2044 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Andreadou et al.
Se-Ben zyl-L-selen ocystein e (8). Se-Benzylselenocysteine
was synthesized according to method B, using 6 mmol of benzyl
bromide instead of ethyl iodide. The product was recrystal-
lized from hot water to give white crystals:23 yield 55%; mp
171-172 °C; 1H-NMR (D2O) δ (ppm) 2.78 (dd, 2H, C-CH2-Se),
3.7 (s, 2H, Ar-CH2-Se), 3.91 (t, 1H, N-CH-CO), 7.12 (m, 5H,
Ar-H); 13C-NMR (D2O), δ (ppm) 27.8 (H-CH2-Se), 31.6 (Ar-CH2-
Se), 56.9 (N-CH-CO), 130.1 (p-Ar-C), 131.77 (o-Ar-C), 133.37
(m-Ar-C), 139.8 (Ar-C-CH2-Se), 182.32 (CH-CO-CO).
Se-(4-Meth ylben zyl)selen ocystein e [L-for m (9) a n d D-
for m (10)]. Se-(4-Methylbenzyl)selenocysteine was synthe-
sized according to method B, using 6 mmol of R-chloro-p-xylene
instead of ethyl iodide. To obtain both the D- and L-isomers
of the selenocysteine conjugates, 1.5 mmol of D- or L-seleno-
cystine was used, respectively. The products were recrystal-
lized by hot water to give white crystals.
2H, Se-CH2-C), 3.3 (t, 1H, N-CH-CO), 7-7.5 (m, 5H, m-,o-Ar-
H); 13C-NMR (D2O) δ (ppm) 21.586 (CH-CH2-Se), 35.207 (CH3-
Ar), 57.105 (N-CH-CO), 126.718 (o-Ar-C), 131.565 (o-Ar-C),
134.63 (Ar-C-Se), 139.46 (p-Ar-C), 182.337 (CH-CO-OH). Anal.
Calcd (C10H13NO2Se): C, 46.52; H, 5.08; N, 5.43. Found: C,
46.40; H, 5.16; N, 5.25.
Se-(4-Met h ylp h en yl)-D-selen ocyst ein e: yield 35%; mp
189 °C; 1H-NMR (D2O) δ (ppm) 2.25 (s, 3H, CH3-Ar), 3.05 (dd,
2H, Se-CH2-C), 3.3 (t, 1H, N-CH-CO), 7-7.5 (m, 4H, m-,o-Ar-
H); 13C-NMR (D2O) δ (ppm) 21.59 (CH-CH2-Se), 35.22 (CH3-
Ar), 57.12 (N-CH-CO), 131.57 (o-Ar-C), 134.64 (o-Ar-C), 139.47
(Ar-C-Se), 182.34 (CH-CO-OH), 199.61 (p-Ar-C). Anal. Calcd
(C10H13NO2Se): C, 46.52; H, 5.08; N, 5.43. Found: C, 46.31;
H, 5.12; N, 5.33.
Se-(4-Meth oxyp h en yl)-L-selen ocystein e (17). The same
procedure as for the preparation of Se-(4-methylphenyl)-
selenocysteine was employed using 1.5 mmol of 4-methoxy-
diphenyl diselenide and 6 mmol of â-chloro-L-alanine. The
product was recrystallized from hot water to give white
crystals: yield 41.5%; mp 173 °C; 1H-NMR (D2O) δ (ppm) 3.1
(dd, 2H, Se-CH2-C), 3.3 (t, 1H, N-CH-CO), 3.7 (s, 3H, CH3O-
Ar), 6.75-7.5 (m, 4H, m-,o-Ar-H); 13C-NMR (D2O) δ (ppm)
35.905 (CH-CH2-Se), 56.87 (N-CH-CdO), 57.036 (Ar-OCH3),
116.549 (m-Ar-C), 120.959 (o-Ar-C), 136.949 (Ar-C-Se), 160.146
(p-Ar-C), 182.333 (CH-CO-OH). Anal. Calcd (C10H13NO3Se):
C, 43.57; H, 4.98; N, 4.90. Found: C, 43.81; H, 4.78; N, 5.11.
