Syntheses and Structural Characterization of Water-Soluble Selenium Reagents for the Redox Control of Protein Disulfide Bonds

Article abstract of DOI:10.1002/hc.1047

A new class of water-soluble redox reagents (1-4) that contain selenium as the active site was developed for the purpose of the redox control of protein structures. The X-ray crystallographic analyses revealed that trans-3,4-dihydroxy-1-selenolane (DHS^red) (1) and its Se-oxide (DHS^ox) (2) have two axial hydroxy groups on the selenolane five-membered ring, whereas trans-1,2-diselenane-4,5-diol (DST^ox) (3), a selenium analog of oxidized dithiotreiotol (DDT^ox), has two equatorial hydroxy groups on the diselenane six-membered ring. According to the vicinal ³J_HH coupling constants observed for 1-3, it was suggested that they adopt similar structures in solution to those in the solid state. Diselenothreitol (DST^red (4), a selenium analog of dithiothreitol (DTT^red), was also synthesized, but it was too air sensitive to be isolated. The reactions of 1-4 with DTT^ox and DTT^red indicated that the oxidizing power of DHS^ox (2) exceeds by far that of DTT^ox, while the reducing power of DST^red (4) exceeds that of DTT^red.

Full text of DOI:10.1002/hc.1047

Heteroatom Chemistry
Volume 12, Number 4, 2001
Syntheses and Structural Characterization of
Water-Soluble Selenium Reagents for the
Redox Control of Protein Disulfide Bonds
Michio Iwaoka, Taro Takahashi, and Shuji Tomoda
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo,
Komaba, Meguro-ku, Tokyo 153-8902, Japan; E-mail: tomoda@selen.c.u-tokyo.ac.jp
Received 18 December 2000; revised 18 January 2001
INTRODUCTION
ABSTRACT: A new class of water-soluble redox re-
agents (1–4) that contain selenium as the active site
was developed for the purpose of the redox control of
protein structures. The X-ray crystallographic anal-
yses revealed that trans-3,4-dihydroxy-1-selenolane
(DHSred) (1) and its Se-oxide (DHSox) (2) have two
axial hydroxy groups on the selenolane five-membered
ring, whereas trans-1,2-diselenane-4,5-diol (DSTox)
(3), a selenium analog of oxidized dithiothreitol
(DTTox), has two equatorial hydroxy groups on the di-
selenane six-membered ring. According to the vicinal
3JHH coupling constants observed for 1–3, it was sug-
gested that they adopt similar structures in solution to
those in the solid state. Diselenothreitol (DSTred) (4),
a selenium analog of dithiothreitol (DTTred), was also
synthesized, but it was too air sensitive to be isolated.
The reactions of 1–4 with DTTox and DTTred indicated
that the oxidizing power of DHSox (2) exceeds by far
that of DTTox, while the reducing power of DSTred (4)
exceeds that of DTTred. ᭧ 2001 John Wiley & Sons,
Inc. Heteroatom Chem 12:293–299, 2001
Disulfide (SS) bonds in proteins are important ele-
ments for the conformational stability of the native
folded state [1]. Proteins lose their native structure
by cleavage of the SS bonds, but the native structure
can be regenerated by formation of the SS bonds for
small monomeric proteins [2]. In such cases, the
protein structures can be reversibly controlled by re-
duction and oxidation of the SS bonds in vitro [3].
To control the redox states of protein SS bonds,
organic sulfur reagents, such as glutathione (GSH)
[4,5], dithiothreitol (DTT) [6,7], 2-mercaptoethanol
[4,6], and cystamine [8] are usually employed in vivo
and in vitro. However, since the redox power of these
sulfur reagents is not reasonably strong, excess
amounts of the reagents as well as a limited pH range
are required to control protein structures by using
such reagents. Barany et al. [9] recently developed
novel solid-phase reagents for facile formation of
peptide SS bonds. Although the active site of the re-
agents includes an SS functional group like the oxi-
dized forms of normal sulfur reagents, the polymer
support enables efficient formation of peptide SS
bonds in a wide pH range. On the other hand, Shi
and Rabenstein [10] reported that platinum complex
trans-[Pt(ethylenediamine)2Cl2]2ם is a highly selec-
tive and efficient reagent for the quantitative for-
mation of intramolecular SS bonds in peptides. This
reagent is different from previous redox reagents in
that a transition metal is involved as the redox
center.
