Production of polyselenodipenicillamines, unique selenium compounds

Article abstract of DOI:10.1248/cpb.58.957

Selenite (H₂SeO₃) reacts with thiol compounds (RSH) under acidic conditions to form selenotrisulfides (RSSeSR, i.e. monoselenodithiols). The stoichiometry of the reaction is proposed as 4RSH+H₂SeO₃→RSSeSR+ RSSR+

Full text of DOI:10.1248/cpb.58.957

July 2010
Note
Chem. Pharm. Bull. 58(7) 957—960 (2010)
957
Production of Polyselenodipenicillamines, Unique Selenium Compounds
Hiroyuki NISHIDA, Motozumi ANDO, Kazuo ITOH, Koji UEDA, Yujiro NISHIDA, Yoshinori OKAMOTO,
Chitose TODA, and Nakao KOJIMA*
Faculty of Pharmacy, Meijo University; 150 Yagotoyama, Tempaku-ku, Nagoya 468–8503, Japan.
Received January 6, 2010; accepted April 12, 2010; published online April 15, 2010
Selenite (H2SeO3) reacts with thiol compounds (RSH) under acidic conditions to form selenotrisulfides
(RSSeSR, i.e. monoselenodithiols). The stoichiometry of the reaction is proposed as 4RSH؉H2SeO3→RSSeSR؉
RSSR؉3H2O. Surprisingly, we found novel polynuclear selenium-containing compounds, i.e. polyselenodipenicil-
lamines (PenSSe2–4SPen), in the reaction of D-penicillamine (PenSH) with H2SeO3. The selenium-centered fea-
1
tures of PenSSe2–4SPen were determined by H-NMR and LC-MS/MS analyses, showing that the selenium iso-
tope abundance patterns of the compounds were in good agreement with the theoretically-calculated ones. In
order to better understand the mechanisms for PenSSe2–4SPen production, various molar ratio of H2SeO3 (1/8 to
4 times of PenSH) was reacted with PenSH, and the concentration of the products was calculated from integral
values of dimethyl proton signals for PenSSe1–2SPen as compared with methyl proton signals for acetic acid (an
internal standard). Total PenSSe1–2SPen concentration was increased with increasing of H2SeO3, in which con-
comitant decrease of PenSSPen (disulfide form of PenSH) was observed. Based on these results, we proposed the
PenSSe2-4SPen production mechanisms being involved in penicillamine selenopersulfides (PenSSe1–2H).
Key words selenium; polyselenodithiol; selenotrisulfide; penicillamine; cancer chemoprevention
Selenium is an essential micronutrient that is known to selenium metabolism, leading to selenium-mediated cancer
play an important role in many physiological functions of the prevention.5) The formation of selenodiglutathione, seleno-
human body. Recent studies have shown that selenium sup- cysteineglutathione, and selenodicysteine was demonstrated
plements in the diet can reduce the risk of cancer and other based on the models of selenium metabolism in cell-free sys-
diseases.1,2) To produce unique selenium compounds leading tems and in rats by Gabel-Jensen et al.6) and by Braga et al.,7)
to the therapeutic application, the reaction chemistry of sele- who proposed the formation of selenide species—selenoper-
