A water-soluble cyclic selenide with enhanced glutathione peroxidase-like catalytic activities

Article abstract of DOI:10.1002/ejoc.200901114

Antioxidative catalytic activities of trans-3,4-dihydroxyselenolane (DHS^red), a water-soluble cyclic selenide, were investigated in the reaction of hydrogen peroxide with three different thiol substrates, monothiol glutathione (GSH), dithiol dithiothreitol (DTT^red), and polythiol reduced ribonuclease A (RNase A) having eight thiol groups along the polypeptide chain. For all the thiol substrates, DHS^red exhibited higher glutathione peroxidase (GPx)-like antioxidative catalytic activities than the corresponding linear selenide, that is, Se(CH₂CH₂OH) ₂. The rate-determining step of the catalytic cycle was unambiguously assigned to the oxidation process of the selenide rather than the reduction process of the selenoxide intermediate. The enhanced catalytic activities of DHS^red can be ascribed, to the cyclic structure, which elevates the HOMO energy level and makes the selenium atom more exposed to the surroundings

Full text of DOI:10.1002/ejoc.200901114

FULL PAPER
DOI: 10.1002/ejoc.200901114
A Water-Soluble Cyclic Selenide with Enhanced Glutathione Peroxidase-Like
Catalytic Activities
[a]
[b]
[b]
[a]
Fumio Kumakura, Beena Mishra, K. Indira Priyadarsini, and Michio Iwaoka*
Keywords: Antioxidants / Enzyme models / Peroxides / Protein folding / Selenium
Antioxidative catalytic activities of trans-3,4-dihydroxyselen-
olane (DHS ), a water-soluble cyclic selenide, were inves-
tivities than the corresponding linear selenide, that is,
Se(CH CH OH) . The rate-determining step of the catalytic
red
2
2
2
tigated in the reaction of hydrogen peroxide with three dif-
ferent thiol substrates, monothiol glutathione (GSH), dithiol
dithiothreitol (DTT ), and polythiol reduced ribonuclease A
cycle was unambiguously assigned to the oxidation process
of the selenide rather than the reduction process of the selen-
oxide intermediate. The enhanced catalytic activities of
DHS can be ascribed to the cyclic structure, which elevates
the HOMO energy level and makes the selenium atom more
exposed to the surroundings.
red
red
(
RNase A) having eight thiol groups along the polypeptide
red
chain. For all the thiol substrates, DHS
exhibited higher
glutathione peroxidase (GPx)-like antioxidative catalytic ac-
Introduction
Organic selenides have frequently been applied as syn-
thetic mimics of glutathione peroxidase (GPx), a selenium-
containing antioxidant enzyme catalyzing the reduction of
hydroperoxides at the expense of two molecules of glutathi-
[
1]
one (GSH). For example, selenomethionine, a selenium
In the present study, the GPx-like antioxidative catalytic
analog of amino acid methionine, was demonstrated to have activities of 1 were investigated in the reactions between
[
2]
an antioxidative catalytic activity and has been widely ap- hydrogen peroxide (H
O
) and various thiol substrates
2
2
plied as an anticancer reagent or an antioxidant.[ Bis(3- (RSH; Scheme 1). The reaction rates were compared with
hydroxypropyl) selenide and the related selenide com- those obtained by using linear analog 3 as a catalyst to
3]
[
8]
pounds were recently found to exhibit strong antioxidative show advantageous features of the cyclic selenide as a redox
[4]
catalytic activities as GPx mimics. Redox reactions of catalyst. The reactions were also carried out in the presence
many other selenides have also been investigated.[ We re- of selenide catalysts 4 and 5. The catalytic reaction
5]
[9]
[8]
red
cently synthesized trans-3,4-dihydroxyselenolane (DHS
,
mechanism as well as the rate-determining step was unam-
[6]
1
), a water-soluble cyclic selenide, applied the correspond- biguously assigned.
ox
ing selenoxide (DHS , 2) to redox-coupled folding experi-
ments of ribonuclease A (RNase A)[ and demonstrated
that 2 is a powerful oxidant for the rapid and quantitative
transformation of the cysteinyl thiol groups into the disul-
fide (SS) bonds over a wide pH range. Thus, redox chemis-
try of selenides and selenoxides has attracted increasing
interest in relation to their biochemical applications.