Se-(4-Ch lor op h en yl)-L-selen ocystein e (18). The same
procedure as for the preparation of Se-(4-methylphenyl)-
selenocysteine was employed, using 1.5 mmol (571.5 mg) of
4-chlorodiphenyl diselenide. The product was recrystallized
from hot water to give white crystals: yield 25.3%; mp 182
°C; 1H-NMR (D2O) δ (ppm) 3.1 (dd, 2H, Se-CH2-C), 3.35 (t, 2H,
C-CH-N), 7.15-7.55 (m, 4H, o-,m-Ar-H); 13C-NMR (D2O) δ
(ppm) 35.145 (CH-CH2-Se), 57.047 (N-CH-CdO), 129.052 (m-
Ar-C), 130.685 (o-Ar-C), 134.307 (Ar-C-Se), 135.695 (p-Ar-C),
182.213 (CH-CO-OH). Anal. Calcd (C9H10ClNO2Se): C, 38.77;
H, 3.63; N, 4.96. Found: C, 38.80; H, 3.62; N, 5.03.
Se-(4-Met h ylb en zyl)-L-selen ocyst ein e: yield 39%; mp
1
175 °C; H-NMR (D2O) δ (ppm) 2.2 (s, 3H, Ar-CH3), 2.7 (dd,
2H, C-CH2-Se), 3.3 (t, 1H, N-CH-CO), 3.7 (s, 2H, Ar-CH2-Se),
7.1 (m, 4H, Ar-H); 13C-NMR (D2O) δ (ppm) 21.77 (Ar-CH3),
28.23 (CH-CH2-Se), 31.25 (Ar-CH2-Se), 59.92 (N-CH-CdO),
130.28 (o-Ar-C), 130.81 (m-Ar-C), 138.35 (Ar-C-CH2-Se), 138.348
(p-Ar-C), 182.42 (CH-CO-OH). Anal. Calcd (C11H15NO2Se):
C, 48.54; H, 5.55; N, 5.15. Found: C, 48.97; H, 5.63; N, 4.99.
Se-(4-Meth ylben zyl)-D-selen ocystein e: yield 42%; mp
1
177 °C; H-NMR (D2O) δ (ppm) 2.3 (s, 3H, Ar-CH3), 2.7 (dd,
2H, C-CH2-Se), 3.35 (t, 1H, N-CH-CO), 3.7 (s, 2H, Ar-CH2-Se),
7.15 (m, 4H, Ar-H).
Se-(4-Ch lor oben zyl)-L-selen ocystein e (12). Se-(4-Chlo-
robenzyl)-L-selenocysteine was synthesized according to method
B, using 6 mmol of 4-chlorobenzyl chloride instead of ethyl
iodide. The product was recrystallized from hot water giving
white crystals: yield 40%; mp 168.5 °C; 1H-NMR (D2O) δ (ppm)
2.7 (dd, 2H, C-CH2-Se), 3.3 (t, 1H, N-CH-CO), 3.7 (s, 2H, Ar-
CH2-Se), 7.2 (s, 4H, Ar-H); 13C-NMR (D2O) δ (ppm) 27.76 (CH-
CH2-Se), 31.57 (Ar-CH2-Se), 56.94 (N-CH-CdO), 130.087 (m-
Ar-C), 131.77 (o-Ar-C), 133.37 (Ar-C-CH2-Se), 139.82 (p-Ar-
C), 182.22 (CH-CO-OH). Anal. Calcd (C10H12ClNO2Se): C,
41.3; H, 4.15; N, 4.68. Found: C, 41.05; H, 4.13; N, 4.79.
Se-(3,4-Dich lor oben zyl)-L-selen ocystein e (13). Se-(3,4-
Dichlorobenzyl)selenocysteine was synthesized according to
method B, using 6 mmol of 3,4-dichlorobenzyl chloride instead
of ethyl iodide. The product was recrystallized from hot water
giving white crystals: yield 45%; mp 159.5 °C; 1H-NMR (D2O)
δ (ppm) 2.7 (dd, 2H, C-CH2-Se), 3.3 (t, 1H, N-CH-CO), 3.6 (s,
2H, Ar-CH2-Se), 7.1 (d, 1H, m-Ar-H), 7.2-7.4 (m, 2H, o-Ar-
H); 13C-NMR (D2O) δ (ppm) 27.44 (CH-CH2-Se), 32.304 (Ar-
CH2-Se), 57.056 (N-CH-CdO), 129.946 (o-Ar-C), 131.61 (m-Ar-
C), 131.87 (m-Ar-C-Cl), 131.875 (Ar-C-CH2-Se), 181.895 (CH-
CO-OH). Anal. Calcd (C10H11Cl2NO2Se): C, 36.68; H, 3.45;
N, 4.25. Found: C, 36.72; H, 3.39; N, 4.28.
Bioch em ica l Assa ys. P r ep a r a tion of Ra t Kid n ey Cy-
tosolic F r a ction . Male Wistar rats (160-180 g) obtained
from Harlan-SD (Zeist, The Netherlands) were fed on a
standard laboratory diet. Unfasted animals were sacrificed
by decapitation, and the kidneys were isolated. After removal
of the ligament, a 50% (w/v) homogenate was prepared in ice-
cold potassium phosphate buffer (50 mM, pH 7.4) using a
Potter-Elvehjem homogenizer. The homogenate was then
centrifuged at 4 °C for 30 min at 9000g to obtain the S9
fraction. Subsequently, the S9 fraction was centrifuged at
100000g for 60 min to give the cytosolic fraction. This fraction
was dialyzed overnight against a 20-fold excess of potassium
phosphate (50 mM, pH 7.4), lyophilized, and finally stored at
-80 °C until use.25
Se-P h en yl-L-selen ocystein e (14) (Meth od C). Diphenyl
diselenide (1.5 mmol) was dissolved in 8 mL of 0.5 N NaOH
and 2 mL of ethanol. At 0 °C, 15 mmol (0.4 g) of NaBH4 was
added, and the mixture was allowed to reach room tempera-
ture. When the color changed from yellow to colorless, the
reaction mixture was cooled to 0 °C, and subsequently 4 mL
of 2 N NaOH and 7.88 mmol of â-chloroalanine were added.
After stirring for 3 h at room temperature, the product was
precipitated by adding concentrated HCl until pH ) 7. The
product was recrystallized from hot water giving white crystals:
24 yield 61%; mp 178.5 °C; 1H-NMR (D2O) δ (ppm) 3.2 (dd, 2H,
C-CH2-Se), 4.05 (t, 1H, N-CH-CO), 7.1 (m, 3H, m,p-Ar-H), 7.4
(m, 2H, o-Ar-H); 13C-NMR (D2O) δ (ppm) 27.832 (CH-CH2-Se),
53.57 (N-CH-CdO), 128.22 (p-Ar-C), 129.8 (o-Ar-C), 130.879
(m-Ar-C), 135.24 (Ar-C-Se), 171.249 (CH-CO-OH).
Purified cysteine conjugate â-lyase was purified from rat
26
renal cytosol as described by Yamauchi et al.
The purified
protein has a molecular weight of ca. 90-95 kDa and is the
major â-lyase in rat renal cytosol.12
In cu ba tion s. The lyophilized rat renal cytosolic fraction
was reconstituted in a Tris-HCl buffer (pH 8.6, 50 mM; final
protein concentration 0.2 mg/mL as determined with the Bio-
Rad assay) and preincubated at 37 °C for 5 min. Because
aromatic and benzylic selenocysteine conjugates had a lower
solubility than their corresponding sulfur analogues, it re-
quired the use of an ultrasonic bath for prolonged times to
prepare a stock solution of 1.5 mM of these substrates.
Incubations were started by addition of substrate solution (in
final concentrations varying from 1 to 0.05 mM) to give a final
volume of 300 µL in the presence of 0.1 mM R-keto-γ-
methiolbutyric acid (KMB). No decomposition was observed
under these conditions. The mixtures were incubated at 37
°C for 10 min. The incubations were terminated by addition
of 1 mL of o-phenyldiamine (OPD) solution (12 mM OPD in 3
M HCl).
Se-(4-Meth ylp h en yl)selen ocystein e [L-for m (15) a n d
D-for m (16)]. Se-(4-Methylphenyl)selenocysteine was syn-
thesized according to method C, using 1.5 mmol of 4-methyl-
diphenyl diselenide instead of diphenyl diselenide and 6 mmol
of â-chloroalanine (D- or L-form). After the mixture stirred for
3 h at room temperature, concentrated HCl was added until
pH ) 6-7. The products were precipitated as a white solid
and recrystallized by hot water to give white crystals.
Se-(4-Meth ylp h en yl)-L-selen ocystein e: yield 39%; mp
Incubations and analysis with purified â-lyase were per-
formed by the same procedures as described above, except that
incubations were performed in the presence of 0.2 µg/mL
purified â-lyase instead of 0.2 mg/mL cytosolic protein.
1
187 °C; H-NMR (D2O) δ (ppm) 2.3 (s, 3H, CH3-Ar), 3.05 (dd,