nium should be studied in greater detail. Selenite (H2SeO3) sulfide (RSSeH), hydrogen selenide (H2Se), and hydrogen se-
reacts with thiol compounds (RSH) under acidic conditions lenide anion (HSeϪ)—through the reduction of RSSeSR. Re-
to form selenotrisulfides (RSSeSR), which was first de- cently, selenodipenicillamine (PenSSeSPen), a chemically
scribed by Painter using cysteine.3) Reactions of glutathione, stable RSSeSR, was isolated and its bioavailability in mice
cysteine, and 2-mercaptoethanol with H2SeO3 in a molar was investigated by Nakayama et al.8,9) In our study aiming
ratio of 4 : 1 under acidic conditions lead to form selen- at discovering novel selenium compounds, the reaction of D-
odiglutathione, selenodicysteine, and selenodi-2-mercap- penicillamine (PenSH) with H2SeO3 was found to yield poly-
toethanol, respectively.4) Painter and Ganther proposed the selenodipenicillamines (PenSSe2–4SPen), which had been
following reaction, 4RSHϩH2SeO3→RSSeSRϩRSSRϩ overlooked previously (Chart 1). We describe herein the syn-
3H2O.
In biological systems, RSSeSR derived from glutathione
and cysteine have been reported to play an important role in
thesis and characterization of PenSSe2–4SPen.
Experimental
Preparation of PenSSe2–4SPen PenSSe2–4SPen were prepared by mix-
ing with 10 mM PenSH (Tokyo Kasei Kogyo, Tokyo, Japan) and 40 mM
H2SeO3 (Wako Pure Chemical, Osaka, Japan) in 400 ml of 2.5 mM HCl.
1
PenSSe2–4SPen production was confirmed using LC-MS/MS and H-NMR.
Products were purified using preparative HPLC equipped with reversed-
phase column (Develosil C-18, 250ϫ20 mm i.d., 5 mm-pore size, Nomura
Chemical, Aichi, Japan) and were analyzed by LC-MS/MS and 1H-NMR.
LC-MS/MS Analysis of PenSSe2–4SPen LC-MS/MS experiments were
conducted using an Agilent 1100 Series HPLC system (Agilent Technolo-
gies Japan Ltd., Tokyo, Japan) coupled to an LCQ DECA XP ion trap mass
spectrometer (Thermo Fisher Scientific, Kanagawa, Japan). LC was carried
out using a reversed-phase column (Develosil C-18, 50ϫ4.6 mm i.d., 3 mm-
pore size, Nomura Chemical). Mobile phase A consisted of 0.05% formic
acid and mobile phase B consisted of methanol. The separation of
PenSSe0–4SPen was performed in 15 min using a linear gradient elution from
A to B at a flow rate of 0.8 ml/min.
1H-NMR Analysis 1H-NMR spectra were obtained using an ECP500
spectrometer (Jeol Ltd., Tokyo, Japan). All NMR spectra were measured in
D2O (Wako) unless otherwise indicated and the signal positions are ex-
pressed in parts per million (ppm) based on the D2O signal as a reference at
4.80 ppm.
Results and Discussion
As shown in Fig. 1, through LC-MS analysis, we found
Chart 1
∗ To whom correspondence should be addressed. e-mail: kojiman@meijo-u.ac.jp
© 2010 Pharmaceutical Society of Japan

