Selenoglucosinolates and their metabolites produced in Brassica spp. fertilised with sodium selenate

Article abstract of DOI:10.1016/j.phytochem.2011.11.021

Glucosinolates are sulphur-containing glycosides found in many Brassica spp. that are important because their aglycone hydrolysis products protect the plant from herbivores and exhibit anti-cancer properties in humans. Recently, synthetically produced selenium analogues have been shown to be more effective at suppressing cancers than their sulphur counterparts. Although selenium is incorporated into a number of Brassica amino acids and peptides, firm evidence has yet to be presented for the presence of selenium in the glucosinolates and their aglycones in planta. In this study broccoli and cauliflower florets, and roots of forage rape, all obtained from plants treated with sodium selenate, were analysed for the presence of organoselenides. GC-MS analysis of pentane/ether extracts identified six organoselenium compounds including selenium analogues of known myrosinase-derived Brassica volatiles: 4-(methylseleno)butanenitrile, 5-(methylseleno)pentanenitrile, 3-(methylseleno)propylisothiocyanate, 4-(methylseleno)butylisothiocyanate, and 5-(methylseleno)pentylisothiocyanate. LC-MS analysis of ethanolic extracts identified three selenoglucosinolates: 3-(methylseleno)propylglucosinolate (glucoselenoiberverin), 4-(methylseleno)butylglucosinolate (glucoselenoerucin), and 5-(methylseleno)pentylglucosinolate (glucoselenoberteroin). LC-MS/MS analysis was used to locate the position of the selenium atom in the selenoglucosinolate and indicates preferential incorporation of selenium via selenomethionine into the methylselenyl moiety rather than into the sulphate or β-thioglucose groups. In forage rape, selenoglucosinolates and their aglycones (mainly isothiocyanates), occurred at concentrations up to 10% and 70%, respectively, of their sulphur analogues. In broccoli, concentrations of the selenoglucosinolates and their aglycones (mainly nitriles) were up to 60% and 1300%, respectively of their sulphur analogues. These findings indicate the potential for the incorporation of high levels of selenium into Brassica glucosinolates.

Full text of DOI:10.1016/j.phytochem.2011.11.021

Phytochemistry 75 (2012) 140–152
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Selenoglucosinolates and their metabolites produced in Brassica spp. fertilised
with sodium selenate
⇑
Adam J. Matich , Marian J. McKenzie, Ross E. Lill, David A. Brummell, Tony K. McGhie, Ronan K.-Y. Chen,
Daryl D. Rowan
The New Zealand Institute for Plant & Food Research Limited (Plant & Food Research), Private Bag 11600, Palmerston North 4442, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2011
Received in revised form 25 November 2011
Available online 23 December 2011
Glucosinolates are sulphur-containing glycosides found in many Brassica spp. that are important because
their aglycone hydrolysis products protect the plant from herbivores and exhibit anti-cancer properties in
humans. Recently, synthetically produced selenium analogues have been shown to be more effective at
suppressing cancers than their sulphur counterparts. Although selenium is incorporated into a number of
Brassica amino acids and peptides, firm evidence has yet to be presented for the presence of selenium in
the glucosinolates and their aglycones in planta. In this study broccoli and cauliflower florets, and roots of
forage rape, all obtained from plants treated with sodium selenate, were analysed for the presence of
organoselenides. GC–MS analysis of pentane/ether extracts identified six organoselenium compounds
including selenium analogues of known myrosinase-derived Brassica volatiles: 4-(methylseleno)butane-
nitrile, 5-(methylseleno)pentanenitrile, 3-(methylseleno)propylisothiocyanate, 4-(methylseleno)butylis-
othiocyanate, and 5-(methylseleno)pentylisothiocyanate. LC–MS analysis of ethanolic extracts identified
three selenoglucosinolates: 3-(methylseleno)propylglucosinolate (glucoselenoiberverin), 4-(methylse-
leno)butylglucosinolate (glucoselenoerucin), and 5-(methylseleno)pentylglucosinolate (glucoselenober-
teroin). LC–MS/MS analysis was used to locate the position of the selenium atom in the
selenoglucosinolate and indicates preferential incorporation of selenium via selenomethionine into the
methylselenyl moiety rather than into the sulphate or b-thioglucose groups. In forage rape, selenogluc-
osinolates and their aglycones (mainly isothiocyanates), occurred at concentrations up to 10% and 70%,
respectively, of their sulphur analogues. In broccoli, concentrations of the selenoglucosinolates and their
aglycones (mainly nitriles) were up to 60% and 1300%, respectively of their sulphur analogues. These find-
ings indicate the potential for the incorporation of high levels of selenium into Brassica glucosinolates.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Broccoli
Cauliflower
Forage rape
Brassica
Organoselenides
3-(Methylseleno)propylglucosinolate
4-(Methylseleno)butylglucosinolate
5-(Methylseleno)pentylglucosinolate
4-(Methylseleno)butanenitrile
5-(Methylseleno)pentanenitrile
3-(Methylseleno)propylisothiocyanate
4-(Methylseleno)butylisothiocyanate
5-(Methylseleno)pentylisothiocyanate
1. Introduction
plants is selenomethionine, the selenium analogue of methionine
(Mounicou et al., 2006; Pedrero et al., 2007), which can be a source
The uptake of selenium (Se) into the sulphur (S) assimilation
pathway of edible Brassica plants provides a possible route to the
increased dietary intake of selenium, which in some countries is
low or below the recommended daily intake (Broadley et al.,
2006). Selenium deficiency is associated with reduced human fer-
tility, reduced immune and cognitive function, heart disease,
hyperthyroidism, and an increased risk of certain types of cancer.
There is evidence that these disorders can be suppressed by sup-
plementing the diet with organoselenides (Finley et al., 2000; Ellis
and Salt, 2003; Broadley et al., 2006; Rayman, 2008). One very po-
tent anti-cancer agent is methylselenol, which is thought to be
biosynthesised from the amino acid derivative Se-methyl seleno-
of stored selenium in the human body (Rayman, 2008).
A number of methylthioalkyl nitriles, thiocyanates, and isothio-
cyanates are reported in Brassica spp. (Verkerk et al., 2009;
Sonderby et al., 2010). Isothiocyanates both prevent the initiation
and retard the progression of carcinogenesis (Rose et al., 2005;
Hayes et al., 2008; Melchini and Traka, 2010), while syn-
thetic selenium-containing isothiocyanates, seleno-sulphoraphane
(4-methylthio) butylisoselenocyanate and several phenylalkylis-
oselenocyanates are reported to be more potent inhibitors of
cancer-cell and tumour growth than their sulphur analogues
(Sharma et al., 2009; Emmert et al., 2010). Incorporation of
selenium from selenomethionine into the above methylthioalkyl
compounds would create methylselenoalkyl nitriles and isothiocy-
anates similar to the methylseleno aldehydes we have previously
identified in genetically modified tobacco (Matich et al., 2009).
Methylselenoalkyl nitriles and isothiocyanates have not been
reported in nature and it is unknown whether these classes of
cysteine and the peptide
c-glutamyl-Se-methyl selenocysteine
(Rayman, 2008). Another product of selenium assimilation in
⇑
Corresponding author. Tel.: +64 6 9537679; fax: +64 6 9537701.
E-mail address: adam.matich@plantandfood.co.nz (A.J. Matich).
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.11.021

A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
141
organoselenides also contribute to the anti-cancer activity of Bras-
sica. Verification of their presence would provide another group of
compounds to be targeted in the study of the anti-cancer activity of
Brassica.
