Simple methanesulfonates are hydrolyzed by the sulfatase carbonic anhydrase activity

Article abstract of DOI:10.3109/14756366.2011.637202

The possible sulfatase activity of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms have been investigated with a series of synthesized methanesulfonate derivatives of phenols. Four α-CA isozymes, i.e. hCA I, hCA II, hCA IV and hCA VI (h=human isoform), were included in the study. We evidenced that the original sulfonate esters are being hydrolyzed effectively to the corresponding phenols which there after act as CA inhibitors. The KI-s of these compounds ranged from 10.24 to 4012 μM against hCA I, 0.10 to 35.42 μM against hCA II, 0.49 to 45.06 μM against hCA IV and 3.27 to 608 μM against CA VI, respectively. The relevant sulfatase activity of CA with these esters is amazing considering the fact that 4-nitrophenyl-sulfate, an activated ester, is not a substrate of these enzymes.

Full text of DOI:10.3109/14756366.2011.637202

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(6): 880–885
2012 Informa UK, Ltd.
©
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.637202
ꢀ
eseaꢀꢁh aꢀTIꢁLe
Simple methanesulfonates are hydrolyzed by the sulfatase
carbonic anhydrase activity
1
,2
1
1
3
Elif Akın Kazancıoğlu , Murat Güney , Murat Şentürk , and Claudiu T. Supuran
1
Department of Chemistry, Art and Science Faculty, Ağrı Ibrahim Çeçen University, Ağrı, Turkey,
Department of Chemistry, Science Faculty, Atatürk University, Erzurum, Turkey, and Polo Scientifico,
2
3
Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Florence, Italy
abꢂtrꢃct
The possible sulfatase activity of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms have been investigated with a
series of synthesized methanesulfonate derivatives of phenols. Four α-CA isozymes, i.e. hCA I, hCA II, hCA IV and hCAVI
(
h=human isoform), were included in the study. We evidenced that the original sulfonate esters are being hydrolyzed
effectively to the corresponding phenols which there after act as CA inhibitors. The K -s of these compounds ranged
I
from 10.24 to 4012 µM against hCA I, 0.10 to 35.42 µM against hCA II, 0.49 to 45.06 µM against hCA IV and 3.27 to 608
µM against CA VI, respectively. The relevant sulfatase activity of CA with these esters is amazing considering the fact
that 4-nitrophenyl-sulfate, an activated ester, is not a substrate of these enzymes.
Kꢄywordꢂ: Methanesulfonate, phenol, carbonic anhydrase, hCA II
Introduction
as edema, glaucoma, obesity, cancer, epilepsy and
osteoporosis .
1
–8

e carbonic anhydrases (CA, EC. 4.2.1.1) are a family
of metalloenzymes, which participate primarily to the
maintenance of pH homeostasis, catalyzing the revers-
Our groups recently investigated the interaction of CA
I and II isozymes with several types of phenols, such as
the simple phenol, hydroxy-/methoxysubstituted benzoic
acids as well as di-/tri-methoxy benzenes, antioxidant
bisphenols and several of its substituted derivatives, e.g.
ible hydration of CO in a two-step reaction, to yield
2
−
+
HCO and H (the physiological reaction in which CAs
3
1
–3).
participate, reaction 1 in Scheme 1 e enzyme plays
an important role in many physiological processes, e.g.
is coupled with anion exchangers to form metabolons
which enhance the chloride and bicarbonate transport
across cell membranes . At least 16 CA isozymes have
been described up to now in mammals . Some of the
9–11
salicyclates and some of their derivatives . Here we
extend these earlier investigations to a series of phenolic
compounds, some of which are widely used as antioxidant
food additives or as drugs and which are reported to pos-
sess anticancer, anti-carcinogenic, antimutagenic, antibac-
1
–3
4
5–11
isozymes are cytosolic (CA I, CA II, CA III, CA VII and
CA XIII), two are mitochondrial (CA VA and CA VB), one
is secreted (CA VI), and others are membrane bound
terial, antiviral or anti-inflammatory activities . Phenol 7,
phenolic compounds 8–16 are widely used prodrugs or
drugs. Vanillin (14) is another phenol derivative which is
frequently used in food and drug industry due to its aroma.