Dedicated to Professor Naoki Inamoto on the occasion of his
72nd birthday.
Correspondence to: Shuji Tomoda.
Contract Grant Sponsor:
Contract Grant Number: Grant-in-Aid for Scientific Research
11740346.
Contract Grant Sponsor: Mitsubishi Chemical Corporation
Fund.
᭧ 2001 John Wiley & Sons, Inc.
293

294 Iwaoka, Takahashi, Tomoda
We have recently developed a new class of redox
reagents (DHSred, 1; DHSox, 2) that contain redox-
active selenium instead of sulfur [11]. The selenium
reagents were designed on the basis of the observa-
tions that the redox activity of selenium is higher
than that of sulfur [12] and that some selenium com-
pounds work as good catalysts for the formation or
the reduction of SS bonds in organic and enzyme-
mimetic reactions [13]. According to the oxidative
regeneration experiments of ribonuclease A having
four intramolecular SS bonds, it was shown that 2
is a rapid and quantitative reagent for formation of
SS bonds in polypeptides in a wide range of pH (at
least from pH 3 to 9) [11].
In this article, synthesis and structural charac-
terization of new water-soluble selenium reagents
(1–4), which have been developed for the purpose of
redox control of protein structures, are presented in
detail [14]. Configurations and conformations of 1–
3 were unambiguously determined in the solid state
by X-ray analysis. According to the vicinal 3JHH cou-
pling constants observed for 1–3, it was suggested
that their conformations in solution are similar to
those in the solid state. The redox properties of 1–4
are also reported briefly on the basis of the reactions
of 1–4 with sulfur reagents DTTox (5) and DTTred (6).
ucts. Obtained 1 and 3 were stable at room tempera-
ture under air at least for a few months.
Treatment of DHSred (1) with m-chloroperben-
zoic acid in dichloromethane (CH2Cl2) generated the
corresponding selenium oxide (DHSox) (2) as a col-
orless precipitate due to the low solubility of 2 in the
solvent; 2 was stable at room temperature under air
and was readily soluble in water. It should be noted
that each hydrogen and carbon atom gives a differ-
ent NMR signal in solution due to the chiral center
at the selenium atom, except for the two hydroxy
(OH) hydrogen atoms.
On the other hand, the reduction of DSTox (3)
with sodium borohydride (NaBH4) followed by the
addition of aqueous HCl afforded diselenothreitol
(DSTred) (4) as a white powder. Compound 4 was too
air sensitive to be isolated presumably due to the
high reactivity of the selenol moieties: the crude
product contained ꢀ20% of an unknown byproduct
(77Se NMR d מ82.8). The open-chain diselenol struc-
1
ture of 4 was explicitly characterized by H NMR [d
3
מ0.51 (t, JHH ס 7.5 Hz, SeH)] and 77Se NMR [d
1
מ78.3 (d, JSeH ס 33.5 Hz)] spectroscopy. The ob-
1
served value of JSeH was slightly smaller than the
1
normal values of JSeH (ꢀ40 to ꢀ65 Hz) [16], sug-
gesting enhanced acidity of the selenol moieties of 4.
Although the molecular formulae of 1–4 could
be easily drawn based on the NMR and/or elemental
analyses, their configurations (trans or cis) and con-
formations (axial or equatorial) as to the two OH
substituents were not obvious only from the spectro-
scopic data. In order to clarify these structural fea-
tures, X-ray crystallographic analysis was performed
for 1–3.