Krumbein et al., 2010) as have some of the organoselenides.
Dimethylselenosulphide (2, CH3SeSCH3) has been reported in garlic
(Cai et al., 1994) and Brassica juncea (Indian mustard) (Meija et al.,
2002; Kubachka et al., 2007) watered with selenium fertilisers.
Dimethyldiselenide (3, CH3Se2CH3) is frequently reported in Brassica
(Meija et al., 2002; Kubachka et al., 2007), while CH3S2SeCH3 (7) has
been identified in genetically modified bacteria and tobacco
(Swearingen et al., 2006; McKenzie et al., 2009) and in selenium-
fertilised green onions (Allium fistulosum) (Shah et al., 2007). These
mixed sulphur–selenium compounds are probably products of the
exchange reaction between CH3Se2CH3 (3), CH3S2CH3 (1), and
CH3S3CH3 (6) (Chasteen, 1993), all of which we identified in the
Brassica extracts (Table 1).
The above nitriles, thiocyanates and isothiocyanates are biosyn-
thetic hydrolysis products of glucosinolates (Verkerk et al., 2009),
characteristic secondary metabolites of Brassica which are associ-
ated with resistance to herbivores (Halkier and Gershenzon,
2006; Mumm et al., 2008). Glucosinolates (Fig. 1, 22–24) are b-thi-
oglucoside N-hydroxysulphates with a side chain derived from one
of eight amino acids that may also have undergone chain elonga-
tion or modification before glucosinolate formation (Halkier and
Gershenzon, 2006). Myrosinase (or thioglucoside glucohydrolase)
hydrolysis of glucosinolates yields glucose and an unstable agly-
cone that spontaneously rearranges to an isothiocyanate. Plants
that contain epithiospecifier proteins (ESPs) can alternatively form
nitriles and thiocyanates from glucosinolates at the expense of iso-
thiocyanates (Halkier and Gershenzon, 2006; Burow et al., 2007;
Mumm et al., 2008; Kissen and Bones, 2009). Trace amounts of
selenium-containing glucosinolates (selenoglucosinolates) have
been reported in the Brassicaceae plants Stanleya pinnata (Prince’s
plume) and Nasturtium officinale (watercress) (Bertelsen et al.,
1988; Wielanek et al., 2005). Theoretically, the glucosinolates in
Brassica might incorporate up to three atoms of selenium: in the
sulphate, the modified amino acid (selenomethionine or methylse-
lenocysteine) side chain (Pedrero et al., 2007), or in the b-thioglu-
cose where it has been reported once in nature (Bertelsen et al.,
1988). Thus, depending where in the glucosinolate molecule the
selenium is incorporated, hydrolysis by myrosinase may produce
a number of selenium containing products.
In the present study, we investigated the incorporation of sele-
nium into the florets of the food Brassica spp., broccoli and
cauliflower (Brassica oleracea L. var. italica and var. botrytis, respec-
tively), and also into the roots of forage rape (Brassica napus) since
this species has been reported to accumulate very high concentra-
tions of organosulphides (McCully et al., 2008). To assist with the
identification of organoselenides that might otherwise be at trace
levels, we treated the plants with sodium selenate fertiliser to
produce organoselenide concentrations well in excess of potential
dietary levels (Finley et al., 2000). Using gas chromatography–mass
spectrometry (GC–MS) and liquid chromatography–mass spec-
trometry (LC–MS), we demonstrate, for the first time, the incorpo-
ration of inorganic selenium into the amino acid-derived
methylselenyl side chain of Brassica selenoglucosinolates.
2.1.1. Selenium volatiles not previously reported in Brassica spp.
Methylselenomethanenitrile (4b, CH3SeCN) was identified from
its mass spectrum (Fig. 2) (Yang et al., 2004) and was found in only
one of the cauliflower extracts. This compound has not previously
been reported in nature, although its sulphur analogue, methylthi-
omethanenitrile (4a, CH3SCN), is reported in broccoli (Jacobsson
et al., 2004; Vidal-Aragón et al., 2009; Krumbein et al., 2010) and
was present in all three Brassica species (Table 1). The sulphur com-
pound 2-(methylthio)ethanal has been reported in tomatoes (Birtic
et al., 2009) and heated onion sprouts (Allium cepa L.) (Takahashi and
Shibamoto, 2008). We recently reported its selenium analogue,
2-(methylseleno)ethanal (5), in genetically modified tobacco over-
expressing a selenocysteine methyltransferase transgene as a possi-
ble metabolite of methylselenocysteine (Matich et al., 2009). In the
present study we detected traces of 5 in both broccoli and forage
rape, but only in solvent extracts stored for several weeks at
À20 °C, and not in extracts stored for shorter periods of time, sug-
gesting this compound may be a storage artefact.
2.1.2. Selenium-containing aldehydes
To our knowledge, the other organoselenides listed in Table 1
(8, 9b, 10b, 11b, 12b, and 13b) have not previously been reported.
Aldehyde 8 was tentatively identified by its fragmentation pattern
and its distinctive selenium isotope pattern which was repeated
for the clusters of ions around m/z 192 (M+), 177 (M+ÀCH3), 163
(M+ÀC2H5), and 135 (M+ÀC2H5ÀCO) (Fig. 3). The fragments m/z
163 and 135 were previously used as identifiers of 4-(methylse-
leno)-2-alkenals (Matich et al., 2009). Herein, CH3Se+H (m/z 96)
indicates the loss of C6H8O (hexa-2,4-dienal) and the distinctive
m/z 81 (CH2C+CHCHCHO) the loss of C2H7Se. 4-(Methylse-
leno)hex-E2-enal (8) was synthesised by the acid hydrolysis of
(2,2-dimethoxyethyl)(methyl)selane in the presence of hex-E2-
enal, as used previously to prepare 4-(methylseleno)non-E2-enal
and 4-(methylseleno)nona-E2,Z6-dienal (Matich et al., 2009). In
the present study, however, the product yield was poor and we
were unable to purify 8, which did, however, have the same mass
spectrum and GC retention time as the compound in the Brassica
extracts. We did not identify the sulphur analogue (4-(methyl-
thio)hex-E2-enal) of compound 8 in our solvent extracts, and could
not find it reported in the literature. Previously, we suggested that
methylseleno aldehydes with structures similar to 8 were artefacts
or arose from spontaneous chemical reactions within the plant
(Matich et al., 2009). In the present study aldehyde 8 occurred at
low concentrations, and was a minor component of the organosel-
enides found in the broccoli and cauliflower samples.
2. Results and discussion
2.1. GC–MS analysis of volatile organoselenides
Organoselenides are readily identifiable by mass spectrometry in
complex plant extracts due to the mass deficiency of elemental sele-
nium (Se80 = 79.9165 Da), the distinctive isotope pattern of the six
major isotopes and the several characteristic selenium-containing
ions (m/z 93, CHSe+; 95, CH3Se+; 96, CH3Se+H; and 109, C2H5Se+)
frequently observed in the mass spectra of organoselenides. The
structures of the volatile organoselenides identified in selenium
supplemented broccoli and cauliflower florets, and forage rape roots
are shown in Fig. 1. Eleven selenium containing volatiles were iden-
tified, including two methylseleno-aldehydes (5 and 8 in Fig. 1),
three methylseleno-nitriles (4b, 9b, and 10b in Fig. 1), and three
methylseleno-isothiocyanates (11b, 12b, and 13b in Fig. 1). For com-
parison, their sulphur analogues identified herein in these species
are also shown. All organosulphides identified in these samples have
previously been reported in Brassica (Hashimoto et al., 1982;
Nakamura et al., 1993; Tulio et al., 2002; de Pinho et al., 2009;
2.1.3. Selenium-containing nitriles
The molecular ion (M+Å) of compound 9b (4-(methylse-
leno)butanenitrile) was m/z 162.9901 (calcd. for C5H9NSe,
162.9900) and its fragmentation pattern (Fig. 2) was the most diag-
nostic of the two nitriles identified, viz. m/z 123 (M+ÀCH2CN), m/z
109 (M+ÀC2H4CN to give CH3SeCH2+), and m/z 95 (CH3Se+). 5-

142
A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
Fig. 1. Structures of selenium and sulphur compounds identified during this and previous studies.