However, vanillin is used not only as a flavouring ingredient
but also to mask undesirable off-flavours developed during
1
–8
(
CA IV, CA IX, CA XII, CA XIV and CA XV ). Many such
CA isozymes involved in these processes are important
therapeutic targets with the potential to be inhibited/
activated for the treatment of a range of disorders such
12
storage of products susceptible to oxidative degradation .
Address for Correspondence: Murat Güney, Department of Chemistry, Art and Science Faculty, Ağrı Ibrahim Çeçen University, Ağrı, Turkey.
Tel: +90-442-2314414. Fax: +90-442-2360948. E-mail: m.guney2000@gmail.com; Murat Şentürk, Department of Chemistry, Art and Science
Faculty, Ağrı Ibrahim Çeçen University, Ağrı, Turkey. Tel: +90-442-2314414. Fax: +90-442-2360948. E-mail: senturkm36@gmail.com;
Claudiu T. Supuran, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Florence, Italy.
Tel: +39-055-4573005. Fax: +39-055-4573385. E-mail: claudiu.supuran@unifi.it
(
Received 12 October 2011; revised 28 October 2011; accepted 29 October 2011)
8
80

Hydrolysis of methanesulfonates 881
Scheme 2. Reactions 11 and 12 catalyzed by α-CAs: 4-nitrophenyl
18
acetate/phosphate hydrolysis .
acetate 1, phsophorus for phosphate) with formation
of a transition state from which the 4-nitrophenoxide
is released. Considering the fact that CAs contain the
−
equivalent of a strong base (hydroxide ions, HO coordi-
nated to the zinc ion) at neutral pH, due to the powerful
activation of H O by the zinc ion from the active site cav-
2
Scheme 1. Reactions (1–10) catalyzed by α-CAs.
ity and the hydrophobic environment of the protein, in
principle, hydrolytic reactions 11–12 of Scheme 2 should
have the same mechanism as the hydrolysis catalyzed
Isoform hCA II, one of the most effective and best
studied isoenzyme, is not only a very effective catalyst
for the physiological reaction, but also shows some
catalytic versatility, participating in other hydrolytic
processes which presumably involve non-physiological
substrates. Some of these reactions include the hydration
2,18
by bases in solution. In these studies, Innocenti et al.
showed that the hydrolytic processes described by Eqs.
2
+
−
11–12 of Scheme 2, involve the active site Zn (OH) func-
tionality of the enzyme, that is, the same one responsible
of the CO hydration activity of α-CAs. Probably, com-
2
1
3
of cyanate to carbamic acid (reaction 2, Scheme 1 ), or
pounds 4–6 are hydrolyzed by carbonic anhydrase II in
the same way in the current study. It is interesting to note
here that the aromatic, methanesulfonate derivatives
investigated here, like the aromatic activated one (Ester
1) indeed act as substrates for CAs. e sulfatase activity
of this enzyme has been in fact discovered earlier with a
14
of cyanamide to urea (reaction 3, Scheme 1 ), the alde-
15
hyde hydration to gem-diols (reaction 4 ); the hydrolysis
1
6,17
of some carboxylic
or sulfonic acid esters (reactions
and 6 ), as well as other less investigated hydrolytic
processes in which aryl halides, chloroformates or sul-
1
8–22
5
1
8–22
19
fonyl chlorides
act as substrates, as described by eq.
cyclic sulfate ester as substrate, by Kaiser and Lo .
7
–9 of Scheme 1. A rather controversial issue regards
We report here an inhibition study of the four cata-
lytically active human isoforms hCA I, II, IV and VI with
compounds 4−16. ey incorporate methanesulfonate
derivatives in their molecules and scaffolds representing
thus an interesting starting point for different chemo-
types belonging to the CA inhibitors (CAIs). In fact, in an
the possible phosphatase activity of CA III, an isozyme
with very low CO hydration activity, which has originally
been reported to be a phosphatase (with 4-nitrophenyl
phosphate as substrate, reaction 10 in Scheme 1 ), but
subsequently this has been retracted , being considered
that the phosphatase activity is due to another protein
impurity present in the CA III preparations used in the
2
1
8–21
22
2
3,24
earlier study
we reported micromolar/submicromo-
lar inhibitors of the cytosolic isoforms hCA I and II with a
library of organic nitrates.
21
earlier investigations .