RESULTS AND DISCUSSION
Synthesis
Following the synthetic procedure of DTTox (5) [15],
a racemic mixture of 1,3-butadiene diepoxide was
allowed to react with NaHSe or Na2Se2 in water
(Schemes 1 and 2). Since the reaction products
(trans-3,4-dihydroxy-1-selenolane [DHSred, 1] and
trans-1,2-diselenane-4,5-diol [DSTox, 3], respectively)
were soluble in both water and ether, continuous ex-
traction with ether was necessary to isolate the prod-
X-ray analysis
Molecular structures of DHSred (1), DHSox (2), and
DSTox (3) determined by X-ray analysis are shown in
Figures 1–3. All atoms including hydrogen atoms
were reasonably located by the difference Fourier
maps, and their atomic parameters were refined by
the least-squares methods without constraints. As
clearly seen in the figures, the two OH substituents
are trans to each other for all cases, indicating that
the reactions of 1,3-butadiene diepoxide with NaHSe
and Na2Se2 (Schemes 1 and 2) proceeded with reten-
tion of configuration at C2 and C3. Thus the config-
urations of 1–3, and hence that of DSTred (4), could
be unambiguously determined.
SCHEME 1
Two crystallographically independent molecules
are present for DHSred (1), one of which is located at
an equivalent position (Figure 1). The two molecules
have similar conformations to each other with two
axial OH substituents on the selenolane five-mem-
bered ring (O1–C2–C3–O2 ס מ171.2ꢁ, O3–C6–C6_2–
O3_2 ס 173.3ꢁ). Similarly, DHSox (2) exists as a diax-
SCHEME 2

Syntheses and Structural Characterization of Water-Soluble Selenium Reagents for the Redox Control of Protein Disulfide Bonds 295
FIGURE 1 ORTEP plot for DHSred (1) at the 50% probability level. Selected bond parameters: Se1–C1 1.953(5), Se1–C4
1.949(5), Se2–C5 1.959(4), O1–C2 1.428(5), O2–C3 1.429(5), O3–C6 1.421(5), C1–C2 1.515(6), C2–C3 1.516(6), C3–C4
1.495(6), C5–C6 1.515(6), C6–C6_2 1.526(7), C4–Se1–C1 89.4(2), C5–Se2–C5_2 89.9(3), C2–C1–Se1 106.9(3), C1–C2–C3
108.5(4), C4–C3–C2 108.3(4), C3–C4–Se1 106.5(3), C6–C5–Se2 106.4(3), C5–C6–C6–_2 108.6(2), C4–Se1–C1–C2
מ9.8(3), Se1–C1–C2–C3 34.4(4), O1–C2–C3–O2 מ171.2(3), C1–C2–C3–C4 מ49.8(5), C2–C3–C4–Se1 40.0(4), C1–Se1–
C4–C3 מ16.8(4), C5_2–Se2–C5–C6 13.24(17), Se2–C5–C6–C6_2 מ37.0(4), O3–C6–C6_2–O3_2 173.3(6), C5–C6–C6_2–
C5_2 49.7(8).
ial conformation as to the two OH substituents in
the solid state (O2–C2–C3–O3 ס 179.4ꢁ) (Figure 2).
Although the relative stability of the diaxial and di-
equatorial conformations may depend on the sub-
stituents on the selenolane ring and the molecular
packing in the solid state, it seems that the diaxial
conformation is intrinsically more stable than the
diequatorial one because diaxial conformations have
been usually observed for analogous thiolane com-
pounds in the solid state [17].
In contrast, DSTox (3) has two equatorial OH
substituents on the diselenane six-membered ring
(O1–C2–C3–O2 ס 53.2ꢁ), which adopts a slightly dis-
torted chair conformation (Figure 3). The diequa-
torial conformation is stabilized by an intramolec-
˚
ular hydrogen bond (O1–H1•••O2; H1•••O2 2.38 A,
O1–H1•••O2 116ꢁ). A similar molecular structure
has been reported for DTTox (5) in the solid state [18].
The bond parameters (bond lengths, bond angles,
and dihedral angles) of the diselenane ring observed
for 3 were entirely consistent with those reported for
1,2-diselenane-3,6-dicarboxylic acid [19].