7]
Scheme 1. A model reaction to examine GPx-like catalytic activities
of selenide catalysts.
Results and Discussion
The GPx-like catalytic activity of 1 was first investigated
[
[
a] Department of Chemistry School of Science,
Tokai University,
by using H O as an oxidant and glutathione (GSH) as a
2
2
Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan
E-mail: miwaoka@tokai.ac.jp
monothiol substrate in the presence of nicotinamide ade-
nine dinucleotide phosphate (NADPH) and glutathione re-
ductase (GR). In this assay system, oxidized glutathione
b] Radiation and Photochemistry Division,
Bhabha Atomic Research Centre,
Trombay, Mumbai 400085, India
(
GSSG), which is produced by reaction of GSH with H O ,
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200901114.
2 2
is reduced by NADPH to GSH with the enzymatic function
440
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 440–445

A Water-Soluble Cyclic Selenide with Enhanced Catalytic Activities
of GR; hence, the reaction progress can be monitored by first assay in water, indicating the presence of strong solvent
the decrease in the UV absorbance at 340 nm due to effects. We found that the high activity for 5 in methanol
[
10]
NADPH.
was due to the free amino groups, which are good base
[
13]
NADPH was completely consumed in ca. 600 s in the catalysts for the reaction given in Scheme 1. Such a cata-
absence of a catalyst, as shown in Figure 1. However, it dis- lytic function of the amino groups should be inhibited in
appeared within 330 s in the presence of 1. Selenide 3 also water because of the protonation. On the other hand, the
showed antioxidative catalytic activity, but the activity was reason for the low catalytic activity for 4 was found to be
lower than that of 1, suggesting that a cyclic selenide is degradation of the selenoxide intermediate in methanol as
more efficient as a GPx mimic than a linear selenide. The discussed later.
catalytic activities of various water-soluble selenides were in
the order 4Ͼ1Ͼ3Ͼ5. The highest activity for 4 would be
due to the ionization of the carboxylic groups in the buffer
–
solution to COO , the negative charge of which should in-
ductively increase the oxidizability of the selenium atom.
Conversely, the lowest activity for 5 would be due to the
+
ionization of the amino groups to NH , which should de-
3
crease the oxidizability of the selenium atom.[ The order
of the catalytic activity strongly suggested that the GPx-
like activities of selenides in water can be controlled by the
electronic effects from the terminal hydrophilic substituents
as well as the steric environments around the selenium
atom.
11]
1
Figure 2. A series of 500 MHz H NMR spectra obtained in the
red
oxidation of DTT (0.15 mmol) with H
2
O
2
(0.15 mmol) in the
OD
presence of a catalytic amount of 1 (0.015 mmol) in CD
1.1 mL) at 25 °C. Unknown byproduct is indicated by ϫ.
3
(
Figure 1. NADPH-coupled GPx assay for water-soluble selenides 1
and 3–5. Reaction conditions: [GSH] = 1.0 m, [H = 2.5 m,
NADPH] = 0.3 m, [GR] = 4 units/mL and [selenide] = 0.2 m
0
2 2 0
O ]
[
0
in pH 7.4 phosphate buffer at room temperature.
Second, the catalytic activity of 1 was investigated by
red
using a dithiol, that is, dithiothreitol (DTT ), instead of a
monothiol substrate. Methanol (CD OD) was selected as a
solvent in this assay because the reaction proceeded too fast
3
red
Figure 3. Percentages of residual DTT as a function of the reac-
red
tion time in the oxidation of DTT with H
2 2
O in the presence of
1
[12]
in water to be monitored by H NMR spectroscopy.
selenide catalysts 1 and 3–5 in CD OD. Reaction conditions:
3
1
red
In the series of the H NMR spectra (Figure 2), the ab- [DTT ] = [H O ] = 0.14 m and [selenide] = 0.014 m at 25 °C.
