Carbonic anhydrase i and II activation with mono- and dihalogenated histamine derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.06.030

Mono- and dihalogenated histamine derivatives incorporating fluorine, chlorine and bromine have been prepared together with the corresponding boc-protected compounds at the aminoethyl group. They have been investigated as activators of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The cytosolic human (h) isoforms hCA I and II were moderately activated by the boc-protected halogenated histamines and very effectively activated by the deprotected ones. Low nanomolar and subnanomolar hCA I and II activators have been detected for the first time, starting from histamine as lead which has an affinity of 2 μM against isoform I and of 125 μM against hCA II.

Full text of DOI:10.1016/j.bmcl.2011.06.030

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4884–4887
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbonic anhydrase I and II activation with mono- and dihalogenated
histamine derivatives
a
b
a
b
a,
⇑
Mohamed-Chiheb Saada , Daniela Vullo , Jean-Louis Montero , Andrea Scozzafava , Jean-Yves Winum ,
Claudiu T. Supuran
a
b,
⇑
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier,
rue de l’Ecole Normale, 34296 Montpellier Cedex, France
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
8
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Mono- and dihalogenated histamine derivatives incorporating fluorine, chlorine and bromine have been
prepared together with the corresponding boc-protected compounds at the aminoethyl group. They have
been investigated as activators of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The cytosolic
human (h) isoforms hCA I and II were moderately activated by the boc-protected halogenated histamines
and very effectively activated by the deprotected ones. Low nanomolar and subnanomolar hCA I and II
activators have been detected for the first time, starting from histamine as lead which has an affinity
Received 26 April 2011
Revised 6 June 2011
Accepted 8 June 2011
Available online 22 June 2011
Keywords:
of 2 lM against isoform I and of 125 lM against hCA II.
Carbonic anhydrase
Enzyme activator
Histamine
Ó 2011 Elsevier Ltd. All rights reserved.
Halogenated histamine
Cytosolic isoforms I and II
Activation of the metalloenzyme carbonic anhydrase (CA, EC
.2.1.1) remains a rather neglected research field, in contrast to
histamine.11 In this and subsequent work it has been shown that
the activator binding site is at the entrance of the enzyme cavity,
far away from the catalytic metal ion (Fig. 1). In such a position,
the activators are able to participate in supplementary proton re-
lease pathways (to the one achieved through His64), with en-
hanced generation of the nucleophilic zinc-hydroxide species of
4
CA inhibition, which is widely investigated in the search of antitu-
mor, antiglaucoma, diuretic, anticonvulsant and antiobesity thera-
1
–6
pies.
All 16 CA mammalian isoforms have been investigated for
their activation with several classes of activators, most of which be-
7
–10
11
long to the amine, amino acid and oligopeptide chemotypes.
the enzyme, leading thus to more efficient catalysis. Many other
The CA activation mechanism is thoroughly understood through
the extensive X-ray crystallographic and kinetic work from several
adducts of activators complexed to various CA isoforms have been
reported in the last period, such as, for example, the human (h)
7
,11–14
7,12–14
groups.
The rate-determining step for the CO
2
hydration reac-
hCA II adducts with
L
-/D
-Phe,
L
-/D
-His,
D-Trp, etc.
Further-
tion catalyzed by CAs is the proton transfer reaction from the water
bound to the Zn(II) ion to the reaction medium, with generation of
the zinc hydroxide species of the enzyme.7
This step is achieved in many a-CA isoforms through the partic-
ipation of the active site residue His64, which being highly flexible
more, CAs possessing a lower catalytic activity (e.g., CA III, XIII,
etc.) were also shown to be highly activatable with many classes
of such activators, which provide alternative pathways to the nat-
ural shuttle residue for the proton release from the active site.1
It has been proposed that CAAs may be effective for enhancing cog-
nition in neurodegenerative diseases (Alzheimer’s disease) or due
to aging, since the level of brain CA isoforms is diminished in such
,11–14
5,16
acts as a proton shuttle between the active site and the reaction
7
,11–14
medium.