FIGURE 2 ORTEP plot for DHSox (2) at the 50% probability
level. Selected bond parameters: Se1–O1 1.679(4), Se1–C1
1.946(6), Se1–C4 1.946(6), O2–C2 1.433(8), O3–C3
1.444(8), C1–C2 1.531(8), C2–C3 1.491(9), C3–C4 1.515(8),
O1–Se1–C1 103.8(2), O1–Se1–C4 104.5(3), C1–Se1–C4
88.9(2), C2–C1–Se1 107.4(4), C3–C2–C1 107.2(5), C2–C3–
C4 107.5(5), C3–C4–Se1 104.8(4), C4–Se1–C1–C2
מ6.1(5), Se1–C1–C2–C3 33.5(6), O2–C2–C3–O3 179.4(4),
C1–C2–C3–C4 מ53.0(7), C2–C3–C4–Se1 46.3(6), C1–
Se1–C4–C3 מ21.9(5).
Stable Conformations in Solution
Since the reactivity of a compound depends on the
structure, it is important to elucidate a major con-
formation in solution. The structures observed for 1–
3 in the solid state are needless to say strong candi-
dates for the major conformations in solution
although they are not necessary the same as those in
the solid state.

296 Iwaoka, Takahashi, Tomoda
yses, it is suggested that the predominant confor-
mations of 1–3 in solution are similar to those in the
solid state. However, the two OH groups of 1–3 may
freely rotate in solution or locate in different posi-
tions from those in the solid state.
Reactivity of DHS and DST
The redox reactivity of DHSred (1) and DHSox (2) were
tested by the reactions with DTTox (5) and DTTred (6)
in various buffer solutions (pH ꢀ4.0–9.0) (Scheme
3). Reverse-phase high-performance liquid chroma-
tography (HPLC) analysis of the mixtures of DHSred
(1) and DTTox (5) at various ratios (5:1 ꢀ 1:200) re-
vealed that the forward reaction of Scheme 3 does
not proceed at all under these conditions. On the
other hand, the 1:1 mixture of DHSox (2) and DTTred
(6) rapidly produced DHSred (1) and DTTox (5) quan-
titatively (within 10 seconds at room temperature).
The results clearly showed that the oxidizing power
of DHSox (2) by far exceeds that of DTTox (5). Indeed,
the oxidation of the reduced form of ribonuclease A,
which has eight SH groups along the polypeptide
chain, with DHSox (2) was extremely faster than that
with DTTox (5) [11].
FIGURE 3 ORTEP plot for DSTox (3) at the 50% probability
level. Selected bond parameters: Se1–Se2 2.3108(9), Se1–
C1 1.945(4), Se2–C4 1.947(4), O1–C2 1.442(5), O2–C3
1.435(4), C1–C2 1.514(5), C2–C3 1.518(5), C3–C4 1.521(5),
O1 ••• O2 2.740(3), H1 ••• O2 2.38, C1–Se1–Se2 94.68(13),
C4–Se2–Se1 95.90(14), C2–C1–Se1 113.3(3), C1–C2–C3
115.4(3), C2–C3–C4 115.1(3), C3–C4–Se2 113.0(3), O1–H1
••• O2 116, C1–Se1–Se2–C4 53.93(19), Se2–Se1–C1–C2
מ63.2(3), Se1–C1–C2–C3 69.8(4), O1–C2–C3–O2 53.2(4),
C1–C2–C3–C4 מ67.0(5), C2–C3–C4–Se2 68.0(4), Se1–
Se2–C4–C3 מ62.5(3).
Similarly, the redox reactivity of DSTox (3) and
DSTred (4) were tested by the reactions with DTTox
(5) and DTTred (6) (Scheme 4). Reverse-phase HPLC
analysis of the mixtures of DSTox (3) and DTTred (6)
at various ratios (1:1 ꢀ 1:100) revealed that the back-
ward reaction of Scheme 4 does not proceed at all at
pH values of ꢀ4.0–9.0. On the other hand, NMR
analysis of a mixture of DSTred (4) and DTTox (5) in
D2O showed facile formation of DSTox (3) and DTTred
The major conformations of 1–3 in solution were
deducible from the observed J coupling constants
between vicinal hydrogen atoms (3JHH) by using the
Karplus equation [20]. For the case of DHSred (1), the
3
values of JHH for the CH–CH2 moiety were 4.0 Hz
and 4.0 Hz, suggesting that the HC–CH dihedral an-
gles are ꢀ60ꢁ or ꢀ120ꢁ. Since the only possible di-
hedral angle is ꢀ60ꢁ, both C–O bonds of 1 would be
axial in the major conformation in solution. Simi-
larly, for the case of DHSox (2), the values of 3JHH for
the one CH–CH2 moiety were 3.0 Hz and 2.5 Hz, and
those for the other were 5.5 Hz and ꢀ0 Hz: the two
CH–CH2 moieties exhibited different NMR behav-
iors due to the chiral center at the selenium atom.
