic
acid. Acta Crystallogr., Part B 1969, 25, 350-354.
(46) Bryden, J . H.; McCullough, J . D. The crystal structure
of benzeneseleninic acid. Acta Crystallogr. 1954, 7, 833-
838.
(47) Bryden, J . H.; McCullough, J . D. The crystal structure
of p-chlorobenzeneseleninic acid. Acta Crystallogr. 1956,
9, 528-533.
(48) Syed, R.; Wu, Z.-P.; Hogle, J . M.; Hilvert, D. Crystal
structure of selenosubtilisin at 2.0-Å resolution. Bio-
chemistry 1993, 32, 6157-6164.
(49) Reich, H. J .; Yelm, K. E.; Wollowitz, S. Kinetics,
thermodynamics, and stereochemistry of the allyl sulf-
oxide-sulfenate and selenoxide-selenenate [2,3]-sigma-
tropic rearrangements. J . Am. Chem. Soc. 1983, 105,
2503-2504.
(50) Sharpless, K. B.; Lauer, R. F. A mild procedure for the
conversion of epoxides to allylic alcohols. The first
organoselenium reagent. J . Am. Chem. Soc. 1973, 95.
2697-2699.
(51) Goto, K.; Okazaki, R. Molecular bowls and capsules with
an endohedral functionality: the stabilization of highly
reactive species in their inner phase. Liebigs Ann./
Recueil 1997, 2393-2407.
(52) Stocking, E. M.; Schwarz, J . N.; Senn, H.; Salzmann,
M.; Silks, L. A. Synthesis of L-selenocystine, L-[77Se]-
selenocystine and L-tellurocystine. J . Chem. Soc., Perkin
Trans. 1 1997, 2443-2447.
(53) Reich, H. J .; Willis, W. W., J r.; Wollowitz, S. “Stable”
selenenic acids. Tetrahedron Lett. 1982, 23, 3319-3322.
(54) Saiki, T.; Goto, K.; Okazaki, R. Isolation and X-ray
crystallographic analysis of a stable selenenic acid.
Angew. Chem., Int. Ed. Engl. 1997, 36, 2223-2224.
(55) Iwaoka, M.; Tomoda, S. A model study of the effect of
an amino group on the antioxidant activity of glu-
tathione peroxidase. J . Am. Chem. Soc. 1994, 116,
2557-2561.
(32) Zainal, H. A.; LaCroix, D. E.; Wolf, W. R. Utilization of
chromatographic and spectroscopic techniques to study
the oxidation kinetics of selenomethionine. Fresenius’
J . Anal. Chem. 1996, 356, 311-314.
1
(33) Isab, A. A. A H NMR study of the reaction of gold(III)
with DL-selenomethionine in aqueous solution. Inorg.
Chim. Acta 1983, 80, L3-L4.
(56) Block, E.; Gillies, J . Z.; Gillies, C. W.; Bazzi, A. A.;
Putman, D.; Revelle, L. K.; Wall, A.; Wang, D.; Zhang,
X. Allium chemistry: identification, mechanism of
formation, synthesis, and reactions of (E,Z)-propanethial
S-oxide, the lachrymatory factor of the onion (Allium
cepa). J . Am. Chem. Soc. 1996, 118, 7492-7501.
(57) Kice, J . L.; McAfee, F.; Slebocka-Tilk, H. The anhydride
structure of several previously reported stable arene-
selenenic acids. Tetrahedron Lett. 1982, 23, 3323-3326.
(58) Reich, H. J .; J asperse, C. P. Organoselenium chemistry.
Preparation and reactions of 2,4,6-tri-tert-butylbenzen-
eselenenic acid. J . Org. Chem. 1988, 53, 2389-2390.
(59) Nigan, S. N.; McConnell, W. B. Seleno amino compounds
from Astragalus bisulcatus. Isolation and identification
of γ-L-glutamyl-Se-methylseleno-L-cysteine and Se-me-
thylseleno-L-cysteine. Biochim. Biophys. Acta 1969, 192,
185-190.
(60) Guo, X.; Wu, L. Distribution of free seleno-amino acids
in plant tissue of Melilotus indica L. grown in selenium-
laden soils. Ecotoxicol. Environ. Saf. 1998, 39, 207-214.
(61) Cone, J . E.; Del Rio, R. M.; Davis, J . N.; Stadtman, T.
C. Chemical characterization of the selenoprotein com-
ponent of clostridial glycine reductase: identification of
selenocysteine as the organoselenium moiety. Proc. Natl.
Acad. Sci. U.S.A. 1976, 73, 2659-2663.
(34) Assmann, A.; Briviba, K.; Sies, H. Reduction of methion-
ine selenoxide to selenomethionine by glutathione. Arch.
Biochem. Biophys. 1998, 349, 201-203.
(35) Assmann, A.; Bonifacic, M.; Briviba, K.; Sies, H.; Asmus,
K.-D. One-electron reduction of selenomethionine oxide.
Free Radical Res. 2000, 32, 371-376.
(36) Wrench, J . J . Selenium metabolism in the marine
phytoplankters Tetraselmis tetrathele and Dunaliella
minuta. Mar. Biol. 1978, 49, 231-236.
(37) Bottino, N. R.; Banks, C. H.; Irgolic, K. J .; Micks, P.;
Wheeler, A. E.; Zingaro, R. A. Selenium containing
amino acids and proteins in marine algae. Phytochem-
istry 1984, 23, 2445-2452.
(38) Peterson, P. J .; Butler, G. W. The uptake and assimila-
tion of selenite by higher plants. Aust. J . Biol. Sci. 1962,
15, 126-139.
