Isolation of a Se-nitrososelenol: A new class of reactive nitrogen species relevant to protein Se-nitrosation

Article abstract of DOI:10.1021/ja0457009

Nitric oxide (NO) is a messenger molecule implicated in a number of physiological processes. Nitrosation of selenoproteins has been suggested as playing an important role in NO-mediated cellular functions such as the inactivation of glutathione peroxidase

Full text of DOI:10.1021/ja0457009

Published on Web 09/25/2004
Isolation of a Se-Nitrososelenol: A New Class of Reactive Nitrogen Species
Relevant to Protein Se-Nitrosation
Keiichi Shimada,† Kei Goto,*,† Takayuki Kawashima,*,† Nozomi Takagi,‡ Yoong-Kee Choe,‡ and
Shigeru Nagase‡
Department of Chemistry, Graduate School of Science, The UniVersity of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Department of Theoretical Studies,
Institute for Molecular Science, Myodaiji, Okazaki 444-8585, Japan
Received July 17, 2004; E-mail: goto@chem.s.u-tokyo.ac.jp; takayuki@chem.s.u-tokyo.ac.jp
Nitric oxide (NO) is a messenger molecule implicated in a
number of physiological processes.1,2 One of the mechanisms by
which NO affects cellular processes is through direct interaction
with cellular proteins by nitrosylation and nitrosation reactions.
S-Nitrosation of cysteine residues to produce S-nitrosothiols (RS-
NOs) is one of the most important NO-mediated modifications of
proteins.3,4 Recently, there have been several reports suggesting
that the interactions of NO (or NO-derived species) with the SeH
groups of selenoproteins are also involved in NO-mediated cellular
Figure 1. Synthesis and reactions of Se-nitrososelenol 2.
functions.5-11 For example, glutathione peroxidase (GPx), an
essential selenium-containing antioxidant enzyme, is inactivated by
treatment with RSNO as well as by endogenous NO,6-11 presumably
through selenocysteine Se-nitrosation.7,9 In contrast to the extensive
studies undertaken on S-nitrosothiols, however, no chemical
information about their selenium analogues has been available to
date, despite their potential physiological importance.12 To elucidate
the mechanism of NO-mediated modification of selenoproteins,
reference data on Se-nitrosated species are indispensable. Here we
report the synthesis of a stable Se-nitrosated derivative of an
organoselenol, a Se-nitrososelenol (RSeNO), and its crystal structure
and spectral properties. This Se-nitrososelenol can be formed by
Figure 2. ORTEP drawing of Se-nitrososelenol 2 (30% probability).
Selected bond lengths (Å), bond angles (deg), and a torsion angle (deg):
O(1)-N(1), 1.162(6); N(1)-Se(1), 2.107(6); Se(1)-C(1), 1.915(3); O(1)-
N(1)-Se(1), 116.2(3); N(1)-Se(1)-C(1), 94.88(16); O(1)-N(1)-Se(1)-
C(1), -2.2(5). In the crystalline state, there is a rotational disorder of the
N-O moiety around the C-Se bond in the ratio of 0.81:0.19, and only the
major component is shown. One molecule of benzene is included in the
asymmetric unit, which is omitted for clarity.
direct transnitrosation from RSNO to a selenol, while it is reduced
to the selenol by treatment with dithiothreitol, as proposed in the
hypothetical pathway for the NO-mediated inactivation of GPx.7,9
Because the selenium-nitrogen bond of the Se-NO group is
considered to be weaker than the sulfur-nitrogen bond of the
S-NO group, it is likely that Se-nitrososelenols are more labile
than S-nitrosothiols, which are already notorious for their very facile
bimolecular decomposition to give disulfides and NO. Previously
we reported that S-nitrosothiols can have a long lifetime if their
bimolecular decomposition is sterically suppressed by the den-
drimer-type substituents,13,14 including a Bpq group14,15 shown in
Figure 1. This methodology is also expected to be effective for the
stabilization of Se-nitrososelenols. Se-Nitrososelenol 2 was prepared
by nitrosation of selenol 1 bearing a Bpq group.16 Treatment of 1
with an excess of ethyl nitrite in degassed CDCl3 led to the
quantitative formation of Se-nitrososelenol 2, which was isolated
as reddish purple crystals with a melting point of 130.0-132.5 °C
(decomp) by recrystallization from toluene-hexane in 89% yield
(Figure 1).16 Formation of 2 was also observed in the reaction of
1 with S-nitrosoglutathione (GSNO) (vide infra). Se-Nitrososelenol
2 is sensitive to atmospheric oxygen but stable toward water. In
CDCl3, no decomposition of 2 was observed after 1 week at room
Figure 2 shows the crystal structure of 2 with selected bond
lengths, bond angles, and a torsion angle. The Se-N bond length
(2.107(6) Å) and the N-O bond length (1.162(6) Å) are consistent
with a selenium-nitrogen single bond and a nitrogen-oxygen
double bond. The observed bond lengths and angles are in good
agreement with the calculated values for a model Se-nitrososelenol,
C6H5SeNO (Se-N, 2.097 Å; N-O, 1.172 Å; O-N-Se, 115.4°),
obtained by density functional theory (DFT) calculation at the
B3LYP/6-31G(d) level,17 indicating that the structure around the
SeNO functionality of 2 is not affected by the two bulky substituents
at the ortho positions. The C-Se-N-O linkage adopts only the