It is known that carboxylate/phosphate esters are
hydrolyzed by α-CAs, although 4-nitrophenyl-sulfate
was shown not to be a substrate for the cytosolic isoforms
hCA I, II and XIII . Recently, one of our groups reported
kinetic study on the hydrolysis of 4-nitrophenyl acetate
Mꢃtꢄriꢃlꢂ ꢃnd mꢄtꢅodꢂ
2,18
Phenol, biphenyl-4-ol, hydroquinone, pyrogallol, catechol,
vanillin, guaiacol, CNBr-activated Sepharose 4B, protein
assay reagents, p-aminobenzene sulfonamide L-tyrosine,
4-nitrophenyl acetate and chemicals for electrophoresis
were purchased from Sigma-Aldrich Co., Germany. All other
chemicals were analytical grade and obtained from Merck.
1
and phsophate in the presence of three cytosolic CA
18
isozymes, hCA I, hCA II and hCA XIII . In solution, these
esters are hydrolyzed by the nucleophilic attack of water
(or hydroxide ions) to the central atom (carbonyl CO for
©
2012 Informa UK, Ltd.

8
82 E.A. Kazancıoğlu et al.
Protein determination
Table 1. Inhibition data (K values, μM) against hCA I,
I
Protein quantity was determined spectrophotometrically
at 595 nm according to the Bradford method during the
purification steps, using bovine serum albumin as the
hCA II, hCA IV and hCA VI of compounds 4–16 and standard
sulfonamide inhibitors (EZA, ZNS and AZA) by an esterase
method with 4-nitrophenyl acetate as substrate.
2
5
Inhibitor
hCA I
27.36
48.54
22.14
10.24
19.54
10.8
hCA II
8.70
12.24
4.23
5.6
hCA IV
15.43
26.48
9.12
hCA VI
105.2
162.1
28.43
210
standard .
4
5
6
7
8
9
Sodium dodecyl sulphate (SDS) polyacrylamide gel
electrophoresis
SDS polyacrylamide gel electrophoresis was performed
after purification of the enzymes. It was carried out in
9.7
7.41
0.10
35.42
28.51
6.45
0.52
9.92
1.32
2.98
0.32
1.07
0.37
12.34
10.8
85.04
524
1
0% and 3% acrylamide for the running and the stack-
11
13
15
10
12
14
16
478.3
196.2
79.13
7.35
45.06
41.64
8.14
458.1
312.7
12.1
ing gel, respectively, containing 0.1% SDS according to
26
Laemmli procedure .
0.49
6.83
General procedure for synthesis of methanesulfonates
Triethylamine (each hydroxyde gruop in phenolic
compound 1 eq.) was added to a solution of pheno-
lic compound (1 eq.) and methanesulfonyl chloride
4012
55.7
10.94
1.84
608
3.27
60.23
3.75
3.56
217
EZA
ZNA
AZA
0.84
1.58
(each hydroxyde gruop in phenolic compound 1 eq.) in
14.8
38.45
0.578
2.42
dichloromethane (10 ml) in an ice bath and the resulting
mixture was stirred between 30 minutes and 2 h at room
temperature. Water was added to the mixture, which was
extracted with dichloromethane. e organic phase was
washed with water, brine, dried (Na SO ), and concen-
36.2
0.34
Mean from at least three determinations. Errors were in the range
of ± 3ꢀ of the reported value (data not shown).
AZA, acetazolamide; EZA, sulfonamides ethoxzolamide; hCA,
human erythrocyte carbonic anhydrase; ZNA, zonisamide.
2
4
trated. e obtained crude mesylates were crystallized
from dichloromethane/hexane (20:1). e correspond-
ing methanesulfonates 4–6, 11, 13, and 15 were obtained
in 80–98% yields. Detailed synthetic procedures for the
preparation of all derivatives can be found in Ref. Morita
diols in 100% yield in 20 min. Prior to analysing the prod-
ucts, the mixture was left for 2 h to be separated from CA
by decantation. e thermal denaturation of the enzyme
(2 min at 80°C) was also tested to ensure the release from
casting site of some possible tightly bound ligands.
2
7
et al. (2005) .
CA purification assay and kinetic studies
ꢀꢄꢂultꢂ ꢃnd diꢂcuꢂꢂion

e purification of hCA isozymes was performed with
a simple one step method by a Sepharose-4B-aniline-
sulfanilamide affinity column, as described earlier .