(Methylseleno)pentanenitrile (10b) had an M+ of m/z 177.0055
(calcd. for C6H11NSe, 177.0057) and a slightly different fragmenta-
tion pattern (Fig. 2), viz. m/z 162 (M+ÀCH3), m/z 135 (M+ÀC2H4N),
m/z 109 (M+ÀC3H6CN to give CH3SeCH2+), m/z 95 (CH3Se+), and m/z

A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
143
Table 1
Semi-quantitative GC–MS analysis (l
g kgÀ1 FW) of selenium-containing volatiles and, where found, of their sulphur analogues in extracts of broccoli and cauliflower floret tissue
and forage rape root tissue from control plants and from plants fertilised with 5 mM sodium selenate (Na2SeO4). Concentrations are the average ( SEM) of three samples, each
sample comprising tissue combined from two separate plants. Chemical structures are given in Fig. 1.
Compound
RT (min)
RI (Wax)a
Broccoli cv. ‘Triathlon’
Cauliflower cv. ‘Liberty’
Forage rape cv. ‘Maxima’
Control
Na2SeO4
Control
Na2SeO4
Control
Na2SeO4
CH3SSCH3 (1)b
4.71
7.2
9.85
1060
1135
1210
1240
1339
1424
1459
1742
1815
1831
1888
1939
1983
2032
2086
2181
2209
2301
2700 (130)
–
–
290 (14)
5800 (1230)
–
–
80 (16)
–
–
1800 (420)
9 (1)
–
–
2200 (510)
310 (73)
13 (2)
280 (44)
3500 (790)
–
2700 (680)
–
–
270 (130)
1600 (250)
–
–
2070 (340)
–
–
160 (74)
170 (130)
–
–
1460 (350)
164 (7)
10 (4)
174 (38)
1150 (420)
34 (34)
23 (11)
1490 (590)
11 (5)
400 (120)
90 (50)
390 (240)
4 (1)
110 (73)
40 (20)
4 (2)
260 (140)
–
–
15 (4)
280 (100)
–
–
80 (20)
104 (38)
16 (4)
13 (2)
98 (34)
–
7 (0.3)
70 (34)
–
26 (13)
620 (125)
63 (10)
260 (140)
46 (13)
6900 (810)
2040 (460)
14800 (800)
320 (83)
k
CH3SeSCH3 (2)c
CH3Se2CH3 (3)b
CH3SCN (4a)d
10.93
14.91
18.39
18.99
28.18
29.59
29.86
30.81
31.62
32.34
33.04
33.82
35.17
35.6
CH3S3CH3 (6)d
CH3SeCN (4b)e
CH3S2SeCH3 (7)f
26 (9)
CH3SC3H6CN (9a)g
C3H6(CH3Se)C3H3O (8)h
CH3SeC3H6CN (9b)g
CH3SC4H8CN (10a)d
CH3SC3H6NCS (11a)d
CH3SeC4H8CN (10b)h
CH3SeC3H6NCS (11b)h
CH3SC4H8NCS (12a)i,j
CH3SeC4H8NCS (12b)h
CH3SC5H10NCS (13a)i
CH3SeC5H10NCS (13b)g
320 (45)
5.7 (0.4)
1350 (160)
1600 (230)
19 (6)
1050 (100)
260 (33)
340 (17)
1000 (44)
–
23 (6)
–
0.9 (0.1)
460 (110)
43 (16)
0.5 (0.1)
–
5700 (1980)
–
9100 (970)
–
320 (70)
45 (10)
–
–
–
–
–
–
–
–
36.81
–
a
b
c
d
e
f
Retention Index on DB-Wax GC column with respect to a series of straight-chain hydrocarbons.
Identified against authentic compounds.
Identified previously (Matich et al., 2009).
Mass spectrum library matching (NIST, 2002).
Yang et al. (2004).
Meija and Caruso (2004), Swearingen et al. (2006) and Shah et al. (2007).
Tentatively identified by MS analysis.
Identified by synthesis of authentic compounds.
Kjaer et al. (1963).
g
h
i
j
Vercammen et al. (2001).
Not detected.
k
82 (C4H8CN+). These fragmentations are analogous to those ob-
served for the sulphur analogues of these compounds (Spencer
and Daxenbichler, 1980). We confirmed the identification of 9b
and 10b by chemical synthesis. These two organoselenides and
their sulphur analogues (9a and 10a) were present in the extracts
of all three Brassica spp. Compounds 9a and 10a have been re-
ported in steam distillates of cabbage, broccoli and cauliflower
(Buttery et al., 1976; Valette et al., 2003). Small amounts of organo-
selenides 9b and 10b were also identified in the forage rape control
samples (no selenium fertiliser used) confirming the natural occur-
rence of these compounds (Table 1). The concentration of organo-
selenide 9b in the selenium-fertilised forage rape samples was ca.
30-fold higher than in the control samples, and for 10b it was ca.
500-fold higher. In total, the concentration of organoselenides (ni-
triles plus isothiocyanates) in the selenised forage rape roots was
ca. 2000-fold higher than in the control samples.
The anti-cancer properties of Brassica nitriles have not received
as much attention as the isothiocyanates and it has been suggested
this is because few bioactive nitriles have been reported (Keck and
Finley, 2004). In some studies nitriles have a similar anti-cancer
activity to the isothiocyanates, but generally they are less active
(Nastruzzi et al., 2000; Keck and Finley, 2004). Another biological
activity reported for the Brassica nitriles is as a plant chemical de-
fence against specialist herbivores. Predation is discouraged by a
combination of reduced attraction of the specialist herbivore to
the plant and the attraction of parasites that feed on the herbivore
(Mumm et al., 2008).
and 149, respectively). All compounds produced m/z 109
(CH3Se+CH2), m/z 93 (Se+CH), and m/z 72 (CH2@N+CS) which is a
well known isothiocyanate fragment (Kjaer et al., 1963). We sur-
mise that M+-74 represents the sum of the loss of HNCS and CH3,
and that these compounds are methylselenoalkylisothiocyanates.
The fragment m/z 101 in compound 11b (vs. m/z 114 in 13b and
m/z 128 in 13b) was not totally consistent with the series, but a
discontinuity in the fragmentation patterns for the sulphur ana-
logues of these compounds is also observed (Kjaer et al., 1963).
This fragment corresponds to a cyclisation and loss of CH2Se_ to
produce the 4,5-dihydro-3H-pyrrole-2-thiol ion (Fig. 4, C4H7N+S).
In contrast, m/z 114 and 128 are probably C4H8NCS+ and
C5H10NCS+, respectively.
