G. Mugesh and K. P. Bhabak
16.51, 43.33, 126.33, 126.55, 127.67, 128.86, 131.77, 133.94, 139.41, 140.17,
166.46 ppm; 77Se NMR ([D6]DMSO): d=852.5 ppm; HRMS (TOF MS):
m/z calcd for C33H27N3O3Se3 [M+Na]+: 775.9446; found: 775.9450.
Lee–Yang–Parr (B3LYP) exchange correlation functional was applied
for DFT calculations.[22] Geometries were fully optimized at the B3LYP
level of theory by using the 6–31G(d) basis sets. The NMR calculations
were done at B3LYP/6–311+G(d,p) level on B3LYP/6–31G(d)-level-op-
C
General procedure for the synthesis of 28–31: 4-Methylbenzenethiol
(0.015 g, 0.124 mmol) was added to a stirred solution of the correspond-
ing ebselen derivative (0.124 mmol in CH2Cl2). The resulting solution was
stirred for 1 h at room temperature, and the solvent was evaporated
under reduced pressure. The product obtained was washed with petrole-
um ether to remove unreacted thiol and disulfide formed during the reac-
tion. The selenenyl sulfides were obtained as a white amorphous solid in
quantitative yield.
X-ray crystallography: X-ray crystallographic studies were carried out on
a Bruker CCD diffractometer with graphite-monochromatized MoKa ra-
diation (l=0.71073 ) controlled by a Pentium-based PC running the
SMART (Version 5.05; Brucker AXS, Madison, WI, 1998) software pack-
age. Single crystals were mounted at room temperature on the ends of
glass fibers, and data were collected at room temperature (291 K). The
structures were solved by direct methods and refined by using the
SHELXTL software package.[25] In general, all non-hydrogen atoms were
refined anisotropically. Hydrogen atoms were assigned at idealized loca-
tions. Empirical absorption corrections were applied to all structures by
using SADABS.[26] The structure was solved by a direct method (SIR-92)
and refined by a full-matrixleast-squares procedure on F2 for all reflec-
tions (SHELXL-97).[27]
1
Compound 28: M.p: 182–1848C; H NMR ([D4]MeOH): d=2.16 (s, 3H),
6.68–6.71 (d, 3J=8.8 Hz, 2H), 6.94–6.96 (d, 3J=8.0 Hz, 2H), 7.24–7.39
(m, 6H) 7.86–7.88 (d, 3J=8 Hz, 1H), 8.07–8.09 ppm (d, 3J=8 Hz, 1H);
13C NMR ([D4]MeOH): d=19.53, 114.90, 123.27, 125.87, 127.64, 127.82,
128.38, 128.91, 129.27, 131.57, 131.73, 133.16, 136.30, 136.51, 154.66,
167.00 ppm; 77Se NMR ([D4]MeOH): d=588.5 ppm; TOF MS: m/z calcd
for C20H17NO2SSe [M+Na]+: 438.0043, found: 438.0021; elemental analy-
sis calcd (%) for C20H17NO2SSe: C 57.97, H 4.14, N 3.38, S 7.74; found:
C 57.63, H 4.52, N 3.25, S 7.62.
Crystal data for 16: C7H5NOSe, Mr =198.08, monoclinic, space group
P21/c, a=14.141(2), b=12.598(17), c=12.612(17) , b=108.431(2)8; V=
2131(5) 3, Z=12, 1calcd =1.852 gcmÀ3, GOF=0.825, R1 =0.037, wR2 =
0.084 [I>2s(I)]; R1 =0.063, wR2 =0.099 (all data).
1
3
Compound 29: H NMR ([D4]MeOH): d=2.28 (s, 3H), 6.59–6.61 (d, J=
6.4 Hz, 1H), 7.14–7.22 (m, 4H), 7.36–7.52 (m, 4H), 7.64–7.68 (t, 3J=
3
3
7.2 Hz, 1H), 8.16–8.18 (d, J=8.0 Hz, 1H), 8.22–8.24 (d, J=7.6 Hz, 1H),
9.56 (s, 1H), 10.49 ppm (s, 1H); 13C NMR ([D4]MeOH): d=22.21, 109.74,
113.40, 128.03, 129.85, 130.62, 130.94, 131.12, 131.58, 131.78, 132.98,
133.68, 134.18, 137.59, 138.24, 139.23, 140.96, 141.42, 159.31, 167.90 ppm;
77Se NMR ([D4]MeOH): d=599.0 ppm; TOF MS: m/z calcd for
C20H17NO2SSe [M+Na]+ 438.0043; found: 438.0060.
Crystal data for 17: C13H9NO2Se, Mr =290.17, monoclinic, space group
P21/c, a=5.5584(10), b=13.8780(26), c=14.5126(27) , b=95.876(3)8;
V=1113(5) 3, Z=4, 1calcd =1.370 gcmÀ3, GOF=1.370, R1 =0.045, wR2 =
0.103 [I>2s(I)]; R1 =0.047, wR2 =0.104 (all data).
Crystal data for 18: C13H9NO2Se, Mr =290.17, orthorhombic, space group
Pna21, a=15.7085(31), b=4.6494(9), c=15.3829(30) ; V=1123(4) 3,
Z=4, 1calcd =1.72 gcmÀ3, GOF=1.032, R1 =0.031, wR2 =0.073 [I>2s(I)];
R1 =0.043, wR2 =0.086 (all data).