Inhibitoryeffectsofcompoundsphenylmethanesulfonate
e CA isozymes play important roles in different
tissues . It is known that CA has been purified many
7
,8
1–8
times from different organisms and the effects of vari-
ous chemicals, pesticides, anions, metal ions and drugs
(4), biphenyl-4-yl methanesulfonate (5), 1,4-phenylene
7
–17,29–33
dimethanesulfonate (6), phenol (7), biphenyl-4-ol (8),
hydroquinone (9), pyrogallol (10), benzene-1,2,3-triyl
trimethanesulfonate (11), catechol (12), 1,2-phenylene
dimethanesulfonate (13), vanillin (14), 4-formyl-2-
methoxyphenyl methanesulfonate (15), and guaiacol
have been investigated on its activity
. In this
study, CA I, II, IV and VI were purified from human
erythrocytes and serum by a simple procedure using
Sephrarose-4B-aniline-sulfanilamide affinity column.
e activity of the effluents was determined by the
(16) on these different isoenzymes. e activities of the
hydratase method, with CO as substrate and further
2
tested compounds (as K values) were calculated from
Lineweaver–Burk plots and are given in Table 1.
kinetic studies were performed using the esterase
activity method, with 4-nitrophenyl acetate (NPA) as
substrate. hCA I was purified, 102.4-fold with a specific
I
28
−
1
Enzyme reactions
Enzyme mediated synthesis of phenols: e reactions
were performed in the presence of hCA II in water at pH
activity of 873 EUmg and overall yield of 54% and the
hCA II enzyme was purified, 724.8-fold with a specific
−
1
activity of 6193.1 EUmg and overall yield of 44%. hCA
IV was purified, 84.7-fold with a specific activity of 742
7
5
.5. A 10-fold excess of the starting methanesulfonates 4,
, 6 was used to limit side reactions. ree reactions were
−
1
EUmg and overall yield of 17.5%. Similarly, hCA VI
was purified, 69.2-fold with a specific activity of 418
performed with and without enzyme in a sodium phos-
phate solution at pH 7.4 (20 mM phosphate buffer). Stock
solution in dimethyl sulfoxide of three methanesulfo-
nates (10 mM) was added to three aqueous solutions in
order to reach the final concentration of 0.08 mM. e
clear mixture was incubated at 25°C for 20 minutes for
−
1
22–37
EUmg and overall yield of 13.2%
.
We report here a study on the inhibitory effects of
methanesulfonate derivatives and some phenolic com-
pounds on the CA esterase activity of isoforms hCA I, II,
IV and VI. Data of Table 1 show the following, regarding
inhibition of hCA I, II, IV and VI with compounds 4–16
4
, 5 and 6. e sulphate esters yielded the corresponding
Journal of Enzyme Inhibition and Medicinal Chemistry

Hydrolysis of methanesulfonates 883
and with positive controls sulfonamides ethoxzolamide
EZA), zonisamide (ZNA) and acetazolamide (AZA):
(
1. Against the slow cytosolic isozyme hCA I, com-
pounds 11–16 behave as weak, micromolar inhibi-
tors, with K values ranging from 60.23 to 4012 µM.
I
Compound 12 was an ineffective hCA I inhibitor (KI
of 4012 µM). A second group of derivative, including
4–6, showed better inhibitory activity as compared to
the previously mentioned compounds 11–16, with KI
values of 22.14–48.54 μM, (Table 1). Data of Table 1
7
–11
also show that similarly to phenolic compounds
most of the investigated methanesulfonates (4, 5 and
) act as competitive inhibitors with 4-NPA as sub-
,
6
strate, i.e. they bind in the same regions of the active
site cavity as the substrate. However the binding site
of 4-NPA itself is unknown, but it is presumed to be
in the same region as that of CO , the physiological
2
9–11
substrate of this enzyme . Similarly to salicylic acid
derivatives and phenolic compounds investigated
8
,26–33
earlier by us
, the investigated compounds act as
competitive inhibitors with 4-NPA as substrate, that
is, they bind in same regions of the active site cavity
as compared to the substrate.
. A better inhibitory activity has been observed with
compounds 14 and 16 investigated here for the inhi-
bition of the rapid cytosolic isozyme hCA II (Table
Scheme 3. e hydrolysis reaction of compound 4, 5 and 6 with
CA II isoenzyme.
2
hydrolysis, the carbonyl group acting as an electronic
withdrawing group might help this hydrolysis sequence
through an increase of the electrophilic character of the
sulfur atom (Figure 1).