Compounds able to participate in such proton
1
7
transfer reactions, of the amine, amino acid and oligopeptide type
conditions.
1
,7
have been shown to act as efficient CA activators (CAAs).
It
As mentioned above, several X-ray crystal structures are avail-
able for adducts of the physiologically dominant isoforms hCA II
with amine and amino acid CAAs. As shown in Figure 1, most of
them bind in the same region of the active site, at its entrance, par-
allel to the natural proton shuttle residue, His64. Indeed, hista-
should be mentioned that the first X-ray crystal structure of an
activator complexed to CA was the adduct of hCA II with
⇑
Correspondence authors. Tel.: +33 4 67 14 72 34; fax: +33 4 67 14 43 44
J.-Y.W.); tel.: +39 055 457 3005; fax: +39 055 457 3385 (C.T.S.).
mine,
denominated the activator binding site A.
ferently, towards the more external part of the active site (and is
L-/D-His and L-/D-Phe bind in this way, in what we have
(
7
–11
Only D-Trp binds dif-
E-mail addresses: jean-yves.winum@univ-montp2.fr (J.-Y. Winum), claudiu.
supuran@unifi.it (C.T. Supuran).
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.030

M.-C. Saada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4884–4887
4885
cytosolic isozyme.7,8,14,18 We have thus used histamine 1 as lead
molecule to design novel activators targeting the cytosolic iso-
forms hCA I and II which we report here. Our goal was to prepare
mono- and dihalogenated histamine derivatives (at the imidazole
ring), since due to their highly electronegative character the halo-
gen atoms usually induce a net change in the physico-chemical
properties of the biologically active molecules containing them.
Such halogenated histamine derivatives have not been tested up
to now for their effects as CAAs.
The different mono and dihalogenated histamines investigated
here were prepared by a four steps synthesis procedure, starting
from commercially available histamine (Scheme 1). Histamine 1
was fully protected by the Boc group, both at the ethylamino and
imidazole nitrogens, leading to 2, which was thereafter selectively
deprotected on the N
potassium carbonate in methanol. The resulting key intermediate
was halogenated with N-halogenosuccinimides in acetonitrile,
leading to a mixture of mono- and dihalogenated compound, of
s of the imidazole ring in the presence of
1
9
3
2
0
types 4 and 5. The difference of polarity between 4 and 5 allowed
their facile separation by silica gel column chromatography. The fi-
nal deprotection step of 4 and 5, in the presence of trifluoroacetic
acid (TFA) afforded the mono- and dihalogenated compounds 6
2
1
and 7 in quantitative yields.
Figure 1. Superposition of the hCA II adducts with histamine (pink, PDB 1AVN)11;
The halogenated protected and deprotected histamine deriva-
tives 4–7 prepared as described above have been tested22 for their
activating properties against isoforms hCA I and II, which are
9
9
L
-His (gold, PDB 2ABE) ;
D
-His (sky blue, PDB 2EZ7) ;
L
-Phe (magenta, PDB
1
0a
10b
10c
2
FMG)
;
D
-Phe (yellow, PDB 2FMZ) ; and
D-Trp (grey, PDB file 3EFI).
The
Zn(II) ion from the enzyme active site is shown as the violet sphere, and is
coordinated by three His residues (His94, 96 and 119 shown as blue and green
sticks) and a water molecule/hydroxide ion (not shown). The natural proton shuttle
residues, His64 is shown in red, whereas the protein backbone is the green ribbon.
among the most physiologically relevant and widely distributed
1–4
CAs in mammals, including humans.
The following structure–
activity relationship (SAR) can be observed from the data of
Table 1:
not superposable to the other activators), in the activator binding
(i) Against hCA I, the boc-protected halogenated derivatives of
1
0c
site B (Fig. 1). Histamine 1, which binds with the imidazole moi-
ety orientated towards the middle of the active site cavity and the
aminoethyl moiety towards its exit, was much used as a lead mol-
type 4 and 5, were less effective than he lead 1 (KA of
1
1
2
l
M),
with activation constants in the range of 5.