syn conformation, which is similar to the aromatic S-nitrosothiols
structurally characterized so far.14,18
The 77Se NMR spectrum (CDCl3) of 2 shows a signal at δ 2229
ppm, about 2100 ppm lower field than that of selenol 1 (δ 135),
suggesting the strong magnetic deshielding effect of the NO moiety.
Such an extreme low-field shift was also found in theoretical
calculation. By the gauge-including atomic orbital (GIAO) calcula-
tion at the B3LYP/6-311G(3d)[Se]:6-311G(d)[C,O,N,H]//B3LYP/
6-31G(d) level, the chemical shift of 2 was estimated to be δ 2449
1
temperature. The structure of 2 was determined by H, 13C, and
77Se NMR spectroscopy, UV-vis and IR spectroscopy, and X-ray
crystallographic analysis.16
† The University of Tokyo.
‡ Institute for Molecular Science.
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10.1021/ja0457009 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
tively reduced Se-nitrososelenol 2 to the parent selenol 1 (Figure
1). The reaction of 2 with an excess of 1-butanethiol led to the
quantitative formation of selenenyl sulfide 4 (Figure 1).20 These
results coincide with the proposed mechanism for the NO-mediated
GPx inactivation described above, and strongly suggest the possible
involvement of Se-nitrosation of selenoproteins by NO-derived
species in redox regulation of cellular functions.
Modification of antioxidant enzymes such as GPx by reactive
nitrogen species has been suggested as playing a pivotal role in
NO-related cellular signaling cascades. The stable Se-nitrososelenol
obtained in this study is expected to serve as a reference compound
for identification of yet unconfirmed Se-nitrosated species in
proteins and understanding of their physiological functions.
Figure 3. UV-vis spectral change during the reaction of selenol 1 (1.6
M) with GSNO (1.8 M) in THF-water (4 mL, THF:water ) 3:1 v/v).
Inset: magnified view for the range of 400-780 nm.
Acknowledgment. This work was partially supported by Grants-
in-Aid for the Scientific Research (14703066 (K.G.) and 15105001
(T.K.)), the 21st Century COE Program for Frontiers in Funda-
mental Chemistry (T.K.), and the Nanotechnology Support Project
(K.G. and S.N.) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan. We also thank Tosoh Finechem
Corporation for the generous gifts of alkyllithiums.
ppm. The UV-vis spectrum of 2 shows the absorption maximum
at 485 nm (ꢀ 150) in chloroform. The time-dependent DFT
calculation for C6H5SeNO at the B3LYP/6-311+G(2d)//B3LYP/
6-31G(d) level shows two characteristic bands at 495 nm (n-π*
transition) and 711 nm (π-π* transition), although the intensity
of the latter is nearly zero. The observed 485 nm band can be
reasonably assigned to the n-π* transition, which shows a
bathochromic shift of about 140 nm compared to that of the
corresponding S-nitrosothiol, BpqSNO (3) (345 nm).14 The π-π*
transition band of 2 is considered to be so weak that it could
possibly be hidden underneath the broad absorption feature of the
n-π* transition. In the IR spectrum of 2, the N-O stretching band
was observed at 1563 cm-1, which is slightly higher than that of
BpqSNO (3) (1548 cm-1).14
Supporting Information Available: Experimental procedures and
characterization data for new compounds (PDF); X-ray crystallographic
data of 2 (CIF). This material is available free of charge via the Internet
at http://pubs.acs.org.
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agent such as dithiothreitol (DTT). The second reaction involves
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in bovine GPx and cannot be reversed by a reducing agent.19 As a
model for this mechanism, the formation of Se-nitrososelenol 2 by
transnitrosation from an S-nitrosothiol to selenol 1 was examined,
and it was revealed that S-nitrosoglutathione (GSNO), one of the
important NO carriers in the biological system, undergoes NO
transfer to selenol 1 (Figure 1). When the reaction of selenol 1
with GSNO in degassed THF-water (THF:water ) 3:1 v/v) at room
temperature in the dark was monitored by UV-vis spectroscopy,
a decrease in the absorbance of GSNO at 334 nm and a corre-
sponding increase in a maximum at 483 nm of Se-nitrososelenol 2
were observed, with clear isobestic points at 328 and 415 nm (Figure
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to the SeH group of 1 to produce Se-nitrososelenol 2. Treatment
with an excess of DTT in the presence of triethylamine quantita-
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(16) See Supporting Information for the details of the synthetic procedures
and spectral data of 1, 2, and 4 as well as the crystallographic data of 2.
(17) All calculations were performed using the Gaussian 98 program (Gaussian
Inc., Pittsburgh, PA, 1998).
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(19) An alternative pathway which includes a selenenic acid (RSeOH) instead
of a Se-nitrososelenol was also suggested in ref 7.
(20) Selenenyl sulfide 4 is not reduced by DTT under the conditions where
Se-nitrososelenol 2 is reduced to selenol 1.
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