Possible combinations of the HC–CH dihedral angles
are ꢀ70ꢁ and ꢀ70ꢁ for the former CH–CH2 moiety
and ꢀ50ꢁ and ꢀ90ꢁ for the latter one, suggesting that
the major conformation of 2 in solution has axial C–
1
(6): according to the H NMR spectrum, DSTred (4)
disappeared completely to form DTTred (6) within 5
minutes at room temperature. Therefore, the reduc-
ing power of DSTred (4) was found to be higher than
that of DTTred (6).
CONCLUSIONS
New water-soluble selenium reagents (1–4) were
synthesized, and their structures were characterized
in the solid state and also in solution. In the solid
state, DHSred (1) and DHSox (2) have two axial OH
groups on the selenolane five-membered ring,
whereas DSTox (3) has two equatorial OH groups on
the diselenane six-membered ring. Similar struc-
tures are probably the major conformations of 1–3
in solution. The reactions of 1–4 with DTTox (5) and
DTTred (6) suggested that DHSox (2) is a strong oxi-
3
O bonds. The observed value of JHH for the central
CH–CH moiety of 2 (3.0 Hz) also supported this as-
signment. On the other hand, DSTox (3) exhibited dif-
ferent behaviors: the values of 3JHH for CH–CH2 were
10.0 Hz and 2.8 Hz, suggesting that the HC–CH di-
hedral angles are ꢀ180ꢁ and ꢀ70ꢁ. Therefore, the
major conformation of 3 in solution would have
equatorial C–O bonds.
According to the previous conformational anal-

Syntheses and Structural Characterization of Water-Soluble Selenium Reagents for the Redox Control of Protein Disulfide Bonds 297
SCHEME 3
SCHEME 4
1
dizing agent for formation of protein SS bonds,
while DSTred (4) is a strong reducing agent for cleav-
age of protein SS bonds. Molecular design of redox
reagents using selenium, instead of sulfur, could be
an effective approach to the redox control of protein
structures.
79–80ꢁC. Spectral data for 1: H NMR (500 MHz in
CDCl3) d 2.10 (d, J ס 5.0, 2H), 2.87 (dd, J ס 4.0 and
11.0, 2H), 3.16 (dd, J ס 4.0 and 11.0, 2H), 4.27 (m,
2H); 13C NMR (125.65 MHz) d 26.8, 78.9; 77Se NMR
(95.35 MHz) d 65.6. Anal. Calcd for C4H8O2Se: C,
28.76; H, 4.83. Found: C, 28.55; H, 4.70.
Synthesis of DL-trans-3,4-dihydroxy-1-selenolane
EXPERIMENTAL
General
oxide (DHSox) (2)
To a CH2Cl2 solution (10 mL) of 1 (55 mg, 0.33
mmol), m-chloroperbenzoic acid (56 mg, 0.33
mmol) dissolved in CH2Cl2 (10 mL) was added
slowly. Precipitated white crystals were collected by
filtration. Obtained 2 was purified by recrystalliza-
tion from MeOH. Yield, 41 mg (69%). Colorless crys-
Commercially available organic and inorganic re-
agents were used without further purification. Di-
chloromethane (CH2Cl2) was dried over calcium hy-
dride and was distilled under nitrogen before use.
Methanol (MeOH) was distilled under nitrogen.
Other organic solvents were used without purifica-
tion. Column chromatography was carried out by us-
ing Fuji Silysia BW-300 silica gel. NMR spectra were
measured on a JEOL ␣500 spectrometer. Dimethyl
selenide (d 0 ppm) in CDCl3 was used as an external
standard for measurement of 77Se NMR chemical
shifts.
