Protein Nitration
FULL PAPER
benzyl chloride (0.82 mL, 6.09 mmol) was added dropwise and the mix-
ture was stirred overnight. The solution was filtered through a celite pad
and solvent was evaporated to obtain brown colored oil, which was puri-
fied by flash chromatography with pre-loaded silica gel cartridges using
petroleum ether and ethyl acetate as eluent. Yield: 0.87 g (61%);
1H NMR (CDCl3): d=3.21 (s, 3H), 3.68 (s, 3H), 4.07 (s, 2H), 6.57 (s,
1H), 6.68–6.70 (d, J=7.6 Hz, 1H), 6.73–6.75 (d, J=8.4 Hz, 1H), 6.83 (s,
1H), 7.09–7.14 ppm (m, 2H); 13C NMR (CDCl3): d=33.1, 40.2, 55.2,
113.4, 113.7, 121.1, 122.5, 129.5, 129.6, 139.3, 140.3, 159.6 ppm; ESI-MS
(HRMS): m/z calcd for C12H14N2OS [M+Na]+: 257.0725; found:
257.0724.
1H); 13C NMR (CDCl3): d=13.7, 15.0, 20.9, 39.2, 52.5, 106.8, 161.9,
162.7, 165.7 ppm; ESI-MS: m/z calcd for C9H14N2OS [M+H]+: 199.08;
found: 199.33.
General procedure for the reaction of inhibitors with PN: To the ice-
cooled solution of the inhibitor in water was added alkaline solution of
excess peroxynitrite (PN) in portions (three times with 3–4 h intervals)
and stirred vigorously for 12 h at room temperature. The reaction mix-
ture was extracted with dichloromethane 3–4 times to remove the un-
reacted starting material and the nonpolar impurities. The aqueous solu-
tion was neutralized with 1n HCl and the solvent was evaporated by lyo-
philization. The solid obtained was dissolved in minimum amount of dry
methanol to remove the excess NaCl. The solution was filtered and the
solvent was evaporated to obtain the product in a reasonably pure form.
Synthesis of compound 19: A solution of nBuLi (3.80 mL, 1.6m in
hexane) was added by syringe to a cooled (À788C) solution of N-methyl-
imidazole (0.48 mL, 6.09 mmol) in freshly distilled THF (50 mL). The re-
action mixture was stirred at this temperature for 30 min and then al-
lowed to warm to room temperature at which time finely powdered ele-
mental selenium (0.72 g, 9.12 mmol) was added under a brisk flow of dry
nitrogen gas and stirred for 5 h. Then 3-methoxybenzyl chloride
(0.82 mL, 6.09 mmol) was added dropwise and the mixture was stirred
overnight. The solution was filtered through a celite pad and solvent was
evaporated to obtain brown colored oil, which was purified by flash chro-
matography with pre-loaded silica gel cartridges using petroleum ether
and ethyl acetate as eluent. Yield: 0.94 g (55%); 1H NMR (CDCl3): d=
3.11 (s, 3H), 3.60 (s, 3H), 4.01 (s, 2H), 6.43 (s, 1H), 6.58–6.60 (d, J=
7.6 Hz, 1H), 6.64–6.66 (d, J=8.4 Hz, 1H), 6.82 (s, 1H), 7.02–7.06 ppm
(m, 2H); 13C NMR (CDCl3): d=33.2, 34.2, 55.2, 113.2, 113.5, 120.9, 123.0,
129.5, 130.6, 134.6, 140.3, 159.5 ppm; 77Se NMR (CDCl3): d=282 ppm.
ESI-MS (HRMS): m/z calcd. for C12H14N2OSe [M+Na]+: 305.0169;
found: 305.0124.
Compound 1 with excess PN: 2-Mercapto-1-methylimidazole (1; 0.25 g,
2.19 mmol); product: colorless oil; yield: 0.10 g (57%); 1H NMR (D2O):
d=3.66 (s, 3H), 7.17 (s, 2H), 8.39 ppm (s, 1H); 13C NMR (D2O): d=
35.2, 119.3, 122.7, 134.8 ppm; ESI-MS: m/z calcd for C4H6N2 [M+H]+:
83.06; found: 82.99.
Compound
2 with excess PN: 1-Methylimidazoleselone (2; 0.10 g,
0.62 mmol); product: colorless oil; yield: 28.5 mg (56%); 1H NMR
(D2O): d=3.65 (s, 3H), 7.17 (s, 2H), 8.40 ppm (s, 1H); 13C NMR (D2O):
d=35.3, 119.3, 122.8, 134.8 ppm; 77Se NMR (D2O): d=1314 ppm; ESI-
MS: m/z calcd for C4H6N2 [M+H]+: 83.06; found: 83.00.
Compound
3 with excess PN: 6-n-Propyl-2-thiouracil (3; 0.25 g,
1.46 mmol); product: colorless oil; yield: 83 mg (41%); 1H NMR (D2O):
d=0.66–0.70 (t, J=8.0 Hz, 3H), 1.40–1.45 (m, 2H), 2.40–2.43 (t, J=
8.0 Hz, 2H), 6.33 (s, 1H), 8.89 ppm (s, 1H); 13C NMR (D2O): d=12.2,
20.0, 33.5, 113.7, 152.0, 158.0, 160.7 ppm; ESI-MS: m/z calcd for
C7H10N2O [M+H]+: 139.07; found: 138.88.
Synthesis of compounds 20 and 22: Powdered potassium hydroxide
(1.37 g, 24.61 mmol) was added to a stirred suspension of 4 (0.70 g,
4.92 mmol) in acetone (25 mL). After 15 min, methyl iodide (1.23 mL,
19.69 mmol) was added to the reaction mixture, which was then stirred
vigorously for 1 h. The acetone solution was then transferred to a sepa-
rating funnel with excess benzene. The organic layer was washed with
water (2ꢁ50 mL) and dried over sodium sulfate. The solvent was evapo-
rated to obtain white semi-solid compound which was purified by flash
chromatography with pre-loaded silica gel cartridges using ethyl acetate
and petroleum ether as eluent. The expected compounds eluted with 30
and 45% ethyl acetate, respectively. For the first fraction, the solvent was
evaporated to obtain 20 as colorless oil. Yield: 0.17 g (21%); 1H NMR
(CDCl3): d=2.35 (s, 3H), 2.54 (s, 3H), 3.94 (s, 3H), 6.22 ppm (s, 1H);
13C NMR (CDCl3): d=13.0, 22.7, 52.5, 100.8, 166.5, 168.5, 170.3 ppm;
ESI-MS (HRMS): m/z calcd for C7H10N2OS [M+Na]+: 193.0412; found:
193.0408. For the second fraction, the solvent was evaporated to obtain
22 as a white solid. Yield: 0.27 g (32%); 1H NMR (CDCl3): d=2.23 (s,
3H), 2.57 (s, 3H), 3.49 (s, 3H), 6.05 ppm (s, 1H); 13C NMR (CDCl3): d=
15.2, 24.0, 30.1, 107.5, 162.2, 162.6, 162.7 ppm; ESI-MS: m/z calcd for
C7H10N2OS [M+H]+; 171.05; found: 170.84.
Compound
4
with excess PN: 6-Methyl-2-thiouracil (4; 0.25 g,
1.77 mmol); product: colorless oil; yield: 88 mg (46%); 1H NMR (D2O):
d=2.15 (s, 3H), 6.31 (s, 1H), 8.88 ppm (s, 1H); 13C NMR (D2O): d=
17.9, 99.1, 114.4, 150.8, 154.6, 160.6 ppm; ESI-MS: m/z calcd for
C5H6N2O [M+H]+: 111.04; found: 110.91.
Compound 5 with excess PN: 1,3-Dimethylimidazolethione (5; 0.25 g,
1.95 mmol); product: colorless oil; yield: 0.10 g (56%); 1H NMR (D2O):
d=3.64 (s, 6H), 7.16 (s, 2H), 8.39 ppm (s, 1H); 13C NMR (D2O): d=
35.4, 123.2, 136.4 ppm; ESI-MS: m/z calcd for C5H9N2 [M]+: 97.07;
+
found: 96.95.