M. It may be observed that for the dihalogenated
4–29.3
l
7
,8,14
ecule to design both low molecular
and nanoparticle type
derivatives 4 the activating effects diminished with the
increase of the atomic weight of the halogen, whereas for
the monohalogenated ones 5, the reverse was true. However,
the boc-deprotected compounds 6 and 7 showed highly
CAAs.1 Although histamine itself is a rather ineffective activator
for hCA II (K of 125 lM), many of its derivatives showed a better
A
8
11
activity towards this isoform and also against hCA I, the slower
NH2
HN Boc
HN Boc
N
Boc O
TEA
N
N
N
2
K
2
CO
3
MeOH
Δ
N
N
Boc
H
H
1
2
3
O
CH CN
3
N X
O
HN Boc
HN Boc
N
N
X
N
H
X
N
H
X
X
Cl 4a, 5a, 6a, 7a
Br 4b, 5b, 6b, 7b
4
6
5
TFA/CH Cl2
2
I
4c, 5c, 6c, 7c
NH2
NH2
N
N
X
N
H
X
N
H
X
7
Scheme 1. Preparation of the halogenated histamine derivatives 4–7.

4
886
M.-C. Saada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4884–4887
Table 1
hCA I and II activation with histamine and halogenated/protected-histamine deriv-
atives 4–7, by a stopped-flow, CO hydrase assay
2
constants of 0.7–21 nM against hCA I and of 1–115 nM against
hCA II. The monohalogenated histamines were generally more ac-
tive than the corresponding dihalogenated derivatives. CA activa-
tion increased with the increase of atomic weight of the halogen.
Some of the compounds reported here are among the most effective
hCA I and II activators reported so far, with low nanomolar or subn-
anomolar activation constants.
2
2
*
No.
X
A
K (lM)
hCA I
hCA II
Histamine
2.0
125
4
4
4
5
5
5
6
6
6
7
7
7
a
b
c
a
b
c
a
b
c
a
b
c
Cl
Br
I
Cl
Br
I
Cl
Br
I
Cl
Br
I
5.4
7.1
29.3
16.5
13.3
12.1
0.021
0.015
0.0009
0.018
0.012
0.0007
50.2
44.5
13.6
24.8
28.5
36.7
0.115
0.096
0.065
0.032
0.008
0.001
References and notes
1.
(a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T. Bioorg. Med.
Chem. Lett. 2010, 20, 3467; (c) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.;
Williams, K.; Kienninger, J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.; Lambin,
P.; Wouters, B. G.; Supuran, C. T.; Poellinger, L.; Ratcliffe, P.; Kanopka, A.;
Görlach, A.; Gasmann, M.; Harris, A. L.; Maxwell, P.; Scozzafava, A. J. Enzyme
Inhib. Med. Chem. 2009, 24, 1.
2.
(a) Wagner, J. M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; Supuran, C. T.;
McKenna, R. Bioorg. Med. Chem. 2010, 18, 4873; (b) Pacchiano, F.; Aggarwal, M.;
Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem.
Commun. (Camb) 2010, 46, 8371; (c) Pastorekova, S.; Parkkila, S.; Pastorek, J.;
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004, 19, 199; (d) Franchi, M.; Vullo,
D.; Gallori, E.; Pastorek, J.; Russo, A.; Scozzafava, A.; Pastorekova, S.; Supuran, C.
T. J. Enzyme Inhib. Med. Chem. 2003, 18, 333.
*
Mean from three different determinations. Errors were in the range of ±10ꢀ of the
reported values.
3
4
.
.
(a) Winum, J. Y.; Rami, M.; Scozzafava, A.; Montero, J. L.; Supuran, C. Med. Res.
Rev. 2008, 28, 445; (b) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev.
enhanced CA activating properties compared to the corre-
2003, 23, 146.
sponding protected ones from which they were obtained
and also compared to the lead 1. Indeed, these CAAs showed
activation constants in the range of 0.7–21 nM (Table 1).