1
tals, m.p. 124–125ꢁC. Spectral data for 2: H NMR
(500 MHz in MeOH-d4) d 2.87 (d, J ס 13.0, 1H), 3.08
(dd, J ס 3.0 and 12.0, 1H), 3.58 (dd, J ס 5.5 and 13.0,
1H), 3.78 (dd, J ס 2.5 and 12.0, 1H), 4.63 (m, 1H),
4.72 (dd, J ס 3.0 and 5.5, 1H), 4.80 (s, 2H); 13C NMR
(125.65 MHz) d 55.0, 58.0, 79.1, 79.5; 77Se NMR
(95.35 MHz) d 941.8. Anal. Calcd for C4H8O3Se: C,
26.24; H, 4.41. Found: C, 26.16; H, 4.17.
Synthesis of DL-trans-3,4-dihydroxy-1-selenolane
Synthesis of DL-trans-1,2-diselenane-4,5-diol
(DHSred) (1)
(DSTox) (3)
To a freshly prepared aqueous solution of NaHSe
[21] (19 mmol), 1,3-butadiene diepoxide (1.2 mL, 16
mmol) was added. The mixture was stirred for 10
minutes at room temperature under nitrogen and
then overnight under air. The resulting mixture was
filtered. The filtrate was extracted with ether for 3
days by using a liquid/liquid continuous extractor.
Selenolane 1 was obtained from the ether layer by
concentration and was purified by silica gel column
chromatography (ether) or recrystallization from
CHCl3. Yield, 1.44 g (55%). Colorless crystals, m.p.
To a freshly prepared aqueous solution of Na2Se2
[21] (38 mmol), 1,3-butadiene diepoxide (1.5 mL, 19
mmol) was added. The mixture was refluxed for 1
hour under nitrogen. The resulting mixture was then
stirred overnight at room temperature under air. Se-
lenium precipitates were filtered off. The filtrate was
extracted with ether for 3 days by liquid/liquid con-
tinuous extraction. Diselenane 3 was obtained from
the ether layer by concentration and was purified by
silica gel column chromatography (ether) or recrys-
tallization from MeOH. Yield, 2.54 g (54%). Yellow

298 Iwaoka, Takahashi, Tomoda
1
crystals, m.p. 116–118ꢁC. Spectral data for 3: H
NMR (500 MHz in DMF-d7) d 3.16 (dd, J ס 10.0 and
12.0, 2H), 3.43 (dd, J ס 2.8 and 12.0, 2H), 3.53 (m,
2H), 5.20 (d, J ס 4.0, 2H); 13C NMR (125.65 MHz) d
31.8, 75.4; 77Se NMR (95.35 MHz) d 268 (broad).
Anal. Calcd for C4H8O2Se2: C, 19.53; H, 3.28. Found:
C, 19.59; H, 3.14.
matized by graphite. Intensity data were collected
using an x-2h scan technique to a maximum 2h value
of 55.0ꢁ. A total of 2629 reflections was collected. The
structure was solved by the direct method (SIR-92)
[22]. The final full-matrix least-squares refinement of
F2 against all unique 1358 reflections (Rint ס 0.0466)
was performed by using the SHELXL-97 program
[23]. The crystal data obtained are as follows: For-
mula, C4H8O3Se; M, 183.06; Space group, mono-
Synthesis of DL-threo-2,3-dihydroxy-1,4-
butanediselenol (diselenothreitol, DSTred) (4)
˚
˚
clinic Pbca (no. 61); a, 9.791(12) A; b, 17.123(6) A; c,
3
7.049(6) A; V, 1181.8(18) A ; Z, 8; l, 6.3 mmמ1; Dc,
2.058 g/mL; T, 296 K; Size, 0.40 ן 0.40 ן 0.30 mm;
Number of variables, 105; R(F2), 7.29; wR(F2), 12.24.
The graphical molecular structure of 2 (Figure 2)
was drawn using the ORTEP-3 program for Win-
dows [24]. Complete lists of atomic coordinates,
thermal parameters, and bond parameters have
been deposited at the Cambridge Crystallographic
Data Centre (No. CCDC-154249).