We confirmed the identity of 4-(methylseleno)butylisothiocya-
nate (12b) by synthesis. Compounds 11b and 13b were therefore
assigned as 3-(methylseleno)propylisothiocyanate and 5-(methyl-
seleno)pentylisothiocyanate, respectively, based upon their GC
retention times and the similarity of their fragmentation patterns
to that of 12b (Fig. 4) and that of their sulphur analogues (Kjaer
et al., 1963). 3-(Methylseleno)propylisothiocyanate (11b), 4-
(methylseleno)butylisothiocyanate (12b) and their sulphur ana-
logues 11a and 12a were identified in all three Brassica extracts,
whilst 5-(methylseleno)pentylisothiocyanate (13b) and its sulphur
analogue 13a were found only in the extracts of forage rape.
Methylselenoalkylisothiocyanates 11b–13b were previously
unknown in nature and their biological activity is yet to be deter-
mined. However their sulphur analogues, and other isothiocya-
nates, exhibit anti-cancer activities (Zhang et al., 1992; Hwang
and Lee, 2006; Melchini and Traka, 2010). Erucin (12a), was origi-
nally identified at high levels in the salad vegetable ‘‘Rocket’’ (Eruca
sativa) and its mode of action as an anti-cancer phytochemical
investigated (Wang et al., 2005; Melchini and Traka, 2010). Iberv-
erin (11a) activates phase II detoxification enzymes, which are
2.1.4. Selenium-containing isothiocyanates
The mass spectra of compounds 11b, 12b and 13b led us to sug-
gest they were homologues (Fig. 4). With M+ differing by 14 amu,
viz. m/z 195, 209, and 223, respectively, they all displayed M+-15
(m/z 180, 194, and 208, respectively), and M+-74 (m/z 121, 135,

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A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
Fig. 2. Mass spectra of methylselenomethanenitrile (CH3SeCN, 4b), 4-(methylseleno)butanenitrile (CH3SeC3H6CN, 9b), and 5-(methylseleno)pentanenitrile (CH3SeC4H8CN,
10b).
Fig. 3. Mass spectrum of 4-(methylseleno)hex-E2-enal (8).

A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
145
Fig. 4. Mass spectra of 3-(methylseleno)propylisothiocyanate (CH3SeC3H6NCS, 11b), 4-(methylseleno)butylisothiocyanate (CH3SeC4H8NCS, 12b), and 5-(methylseleno)penty-
lisothiocyanate (CH3SeC5H10NCS, 13b).
known to protect against chemical carcinogenesis (Munday and
Munday, 2004). As a chemical class, isothiocyanates seem to be
generally active against cancers (Russo et al., 2010). Therefore,
compounds 11b–13b may also show such activity, and given the
suggestion that some selenium compounds are more potent anti-
cancer compounds than their sulphur analogues (Sharma et al.,
2009; Emmert et al., 2010), the discovery of these compounds pro-
vides new opportunities for understanding and improving the can-
cer-preventative properties of Brassica.
analogues of compounds that are produced by the Brassica sulphur
assimilation pathway.
The selenium-containing nitriles 9b and 10b, were at their
highest concentrations in the broccoli samples at around 1 mg kgÀ1
FW (Table 1), with concentrations at similar orders of magnitude to
those of their sulphur analogues (9a and 10a), and with 9b
(1.35 mg kgÀ1 FW) 4-fold more concentrated than its sulphur ana-
logue 9a (0.32 mg kgÀ1 FW). In broccoli both selenium-containing
isothiocyanates (11b and 12b) were also at higher concentrations
than their sulphur analogues (11a and 12a). Concentrations of
these compounds in cauliflower were lower than in the broccoli
and the selenium compounds were at lower concentrations than
their sulphur counterparts (Table 1). Cauliflower florets were how-
ever, the only plant tissue to contain methylselenomethanenitrile
(4b), even though all three Brassica spp. contained its sulphur ana-
logue (4a) (Table 1).
2.1.5. Concentrations of the organoselenides and their sulphur
analogues
Table 1 gives semi-quantitative measurements of the tissue
concentrations of the selenium compounds, and their sulphur ana-
logues, identified in the solvent extracts of Brassica fertilised with
sodium selenate. Table 2 lists the sulphur compounds for which
the corresponding selenium analogues were not identified. These
tables do not represent an exhaustive identification of all sulphur
compounds present in the solvent extracts, just those easily iden-
tifiable and, in Table 1, of biosynthetic relevance to the selenium
compounds discovered. The compounds of greatest interest were
the isothiocyanates and the nitriles, because they are selenium
The selenium compound at highest concentration in forage rape
roots was 4-(methylseleno)butylisothiocyanate (12b) at 2 mg kgÀ1
,
which was 30% of the concentration of its sulphur analogue (12a).
The forage rape roots had the highest concentrations of sulphur
aglycones, in the form of the isothiocyanates 12a and 13a (ꢀ7
and ꢀ15 mg kgÀ1 FW, respectively, Table 1). However, the concen-

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A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
Table 2
Semi-quantitative analysis of sulphur-containing volatiles, measured by GC–MS (l
g kgÀ1 FW), in extracts of broccoli and cauliflower floret tissue and forage rape root tissue from
control plants and plants fertilised with 5 mM sodium selenate (Na2SeO4), for which selenium analogues were not identified. Compound identification is by mass spectrum library
matching (NIST, 2002). Concentrations are the average ( SEM) of three samples, each sample comprising tissue combined from two separate plants. Chemical structures are given
in Fig. 1.
Compound
RT (min)
RI (Wax)a
Broccoli cv. ‘Triathlon’
Cauliflower cv. ‘Liberty’
Forage rape cv. ‘Maxima’
Control
Na2SeO4
Control
Na2SeO4
Control
Na2SeO4
CH2@CHC2H4NCS (14)
C6H13NCS (15)
C7H15NCS (16)
CH3S(O)SCH3 (17)
CH3S4CH3 (18)
CH3S(O2)SCH3 (19)
PhC2H4CN (20)
18.27
23.38
25.06
25.25
27.11
31.54
32.50
35.05
1425
1555
1609
1610
1687
1932
1994
2175
4 (1)
31 (4)
16 (3)
–
5700 (890)
200 (35)
75 (34)
84 (10)
340 (26)
2.1 (0.5)
–
–
5900 (1500)
180 (60)
360 (170)
60 (20)
100 (24)
1 (0.1)
–
–
4200 (140)
70 (23)
280 (230)
15 (7)
60 (25)
360 (40)
25 (4)
65 (18)
123 (36)
13 (1)
105 (35)
1600 (190)
80,000 (11,800)
230 (46)
40 (7)
110 (14)
72 (16)
12 (4)
76 (52)
23 (4)
b
–
7100 (1570)
430 (210)
40 (25)
220 (10)
560 (21)
2100 (550)
83,000 (7660)
PhC2H4NCS (21)
a
Retention Index on DB-Wax GC column with respect to a series of straight-chain hydrocarbons.
Not detected.
b
trations of their corresponding seleno-aglycones (12b and 13b)
were not similarly high, and were at around the same concentra-
tions as those of the selenium-containing nitriles and isothiocya-
nates in the broccoli samples. Generally, the forage rape samples
had higher concentrations of isothiocyanates than of nitriles, while
in the broccoli and cauliflower the reverse was observed. The
concentration of 5-(methylseleno)pentylisothiocyanate (13b) in
forage rape root was around 16% of the concentration of 4-(meth-
ylseleno)butylisothiocyanate (12b), even though there was 2-fold
more of 5-(methylthio)pentylisothiocyanate (13a) present than
there was of 4-(methylthio)butylisothiocyanate (12a). Apparently,
the plant was more likely to produce the selenium analogue of 12a
than 13a, even though these compounds differ in structure by only
one methylene group.
to be observed for 21, which is the highest concentration isothiocy-
anate we identified in these samples. In the present study we were
able to identify seleno-aglycones at concentrations as low as ca.