958
Vol. 58, No. 7
Fig. 2. 1H-NMR Spectra of the Reaction Mixtures ([PenSH : H2SeO3]ϭ
8 : 1 or 1 : 4)
Dimethyl group of PenSH (d 1.53/d 1.60 ppm), two dimethyl groups of PenSSPen (d
1.51/d 1.58 ppm), PenSOSPen (d 1.14/d 1.23 ppm), PenSSeSPen (d 1.55/d 1.65 ppm),
and peak A (PenSSe2SPen, d 1.57/d 1.67 ppm) were observed as a twin signal.
m/z 228 [PenSSe]ϩ; peak A, m/z 150 [PenSHϩH]ϩ, m/z 228
[PenSSe]ϩ, m/z 341 [PenSSe2SHϩH]ϩ; peak B, m/z 150
[PenSHϩH]ϩ, m/z 228 [PenSSe]ϩ, m/z 388 [PenSSe3]ϩ; peak
C, m/z 468 [PenSSe4]ϩ, m/z 501 [PenSSe4SHϩH]ϩ.
PenSSPen (a disulfide form of PenSH) gave typical fragment
ion [PenSS]ϩ (m/z 180), whereas [PenSS]ϩ was not found in
LC-MS/MS spectra of peaks A—C. These results suggest
that PenSSeSPen and peaks A—C would share common
basic structures. In 1H-NMR analyses, the dimethyl group of
PenSH [R–C(CH3)2–SH, R: CH(NH2)COOH] were observed
—
2). PenSSPen [R-C(C—H3)2–SS–C(—CH3)2–R] and PenSSeSPen
as a twin (nonequivalent) signal at d 1.53/d 1.60 ppm (Fig.
[mRe–trCic(C—alHc3e)n2–teSrS, esSh–oCw(eC—dHa3)t2w–iRn],sibgontahl oaft wdh1ic.5h1h/dav1e.5a8spypmm-
and d 1.55/d 1.65 ppm, respectively. Also, two dimethyl
groups of peak A were observed as a twin signal at d 1.57/
d 1.67 ppm. These results indicate that peak A has a sym-
metrical structure about linearly-arranged 2Se atoms,
PenSSe2SPen [R–C(C—H3)2–SSe2S–C(—CH3)2–R]. The proton
signals of peaks B and C were lower than detection limit due
Fig. 1. LC-MS Data of the Reaction Mixture of 10 mM PenSH and 40 mM
H2SeO3
to their rather small yields. However, in combination with the
1H-NMR data of peak A and the fragment ion patterns of
PenSSeSPen and peaks A—C, it can be deduced that peaks B
and C have a symmetrical structure like PenSSe3SPen and
PenSSe4SPen, respectively. The product having the nonequiv-
(a) Total ion chromatogram (TIC). Mass spectra of PenSSeSPen (b), peak A (c), peak
B (d), and peak C (e) indicate the isotope patterns of Se, 2Se, 3Se, and 4Se, respec-
tively.
that the reaction of PenSH with H2SeO3 under acidic condi- alent methyl signals at d 1.14/d 1.23 ppm was deduced to be
tions yielded PenSSeSPen (m/z 377) as well as unique sele- dipenicillamine ether (PenSOSPen, bridging two sulfides via
nium-containing compounds (indicated by peaks A—C). The oxygen atom) from LC-MS/MS analysis.
mass spectra of peaks A—C gave [MϩH]ϩ ions at m/z 457,
In order to elucidate the mechanism for PenSSe2SPen pro-
537, and 617, respectively, corresponding to the addition of duction, reaction mixtures consisting of 10 mM PenSH and
80 (Se), 160 (2Se), and 240 (3Se) atomic mass units of H2SeO3 at various molar ratios (1 : 0, 8 : 1, 4 : 1, 2 : 1, 1 : 1,
PenSSeSPen. The observed isotope patterns were in good 1 : 2, and 1 : 4) in 2.5 mM HCl and 10 mM acetic acid (an in-
agreement with the theoretically-calculated ones assuming ternal standard for the calculation of the molar concentra-
1
two, three, and four selenium atoms. LC-MS/MS analyses tion) were prepared in D2O and directly used for H-NMR
showed typical fragment ion patterns of PenSSeSPen and analysis. The molar concentrations of PenSH, PenSSPen,
peaks A—C as follows: PenSSeSPen, m/z 150 [PenSHϩH]ϩ, PenSSeSPen, PenSSe2SPen, and PenSOSPen were calculated