sorptions at δ = 2.63, 3.67 ppm due to DTT decreased
0
2
2 0
red
with increasing reaction time, whereas those at δ = 2.87,
Third, reduced RNase A, which has eight thiol groups
ox
3
.03, 3.49 ppm due to DTT increased. In the absence of a along the peptide chain, was treated with H O in the pres-
2 2
red
catalyst, 86% of DTT remained unreacted after 300 min ence of 1 as a catalyst. RNase A with four native SS link-
Figure 3). In contrast, the oxidation reaction was complete ages is a typical protein that spontaneously folds into the
in 120 min when 1 was added as a catalyst. Again, linear native structure from the reduced unfolded state (R) by oxi-
(
[
14]
selenide 3 showed a lower catalytic activity than cyclic 1, dation and subsequent SS rearrangement. The oxidation
suggesting the importance of the cyclic structure. However, reaction was initiated by the addition of H O to the acetate
2
2
the order of the catalytic activity for other selenides, that is, buffer solution at pH 4.0 containing R and 1 and was
Ͼ1Ͼ3≈4, was very different from that observed in the quenched by the addition of 2-aminoethyl methanethio-
5
Eur. J. Org. Chem. 2010, 440–445
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
441

F. Kumakura, B. Mishra, K. I. Priyadarsini, M. Iwaoka
FULL PAPER
sulfonate (AEMTS), a thiol blocking reagent.[15] The fold-
ing intermediates were then analyzed by HPLC by using a
cation exchange column.
The typical HPLC chromatograms are shown in Fig-
ure 4. In the presence of catalyst 1 (1 equiv.), SS formation
was complete within 180 min, via transient generation of
1S, 2S, and 3S intermediates, corresponding to the interme-
diate ensembles with one, two, and three SS bonds, respec-
tively, and final generation of a scrambled intermediate en-
semble with four SS bonds (4S). On the other hand, in the
absence of 1, formation of SS bonds was very slow with
generation of a number of byproducts probably due to side-
chain modification.
Figure 4. HPLC chromatograms obtained from oxidation of re-
duced RNase A (R) with H
Reaction conditions: [R] = 20 µ, [H
0 µ. (a) The reaction time was 10 min, (b) 60 min, and (c)
80 min. (d) The reaction time was 180 min in the absence of the
2
O
2
catalyzed by 1 at 25 °C and pH 4.0.
Figure 5. Percentages of the residual cysteinyl thiol groups as a
function of the reaction time in the oxidation of reduced RNase A
0
2 2 0
O ]
= 2.0 m and [1] =
2
1
(R) with H
2
O
2
in the presence of selenide catalysts 1 (top) and 3
(bottom). Reaction conditions: [R]
0
= 20 µ and [H = 2.0 m
2 2 0
O ]
catalyst.
at 25 °C and pH 4.0. Amounts of the catalyst are indicated as the
equivalents with respect to R.
Figure 5 shows percentages of the remaining cysteinyl
thiol groups of R as a function of the reaction time in the 925 ppm, accompanied with the slight transformation of 3
presence of variable equivalents of catalysts 1 and 3. When into 6 (Figure 6). Similar competition experiments revealed
selenide 3 was applied as a catalyst, acceleration of SS for- the order of reduction abilities for the selenides as
mation was also observed, but the catalytic activity was 4Ͼ1Ͼ3Ͼ5 in both water and methanol. The order was in
again lower than that of 1. It should be noted that the ac- complete agreement with their catalytic activities in water.
tivity of the selenide catalyst at neutral or higher pH values On the other hand, when a 1:1 mixture of isolated sele-
was not so obvious as that observed at pH 4.0 because di- noxides 2 and 6 was treated with one equivalent of DTTred
rect oxidation of RSH with H O is much faster in basic in D O, selenide 1 was quantitatively obtained (Figure 7).
2
2
2
solutions (see also Figure 1).
The order of oxidation abilities for various selenoxides was
The group of Hilvert recently reported that a similar oxi- revealed to be 8Ͼ2Ͼ6Ͼ7, which corresponds to
dation reaction of R by using oxidized selenoglutathione 5Ͼ1Ͼ3Ͼ4 for the selenide counterparts. The results
(
GSeSeG), a tripeptide dimer with a diselenide bond, as a clearly demonstrate that only selenide and selenoxide spe-
[
16]
catalyst finally produced the native folded protein.