1
). Six derivatives, i.e. 4–8, 12 and 15 showed mod-
erate hCA II inhibitory activity with K -s in the range
I
of 4.23–12.24 µM (Table 1), whereas the remaining
two derivatives were quite effective hCA II inhibi-
In recent studies, it was reported that phenols and
natural phenolic compounds act as CAIs, and could rep-
resent the starting point for a new class of inhibitors that
may have advantages for patients with sulfonamide aller-
tors, with K -s in the range of 0.1–0.52 µM, (Table 1).
I
Structure–activity relationship is thus quite sharp for
this small series of methanesulfonates scaffold com-
pounds (4, 5 and 6) are ineffective leads. e best
hCA II inhibitor in this series of derivatives 9.
. Compound 11 and some of its congener such as
compound 13 are also weak inhibitors of CA IV, with
7
–11,13–17,23,24
gies (thioxolone acts as a prodrug
).
Several multiple sulfated compounds have been found
in biologically active compounds and marine organ-
3
4
3
8
isms . For instance, sulfated sterols have exhibited effects
such as anti-HIV, antiviral activity, and inhibition of pro-
K -s of 41.64 µM. However, again compounds 4–9,
I
3
8–40
11–13, 15, 16 and ZNA are medium potency inhibi-
tein tyrosine kinases . Recent studies also showed that
sulfo-containing salicylic acid derivatives have inhibitory
tors (K -s of 3.56–38.45 µM), and compounds 10, 14,
I
3
8
EZA and AZA show a higher affinity for this isozyme,
with inhibition constant in the range of 0.49–1.84 µM
effects on CA I and II isoenzymes . However, it is criti-
cally important to explore further classes of potent CAIs
in order to detect compounds with a different inhibition
profiles as compared to the sulfonamides and their bio-
isosteres and to find novel applications for the inhibitors
of these widespread enzymes, in particular tumor-re-
lated isoforms. Studies regarding interactions of differ-
ent proteins and enzymes are of particular interest for
(
Table 1).
. Phenol 12 and some of its congeners such as 4, 5,
–10, 11–13 and 16 are also weak inhibitors of the
secreted isozyme hCA VI, with K -s of 85.04–608
7
I
1
0
µM . However, again the compounds 6 and 14 are
medium potent inhibitors (K of 12.1–28.43 µM), and
I
4
1–48
derivatives 10, 14, EZA, ZNA and AZA show higher
affinity for this isozyme, with inhibition constants in
the range of 0.34–6.83 µM (Table 1).
toxicologists and medicinal chemists
and many such
4
9–55
investigations exist in the literature . e discovery of
novel inhibitors of CAs has also gained great attention in
the recent years.
We hypothesize that CAs (which as we show above, pos-
sess esterase activity against several substrates) hydroly-
ses these methanesulfonates leading to methanesulfonic
acid and related phenols, as illustrated in Scheme 3.
When (13) giving 12 and (15) giving 14 are compared by
Phenolic compounds 7, 8 and 9 were synthesized
from methanesulfonates 4, 5 and 6 using carbonic
anhydrase II isoenzyme. Compounds 7–10, 12, 14, and
16 affect the activity of CA isozymes due to the pres-
ence of the functional group (OH) in their aromatic
©
2012 Informa UK, Ltd.

8
84 E.A. Kazancıoğlu et al.
Figure 1. Structure of tested compounds 10–16 and some medical used sulfonamides.
scaffold. Our findings indicate thus another class of
possible CAIs of interest, in addition to the well-known
sulfonamides/sulfamates/sulfamides, although their
mechanism of CA inhibition remains rather elusive at
this moment. Indeed, some methanesulfonates investi-
gated here showed moderate CA I, II, IV and VI inhibi-
tory activity in the micromolar range by the esterase
assay method. ese findings point out that substituted
methanesulfonates may be used as leads for generating
more potent CAIs eventually targeting other isoforms
which have not been assayed yet for their interactions
with such agents.
9. Nair SK, Ludwig PA, Christianson DW. Two-site binding of
phenol in the active site of human carbonic anhydrase II:
Structural implications for substrate association. J Am Chem Soc
1994;116:3659–3660.
1
0. Innocenti A, Vullo D, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors: Interactions of phenols with the 12
catalytically active mammalian isoforms (CA I-XIV). Bioorg Med
Chem Lett 2008;18:1583–1587.