Comparable activity was observed both for the mono- as
well as dihalogenated compounds. In all cases, the iodine
derivatives were more active than the corresponding bro-
mine ones which in turn were more active than the chlori-
nated histamines. Indeed, the di-iodinated histamine 6c
and the mono-iodinated one 7c are among the most effec-
(a) De Simone, G.; Di Fiore, A.; Menchise, V.; Pedone, C.; Antel, J.; Casini, A.;
Scozzafava, A.; Wurl, M.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 2315;
(b) Casini, A.; Antel, J.; Abbate, F.; Scozzafava, A.; David, S.; Waldeck, H.;
Schafer, S.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2003, 13, 841; (c) Abbate, F.;
Casini, A.; Owa, T.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004,
1
4, 217; (d) Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2004, 14, 2357; (e) Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.;
Scozzafava, A.; Pastorekova, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2003,
1
8, 403.
5
.
(a) Buller, F.; Steiner, M.; Frey, K.; Mircsof, D.; Scheuermann, J.; Kalisch, M.;
Bühlmann, P.; Supuran, C. T.; Neri, D. ACS Chem. Biol. 2011, 6, 336; (b) Kivelä, A.
J.; Parkkila, S.; Saarnio, J.; Karttunen, T. J.; Kivelä, J.; Parkkila, A. K.; Pastoreková,
S.; Pastorek, J.; Waheed, A.; Sly, W. S.; Rajaniemi, H. Histochem. Cell Biol. 2000,
A
tive hCA I activators ever reported (K s of 0.7–0.9 nM).
(
ii) All compounds investigated here, of type 4–7, were more
potent as hCA II activators compared to histamine 1, which
1
14, 197; (c) Swietach, P.; Wigfield, S.; Cobden, P.; Supuran, C. T.; Harris, A. L.;
Vaughan-Jones, R. D. J. Biol. Chem. 2008, 283, 20473.
1
1
6. (a) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.;
Dedhar, S.; Supuran, C. T. J. Med. Chem. 2011, 54, 1896; (b) Lou, Y.; McDonald, P.
C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.;
Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland, B. W.; Waterhouse, D.; Bally,
M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton, A.; Pacchiano, F.; Carta, F.;
Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Cancer Res.
is a quite weak activator of this isoforms (K
The boc-protected compounds 4 and 5 were again less effec-
tive CAAs, with K s in the range of 13.6–50.2 M. For the
A
of 125 lM).
A
l
dihalogenated compounds 4, activity increased with the
increase in the atomic weight of the halogen atom, whereas
for the monohalogenated, boc-protected derivatives 5, the
most active compound was the chlorine derivative and the
least active one the iodinated one. However, all of them
are medium potency-weak hCA II activators. The deprotec-
ted derivatives 6 and 7 on the other hand showed an
2011, 71, 3364.
7. (a) Ilies, M.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase activators. In
Carbonic Anhydrase – Its Inhibitors and Activators; Supuran, C. T., Scozzafava, A.,
Conway, J., Eds.; CRC Press: Boca Raton, 2004; pp 317–352; (b) Temperini, C.;
Scozzafava, A.; Supuran, C. T. Drug design studies of carbonic anhydrase
activators. In Drug Design of Zinc-Enzyme Inhibitors – Functional, Structural, and
Disease Applications; Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken, 2009;
pp 473–486.
enhanced activating power, with K
A
s in the range of
8
9
.
.
Temperini, C.; Scozzafava, A.; Supuran, C. T. Curr. Pharm. Des. 2008, 14, 708.
(a) Temperini, C.; Scozzafava, A.; Puccetti, L.; Supuran, C. T. Bioorg. Med. Chem.
Lett. 2005, 15, 5136; (b) Temperini, C.; Scozzafava, A.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2006, 16, 5152.
1
–115 nM (Table 1). The dihalogenated histamines 6a–c
showed activation constants of 65–115 nM, with activity
increasing from chlorine to iodine-containing compounds.