5 l
g kgÀ1 which is less than 0.01% of the concentration of the 2-
phenylethylisothiocyanate in the selenium-treated forage rape
sample (Table 2). Despite this, we did not identify any isoselenocy-
anates in our Brassica extracts. However, it cannot be conclusively
determined that the plants in this study did not produce these
compounds, because we examined only one tissue type from the
plants and some isoselenocyanates are unstable under light and
in aqueous environments (Sonoda et al., 1972; Kjaer and
Skrydstrup, 1987).
2.2. LC–MS analysis of selenoglucosinolates
The summed concentrations of the selenoglucosinolate agly-
cones 9b–13b in the forage rape roots was around 2.7 mg kgÀ1
(12% of the total concentration of the sulphur analogues 9a–13a).
For broccoli, the seleno-aglycones 9b–12b totalled 3.7 mg kgÀ1
(160% of 9a–12a), and for the cauliflower the seleno-aglycones
4b, 9b–12b totalled 0.6 mg kgÀ1 (25% of 4a and 9a–12a). Thus,
there was a greater percentage incorporation of selenium into
the broccoli and cauliflower glucosinolate aglycones than there
was into those of forage rape. The sum of all selenium compounds
in the broccoli (4 mg kgÀ1), cauliflower (0.76 mg kgÀ1), and forage
rape (2.8 mg kgÀ1) samples constituted 21%, 7.5% and 2.5%, respec-
tively of the combined total of sulphur and selenium compounds
identified (Tables 1 and 2). In part, the forage rape samples had
the lowest percentage incorporation of selenium into the sulphur
assimilation pathway because the major forage rape root aglycone
is 2-phenylethylisothiocyanate (21) (McCully et al., 2008). This
compound constituted 75% of the combined total of the sulphur
and selenium compounds present in the forage rape roots but no
selenium analogue of this compound was present.
Most of the compounds in Table 2 do not contain the methyl-
thio moiety, and therefore seleno-amino acid-derived analogues
of these compounds (14, 15, 16, 20, and 21) do not appear to exist.
This is consistent with the exclusive incorporation of selenium into
the selenoglucosinolates via selenomethionine. The selenium ana-
logues of 17 and 19 have not been reported in the literature, pos-
sibly because at room temperature they are unstable and undergo
the selenoxide b-elimination reaction (March, 1985). The 2-pheny-
lethylisothiocyanate (21), found in the forage rape root extract
(Table 2), was at high concentrations of around 83 mg kgÀ1 (FW).
Concentrations of grams per kg of 21 have previously been re-
ported in forage rape roots (McCully et al., 2008) and in Brassica-
ceae such as watercress (Newman et al., 1992). If there was
incorporation of selenium into the isothiocyanate (R-NCS) group
to produce isoselenocyanates (R-NCSe), then it would most likely
Having identified their hydrolysis products, we next sought to
identify the parent selenoglucosinolates directly using high resolu-
tion LC–MS. The LC–MS chromatograms of the ethanolic extracts of
ground, frozen broccoli and forage rape tissues contained chro-
matographic peaks whose high resolution pseudomolecular ions
and isotope patterns matched those reported previously for gluco-
sinolates in these Brassica spp. (Tian et al., 2005; Bialecki et al.,
2010; Cataldi et al., 2010). These nonselenium-containing gluco-
sinolates constituted the majority of the compounds in the etha-
nolic extracts.
Small amounts of three selenium-containing glucosinolates
(22–24 in Fig. 1) were identified in the ethanolic extracts of
broccoli and forage rape tissues. We have named these compounds
glucoselenoiberverin (22), glucoselenoerucin (23), and glucosele-
noberteroin (24): they are the selenium analogues of the well
known glucosinolates glucoiberverin, glucoerucin, and glucober-
teroin, respectively (Nugon-Baudon and Rabot, 1994). All three
selenoglucosinolates were clearly identified in the forage rape
sample, and glucoselenoiberverin and glucoselenoerucin were also
identified in the broccoli sample (Table 3).
2.2.1. LC–MS identification of the selenoglucosinolates
Glucoselenoerucin (23) (RT = 7.0 min, Fig. 5) was the most
abundant of the three selenoglucosinolates and there was suffi-
cient compound present to observe a good selenium isotope pat-
tern and obtain high-resolution mass spectral data. Several
diagnostic daughter ions were also measured from fragmentation
of the pseudomolecular ion m/z 467.9899 ([MÀH]À, À0.7 mDa,
calcd. for C12H22NO9S2Se, 467.9906). Most daughter ions were
diagnostic for glucose (Fig. 6), viz. m/z 275 (C6H11O8S2À) from rear-
rangement of [MÀH]À and neutral loss of CH3SeC4H8N@C@O (m/z
193); m/z 259 (C6H11O9SÀ) from rearrangement and neutral loss
of CH3SeC4H8N@C@S (209); and the thioglucose group (C6H11O5SÀ)

A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
147
with pseudomolecular ion m/z 434.0618 ([MÀH]À, +0.5 mDa, calcd.
for C13H24NO9S3, 434.0613) and a distinctive S3 isotope pattern.
Identification of glucoselenoiberverin 22 (RT = 5.5 min) was less
definitive. This minor compound gave m/z 453.9732 ([MÀH]À,
À1.7 mDa, calcd. for C11H20NO9S2Se, 453.9745), but neither its
selenium isotope pattern nor its daughter ions could be measured.
The sulphur analogue, 3-(methylthio)propylglucosinolate, was
identified eluting 0.65 min before 22 (RT = 4.85 min), and was
identified from its pseudomolecular ion at m/z 406.0272 ([MÀH]À;
À3.4 mDa; calcd. for C11H20NO9S3, 406.0306) and its distinct S3 iso-
tope pattern.
Table 3
Percentage composition (LC–MS peak area of m/z [MÀH]À) of glucosinolates for which
selenium analogues were detected in ethanolic extracts, and ratio of incorporation of
selenium and sulphur (Se:S) into the methylseleno/methylthio group of these
compounds.
Compound
Broccoli florets
cv. ‘Triathlon’
Forage rape roots
cv. ‘Maxima’
Glucoiberverin
Glucoselenoiberverin (22)
Glucoerucin
Glucoselenoerucin (23)
Glucoberteroin
Glucoselenoberteroin (24)
7
0.3
4.2 (38:62)
76
0.03 (9:81)
26
11.3 (13:87)
1.4
1.2 (4:96)
72
nd (0:100)
0.4 (0.6:99.4)
2.2.2. Previous reports of selenoglucosinolates
There appear to be only three previous reports of selenogluco-
sinolates; an organic synthesis of benzyl- and methyl-selenogluco-
sinolate (Kjaer and Skrydstrup, 1987), an unspecified incorporation
of selenium into 2-phenylethylglucosinolate in root cultures of
Prince’s plume (Wielanek et al., 2005) and one of trace levels of
3-butenylselenoglucosinolate in watercress fertilised with sele-
nium fertilisers (Bertelsen et al., 1988). For the two studies in
which the position of selenium incorporation was reported, it
at m/z 195. The bisulphate ion was also identified at m/z 97
(HSO4À). These fragmentation ions agree with those reported
(Cataldi et al., 2010; Clarke, 2010) for the sulphur glucosinolates
and were used to identify the sulphur analogue of 23 (4-(methyl-
thio)butylglucosinolate, glucoerucin), which eluted 0.8 min
(RT = 6.2 min) before the selenium compound with [MÀH]À m/z
420.0460 (calcd. for C12H22NO9S3, 420.0462) and a distinctive S3
isotope pattern.