1
1. Durdagi S, Sentürk M, Ekinci D, Balaydin HT, Göksu S, Küfrevioglu
ÖI,et al. Kinetic and docking studies of phenol-based inhibitors
of carbonic anhydrase isoforms I, II, IX and XII evidence a new
binding mode within the enzyme active site. Bioorg Med Chem
2011;19:1381–1389.
1
1
1
2. BurriJ,GrafM,LambeletP,LoligerJ.,Vanillin.Morethanaflavouring
agent-a potent antioxidant. J Sci Food Agric 1989;48:49–56.
3. Supuran CT, Conroy CW, Maren TH. Is cyanate a carbonic
anhydrase substrate? Proteins 1997;27:272–278.
4. Briganti F, Mangani S, Scozzafava A, Vernaglione G, Supuran
CT. Carbonic anhydrase catalyzes cyanamide hydration to urea:
Is it mimicking the physiological reaction? J Biol Inorg Chem
Dꢄclꢃrꢃtion of intꢄrꢄꢂt

is study was financed by Turkish Republic Prime
Ministry State Planning Organization (DPT), (Project
no: 2010K120440) for (MS). Work from Supuran lab was
financed by an FP 7 EU grant (Metoxia project).
1
999;4:528–536.
15. Pocker Y, Dickerson DG. e catalytic versatility of erythrocyte
carbonic anhydrase. V. Kinetic studies of enzyme-catalyzed
hydrations of aliphatic aldehydes. Biochemistry 1968;7:1995–2004.
1
6. Pocker Y, Meany JE. e catalytic versatility of erythrocyte carbonic
anhydrase. II. Kinetic studies of the enzyme-catalyzed hydration of
pyridine aldehydes. Biochemistry 1967;6:239–246.
ꢀ
ꢄfꢄrꢄncꢄꢂ
1
. Supuran CT. Carbonic anhydrases: Novel therapeutic applications
for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–181.
. Innocenti A, Scozzafava A, Parkkila S, Puccetti L, De Simone G,
Supuran CT. Investigations of the esterase, phosphatase, and
sulfatase activities of the cytosolic mammalian carbonic anhydrase
isoforms I, II, and XIII with 4-nitrophenyl esters as substrates.
Bioorg Med Chem Lett 2008;18:2267–2271.
17. Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human
carbonic anhydrases B and C. J Biol Chem 1967;242:4221–4229.
18. Innocenti A, Supuran CT. Paraoxon, 4-nitrophenyl phosphate
and acetate are substrates of a- but not of β-, γ- and ζ-carbonic
anhydrases. Bioorg Med Chem Lett 2010;20:6208–6212.
19. Kaiser ET, Lo KW. e Carbonic anhydrase catalyzed hydrolysis of
2-hydroxy-5-nitro-α-toluenesuIfonic acid sultone. J Am Chem Soc
1969;91:4912–4918.
20. Henkart P, Guidotti G, Edsall JT. Catalysis of the hydrolysis of
1-fluoro-2,4-dinitrobenzene by carbonic anhydrase. J Biol Chem
1968;243:2447–2449.
21. Register AM, Koester MK, Noltmann EA. Identification of pseudo-
isoenzymic subforms of muscle carbonic anhydrase. J Biol Chem
1978;253:4143–4152.
22. Kim G, Selengut J, Levine RL. Carbonic anhydrase III: e
phosphatase activity is extrinsic. Arch Biochem Biophys
2000;377:334–340.
23. Ekinci D, Cavdar H, Talaz O, Sentürk M, Supuran CT. NO-releasing
esters show carbonic anhydrase inhibitory action against human
isoforms I and II. Bioorg Med Chem 2010;18:3559–3563.
24. Ekinci D, al-Rashida M, Abbas G, Senturk M, Supuran CT.
Chromone containing sulfonamides as potent carbonic
anhydrase inhibitors. J Enzyme Inhib Med Chem 2011. DOI:
10.3109/14756366.2011.614607.
2
3
4
5
. Neri D, Supuran CT. Interfering with pH regulation in tumours as a
therapeutic strategy. Nat Rev Drug Discov 2011;10:767–777.
. Supuran CT, Scozzafava A. Carbonic anhydrases as targets for
medicinal chemistry. Bioorg Med Chem 2007;15:4336–4350.
. Parkkila S, Parkkila AK. Carbonic anhydrase in the alimentary
tract. Roles of the different isozymes and salivary factors in the
maintenance of optimal conditions in the gastrointestinal canal.
Scand J Gastroenterol 1996;31:305–317.