Compound 30: M.p: 150–1528C; 1H NMR ([D1]CHCl3): d=2.28 (s, 3H),
7.03–7.05 (d, 3J=8 Hz, 2H), 7.29–7.32 (t, 3J=8 Hz, 1H), 7.39–7.41 (d,
3J=8 Hz, 1H), 7.45–7.52 (m, 5H), 7.67–7.68 (d, 3J=4 Hz, 1H), 7.99 (s,
1H), 8.25–8.27 ppm (d, 3J=8 Hz, 1H); 13C NMR ([D1]CHCl3): d=21.01,
117.83, 122.11, 126.22, 126.61, 129.10, 129.58, 129.75, 131.03, 132.20,
132.54, 133.05, 136.31, 136.89, 137.88, 166.01 ppm; 77Se NMR
([D1]CHCl3): d=601.6 ppm; TOF MS: m/z calcd for C20H16NOSBrSe
[M+Na]+: 499.9199; found: 499.9220.
Crystal data for 19: C13H8NOBrSe, Mr =353.1, monoclinic, space group
P21/n, a=4.0640(19), b=25.7348(12), c=12.3811(6) ; b=99.226(5)8;
V=1278(15) 3, Z=4, 1calcd =1.83 gcmÀ3, GOF=1.104, R1 =0.064, wR2 =
0.164 [I>2s(I)]; R1 =0.091, wR2 =0.177 (all data).
Compound 31: m.p: 102–1048C; 1H NMR ([D4]MeOH): d=2.34 (s, 3H),
3.61 (s, 2H), 3.81 (s, 2H), 7.09–7.11 (d, J=8 Hz, 2H), 7.37–7.44 (m, 3H),
Crystal data for 20: C9H9NO2Se, Mr =242.11, monoclinic, space group
P21/c, a=7.275(13), b=8.857(16), c=14.022(26) , b=102.127(3)8; V=
883(6) 3, Z=4, 1calcd =1.852 gcmÀ3, GOF=0.852, R1 =0.022, wR2 =0.056
[I>2s(I)]; R1 =0.024, wR2 =0.057 (all data).
3
7.51–7.52 (m, 2H), 7.87–7.89 (d, 3J=8 Hz, 1H), 8.24–8.26 ppm (d, 3J=
8 Hz, 1H); 13C NMR ([D4]MeOH): d=19.52, 41.60, 59.71, 125.12, 126.59,
127.11, 128.32, 128.85, 129.98, 131.03, 132.44, 135.69, 168.18 ppm; 77Se
NMR ([D4]MeOH): d=594.0 ppm; TOF MS: m/z calcd for
C16H17NO2SeS [M+Na]+: 390.0043, found: 390.0061; elemental analysis
calcd (%) for C16H17NO2SeS: C 52.46, H 4.68, N 3.82, S 8.75; found: C
52.32, H 4.96, N 3.85, S 8.65.
Crystal data for 22: C33H27N3O3Se3, Mr =750.50, monoclinic, space group
P21/c, a=8.6387(6), b=24.1949(17), c=17.4994(13) , b=119.581(0)8;
V=3180.86(4) 3, Z=4, 1calcd =1.57 gcmÀ3
, GOF=0.812, R1 =0.040,
wR2 =0.068 [I>2s(I)]; R1 =0.073, wR2 =0.071 (all data).
GSH–GSSG coupled assay: The GPxactivity was followed spectrophoto-
metrically. The test mixture contained thiol, EDTA (1 mm), glutathione
disulfide reductase (1 unitmLÀ1), and NADPH (0.4 mm) in 0.1m potassi-
um phosphate buffer (pH 7.5). GPxsamples (80 mm) were added to the
test mixture at room temperature and the reaction was started by the ad-
dition of peroxide (1.6 mm). The initial reduction rates were calculated
from the rate of NADPH oxidation at 340 nm in a GSH assay. Each ini-
tial rate was measured at least three times and calculated from the first
5–10% of the reaction by using 6.22 mmÀ1 cmÀ1 as the molar extinction
coefficient for NADPH. For the peroxidase activity, the rates were cor-
rected for the background reaction between peroxide and thiol.
Acknowledgements
This study was supported by the Department of Science and Technology
(DST), New Delhi (India). We also thank the DST for the CCD single-
crystal X-ray diffraction facility. We are grateful to the Alexander von
Humboldt Foundation, Bonn, Germany for the donation of an automated
flash chromatography system. G.M. acknowledges the DST for the Ram-
anna Fellowship and K.P.B. thanks the Council of Scientific and Industri-
al Research (CSIR), New Delhi, (India) for a research fellowship.
HPLC assay: We employed a mixture containing a 2:1 molar ratio of
PhSH and peroxide in methanol at room temperature as our model
system. Assays with and without catalyst were carried out under the
same conditions. Periodically, aliquots were injected into the reverse
phase column and eluted with methanol and water (90:10), and the con-
centrations of the product diphenyl disulfide (PhSSPh) were determined
at 254 nm by using pure PhSSPh as an external standard. The amount of
disulfide formed during the course of the reaction was calculated from
the calibration plot for the standard (PhSSPh).
[1] T. C. Stadtman, Annu. Rev. Biochem. 1996, 65, 83–100.
[2] a) L. Flohe, E. A. Günzler, H. H. Schock, FEBS Lett. 1973, 32, 132–
134; b) J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson,
D. G. Hafeman, W. G. Hoekstra, Science 1973, 179, 588–590.
[3] a) J. R. Arthur, F. Nicol, G. J. Beckett, Biochem. J. 1990, 272, 537–
540; b) M. J. Berry, L. Banu, P. R. Larsen, Nature, 1991, 349, 438–
440; c) J. Kçhrle, Methods Enzymol. 2002, 347, 125–167; d) A. C.
Bianco, D. Salvatore, B. Gereben, M. J. Berry, P. R. Larsen, Endocr.
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Computational methods: All calculations were performed by using the
Gaussian98 suite[21] of quantum chemical programs. The hybrid Becke3–
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Chem. Eur. J. 2007, 13, 4594 – 4601