was identified as D-selenoglucose. This contrasts with compounds
The above glucoselenoerucin ions indicate the presence of two
sulphur atoms in the glucosinolate, with one in the sulphate group
and the second as the bridge between glucose and the rest of the
molecule. Therefore, the selenium atom is probably located in
the amino acid-derived side chain. In the sulphur analogue (erucin)
we observed the neutral loss MÀHÀGlcÀSO3 to produce m/z
178.0372 (+1.1 mDa, calcd. for C6H12NOS2, 178.0361) with the
structure CH3SC4H8C(SÀ)@NOH (Clarke, 2010). In selenium com-
pound 23 we also observed this ion, but containing selenium with
m/z 225.9813 (+0.8 mDa, calcd. for C6H12NOSSe, 225.9805) and, by
analogy, with the structure CH3SeC4H8C(SÀ)@NOH (Fig. 6). This
confirmed that the selenium atom was in the side chain of the
glucosinolate molecule and accords with our GC–MS identification
of a number of methylselenoalkly isothiocyanates and nitriles in
the solvent extracts.
4b, 9b, 10b–13b, 22–24 identified here where the selenium was lo-
cated in the amino acid-derived side chain as a methylseleno
group.
2.2.3. Relative concentrations of the selenoglucosinolates
Integration of the [MÀH]À peaks (Fig. 5), for the three selenog-
lucosinolates and their three sulphur analogues in the ethanolic
extract of the forage rape root sample, indicated that the sul-
phur-glucosinolates were the major analogues present (Table 3),
as were their hydrolysis products, the methylthio nitriles and iso-
thiocyanates by GC–MS (Table 1). The major selenoglucosinolate
was glucoselenoerucin (23) at around 4.5% of the concentration
of glucoerucin. These relative amounts were reflected in the agly-
cone hydrolysis products in which 4-(methylseleno)isothiocyanate
(12b) was at the highest concentration and was at around 30% of
the concentration of 4-(methylthio)isothiocyanate (12a, erucin)
(Table 1). The selenoglucosinolates in the forage rape roots consti-
tuted approximately 1.6% of the six glucosinolates in Table 3, and
Glucoselenoberteroin (24) (RT = 8.9 min) was identified from its
pseudomolecular ion at m/z 482.0067 ([MÀH]À, À0.4 mDa, calcd.
for C13H24NO9S2Se, 482.0063) with a distinctive selenium isotope
pattern. The sulphur analogue eluted 1 min earlier (RT = 7.92 min)
Fig. 5. Reconstructed ion chromatogram (RIC) of the pseudomolecular ions [MÀH]À of methylseleno and corresponding methylthio glucosinolates identified in an ethanolic
extract of ‘Maxima’ forage rape root.

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A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
Fig. 6. Fragmentation of the pseudomolecular ion ([MÀH]À, m/z 468) of 4-(methylseleno)butylglucosinolate (glucoselenoerucin, 23) identified in an ethanolic extract of
‘Maxima’ forage rape root.
9% of their aglycones were organoselenides (Table 1). Incorpora-
tion of selenium into these broccoli glucosinolates, and their agly-
cones, was considerably higher. The broccoli glucosinolates shown
in Table 3, were 15.5% organoselenides, and their aglycones
(Table 1) were 78% organoselenides. We did not measure the gluc-
osinolates in the cauliflower samples, but approximately 21% of
their aglycones contained selenium.
Incorporation of selenium into the broccoli and forage rape
glucosinolates was higher for the short-chain aglycone compounds
(Table 3). In the forage rape sample the Se:S ratio decreased with
increasing chain length from 9:81 to 0.6:99.4, and for the broccoli
sample the ratios decreased from 60:40 to 0:100. The Se:S ratio of
the isothiocyanate aglycones showed the same behaviour. The
broccoli Se:S ratio decreased from 93:7 to 75:25, for cauliflower
from 23:77 to 8:92, and for forage rape the ratios were 42:58,
23:77, and 2:98.
from selenomethionine. The sulphur atom in the isothiocyanate
moiety of glucosinolates is derived from glutathione (Sonderby
et al., 2010), and so by analogy isoselenocyanates would be
expected to be derived from selenoglutathiones. However, no
isoselenocyanates (R-NCSe) were found in the present study. Isose-
lenocyanates are not well known in nature with only one report in
which trace amounts of 3-butenylselenoglucosinolate were re-
ported in Brassicaceae (S. pinnata) treated with high concentra-
tions of selenium fertiliser (Bertelsen et al., 1988). Bertelsen et al.
proposed that the poor incorporation of selenium into the gluco-
sinolates indicated the ability of the plants to detect the chemical
non-equivalency of sulphur and selenium.
Methylthio and methylseleno alkyl nitriles and isothiocyanates
were identified in all extracts, but the nitriles occurred at higher
concentrations in the broccoli and cauliflower while the isothiocy-
anates were the major species in the forage rape roots. The
nitrile:isothiocyanate ratios in the three different plants were
70:30, 80:20, and 3:97, respectively. Thus while all plants showed
myrosinase activity, the forage rape roots produced the largest
quantities of isothiocyanates in agreement with previous reports
that the major glucosinolate hydrolysis products in B. napus roots
are isothiocyanates (Brown and Morra, 1996; McCully et al., 2008).
Previous reports of glucosinolate hydrolysis products report
either low molecular weight nitriles (Vidal-Aragón et al., 2009;
Krumbein et al., 2010), or significant amounts of both nitriles
2.3. The biosynthetic origins of the organoselenides
In Brassica, methylthioalkyl nitriles and isothiocyanates are
myrosinase hydrolysis products of methylthioalkyl glucosinolates,
with the sulphur being derived from methionine (Halkier and
Gershenzon, 2006; Kissen and Bones, 2009). Thus in the present
work the methylselenoalkyls would be hydrolysis products of
methylselenoalkyl glucosinolates, with the selenium being sourced

A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
149
and isothiocyanates (Buttery et al., 1976; Di Cesare et al., 2001,
2002; Valette et al., 2003). The studies that identified the low
molecular weight nitriles involved headspace analyses, whereas
the other studies involved thermal extraction methods such as
hydrodistillation. Damage to broccoli tissue at room temperature
produces nitriles as the primary hydrolysis products, as a result
of intercession by epithiospecifier proteins (Matusheski et al.,
2004, 2006). However, heating the broccoli inactivates the epithio-
specifier proteins and allows production of isothiocyanates. In the
present study, solvent extraction of all tissue samples was per-
formed at low temperatures, which is consistent with the identifi-
cation of nitriles as the main glucosinolate hydrolysis products in
broccoli and cauliflower.
harvested from the forage rape plants. For the forage rape tissue
the outer fibrous ring was removed and the inner pith sampled.
All material was immediately frozen in liquid nitrogen before stor-
age at À80 °C until extraction.