A further increase of activity has been observed for the
1
1
0. (a) Temperini, C.; Scozzafava, A.; Vullo, D.; Supuran, C. T. Chem. Eur. J. 2006, 12,
057; (b) Temperini, C.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med. Chem.
7
A
monohalogenated derivatives 7, which had K s in the range
2006, 49, 3019; (c) Temperini, C.; Innocenti, A.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. 2008, 16, 8373.
1
–32 nM, with activity varying in the same manner with the
1. Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran, C.
T. Biochemistry 1997, 36, 10384.
nature of the halogen atom as for the dihalogenated ones.
Thus, generally the best activity has been observed for com-
pounds incorporating only one halogen atom, and the hea-
vier this was, better the CA activating properties were.
12. Becker, H. M.; Klier, M.; Schüler, C.; McKenna, R.; Deitmer, J. W. Proc. Natl. Acad.
Sci. U.S.A. 2011, 108, 3071.
1
3. (a) Elder, I.; Tu, C.; Ming, L. J.; McKenna, R.; Silverman, D. N. Arch. Biochem.
Biophys. 2005, 437, 106; (b) Tripp, B. C.; Ferry, J. G. Biochemistry 2000, 39, 9232.
4. (a) Dave, K.; Ilies, M. A.; Scozzafava, A.; Temperini, C.; Vullo, D.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2011, 21, 2764; (b) Dave, K.; Scozzafava, A.; Vullo, D.;
Supuran, C. T.; Ilies, M. A. Org. Biomol. Chem. 2011, 9, 2790.
1
In conclusion, we prepared ring mono- and dihalogenated hista-
mine derivatives incorporating chlorine, bromine and iodine. The
corresponding boc-protected derivatives (at the aminoethyl group)
ere also obtained. All these histamines have been assayed for the
activation of the cytosolic CA isoforms hCA I and II. Very net SAR
has been obtained for these enzyme activators: against both iso-
forms the boc-protected histamines were moderately active, with
1
5. (a) Parkkila, S.; Vullo, D.; Puccetti, L.; Parkkila, A. K.; Scozzafava, A.; Supuran, C.
T. Bioorg. Med. Chem Lett. 2006, 16, 3955; (b) Vullo, D.; Nishimori, I.; Scozzafava,
A.; Supuran, C. T. Bioorg. Med. Chem. Lett 2008, 18, 4303.
16. (a) Elder, I.; Fisher, Z.; Laipis, P. J.; Tu, C.; McKenna, R.; Silverman, D. N. Proteins
2007, 68, 337; (b) Duda, D. M.; Tu, C.; Fisher, S. Z.; An, H.; Yoshioka, C.;
Govindasamy, L.; Laipis, P. J.; Agbandje-McKenna, M.; Silverman, D. N.;
McKenna, R. Biochemistry 2005, 44, 10046.
1
7. Supuran, C. T.; Scozzafava, A. Carbonic anhydrase activators as potential anti-
Alzheimer’s disease agents. In Protein Misfolding in Neurodegenerative Diseases:
Mechanisms and Therapeutic Strategies; Smith, H. J., Simons, C., Sewell, R. D. E.,
Eds.; CRC Press: Boca Raton (FL)., 2007; pp 265–288.
activation constants of 5.4–29.3
0.2 M against hCA II. The boc-deprotected halogenated hista-
mines showed a enhanced activity as CAAs, with activation
lM against hCA I, and of 13.6–
5
l

M.-C. Saada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4884–4887
4887
1
1
8. Saada, M. C.; Montero, J. L.; Winum, J. Y.; Vullo, D.; Scozzafava, A.; Supuran, C. T.
J. Med. Chem. 2011, 54, 1170.
9. Abdo, M. R.; Joseph, P.; Boigegrain, R. A.; Liautard, J. P.; Montero, J. L.; Köhler, S.;
Winum, J.-Y. Bioorg. Med. Chem. 2007, 15, 4427.