July 2010
959
from their integral values based on the methyl proton quite slow, and it allows time for much of t-BuSOH to un-
value of 10 mM acetic acid (d 2.10), as shown in Fig. 3. In dergo another reaction yielding thiosulfonate (t-BuSO2SBu-
the molar ratio range from 2 : 1 to 1 : 4 (PenSH to t). Therefore, the amount of disulfide (t-BuSSBu-t) is lower
H2SeO3), PenSH (originally 10 mM) was converted to the de- than that of RSSeSR (t-BuSSeSBu-t).
tected symmetric dipenicillamines (PenSSPen, PenSSeSPen,
PenSH has a 1-methylethanethiol group in a manner simi-
PenSSe2SPen, and PenSOSPen: 5 mM in total with a theoreti- lar to a tert-butyl system such as t-BuSH. Therefore, it is be-
cal stoichiometry, see Fig. 3). Kice et al. proposed the reac- lieved that the reaction of PenSH with H2SeO3 proceeds via
tion of RSH compounds with H2SeO3 using 1-butanethiol (n- two attacks on S and on O, and the stoichiometry is similar to
BuSH) and its isomer with a sterically shielded sulfur atom, the reaction of t-BuSH with H2SeO3. We now consider how
2-methyl-2-propanethiol (t-BuSH), as summarized in Chart PenSSe2–4SPen are formed in Chart 3. Figure 3 indicates the
2.10) In the chart, the reaction of RSH compounds with a se- decreasing yields of PenSSPen and increasing ones of
lenium intermediate, RSSe(O)SR, proceeds via two possible PenSSe1–2SPen with decreasing molar ratios of [PenSH]/
pathways (attacks on S and on O) depending on the steric [H2SeO3]. The yield of PenSSPen at a molar ratio of 4 : 1
effect of the R-group. The reaction of n-BuSH (no steric (Painter/Ganther reaction) decreased during the changes of
hindrance) with RSSe(O)SR proceeds via an attack on S molar ratios from 4 : 1 to 1 : 4, whereas that of PenSSe1–2SPen
(Painter/Ganther reaction), and the amount of disulfide (n- increased during the same changes. Inorganic selenium gen-
BuSSBu-n) is equal to that of RSSeSR (n-BuSSeSBu-n). erally exists as several chemical species such as H2Se, HSeϪ,
However, the reaction of t-BuSH proceeds via two attacks on Se0, H2SeO3, HSeO3Ϫ, SeO32Ϫ, HSeO4Ϫ, and SeO42Ϫ, corre-
S and on O (Kice reaction in addition to Painter/Ganther re- sponding to different redox potential levels, of which three,
action) due to steric hindrance by the tert-butyl group in the H2Se, H2SeO3, and Se0, are the main components in acidic
nucleophilic attack on S, leading to the formation of RSSeSR conditions.11) H2Se may allow the following two reactions:
(t-BuSSeSBu-t) and sulfenic acid (t-BuSOH). The reaction i) reduction of PenSSPen into 2 PenSH, which then reacts
of t-BuSOH with t-BuSH to form disulfide (t-BuSSBu-t) is with H2SeO3 again; this reaction results in a decrease in
PenSSPen and increase in PenSSeSPen, ii) reduction of
sulfenic acid (RSOH) and thioselenic acid (RSSeOH) by
the Painter/Ganther reaction and Kice reaction, respectively;
this reaction would lead to the formation of selenoper-
sulfides (PenSSe1–2H), which are key intermediates in the
formation of PenSSe2–4SPen. PenSSe1–2H could react with
PenSSe(O)SPen, PenSOH, and PenSSe1–2OH in competition
with PenSH and produce PenSSe1–4SPen, as shown in Chart
3.
We have discovered unique polynuclear selenium-contain-
ing compounds, PenSSe2–4SPen, in the reaction of PenSH
with H2SeO3. We will be reporting their beneficial effects
such as protection from oxidative DNA damage and inhibi-
tion of cancer cell proliferation. Moreover, a series of non-
symmetric polyselenodisulfides (RSSe2–4SRЈ) are also being
synthesized. Organic selenium compounds such as Se-
Fig. 3. Yields of PenSH, PenSSPen, PenSSeSPen, PenSSe2SPen, and Pen-
methylselenocysteine have been reported to be more effective
SOSPen in the Reaction between 10 mM PenSH and H2SeO3 at Their Various
and safer than inorganic selenium in animal models of cancer
Molar Ratios
chemoprevention.12) Therefore, our efforts are currently fo-
ᮀ, Sum of PenSSPen, PenSOSPen, PenSSeSPen, and PenSSe2SPen; ᭹, PenSSeSPen;
᭡, PenSOSPen; ᭜, PenSSPen; ᭿, PenSSe2SPen.
cused on chemical/biological studies of a series of diverse
polyselenodisulfides aiming at achieving significantly effec-
tive chemoprevention and reducing the toxic side effects as
well as providing a useful tool for new research on the me-
tabolism of selenium.
Chart 2
Chart 3

960
Acknowledgments This research was supported in part by Academic
Research Institute of Meijo University and by Academic Frontier Project for
Private Universities; matching fund subsidy from MEXT (Ministry of Edu-
cation, Culture, Sports, Science and Technology), 2007—2011.
Vol. 58, No. 7
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