4.1.1. Extraction of volatile compounds for GC–MS analysis
Triplicate extractions were performed, each replicate compris-
ing tissue from two separate plants. For each replicate 10–15 g
FW was accurately weighed and ground, with dry ice, to a powder
in a coffee grinder. The sample was combined with 2 vol. (w/v) of
pre-chilled 50:50 pentane/diethyl ether on ice in a Schott bottle,
sealed and left at 1 °C for a week with daily mixing before the solid
material was frozen at À20 °C and the solvent poured off. The sol-
vent extracts were dried (MgSO4), and their volumes reduced to ca.
1.5 ml under a gentle stream of nitrogen.
3. Concluding remarks
4.1.2. Extraction of glucosinolates for LC–MS analysis
Brassica treated with selenate fertiliser incorporate selenium via
the sulphur assimilation pathway to produce selenoglucosinolates,
selenium analogues of well known Brassica compounds. Selenium
in the selenoglucosinolates was present as a methylselenoalkyl
group that was probably derived from selenomethionine. There
was no evidence for incorporation of selenium into either the sul-
phate or the thioglucose moieties of the glucosinolate.
There was a significant incorporation of selenium into roots of
forage rape, with selenoglucosinolates present at up to 10% of
the concentration of their sulphur analogues. The concentrations
of the selenium-containing aglycones of the three glucosinolates
were as high as 70% of the concentrations of their sulphur ana-
logues. The broccoli and cauliflower samples showed a consider-
ably higher relative incorporation of selenium. The broccoli
selenoglucosinolates reached 60% of the concentrations of their
sulphur analogues, and the selenium aglycone concentrations were
up 1300% of their sulphur analogues. The cauliflower aglycones
also had high concentrations of selenium, with an Se:S ratio as high
as 0.3:1. Incorporation of selenium into the glucosinolates and
their hydrolysates appeared to be dependent upon the length of
the side chain derived from selenomethionine. However, overall
these results indicate the potential for the production of significant
concentrations of organoselenium compounds by Brassica species.
Frozen floret tissue (1 g) of broccoli, fertilised with 5 mM Na2-
SeO4, was ground in liquid nitrogen in a mortar and pestle and
poured into 80% ethanol (10 ml) preheated and then held at
80 °C for 5 min in order to inactivate myrosinase (Tian et al.,
2005). Samples were cooled at 5 °C for 30 min, filtered, and the fil-
trate stored at À20 °C. Frozen forage rape root tissue (1 g), partially
ground with sand in liquid nitrogen, was quenched into 80% etha-
nol (20 ml) held at 80 °C for 5 min, and combined with a further
20 ml of 80% ethanol used to rinse the mortar.
4.2. GC–MS analysis of volatile compounds
Separations were performed on an Agilent 6890N GC coupled to a
Waters GCT time of flight (ToF) mass spectrometer with an EI energy
of 70 eV and a scan time of 0.4 s. One microlitre split (5:0.9) injec-
tions were made at 220 °C onto a 20 m  0.18 mm i.d.  0.18
lm
film thickness DB-Wax (J & W, Scientific) capillary column with a
He flow of 0.9 ml minÀ1. The oven temperature programme was
1 min at 35 °C, 3 °C minÀ1 to 100 °C, 7 °C minÀ1 to 240 °C, hold
5 min. External standards used for semi-quantification (using total
ion current peak areas) were CH3SeSeCH3 (3) for compounds 1–3,
6–8 and 17–19, and CH3SeC3H6CN (9b) for the nitriles and isothiocy-
anates, at concentrations of 0.0005, 0.00005 and 0.000005 l
l mlÀ1 in
order to cover the range of concentrations of compounds measured
in the solvent extracts. The external standards and the plant extracts
were spiked with internal standards (undecane, tridecane, and
tetradecane) at concentrations covering three orders of magnitude.
The hydrocarbon standards enabled correction for the plant extract
sample volumes and for GC–MS system responses. Retention indices
of compounds were determined against a standard containing
straight-chain hydrocarbons (C10H22–C26H54).
4. Experimental
4.1. Plant, growth and harvesting
One cultivar each of broccoli (B. oleracea L. var. italica cv. ‘Triath-
lon’), cauliflower (B. oleracea L. var. botrytis cv. ‘Liberty’), and forage
rape (B. napus cv. ‘Maxima’) were grown in 8 l bags of potting mix
at Plant & Food Research, Palmerston North. Plants were watered
once daily via watering spikes for 1 min. Selenium feeding was
commenced for broccoli and cauliflower following the emergence
of immature floret material from the meristem and for forage rape
once the plants had a well established stem (ca. 16 cm from soil to
top of meristem) and approximately 10 fully expanded leaves.
Plants were fed via the soil with 20 ml of 5.0 mM sodium selenate
twice weekly, for four weeks, after watering. We estimate this sele-
nium application to be equivalent to ca. 15 kg haÀ1 which is very
high compared with the 2–20 g haÀ1 reported for agricultural and
forage crops (Pulsipher et al., 2004; Broadley et al., 2006; Eich-
Greatorex et al., 2007). Previous workers reported that broccoli
produced at this higher rate of selenium application contained at
least 500-fold more selenium than control samples (Finley et al.,
2000) but suffered no adverse effects upon its growth. Control
plants received no added selenium. Following the selenium feed-
ing, mature floret tissue was harvested from the broccoli and cau-
liflower plants, and the lower stem/tap root (3–4 cm long) was
4.3. LC–MS analysis of glucosinolates
LC–MS analysis enabled identification of three selenoglucosino-
lates: 3-(methylseleno)propylglucosinolate (22, glucoselenoiberv-
erin), 4-(methylseleno)butylglucosinolate (23, glucoselenoerucin),
and 5-(methylseleno)pentylglucosinolate (24, glucoselenobertero-
in), and their known sulphur analogues. Separations were per-
formed on a Dionex UltimateÒ 3000 Rapid Separation LC with a
micrOTOF-Q II mass spectrometer (Bruker Daltonics, Germany) fit-
ted with an electrospray source operating in negative mode. Sam-
ple injections (2
l
l
l), were made onto
m (Agilent) analytical column maintained at
l minÀ1. Solvent A was 10:90
a Zorbax™ SB-C18
2.1 Â 100 mm, 1.8
50 °C and with a flow of 400
l
H2O:MeOH, and Solvent B was 0.5:99.5 HCO2H:H2O. The solvent
ramp was 1:99 (Solvent A:Solvent B) from injection to 0.5 min, fol-
lowed by a linear gradient to 30:70 (0.5–8 min), to 75:25 (8–
13 min), to 100:0 (13–15 min), hold at 100:0 (15–17 min). The

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A.J. Matich et al. / Phytochemistry 75 (2012) 140–152
micrOTOF-Q II source parameters were: temp. 200 °C; drying N2
flow 8 l minÀ1
nebuliser N2 4 bar (400 kPa), endplate offset
(16), 101 [MÀSeCH2]+ (65), 100 [MÀSeCH3]+ (14), 95 [SeCH3]+
(21), 94 (19), 93 (27), 72 [CH2NCS]+ (38).
;
À500 V, capillary voltage +3500 V; mass range 100–1500 Da at 2
scans sÀ1. Post-acquisition internal mass calibration used sodium
formate clusters with the sodium formate delivered by a syringe
pump at the start of each analysis. Mass spectral data were pro-
cessed using Compass DataAnalysis (Bruker Daltonics).