0.48 (DCM 9.5/MeOH 0.5); mp: 68–70 °C; 1H NMR (400 MHz, DMSO): d ppm
12.76 (s, 1H), 6.90 (t, J = 5.6 Hz, 1H), 3.04–3.09 (m, 2H), 2.58 (t, J = 7.1 Hz, 2H),
1.37 (s, 9H); 13C NMR (101 MHz, DMSO): d ppm 155.44, 136.18, 77.60, 40.15,
+
ꢀ
+
+
28.23, 26.04; MS ESI /ESI : m/z 464.12 (M+H) , 486.12 (M+Na) , 462.20
ꢀ
2
2
0. Jai, R.; Avramovitch, B.; Cohen, L. A. Tetrahedron 1998, 54, 3235.
(MꢀH) .
1. tert-Butyl
4-(2-(tert-butoxycarbonylamino)ethyl)-1H-imidazole-1-carboxylate
tert-Butyl 2-(5-iodo-1H-imidazol-4-yl)ethylcarbamate (5c): Yield: 38ꢀ; Rf: 0.26
1
1
(2): Yield: 38ꢀ; Rf: 0.38 (DCM 9.5/MeOH 0.5); mp: 129.5–130.5 °C; H NMR
(DCM 9.5/MeOH 0.5); mp: 55–57 °C; H NMR (400 MHz, DMSO): d ppm 12.27
(
400 MHz, DMSO): d ppm 8.09 (d, J = 1.2 Hz, 1H), 7.25 (d, J = 0.9 Hz, 1H), 6.85 (t,
(s, 1H), 7.58 (s, 1H), 6.92 (t, J = 5.5 Hz), 3.07 (m, 2H), 2.60 (t, J = 7.4 Hz, 2H), 1.37
J = 5.5 Hz, 1H), 3.15 (dd, J = 13.1 Hz, J = 7.0 Hz, 2H), 2.57 (t, J = 7.1 Hz, 2H), 1.55
(
1
(s, 9H); 13C NMR (101 MHz, DMSO): d ppm 155.37, 130.19, 77.62, 40.01, 28.30,
1
3
+
ꢀ
+
ꢀ
s, 9H), 1.36 (s, 11H); C NMR (101 MHz, DMSO): d ppm 155.59, 146.82,
25.96; MS ESI /ESI : m/z 338.36 (M+H) , 336.34 (MꢀH) .
+
ꢀ
+
ꢀ
2-(2,5-Diiodo-1H-imidazol-4-yl)ethanamine (6c): Yield: 100ꢀ; 1H NMR
(400 MHz, DMSO): d ppm 11.08 (s, 1H), 7.88 (s, 2H), 2.99 (m, 2H), 2.78 (t,
40.97, 85.06, 83.00, 77.59. MS ESI /ESI : m/z 312.33 (M+H) , 310.04 (MꢀH) .
tert-Butyl 2-(1H-imidazol-4-yl)ethylcarbamate (3): Yield: 19ꢀ; Rf: 0.19 (DCM 9/
MeOH 1); 1H NMR (400 MHz, CDCl
): d ppm 7.44 (s, 1H), 6.78 (s, 1H), 6.75 (s,
H), 6.71 (s, 1H), 3.79 (s, 6H), 3.69 (s, 2H), 2.86 (t, 2H, J = 6.5 Hz), 2.73 (t, 2H,
13
3
J = 7.7 Hz, 2H); C NMR (101 MHz, DMSO): d ppm 137.58, 120.16, 37.99,
+
+
2
24.11; MS ESI : m/z 364.10 (M+H) .