6
7
8
. Pastorekova S, Parkkila S, Pastorek J, Supuran CT. Carbonic
anhydrases: Current state of the art, therapeutic applications and
future prospects. J Enzyme Inhib Med Chem 2004;19:199–229.
. Mercan L, Okumus A, Sentürk M, Ekinci D. In vitro enzymatic
response of Turkish native chicken “Gerze” to heavy metal
exposure. J Enzyme Inhib Med Chem 2011. (In Press).
. Cavdar H, Ekinci D, Talaz O, Saraçoglu N, Sentürk M, Supuran CT.
a-Carbonic anhydrases are sulfatases with cyclic diol monosulfate
esters. J Enzyme Inhib Med Chem 2011. (In Press).
Journal of Enzyme Inhibition and Medicinal Chemistry

Hydrolysis of methanesulfonates 885
2
5. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-
dye binding. Anal Biochem 1976;72:248–254.
42. Balaydin HT, Durdagi S, Ekinci D, Senturk M, Goksu S, Menzek
A. Inhibition of human carbonic anhydrase isozymes I, II and VI
with a series of bisphenol, metoxy and bromophenol compounds.
J Enzyme Inhib Med Chem 2011. DOI:10.3109/14756366.2011.596
836.
43. Balaydin HT, Soyut H, Ekinci D, Goksu S, Beydemir S, Menzek A,
Sahin E. Synthesis and carbonic anhydrase inhibitory properties of
novel bromophenols including natural products. J Enzyme Inhib
Med Chem 2011. DOI: 10.3109/14756366.2011.574131..
44. Ceyhun SB, Sentürk M, Ekinci D, Erdogan O, Ciltas A, Kocaman
EM. Deltamethrin attenuates antioxidant defense system and
induces the expression of heat shock protein 70 in rainbow
trout. Comp Biochem Physiol C Toxicol Pharmacol 2010;152:
215–223.
2
2
2
2
6. Laemmli DK. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 1970;227:680–685.
7. Morita J, Nakatsuji H, Misaki T, Tanabe Y. Green Chem
2
005;7:711–715.
8. Lineweaver H, Burk D. e determination of enzyme dissocation
constants. J Am Chem Soc 1934;56:658–666.
9. Ekinci D, Kurbanoglu NI, Salamci E, Senturk M, Supuran
CT. Carbonic anhydrase inhibitors: Inhibition of human and
bovine isoenzymes by benzenesulfonamides, cyclitols and
phenolic compounds. J Enzyme Inhib Med Chem 2011. DOI:
1
0.3109/14756366.2011.621122.
3
0. Alp C, Ekinci D, Gültekin MS, Sentürk M, Sahin E, Küfrevioglu OI. A
novel and one-pot synthesis of new 1-tosyl pyrrol-2-one derivatives
and analysis of carbonic anhydrase inhibitory potencies. Bioorg
Med Chem 2010;18:4468–4474.
45. Ekinci D, Sentürk M, Beydemir S, Küfrevioglu OI, Supuran CT. An
alternative purification method for human serum paraoxonase
1 and its interactions with sulfonamides. Chem Biol Drug Des
2010;76:552–558.
3
3
1. Ekinci D, Senturk M. Assesment of metal inhibition of antioxidant
enzyme glutathione reductase from rainbow trout liver. J Enzyme
Inhib Med Chem 2011. DOI: 10.3109/14756366.2011.615745.
2. Ekinci D, Ceyhun SB, Sentürk M, Erdem D, Küfrevioglu OI,
Supuran CT. Characterization and anions inhibition studies of an
a-carbonic anhydrase from the teleost fish Dicentrarchus labrax.
Bioorg Med Chem 2011;19:744–748.
3. Ceyhun SB, Sentürk M, Yerlikaya E, Erdogan O, Küfrevioglu OI,
Ekinci D. Purification and characterization of carbonic anhydrase
from the teleost fish Dicentrarchus labrax (European seabass) liver
and toxicological effects of metals on enzyme activity. Environ
Toxicol Pharmacol 2011;32:69–74.
4. Wistrand PJ, Carter ND, Conroy CW, Mahieu I. Carbonic
anhydrase IV activity is localized on the exterior surface of human
erythrocytes. Acta Physiol Scand 1999;165:211–218.
5. Whitney PL, Briggle TV. Membrane-associated carbonic anhydrase
purified from bovine lung. J Biol Chem 1982;257:12056–12059.