Our cies are involved in the catalytic cycle as the stable interme-
results demonstrated that similar catalytic oxidative protein diates. In methanol, however, selenoxide 7 gradually decom-
folding would also be possible by using rather simple water- posed into unknown compounds: such degradation prod-
1
[12]
soluble monoselenides as a catalyst in the presence of per- ucts were previously observed in the H NMR spectra.
oxides. The degradation would be responsible for the low catalytic
The mechanism of the catalytic reaction was delineated activity of 4 in methanol (see Figure 3) despite the largest
by 77Se NMR spectroscopy. When a 1:1 mixture of sele- reduction ability among the selenides. It is of particular
nides 1 and 3 was treated with one equivalent of H O in interest that cyclic selenide 1 can be oxidized more easily
2
2
D O, the absorption due to 1 (δ = 74 ppm) decreased and than linear selenide 3 and resulting cyclic selenoxide 2 can
2
a new peak, corresponding to selenoxide 2, appeared at δ = be reduced more easily than linear selenoxide 6.
442
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 440–445

A Water-Soluble Cyclic Selenide with Enhanced Catalytic Activities
Figure 8. HOMOs of 1 (left) and 3 (right) calculated at HF/6-
1G(d). The structures were optimized in water at the same level
3
by using the conductor-like solvation model (CPCM).
Figure 6. 77Se NMR spectra obtained by addition of H
solution containing selenides 1 and 3 (0.025 mmol each) in D
1.0 mL) at 25 °C.
2
2
O to the
2
O
be extremely reactive and momentarily capture the second
RSH molecule. Similar catalytic cycles would be applicable
for 3–5 also. In the case of polythiol substrates like DTT
(
red
and R, the catalytic cycle would proceed more smoothly
than the case of a monothiol substrate, because the reaction
with a second RSH molecule becomes a unimolecular pro-
cess.
Figure 7. 77Se NMR spectra obtained by addition of DTTred to the
solution containing selenoxides 2 and 6 (0.025 mmol each) in D
1.0 mL) at 25 °C.
2
O
(
The above 77Se NMR study strongly suggested that the
rate-determining step in the catalytic cycle is the oxidation
process from a selenide to the selenoxide. To confirm the
assignment, the second-order rate constant (k ) for the
ox
Figure 9. A catalytic cycle of DHSred (1) in the reduction of H
with RSH.
2
O
2
oxidation of 1 with H O was determined at 25 °C in water.
2
2
–
1 –1
The obtained value (kox = 0.57Ϯ0.03  s ) was signifi-
cantly smaller than the second-order rate constant for re-
–1 –1
duction of 2 with a dithiol at pH 8.0 (kred = 7300  s ),
which was estimated from stoichiometric oxidative folding Conclusions
experiments of RNase A by using 2.[
7b]
Water-soluble cyclic selenide 1 exhibited higher GPx-like
catalytic activities than linear analog 3 for all mono-, di-,
and polythiol substrates. The enhanced catalytic activities
can be ascribed to the cyclic structure, which elevates the
HOMO energy level and makes the selenium atom more
exposed to the surroundings. Although the observed GPx-
like catalytic activities may not be so remarkable as those
The enhanced catalytic activity of cyclic 1 can reasonably
be explained by properties of the HOMO (Figure 8). Ab
initio calculations in water[
17,18]
revealed that the HOMO
energy level of 1 is –8.66 eV, which is 0.27 eV higher than
that of 3 in water. Moreover, the HOMO had a dominant
contribution from a 4p orbital of the selenium atom: Fig-
ure 8 clearly shows that the HOMO of cyclic selenide 1 is
completely exposed to its surroundings, permitting easier
access of an oxidant, whereas the oxidant access should be
slightly hindered for linear selenide 3 due to conformational
freedom of the side chains.
[
1,4]
of previous GPx model compounds,
the features of the
water-soluble cyclic selenide will be useful not only for the
molecular design of new GPx mimics with enhanced anti-
oxidative catalytic activities but also for biological applica-
tions as an efficient catalyst for the formation of SS linkages
Considering all the results, the catalytic mechanism for 1
can be summarized as follows (Figure 9). First, 1 is oxidized
with H O to the corresponding selenoxide 2. This step
[
16]
in proteins.