+
+
2-(5-Iodo-1H-imidazol-4-yl) ethanamine (7c): Yield: 100ꢀ; 1H NMR (400 MHz,
DMSO): d ppm 9.07 (s, 1H), 8.05 (s, 2H), 3.14–3.02 (m, 2H), 2.93 (t, J = 7.4 Hz,
J = 6.5 Hz). MS ESI : m/z 212.22 (M+H) .
tert-Butyl 2-(2,5-dichloro-1H-imidazol-4-yl)ethylcarbamate (4a): Yield: 40ꢀ; Rf:
0
1
13
+
.34 (DCM 9.5/MeOH 0.5); H NMR (400 MHz, DMSO): d ppm 7.95 (s, 1H), 6.92
2H); C NMR (101 MHz, DMSO): d ppm 137.75, 120.37, 37.60, 23.25; MS ESI :
m/z 238.15 (M+H) .
13
+
(
t, J = 5.8 Hz, 1H), 3.09 (m, 2H), 2.58 (t, J = 7.1 Hz, 2H), 1.35 (s, 9H); C NMR
+
(
101 MHz, DMSO): d ppm 155.53, 126.59, 77.70, 38.89, 28.26, 24.35; MS ESI /
22. Khalifah, R.G. J. Biol. Chem. 1971, 246, 2561. An applied photophysics stopped-
ꢀ
+
+
ESI : m/z 280.3–282.29 (M+H) , 302.27–304.27 (M+Na) , 278.17–280.16
(
tert-Butyl 2-(5-chloro-1H-imidazol-4-yl)ethylcarbamate (5a): Yield: 49ꢀ; Rf:
0
2
flow instrument was used for assaying the CA catalyzed CO hydration activity.
Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M
ꢀ
MꢀH) .
1
.18 (DCM 9.5/MeOH 0.5); H NMR (400 MHz, DMSO): d ppm 7.58 (s, 1H, H-
), 6.90 (t, J = 5.5 Hz, 1H), 3.14–3.05 (m, 2H), 2.63 (t, J = 7.3 Hz, 2H), 1.36 (s, 9H).
C NMR (101 MHz, DMSO): d ppm 155.47, 133.21, 122.65, 77.60, 39.15, 28.22,
Na
CO
2
SO
4
(for maintaining constant ionic strength), following the CA-catalyzed
concentrations
1
2
hydration reaction for a period of 10 s at 25 °C. The CO
2
1
3
ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
activation constants. For each activator at least six traces of the initial 5–10ꢀ of
the reaction have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and subtracted from
the total observed rates. Stock solutions of activators (10 mM) were prepared
in distilled-deionized water and dilutions up to 0.1 nM were done thereafter
with distilled-deionized water. Activator and enzyme solutions were
preincubated together for 15 min (standard assay at room temperature, or
for prolonged periods of 24–72 h, at 4 °C) prior to assay, in order to allow for
+
ꢀ
+
ꢀ
2
2
4.11; MS ESI /ESI : m/z 246.31–248.30 (M+H) , 244.31–246.30 (MꢀH) .
1
-(2,5-Dichloro-1H-imidazol-4-yl) ethanamine (6a): Yield: 100ꢀ;
H NMR
(
400 MHz, DMSO): d ppm 11.07 (s, 1H), 7.99 (t, J = 5.7 Hz, 1H), 3.06–2.96 (m,
1
3
2
1
H), 2.81 (t, J = 7.6 Hz, 2H); C NMR (101 MHz, DMSO): d ppm 136.51, 127.29,
+ +
22.36, 37.51, 22.10; MS ESI : m/z 180.15–182.14 (M+H) , 202.19–204.24
+
(
M+Na) .
2
-(5-Chloro-1H-imidazol-4-yl)ethanamine (7a): Yield: 100ꢀ; 1H NMR (400 MHz,
13
DMSO): d ppm 7.97 (s, 1H), 3.09–2.97 (m, 2H), 2.87 (t, J = 7.7 Hz, 2H); C NMR
(
1
+
101 MHz, DMSO): d ppm 121.77, 117.24, 37.64, 21.72; MS ESI : m/z 146.13–
the formation of the E–A complex. The activation constant (K
A
), defined
+
48.18 (M+H) .