4-(Methylseleno)butylisothiocyanate (12b, CH3SeC4H8NCS)
synthesis: 4-(methylseleno)butanenitrile was synthesised, as de-
scribed above, from 500 mg (2.6 mmol) of CH3Se2CH3 and 0.79 g
(5.33 mmol) of 4-bromobutanenitrile. The product was dissolved
in dry Et2O (25 ml) and added drop-wise to a slurry of LiAlH4
(200 mg, 5.3 mmol) in dry Et2O (25 ml) under N2 over 5 min fol-
lowed by 15 min of stirring. Wet Et2O (25 ml) was added, 5 ml of
NH4Cl (sat. aq.), and 1 ml of conc. ammonia to solubilise the amine
in the organic phase which was dried (MgSO4) and removed to give
4-(methylseleno)butamine. EI-MS, m/z (rel. int.): 167 [M]+ (24),
152 [MÀCH3]+ (7), 135 [MÀCH3ÀNH3]+ (13), 122 [MÀCH3ÀCH2
NH3]+ (4), 109 [C2H5Se]+ (9), 93 [CHSe]+ (9), 72 [C4H8NH2]+ (100),
55 (33), 43 (62) (Budzikiewicz and Pesch, 1974). Thiophosgene
4.4. Chemical synthesis and mass spectral data
4-(Methylseleno)hex-E2-enal (8, C3H6(CH3Se)C3H3O) synthesis:
CH3Se2CH3 (100 mg, 0.53 mmol, Aldrich) was added to a solution
of NaBH4 (60 mg, 1.59 mmol) in absolute EtOH (5 ml) under N2.
After 5 min of vigorous reaction the colour of the solution changed
from yellow to white. Bromoacetaldehyde dimethylacetal (0.127 g,
0.75 mmol, Aldrich) was added and stirred for 21 h. Water (10 ml)
was added and the organic phase extracted with Et2O (4 Â 25 ml)
and dried (MgSO4). The solvent was removed and the crude prod-
(CSCl2, 750 ll, Janssen) in dry dichloromethane (25 ml) was added
drop wise to the above amine in dry dichloromethane (25 ml) con-
taining diethylamine (0.5 ml) under N2. After stirring for 23 h,
2.5 ml of NaHCO3 (sat. aq.) was added with vigorous stirring, the
mixture was filtered through a plug of silica and dried (MgSO4).
The solvent was removed, the product taken up in dry Et2O, filtered
and the solvent removed to give 12b as a yellow oil. EI-ToF-MS
(Fig. 4), m/z (rel. int.): 209 [M]+ (84), 194 [MÀCH3]+, 135
[CH3SeC2H4]+ (25), 114 [M+ÀSeCH3]+ (57), 109 [CH3SeCH2]+ (19),
93 [SeCH]+ (11), 85 [C2H3NCS]+ (19), 72 [CH2NCS]+ (95), 55 (100),
41 (25). 1H NMR (500 MHz, CDCl3): d1.81 (m, 4H, CH2CH2), 2.01
(s, 3H, CH3Se), 2.57 (t, 2H, J = 6.48 Hz, SeCH2), 3.56 (t, 2H,
J = 6.18 Hz, CH2N).
uct taken up in tetrahydrofuran (3 ml). Water (200
ll), HCl (25 ll
conc.) and hex-E2-enal (15 l, Aldrich) were added and the solu-
l
tion stirred for 20 h. NaHCO3 (3 ml, sat. aq.) was added and the or-
ganic phase extracted with dichloromethane (4 Â 25 ml). Attempts
to purify the desired product by silica flash chromatography were
unsuccessful. GC–MS analysis showed the major product was 2,2-
bis(methylseleno)acetaldehyde with 8 also present. EI-ToF-MS
(Fig. 3), m/z (rel. int.): 192 [M]+ (100), 190 ([M]+, Se78) (24), 177
[MÀCH3]+ (8), 163 [C5H7SeO]+ (16), 135 (4), 97 (20), 96
[MÀSeCH4]+ (38), 95 [CH3Se]+ (17), 94 (10), 93 (25), 81 [C5H5O]+
(31), 69 (5), 67 (8).
5-(Methylseleno)pentylisothiocyanate (13b, CH3SeC5H10NCS)
EI-ToF-MS (Fig. 4), m/z (rel. int., fragment): 223 ([M]+, Se80
)
4-(Methylseleno)butanenitrile (9b, CH3SeC3H6CN) synthesis:
CH3Se2CH3 (100 mg, 0.53 mmol, Aldrich) was added to a solution
of NaBH4 (60 mg, 1.59 mmol) in 5 ml of absolute EtOH under N2.
After 5 min of vigorous reaction the colour of the solution changed
from yellow to white. 5-Bromobutanenitrile (0.157 g, 1.06 mmol,
Acros) was then added and stirred for 2.5 h. Et2O (10 ml) was
added, the reaction mixture was washed with 5 ml each of water
and brine, extracted with 3 Â 25 ml of Et2O, and dried (MgSO4).
The solvent was removed and the crude product purified by flash
chromatography on silica (petroleum ether:Et2O, 90:10 and
85:15) to give 9b as a yellow oil (87 mg, 50%). EI-ToF-MS (Fig. 2),
m/z (rel. int.): 163 [M]+ (100), 161 (42), 123 (14), 121 (12), 109
[CH3SeCH2]+ (32), 107 (22), 96 [CH3SeH]+ (26), 95 [CH3Se]+ (19),
94 [CH2Se]+ (25), 93 [CHSe]+ (31), 68 (6). 1H NMR (500 MHz,
CDCl3): d2.01 (s, 3H, CH3Se), 2.01 (q, 2H, J = 7.1 Hz, CH2CH2CH2),
2.53 (t, 2H, J = 7.1 Hz, CH2CN), 2.66 (t, 2H, J = 7.0 Hz, CH2Se).
5-(Methylseleno)pentanenitrile (10b, CH3SeC4H8CN) synthesis:
CH3Se2CH3 (100 mg, 0.53 mmol, Aldrich) was added to a solution
of NaBH4 (60 mg, 1.59 mmol) in 5 ml of absolute EtOH under N2.
After 5 min of vigorous reaction the colour of the solution changed
from yellow to white. 5-Bromopentanenitrile (0.17 g, 1.06 mmol,
Acros) was then added and stirred for 2.5 h. Et2O (10 ml) was
added, the reaction mixture was washed with 5 ml each of water
and brine, extracted with 2 Â 25 ml of Et2O, and dried (MgSO4).
The solvent was removed to give 10b as a yellow oil (107 mg,
57% yield). EI-ToF-MS (Fig. 2), m/z (rel. int.): 177 [M]+ (100), 175
(28), 173 (15), 135 (10), 109 [CH3SeCH2]+ (33), 107 (17), 96
[CH3SeH]+ (27), 94 [CH2Se]+ (21), 93 [CHSe]+ (25), 82 (22), 55 (4).
1H NMR (500 MHz, CDCl3): d1.75–1.88 (m, 4H, CH2CH2), 2.00 (s,
3H, CH3Se), 2.38 (t, 2H, J = 6.8 Hz, CH2CN), 2.57 (t, 2H, J = 6.9 Hz,
CH2Se).
(100), 221 ([M]+, Se78) (42), 208 [MÀCH3]+ (9), 164 [MÀHNCS]+
(2), 149 [SeC5H9]+ (6), 128 [MÀCH3Se]+ (33), 109 [CH3Se80CH2]+
(25), 107 [CH3Se78CH2]+ (15), 93 [SeCH]+ (12), 72 [CH2NCS]+ (25),
69 [C5H9]+ (10).
Acknowledgements
We are grateful to Stuart Gowers (Plant & Food Research) for
helpful discussions at the beginning of this project and for provi-
sion of the forage rape seeds, and Julie Ryan (Plant & Food Re-
search) for care of the plants.
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