(39) Hamilton, J . W. Chemical examination of seleniferous
cabbage Brassica oleracea capitata. J . Agric. Food Chem.
1975, 23, 1150-1152.
(40) Caldwell, K. A.; Tappel, A. L. Reactions of seleno- and
sulfoamino acids with hydroperoxides. Biochemistry
1964, 3, 1643-1647.
(41) Olson, O. E.; Novacek, E. J .; Whitehead, E. I.; Palmer,
I. S. Investigations on selenium in wheat. Phytochem-
istry 1970, 9, 1181-1188.
(42) Ramadan, S. E.; Razak, A. A.; Yousseff, Y. A.; Sedky,
N. M. Selenium metabolism in a strain of Fusarium.
Biol. Trace Elem. Res. 1988, 18, 161-170.
(43) Maher, W.; Deaker, M.; J olley, D.; Krikowa, F.; Roberts,
B. Selenium occurrence, distribution and speciation in
the cockle Anadara trapezia and the mullet Mugil
cephalus. Appl. Organomet. Chem. 1997, 11, 313-
326.
(62) Sparnins, V. L.; Barany, G.; Wattenberg, L. W. Effects
of organosulfur compounds from garlic and onions on
benzo[a]pyrene-induced neoplasia and glutathione S-
transferase activity in the mouse. Carcinogenesis 1988,
9, 131-134.
(63) Ganther, H. E. Selenium metabolism, selenoproteins
and mechanisms of cancer prevention: complexities
with thioredoxin reductase. Carcinogenesis 1999, 20,
1657-1666.
(44) Kotrebai, M. Ph.D. Thesis, University of Massachusetts,
Amherst, MA, 2000.

470 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Block et al.
(64) Thompson, H. J .; Wilson, A.; Lu, J .; Singh, M.; J iang,
C.; Upadhyaya, P.; el Bayoumy, K.; Ip, C. Comparison
of the effects of an organic and an inorganic form of
selenium on a mammary carcinoma cell line. Carcino-
genesis 1994, 15, 183-186.
(65) Ip, C. Lessons from basic research in selenium and
cancer prevention. J . Nutr. 1998, 128, 1845-1854.
(66) Zhu, Z.; J iang, W.; Ganther, H. E.; Ip, C.; Thompson,
H. J . In vitro effects of Se-allylselenocysteine and Se-
propylselenocysteine on cell growth, DNA integrity
and apoptosis. Biochem. Pharmacol. 2000, 60, 1467-
1473.
(67) Rooseboom, M.; Commandeur, J . N. M.; Floor, G. S.;
Rettie, A. E.; Vermeulen, N. P. E. Selenoxidation by
flavin-containing monooxygenases as a novel pathway
for â-elimination of selenocysteine Se-conjugates. Chem.
Res. Toxicol. 2001, in press.
(68) Ip, C.; Thompson, H. J .; Zhu, Z.; Ganther, H. E. In vitro
and in vivo studies of methylseleninic acid: evidence
that a monomethylated selenium metabolite is critical
for cancer chemoprevention. Cancer Res. 2000, 60,
2882-2886.
rat renal proximal tubular cells. Toxicol. Appl. Phar-
macol. 1996, 141, 278-287.
(72) Rooseboom, M.; Vermeulen, N. P. E.; Andreadou, I.;
Commandeur, J . N. M. Evaluation of the kinetics of
â-elimination reactions of selenocysteine Se-conjugates
in human renal cytosol: possible implications for the
use as kidney selective prodrugs. J . Pharmacol. Exp.
Ther. 2000, 294, 762-769.
(73) Dong, Y.; Lisk, D.; Block, E.; Ip, C. Characterization of
the Biological Activity of γ-Glutamyl-Se-methylseleno-
cysteine: A novel naturally occurring anticancer agent
from garlic. Cancer Res. 2000, submitted for publication.
(74) McSheehy, S.; Yang, W.; Pannier, F.; Szpunar, J .;
Lobinski, R.; Auger, J .; Potin-Gautier, M. Speciation
analysis of selenium in garlic by two-dimensional high-
performance liquid chromatography with parallel in-
ductively coupled plasma mass spectrometric and elec-
trospray tandem mass spectrometric detection. Anal.
Chim. Acta 2000, 421, 147-153.
(75) Whanger, P. D.; Ip, C.; Polan, C. E.; Uden, P. C.;
Welbaum, G. Tumorigenesis, metabolism, speciation,
bioavailability, and tissue deposition of selenium in
selenium-enriched ramps (Allium tricoccum). J . Agric.
Food Chem. 2000, 48, 5723-5730.
(69) Mlochowski, J .; Kloc, K.; Syper, L.; Inglot, A. D. Recent
developments in the chemistry and biology of disele-
nides, benzisoselenazolones and seleninic acids. Phos-
phorus, Sulfur Silicon Relat. Elem. 1991, 59, 267-270.
(70) J iang, W.; Zhu, Z.; Ganther, H. E.; Ip, C.; Thompson,
H. J . Molecular mechanisms associated with Se-allylse-
lenocysteine regulation of cell proliferation and apop-
tosis. Cancer Lett. 2000, in press.
Received for review September 6, 2000. Accepted October 31,
2000. This work was supported by Grant CA45164 from the
National Cancer Institute, by National Science Foundation
Grants CHE 96-28774 and CHE 99-06566, and by NRI
Competitive Grants Program/USDA Grant 96-355003351.
(71) Andreadou, I.; van de Water, B.; Commandeur, J . N.
M.; Nagelkerke, F. J .; Vermeulen, N. P. E. Comparative
cytotoxicity of 14 novel selenocysteine Se-conjugates in
J F001097B