similarly with the inhibition constant K , can be obtained by considering the
I
tert-Butyl 2-(2,5-dibromo-1H-imidazol-4-yl)ethylcarbamate (4b): Yield: 37ꢀ; Rf:
classical Michaelis–Menten equation (Eq. 1), which has been fitted by
nonlinear least squares by using PRISM 3:
1
0
.38 (DCM 9.5/MeOH 0.5); mp: 100–102 °C; H NMR (400 MHz, CDCl
3
): d ppm
1
1.06 (s, 1H), 6.92 (t, J = 5.6 Hz, 1H), 3.09 (dd, J = 13.2 Hz, J = 5.8 Hz, 2H), 2.58 (t,
1
3
J = 13.3 Hz, 2H), 1.36 (s, 9H); C NMR (101 MHz, DMSO): d ppm 155,47, 77.62,
+
ꢀ
+
v
¼
vmax=f1 þ KM=½Sꢁð1 þ ½Aꢁ =K Þg
ð1Þ
3
8.96, 28.25, 25.04; MS ESI /ESI : m/z 389.42–391.38 (M+Na) , 366.12–
f
A
ꢀ
3
68.10–370.07 (MꢀH) .
tert-Butyl 2-(5-bromo-1H-imidazol-4-yl)ethylcarbamate (5b): Yield: 50ꢀ; Rf:
1
where [A] is the free concentration of activator.
f
0
.16 (DCM 9.5/MeOH 0.5); mp: 53–55 °C; H NMR (400 MHz, DMSO): d ppm
1
2.32 (s, 1H), 7.55 (s, 1H), 6.92 (t, J = 5.6 Hz, 1H), 3.14–2.98 (m, 2H), 2.61 (t,
Working at substrate concentrations considerably lower than K
considering that [A] can be represented in the form of the total concentration of
f
M
([S] ꢂ K
M
), and
1
3
J = 7.4 Hz, 2H), 1.37 (s, 9H); C NMR (101 MHz, DMSO): d ppm 155.49, 134.73,
7
3
+
ꢀ
ꢀ
+
7.63, 38.88, 28.27, 24.82; MS ESI /ESI : m/z 290.25–292.24 (M+H) , 312.18–
t t
the enzyme ([E] ) and activator ([A] ), the obtained competitive steady-state
+
equation for determining the activation constant is given by Eq. 2:14,18
13.29 (M+Na) , 289.33–290.32 (MꢀH) .
2-(2,5-Dibromo-1H-imidazol-4-yl)ethanamine (6b): Yield: 100ꢀ; mp: 165–
1
1
2
3
67 °C; H NMR (400 MHz, DMSO): d ppm 11.08 (s, 1H), 7.96 (s, 2H), 3.10–
13
2
v
¼
v
0
:K
A
=fK
A
þ ð½Aꢁ ꢀ 0:5fð½Aꢁ þ ½Eꢁ þ K
A
Þ ꢀ ð½Aꢁ þ ½Eꢁ þ K Þ
A
.93 (m, 2H), 2.80 (t, J = 7.7 Hz, 2H); C NMR (101 MHz, DMSO): d ppm 114.86,
t
t
t
t
t
+
+
1=2
7.60, 24.04; MS ESI : m/z 268.17–270.19–272.15 (M+H)
ꢀ
4½Aꢁ :½EꢁtÞ gg
ð2Þ
t
2
-(5-Bromo-1H-imidazol-4-yl)ethanamine (7b): Yield: 100ꢀ; 1H NMR (400 MHz,
DMSO): d ppm 10.28 (s, 1H), 8.42 (s, 1H), 7.98 (s, 2H), 3.12–2.98 (m, 2H), 2.89
(
3
t, J = 13.6 Hz, 2H); 13C NMR (101 MHz, DMSO): d ppm 135.43, 125.89, 107.35,
7.52, 22.23; MS ESI+: m/z 190.15–192.20 (M+H) .
where
absence of activator.
0
v represents the initial velocity of the enzyme-catalyzed reaction in the
14,18
+
tert-Butyl 2-(2,5-diiodo-1H-imidazol-4-yl)ethylcarbamate (4c): Yield: 35ꢀ; Rf: