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M. Sßentürk et al. / Bioorg. Med. Chem. 17 (2009) 3207–3211
was dissolved in ethanol, and heated with Na2S2O4 (6.0 g,
34.48 mmol) for 20 min. The separated precipitate was collected
on a filter and was crystallized from hexane to give the dimeric
propofol, dipropofol (2.94 g, 74%) as a white solid. 1H NMR
(400 MHz, CDCl3, d, ppm): 7.33 (s, 4H, Haryl), 4.99 (m, 2H, OH),
3.35 (h, J = 6.8 Hz, 4H, CH), 1.46 (d, J = 6.8 Hz, 12H, CH3). 13C NMR
(100 MHz, CDCl3, d, ppm): 149.46, 135.13, 134.19, 122.74, 27.77,
23.21. Anal. Calcd for C24H34O2: C, 81.31; H, 9.67. Found: C,
79.62; H, 9.70.
effects of 2,6-dimethylphenol (DMP), 2,6-diisopropylphenol (DIP,
Propofol), 2,6-di-tbutylphenol (DTP), butylated hydroxytolu-
ene(BHT), butylated hydroxyanisole (BHA), vanillin, guaiacol,
di(2,6-dimethylphenol) (Di-DMP), di(2,6-diisopropylphenol) (Di-
DIP, dipropofol), di(2,6-di-tbutylphenol) (Di- DTP), and acetazola-
mide were examined. All compounds were tested in triplicate at
each concentration used. Different inhibitor concentrations were
used. HCA-I enzyme activities were measured for 2,6-dimethyl-
phenol (DMP)(19.95–199.5
pofol) (13.66–95.6 M), 2,6-di-tbutylphenol (DTP) (0.05–0.2
butylated hydroxytoluene(BHT) (0.047–0.18 M), butylated
hydroxyanisole (BHA) (10.3–51.5 M), vanillin (5.1–51.2 M),
guaiacol (5.48–43.9 M), di(2,6-dimethylphenol) (Di-DMP) (10–
50 M), di(2,6-diisopropylphenol) (Di-DIP, dipropofol) (6.87–
68.78 M), di(2,6-di-t-butylphenol) (Di-DTP)(2.96–29.63 M) and
acetazolamide (2.6–13.4 M) at cuvette concentrations and HCA-
II enzyme activities were measured for 2,6-dimethylphenol
(DMP)(9.97–99.75 M), 2,6-diisopropylphenol (DIP, propofol)
(3.03–21.24 M), 2,6-di-tbutylphenol (DTP) (0.09–0.36 M), butyl-
ated hydroxytoluene(BHT) (0.049–0.396 M), butylated hydroxy-
anisole (BHA) (0.078–0.39 M), vanillin (0.17–1.7 M), guaiacol
(0.27–2.68 M), di(2,6-dimethylphenol) (Di-DMP) (0.37–
1.85 M), di(2,6-diisopropylphenol) (Di-DIP, dipropofol) (0.163–
1.637 M), di(2,6-di-tbutylphenol) (Di-DTP)(0.027–0.269 M) and
acetazolamide (0.065–0.33 M) at cuvette concentrations. Control
lM), 2,6-diisopropylphenol (DIP, Pro-
l
lM),
4.2.3. Synthesis of di(2,6-di-t-butylphenol) (Di-DTP) 10
DTP 3 (5.0 g, 24.27 mg) was dissolved in CH2Cl2 (100 mL) and
stirred with CuCl(OH)ꢁTMEDA (160 mg, 0.34 mmol) for 24 h at
room temperature. The reaction product was extracted with AcOEt.
After removal of solvent, the reactant was dissolved in ethanol, and
heated with Na2S2O4 (6.0 g, 34.48 mmol) for 2 h. The precipitate
was collected on a filter and crystallized from hexane to give
di(2,6-di-t-butylphenol) (3.58 g, 72%) as a white solid. 1H NMR
(400 MHz, CDCl3, d, ppm): 7.33 (s, 4H, Haryl), 5.22 (s, 2H, OH),
1.52 (s, 36H, CH3), 13C NMR (100 MHz, CDCl3, d, ppm): 153.06,
136.15, 134.18, 124.38, 34.70, 30.61. Anal. Calcd for C28H42O2: C,
81.90; H, 10.31. Found: C, 81.50; H, 10.27.
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
4.3. Purification of carbonic anhydrase isozymes from human
erythrocytes by affinity chromatography
l
l
l
cuvette activity in the absence of inhibitor was taken as 100%. For
each inhibitor an Activity (%)-[Inhibitor] graphs were drawn. To
determine Ki values, three different inhibitor concentrations were
tested. In these experiments, 4-nitrophenylacetate was used as
substrate at five different concentrations (0.15–0.75 mM). The
Lineweaver–Burk curves were drawn.17
Erythrocytes were purified from fresh human blood obtained
from the Blood Centre of the Research Hospital at Atatürk Univer-
sity. The blood samples were centrifuged at 1500 rpm for 15 min
and the plasma and buffy coat were removed. The red cells were
isolated and washed twice with 0.9% NaCl, and hemolyzed with
1.5 volumes of ice-cold water. The ghost and intact cells were re-
moved by centrifugation at 20,000 rpm for 30 min at 4 °C. The
pH of the hemolysate was adjusted to 8.7 with solid Tris.9a Firstly,
Sepharose-6B oxidized by KMnO4 and subsequently activated by
SOCl2. After that, aniline attached to the activated gel as a spacer
arm and finally diazotized sulfanilamide clamped to the para posi-
tion of aniline molecule as ligant. The hemolysate was applied to
the prepared Sepharose 6B-aniline-sulfanylamide affinity column
equilibrated with 25 mM Tris–HCl/0.1 M Na2SO4 (pH 8.7). The
affinity gel was washed with 25 mM Tris–HCl/22 mM Na2SO4 (pH
8.7). The human carbonic anhydrase (hCA I and hCA II) isozymes
were eluted with 1 M NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M
CH3COONa/0.5 M NaClO4 (pH 5.6), respectively. All procedures
were performed at 4 °C.12
4.6. Protein determination
Protein during the purification steps was determined spectro-
photometrically at 595 nm according to the Bradford method,
using bovine serum albumin as the standard.15
4.7. SDS polyacrylamide gel electrophoresis
SDS polyacrylamide gel electrophoresis was performed after
purification of the enzymes. It was carried out in 10% and 3% acryl-
amide for the running and the stacking gel, respectively, containing
0.1% SDS according to Laemmli procedure. A 20 mg sample was ap-
plied to the electrophoresis medium. Gels were stained for 1.5 h in
0.1% Coommassie Brilliant Blue R-250 in 50% methanol and 10%
acetic acid, then destained with several changes of the same sol-
vent without the dye.16 The electrophoretic pattern was photo-
graphed (data not shown).
4.4. Hydratase activity assay
Carbonic anhydrase activity was assayed by following the
hydration of CO2 according to the method described by Wilbur
and Anderson.13 CO2-hydratase activity as an enzyme unit (EU)
was calculated by using the equation (to ꢀ tc/tc) where t0 and tc
are the times for pH change of the non-enzymatic and the enzy-
matic reactions, respectively.
Acknowledgments
This research was financed by grants from of the Scientific and
Technological Research Council of Turkey (Project No: TBAG-HD-
107T882) to IG, and by an EU project of the 6th framework pro-
gramme (DeZnIT project) to CTS.
4.5. Esterase activity assay
Carbonic anhydrase activity was assayed by following the
change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to
4-nitrophenylate ion over a period of 3 min at 25 °C using a spec-
trophotometer (CHEBIOS UV–vis) according to the method de-
scribed by Verpoorte et al.14 The enzymatic reaction, in a total
volume of 3.0 mL, contained 1.4 mL 0.05 M Tris–SO4 buffer (pH
7.4), 1 mL 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL en-
zyme solution. A reference measurement was obtained by prepar-
ing the same cuvette without enzyme solution. The inhibitory
References and notes
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