Compound 6 with excess PN: 1,3-Dimethylimidazoleselone (6; 0.25 g,
1.42 mmol); product: colorless oil; yield: 80 mg (58%); 1H NMR (D2O):
d=3.66 (s, 6H), 7.18 (s, 2H), 8.44 ppm (s, 1H); 13C NMR (D2O): d=
35.5, 123.2, 136.4 ppm; 77Se NMR (D2O): d=1314 ppm; ESI-MS
(HRMS): m/z calcd for C5H9N2 [M]+: 97.0760; found: 97.0771.
+
Compound 8 with excess PN: Compound 8 (0.25 g, 0.88 mmol); product:
colorless oil; yield: 93 mg (52%); 1H NMR (D2O): d=3.69 (s, 3H), 3.74
(s, 3H), 5.21 (s, 2H), 6.85–6.91 (m, 3H), 7.25–7.32 ppm (m, 3H);
13C NMR (D2O): d=35.7, 52.6, 55.4, 114.1, 114.7, 121.1, 122.2, 123.8,
130.6, 135.1, 136.0, 159.4 ppm; 77Se NMR (D2O): d=1314 ppm; ESI-MS:
Synthesis of compounds 21 and 23: Powdered potassium hydroxide
m/z calcd for C12H15N2 [M]+: 203.11; found: 202.85.
+
(0.50 g, 8.81 mmol) was added to
a stirred suspension of 3 (0.30 g,
1.76 mmol) in acetone (20 mL). After 15 min, methyl iodide (0.44 mL,
7.05 mmol) was added and the reaction mixture was stirred vigorously
for 1 h. The solution was then transferred to a separating funnel with
excess benzene. The organic layer was washed with water (2ꢁ50 mL)
and dried over sodium sulfate. The solvent was evaporated to obtain col-
orless oil which was purified by flash chromatography with pre-loaded
silica gel cartridges using ethyl acetate and petroleum ether as eluent.
The expected compounds eluted with 25 and 40% ethyl acetate, respec-
tively. For the first fraction, the solvent was evaporated to obtain 21 as
Synthesis of peroxynitrite (PN): Peroxynitrite was synthesized by follow-
ing the literature method with minor modifications.[34] A solution of 30%
(ꢀ8.8m) H2O2 (5.7 mL) was diluted to 50 mL with water, chilled to
about 48C in an ice/water mixture, added to NaOH (5n, 30 mL) and
DTPA (0.04m 5 mL) in NaOH (0.05n) with gentle mixing, and then di-
luted to a total volume of 100 mL. The concentration of H2O2 in the final
solution was 0.5m with the pH ranging from 12.5 to 13.0. The buffered
H2O2 was stirred vigorously with an equimolar amount of isoamyl nitrite
(0.05m or 6.7 mL) for 3–4 h at room temperature. The reaction was moni-
tored by withdrawing aliquots at an interval of 15 or 30 min, and assaying
for peroxynitrite at 302 nm by UV/Vis spectrophotometer. When the
1
colorless oil. Yield: 91 mg (26%); H NMR (CDCl3): d=0.92–0.96 (t, J=
8.0 Hz, 3H), 1.65–1.74 (m, 2H), 2.53 (s, 3H), 2.54–2.58 (t, J=8.0 Hz,
2H), 3.94 ppm (s, 3H), 6.20 (s, 1H); 13C NMR (CDCl3): d=12.7, 13.0,
20.8, 38.8, 52.5, 100.3, 168.5, 170.2, 170.3 ppm; ESI-MS: m/z calcd for
C9H14N2OS [M+H]+: 199.08; found: 198.86. For the second fraction, the
solvent was evaporated to obtain 23 as a faint yellow solid. Yield: 0.13 g
yield of peroxynitrite reached
a maximum, the aqueous phase was
washed with (3ꢁ100) volume of dichloromethane, chloroform, and
hexane in a separatory funnel to remove the contaminating isoamyl alco-
hol and isoamyl nitrite. The unreacted H2O2 was removed by passing the
aqueous phase through a column filled with 25 g of granular MnO2. The
concentration of the stock solution of peroxynitrite was measured after
1
(36%); H NMR (CDCl3): d=0.92–0.96 (t, J=8.0 Hz, 3H), 1.63–1.74 (m,
2H), 2.40–2.44 (t, J=8.0 Hz, 2H), 2.55 (s, 3H), 3.47 (s, 3H), 6.02 ppm (s,
Chem. Eur. J. 2010, 16, 1175 – 1185
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1183