2
2
should be the rate-determining step. Second, 2 is rapidly
reduced with RSH to produce an intermediate having a Se–
Experimental Section
S linkage. Because this kind of intermediate could not be General Methods: Selenides 1,[6] 3,[8] 4,[9] and 5[8] and selenoxide 2[6]
observed in any NMR experiments, the intermediate must were synthesized according to literature methods. Selenoxides 6–8
Eur. J. Org. Chem. 2010, 440–445
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
443

F. Kumakura, B. Mishra, K. I. Priyadarsini, M. Iwaoka
80 µ) and aqueous H solution (100 µL, 8.0 m). The reaction
FULL PAPER
were quantitatively obtained in situ within 1 h by the reaction of
2 2
O
the corresponding selenides with H
cording to the procedure described below for 6 and 8. Selenoxide
2
O
2
in water or synthesized ac-
solution was incubated in a dry thermo bath regulated at
25.0Ϯ0.1 °C. After standing for a certain period of time (1 to
300 min), the solution was added to an aqueous AEMTS solution
(≈7 mg/mL, 600 µL) to quench the reaction. The collected sample
solutions were acidified with acetic acid, desalted into a 0.1  acetic
acid solution and analyzed by HPLC by using a Tosoh TSKgel SP-
5PW strong cation exchange column (7.5ϫ75 mm). A gradient of
[
12]
7
was stable only in solution and could not be isolated.
Other
chemicals and enzymes were purchased from chemical companies
and used without purification. 1H, C, and Se NMR spectra
13
77
were recorded at 500, 125.77, and 95.43 MHz, respectively, at 25 °C
by using CHCl
H, C, and Se, respectively.
3
77
, CDCl
3
, and (PhSe)
2
as the external standards for
1
13
sodium sulfate (Na
buffer A (25 m HEPES/1 m EDTA at pH 7.0) and buffer B
buffer A + 0.5  Na SO ) from 100:0 to 55:45 over 50 min at a
2 4
SO ) was applied by changing the ratios of
Bis(2-Hydroxyethyl) Selenoxide (6): Selenide
.18 mmol) was dissolved in H O (1.0 mL). The solution was added
with aqueous H (0.18 mmol) at room temperature. After 1 h,
3
(30.0 mg,
(
2
4
0
2
flow rate of 0.5 mL/min. A wavelength of the UV detector was set
to 280 nm.
2
O
2
the solution was frozen and then lyophilized. The residue was
recrystallized from ethanol to give selenoxide 6 as white crystals.
Kinetic Analysis: The velocity of the reaction between H O
2
2
1
H NMR (500 MHz, D
), 3.94 (m, 2 H, OCH
O, 25 °C): δ = 50.0 (SeCH
Se NMR (95.43 MHz, D O, 25 °C): δ = 838.5 ppm. C
185.08): calcd. C 25.96, H 5.45; found C 26.16, H 5.38.
2 2
O, 25 °C): δ = 3.05 (m, 1 H, SeCH ), 3.27 (2.0 m) and 1 (0.3 m) was measured at 25 °C in water by follow-
13
(
m, 1 H, SeCH
125.77 MHz, D
2
2
) ppm. C NMR ing the UV absorption change at 225 nm. The measurement was
(
2
2
), 55.2 (OCH
2
) ppm.
repeated five times to determine the kox value.
7
7
2
H O Se
4 10 3
Ab Initio Calculation: A Gaussian 03 software package (revision
B.04)[ was employed. To obtain the global energy minimum
structures for 1 and 3 in water (ε = 78.39), systematic conformer
(
17]
Bis(2-Aminoethyl) Selenoxide (8): Selenide 5 (140.4 mg, 0.84 mmol)
was dissolved in H O (1 mL). The solution was added with 1 
HBr (1 mL) and then aqueous H (0.84 mmol) at room tempera-
ture. After 1 h, the solution was frozen and then lyophilized. The
residue was recrystallized from 2-propanol to give 8·2HBr·3H O as
O, 25 °C): δ = 3.07–3.27 (m,
2
search with geometry optimization was performed in water at HF/
O
2
[18]
2
6-31G(d) by using the conductor-like solvation model (CPCM).
Accuracy of the calculation results was verified by comparing the
bond lengths and angles between the calculated global energy mini-
mum structure of 1 and the experimental structure determined by
2
1
white crystals. H NMR (500 MHz, D
H, SeCH ), 3.37–3.51 (m, 2 H, NCH
125.77 MHz, D O, 25 °C): δ = 34.5 (NCH ), 41.8 (SeCH
7Se NMR (95.43 MHz,
O, 25 °C): 872.2 ppm.
OSe·2HBr·3H O (398.98): calcd. C 12.04, H 5.05, N 7.02;
2
13
2
2
2
) ppm. C NMR
) ppm.
[6]
X-ray analysis: the deviations were within Ϯ0.01 Å for the dis-
(
2
2
2
tances and Ϯ0.5° for the angle relevant to the Se atom.
7
D
2
δ
=
Supporting Information (see footnote on the first page of this arti-
C
4
H
12
N
2
2
1
13
77
cle): H, C, and Se NMR spectra of 6 and 8; kinetic analysis
found C 11.86, H 4.73, N 7.04.
red
2 2
data of the reaction of DTT with H O ; Cartesian coordinates
Activity Measurements: Catalytic activities of the selenides were
of 1 and 3 obtained by ab initio calculations.
red
measured as follows by using GSH, DTT , and reduced RNase A
(R) as a thiol substrate according to the literature methods.
In the NADPH and glutathione reductase (GR)-coupled assay,[10]
a test solution was prepared by mixing a 100 m phosphate/6 m
EDTA buffer solution (1941 µL) at pH 7.4 containing NADPH
Acknowledgments
Support has been provided by the India-Japan Collaborative Sci-
ence Programme (IJCSP) (No. 08039221-000181).
(2.0 µmol) and GSH (6.8 µmol) with a GR solution (453 U/mL,
5
1
9 µL). An aliquot (300 µL) of the test solution was added to a
.0 m selenide solution (200 µL) in 100 m phosphate buffer at
[
1] a) G. Mugesh, W.-W. du Mont, H. Sies, Chem. Rev. 2001, 101,
pH 7.4 and the phosphate buffer solution (430 µL). The reaction
was initiated by addition of a 36 m aqueous H solution
70 µL) to the mixture solution. The reaction progress was moni-
tored by absorption change at 340 nm due to consumption of
NADPH.
2125–2179; b) B. K. Sarma, G. Mugesh, Org. Biomol. Chem.
2
O
2
2008, 6, 965–974.
(
[2] R. Walter, J. Roy, J. Org. Chem. 1971, 36, 2561–2563.
[3] a) L. C. Clark, G. F. Combs Jr., B. W. Turnbull, E. H. Slate,
D. K. Chalker, J. Chow, L. S. Davis, R. A. Glover, G. F. Gra-
ham, E. G. Gross, A. Krongrad, J. L. Lesher Jr., H. K. Park,
B. B. Sanders Jr., C. L. Smith, J. R. Taylor, J. Am. Med. Assoc.
In the NMR assay,[12] DTTred (0.15 mmol) and selenide
(
0.015 mmol) were dissolved in CD
was added to 30% H (17 µL, 0.15 mmol) to start the reaction.
H NMR spectra were measured at a variable reaction time at
3
OD (1.1 mL), and the solution
1
996, 276, 1957–1963; b) G. N. Schrauzer, J. Nutr. 2000, 130,
2
O
2
1653–1656.
1
[4] a) T. G. Back, Z. Moussa, J. Am. Chem. Soc. 2003, 125, 13455–
13460; b) T. G. Back, Z. Moussa, M. Parvez, Angew. Chem.
Int. Ed. 2004, 43, 1268–1270.
red
ox
2
5 °C. The relative populations of DTT and DTT were deter-
1
mined by integration of the H NMR absorptions that were well
isolated on the spectra.
[
5] a) I. A. Cotgreave, P. Moldéus, R. Brattsand, A. Hallberg,
C. M. Andersson, L. Engman, Biochem. Pharmacol. 1992, 43,
793–802; b) V. D. Silva, M. M. Woznichak, K. L. Burns, K. B.
Grant, S. W. May, J. Am. Chem. Soc. 2004, 126, 2409–2413; c)
Z. Dong, X. Huang, S. Mao, J. Liu, G. Luo, J. Shen, Chem.
Lett. 2005, 34, 820–821; d) H. Moroder, C. Kreutz, K. Lang,
A. Serganov, R. Micura, J. Am. Chem. Soc. 2006, 128, 9909–
In the catalytic oxidation of R,[ RNase A (5–10 mg) was reduced
7b]
red
with DTT (7–12 mg) in a 100 m Tris–HCl/1 m EDTA buffer
solution (0.5 mL) at pH 8.0 containing 4  guanidine thiocyanate
as a denaturant. Reduced RNase A (R) was desalted to 200 m
acetate buffer at pH 4.0 by using a Sephadex G25 resin column,
9
918; e) Y. Saito, D. Umemoto, A. Matsunaga, T. Sato, M.
and the concentration was determined by UV absorbance at
Chikuma, Biomed. Res. Trace Elem. 2006, 17, 423–426; f) E. E.
Alberto, L. C. Soares, J. H. Sudati, A. C. A. Borges, J. B. T. Ro-
cha, A. L. Braga, Eur. J. Org. Chem. 2009, 4211–4214.
–1
2
75 nm (ε = 8600 –1 cm ). The R solution was immediately di-
luted with the acetate buffer so that the concentration became
0 µ. An aliquot (200 µL) of the diluted solution was mixed with
the acetate buffer solution (100 µL) containing a selenide (8, 40, or
4
[6] M. Iwaoka, T. Takahashi, S. Tomoda, Heteroat. Chem. 2001,
12, 293–299.
444
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 440–445

A Water-Soluble Cyclic Selenide with Enhanced Catalytic Activities
[
7] a) M. Iwaoka, S. Tomoda, Chem. Lett. 2000, 1400–1401; b) M.
Iwaoka, F. Kumakura, M. Yoneda, T. Nakahara, K. Henmi,
H. Aonuma, H. Nakatani, S. Tomoda, J. Biochem. 2008, 144,
Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N.
Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y.
Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P.
Hratchian, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R.
Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morok-
uma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzew-
ski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K.
Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V.
Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B.
Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.
L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng,
A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson,
W. Chen, M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian 03,
revision B.04, Gaussian, Inc., Wallingford, CT, 2004.
121–130.
[
[
[
[
8] M. D. Milton, S. Khan, J. D. Singh, V. Mishra, B. L. Khandel-
wal, Tetrahedron Lett. 2005, 46, 755–758.
9] D. K. Laing, L. D. Pettit, J. Chem. Soc., Dalton Trans. 1975,
2297–2301.
10] P. Pascual, E. Martinez-Lara, J. A. Bárcena, J. López-Barea, F.
Toribio, J. Chromatogr. 1992, 581, 49–56.
11] B. Mishra, A. Barik, A. Kunwar, L. B. Kumbhare, K. I. Priyad-
arsini, V. K. Jain, Phosphorus Sulfur Silicon Relat. Elem. 2008,
183, 1018–1025.
[
[
[
12] M. Iwaoka, F. Kumakura, Phosphorus Sulfur Silicon Relat.
Elem. 2008, 183, 1009–1017.
13] The reaction was completed within 60 min when triethylamine
(0.014 m) was used as a catalyst in methanol instead of 5.
14] a) C. B. Anfinsen, Science 1973, 181, 223–230; b) D. M. [18]
Rothwarf, H. A. Scheraga, Biochemistry 1993, 32, 2671–2679.
15] T. W. Bruice, G. L. Kenyon, J. Protein Chem. 1982, 1, 47–58.
16] J. Beld, K. J. Woycechowsky, D. Hilvert, Biochemistry 2008, 47,
a) V. Barone, M. Cossi, J. Phys. Chem. A 1998, 102, 1995–
2
001; b) M. Cossi, N. Rega, G. Scalmani, V. Barone, J. Comput.
[
[
Chem. 2003, 24, 669–681.
Received: October 1, 2009
6985–6987.
Published Online: December 3, 2009
[
17] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, J. A. Montgomery, Jr., T.
Eur. J. Org. Chem. 2010, 440–445
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
445