6. Kivelä J, Parkkila S, Waheed A, Parkkila AK, Sly WS, Rajaniemi H.
Secretory carbonic anhydrase isoenzyme (CA VI) in human serum.
Clin Chem 1997;43:2318–2322.
46. Ekinci D, Ceyhun SB, Aksakal E, Erdogan O. IGF and GH mRNA
levels are suppressed upon exposure to micromolar concentrations
of cobalt and zinc in rainbow trout white muscle. Comp Biochem
Physiol C Toxicol Pharmacol 2011;153:336–341.
47. Erdogan O, Ceyhun SB, Ekinci D, Aksakal E. Impact of deltamethrin
exposure on mRNA expression levels of metallothionein A,
B and cytochrome P450 1A in rainbow trout muscles. Gene
2011;484:13–17.
48. Cakmak R, Durdagi S, Ekinci D, Sentürk M, Topal G. Design,
synthesis and biological evaluation of novel nitroaromatic
compounds as potent glutathione reductase inhibitors. Bioorg
Med Chem Lett 2011;21:5398–5402.
49. Ceyhun SB, Aksakal E, Ekinci D, Erdogan O, Beydemir S. Influence
of cobalt and zinc exposure on mRNA expression profiles of
metallothionein and cytocrome P450 in rainbow trout. Biol Trace
Elem Res 2011. DOI: 10.1007/s12011-011-9068-z.
3
3
3
3
50. Aksakal E, Ekinci D, Erdogan O, Beydemir S, Alim Z, Ceyhun
SB. Increasing stocking density causes inhibition of metabolic-
antioxidant enzymes and elevates mRNA levels of heat shock
protein 70 in rainbow trout. Livest Sci 2011;141:69–75.
3
7. Karhumaa P, Leinonen J, Parkkila S, Kaunisto K, Tapanainen J,
Rajaniemi H. e identification of secreted carbonic anhydrase
VI as a constitutive glycoprotein of human and rat milk. Proc Natl
Acad Sci USA 2001;98:11604–11608.
51. Siktar E, Ekinci D, Siktar E, Beydemir S, Gülçin I, Günay M.
Protective role of l-carnitine supplementation against exhaustive
exercise induced oxidative stress in rats. Eur J Pharmacol
2011;668:407–413.
3
3
8. Kornprobst JM, Sallenave C, Barnathan G. Sulfated compounds
from marine organisms. Comp Biochem Physiol B, Biochem Mol
Biol 1998;119:1–51.
9. McKee TC, Cardellina JH 2nd, Riccio R, D’Auria MV, Iorizzi M,
Minale L et al. HIV-inhibitory natural products. 11. Comparative
studies of sulfated sterols from marine invertebrates. J Med Chem
52. Ekinci D, Beydemir S, Küfrevioglu OI. In vitro inhibitory effects of
some heavy metals on human erythrocyte carbonic anhydrases.
J Enzyme Inhib Med Chem 2007;22:745–750.
53. Senturk M, Ekinci D, Alici HA, Beydemir S. Paraoxonase-1, an
organophosphate detoxifier and cardioprotective enzyme, is
inhibited by anesthetics: An in vitro and in vivo insight. Pestic
Biochem Physiol 2011. DOI:10.1016/j.pestbp.2011.09.007.
54. Sentürk M, Talaz O, Ekinci D, Cavdar H, Küfrevioglu OI. In vitro
inhibition of human erythrocyte glutathione reductase by some
new organic nitrates. Bioorg Med Chem Lett 2009;19:3661–3663.
55. Aksakal E, Ceyhun SB, Erdogan O, Ekinci D. Acute and long-
term genotoxicity of deltamethrin to insulin-like growth factors
and growth hormone in rainbow trout. Comp Biochem Physiol C
Toxicol Pharmacol 2010;152:451–455.
1
994;37:793–797.
4
4
0. Anderson CJ, Lucas LJH, Widlanski TS. Molecular recognition
in biological systems: Phosphate esters vs. sulfate esters and
the mechanism of action of steroid sulfatases. J Am Chem Soc
1
995;117:3889–3890.
1. Senturk M, Ekinci D, Goksu S, Supuran CT. Effects of dopaminergic
compounds on carbonic anhydrase isozymes I, II, and VI. J Enzyme
Inhib Med Chem 2011. DOI: 10.3109/14756366.2011.591290.
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2012 Informa UK, Ltd.