˚
˚
To the yellow solution of 3 (69 mg, 0.28 mmol) in
MeOH (0.5 mL), NaBH4 was added under a nitrogen
atmosphere until the solution color disappeared. Six
milliliters of 6 M HCl was added to the solution. The
resulting solution was then combined with 80 mL of
ether and was stirred vigorously under nitrogen for
1 hour to extract 4 in ether. The ether layer was col-
lected by using a syringe. The extraction was carried
out two times. The combined ether layer was dried
over MgSO4. After decantation and concentrated un-
der reduced pressure, 4 was obtained as a white
powder. Diselenol 4 was air sensitive and contained
ꢀ20% of an unknown byproduct (77Se NMR d
X-Ray Analysis of DSTox (3)
A Rigaku AFC6S diffractometer was employed with
˚
the Mo K␣ (k ס 0.71073 A) radiation monochro-
1
מ82.8). Spectral data for 4: H NMR (500 MHz in
matized by graphite. Intensity data were collected
using an x-2h scan technique to a maximum 2h value
of 55.0ꢁ. A total of 3163 reflections was collected. The
structure was solved by the direct method (SIR-92)
[22]. The final full-matrix least-squares refinement of
F2 against all unique 1584 reflections (Rint ס 0.0459)
was performed by using the SHELXL-97 program
[23]. The crystal data obtained are as follows: For-
mula, C4H8O2Se2; M, 246.02; Space group, mono-
CDCl3) d מ0.51 (t, J ס 7.5, 2H), 2.82 (m, 6H), 4.27
(m, 2H); 13C NMR (125.65 MHz) d 22.6, 80.7; 77Se
NMR (95.35 MHz) d מ78.3 (d, 1JSeH ס 33.5).
X-Ray Analysis of DHSred (1)
A Rigaku AFC6S diffractometer was employed with
˚
the Mo K␣ (k ס 0.71073 A) radiation monochro-
matized by graphite. Intensity data were collected
using an x-2h scan technique to a maximum 2h value
of 60.0ꢁ. A total of 2674 reflections was collected. The
structure was solved by the direct method (SIR-92)
[22]. The final full-matrix least-squares refinement of
F2 against all unique 2596 reflections (Rint ס 0.0745)
was performed by using the SHELXL-97 program
[23]. The crystal data obtained are as follows: For-
mula, C4H8O2Se; M, 167.06; Space group, mono-
˚
˚
clinic P21/a (no. 14); a, 8.386(3) A; b, 8.5483(19) A;
3
˚
˚
c, 9.9690(18) A; b, 105.06(2)ꢁ; V, 690.1(3) A ; Z, 4; l,
10.6 mmמ1; Dc, 2.368 g/mL; T, 296 K; Size, 0.40 ן
0.30 ן 0.30 mm; Number of variables, 105; R(F2),
5.91; wR(F2), 9.63. Although numerical absorption
corrections were recommended for this crystal [l ן
size(mid) ꢂ 3.0], psi-scan corrections were applied
due to technical difficulty in the numerical correc-
tions. Resulting bond parameters (bond lengths,
bond angles, and dihedral angles) were consistent
with the normal values, except for the short bond
˚
˚
clinic C2/c (no. 15); a, 17.052(4) A; b, 10.237(3) A; c,
3
˚
˚
11.658(3) A; b, 119.055(13)ꢁ; V, 1778.9(8) A ; Z, 12; l,
6.2 mmמ1; Dc, 1.871 g/mL; T, 296 K; Size, 0.50 ן 0.40
ן 0.40 mm; Number of variables, 144; R(F2), 11.93;
wR(F2), 13.88. The graphical molecular structure of
1 (Figure 1) was drawn using the ORTEP-3 program
for Windows [24]. Complete lists of atomic coordi-
nates, thermal parameters, and bond parameters
have been deposited at the Cambridge Crystallo-
graphic Data Centre (No. CCDC-154248).
˚
length between O1 and H1 (0.67 A) The graphical
molecular structure of 3 (Figure 3) was drawn using
the ORTEP-3 program for Windows [24]. Complete
lists of atomic coordinates, thermal parameters, and
bond parameters have been deposited at the Cam-
bridge Crystallographic Data Centre (No. CCDC-
154250).
X-Ray Analysis of DHSox (2)
Reactions of DHS or DST with DTT
A Rigaku AFC6S diffractometer was employed with
the Mo K␣ (k ס 0.71073 A) radiation monochro-
For the reactivity tests, reverse-phase HPLC or NMR
analysis was applied; 0.1 M stock solutions of DHSred
˚

Syntheses and Structural Characterization of Water-Soluble Selenium Reagents for the Redox Control of Protein Disulfide Bonds 299
(1), DHSox (2), DTTox (5), and DTTred (6) and a 0.05
[9] Annis, I.; Chen, L.; Barany, G. J Am Chem Soc 1998,
120, 7226–7238.
[10] Shi, T.; Rabenstein, D. L. J Am Chem Soc 2000, 122,
M stock solution of DSTox (3) dissolved in degassed
0.1 M acetic acid were prepared. Two stock solu-
6809–6815.
tions, for example DHSred and DTTox, were mixed in
[11] Iwaoka, M.; Tomoda, S. Chem Lett 2000, 1400–1401.
[12] (a) Back, T. G., Ed. Organoselenium Chemistry: A
Practical Approach; Oxford University Press: Oxford,
1999; (b) With, T., Ed. Organoselenium Chemistry:
Modern Developments in Organic Synthesis; In Top-
ics in Current Chemistry; 2000, Vol. 208.
[13] (a) Ogawa, A.; Nishiyama, Y.; Kambe, N.; Murai, S.;
Sonoda, N. Tetrahedron Lett 1987, 28, 3271–3274; (b)
Iwaoka, M.; Tomoda, S. J Am Chem Soc 1994, 116,
2557–2561.
[14] Preliminary results were reported in Ref. [1].
[15] (a) Cleland, W. W. Biochemistry 1964, 3, 480–482; (b)
Carmack, M.; Kelley, C. J. J Org Chem 1968, 33, 2171–
2173.
[16] Klapo¨tke, T. M.; Broschag, M. Compilation of Re-
ported 77Se NMR Chemical Shifts: Up to the Year
1994; John Wiley & Sons: Chichester, 1996.
[17] (a) Clegg, W. Acta Crystallogr 1975, B31, 2722–2724;
(b) Benz, G.; Born, L.; Brieden, M.; Grosser, R.; Kurz,
J.; Paulsen, H.; Sinnwell, V.; Weber, B. Liebigs Ann
Chem 1984, 1408–1423; (c) Nawata, Y.; Ishitani, Y.;
Matsuura, I.; Oishi, H.; Ando, K. Acta Crystallogr
1989, C45, 1112–1113; (d) Rao, A. V. R.; Reddy, K. A.;
Srinivas, N. R.; Gurjar, M. K.; Padmaja, N.; Ramak-
umer, S.; Viswamitra, M. A.; Swapra, G. V. T.; Jagan-
nadh, B.; Kunwar, A. C. J Chem Soc Perkin Trans 1
1993, 1255–1259.
a buffer solution (a sodium acetate buffer at pH 4.0,
a phosphate buffer at pH 7.0, or a tris buffer at pH
9.0) at various ratios. The dilution ratio was ꢀ1000.
The reaction mixture was then analyzed by use of a
Hewlett Packard HP1100 Series HPLC system
equipped with a Hewlett Packard ODS Hypersil col-
umn (4 ן 125 mm). The system was equilibrated
with 0.09% trifluoroacetic acid at the flow rate of 0.5
mL/min. After sample injection, a gradient was ap-
plied by increasing the ratio of acetonitrile, contain-
ing 0.09% trifluoroacetic acid, from 0 to 15% in 25
minutes. For the reaction between DSTred (4) and
DTTox (5), DSTred prepared according to the afore-
mentioned procedure was dissolved in D2O and the
solution was added with DTTox. NMR spectra of the
mixture was then measured. Formation of DSTox (3)
and DTTred (6) was clearly observed in the 1H and 13
C
NMR spectra.
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