Carbonic anhydrase activators: Gold nanoparticles coated with derivatized histamine, histidine, and carnosine show enhanced activatory effects on several mammalian isoforms

Article abstract of DOI:10.1021/jm101284a

Lipoic acid moieties were attached to amine or amino acids showing activating properties against the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The obtained lipoic acid conjugates of histamine, l-histidine methyl ester, and l-carnosine methyl ester were attached to gold nanoparticles (NPs) by reaction with Au(III) salts in reducing conditions. The CA activators (CAAs)-coated NPs showed low nanomolar activation (K_As of 1-9 nM) of relevant cytosolic, membrane-bound, mitochondrial, and transmembrane CA isoforms, such as CA I, II, IV, VA, VII, and XIV. These NPs also effectively activated CAs ex vivo, in whole blood experiments, with an increase of 200-280% of the CA activity. This is the first example of enzyme activation with nanoparticles and may lead to biomedical applications for conditions in which the CA activity is diminished, such as aging, Alzheimer's disease, or CA deficiency syndrome.

Full text of DOI:10.1021/jm101284a

ARTICLE
pubs.acs.org/jmc
Carbonic Anhydrase Activators: Gold Nanoparticles Coated with
Derivatized Histamine, Histidine, and Carnosine Show Enhanced
Activatory Effects on Several Mammalian Isoforms
Mohamed-Chiheb Saada,† Jean-Louis Montero,† Daniela Vullo,‡ Andrea Scozzafava,‡ Jean-Yves Winum,*,†
and Claudiu T. Supuran*,‡
†Institut des Biomolꢀecules Max Mousseron (IBMM), UMR 5247, CNRS-UM1-UM2, B^atiment de Recherche Max Mousseron,
Ecole Nationale Supꢀerieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
‡Universitꢁa degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
I-50019 Sesto Fiorentino (Firenze), Italy
ABSTRACT: Lipoic acid moieties were attached to amine or amino
acids showing activating properties against the zinc enzyme carbonic
anhydrase (CA, EC 4.2.1.1). The obtained lipoic acid conjugates of
histamine, L-histidine methyl ester, and L-carnosine methyl ester were
attached to gold nanoparticles (NPs) by reaction with Au(III) salts in
reducing conditions. The CA activators (CAAs)-coated NPs showed
low nanomolar activation (KAs of 1-9 nM) of relevant cytosolic,
membrane-bound, mitochondrial, and transmembrane CA isoforms,
such as CA I, II, IV, VA, VII, and XIV. These NPs also effectively
activated CAs ex vivo, in whole blood experiments, with an increase of
200-280% of the CA activity. This is the first example of enzyme
activation with nanoparticles and may lead to biomedical applications
for conditions in which the CA activity is diminished, such as aging,
Alzheimer’s disease, or CA deficiency syndrome.
’ INTRODUCTION
far)5,6 by means of a novel mechanism of inhibition, i.e.,
occlusion of the enzyme active site.4,7
In a recent work,1 we have reported the first nanoparticles
(NPs) coated with sulfonamide inhibitors of the metalloenzyme
carbonic anhydrase (CA, EC 4.2.1.1). NPs coated with biologically
active compounds show interesting biomedical applications both for
the site-specific delivery of drugs2 or for imaging purposes.2,3 Several
recent such examples include HIV inhibition with a CCR5 antago-
nist attached to multivalent gold nanoparticles, tumor targeting with
nanoparticles loaded with hydroxycamptothecin, paclitaxel, or fu-
magillin, as well as magnetic resonance imaging (MRI) techniques
of tumors based on integrins targeting with Fe3O4 nanoparticles,
computer tomography, or MRI imaging with gadolinium chelate
gold nanoparticles, etc.2 Furthermore, disassembly driven fluores-
cent nanoprobes for selective protein detection, for various CA
isoforms as examples, were recently reported by Hamachi’s group.3
The field of nanoscale enzyme inhibition is also important for better
understanding at molecular level drug-protein interactions, as
shown for example in a recent work in which fullerene
derivatives (the prototypical NP material) were reported to
act as efficient CA inhibitors (CAIs)4 against all physiologically
relevant mammalian CA isoforms (16 of which are described so
The CA activators (CAAs), unlike most classes of CAIs, bind
at the entrance of the enzyme active site participating in
facilitated proton transfer processes between the active site and
the reaction medium (the rate determining step of the CA
catalytic cycle), as shown by kinetic and X-ray crystallographic
work from this and other groups.8-12 The most extensively
investigated CAAs belong to the amine and amino acid class.8-12
Several high resolution X-ray crystal structures of isoforms CA I
and II in adduct with activators such as histamine, L-/D-histidine,
L-/D-phenylalanine, D-tryptophan, etc.,8-12 and synthetic/screening
work on various CA isoforms,13,14 allowed for a rather detailed
understanding of the activation mechanism and of the structure-
activity relationship governing amine/amino acid type of activators.
The report15 that some CAAs (such as phenylalanine and
imidazole) administered to experimental animals may produce
an important pharmacological enhancement of synaptic efficacy,
Received: July 31, 2010
Published: February 03, 2011
r
2011 American Chemical Society
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Among the investigated activators, only D-tryptophan binds in
a slightly different manner compared to other amines/amino
acids,8b toward a more external part of the active site, but
probably the activation mechanism is the same as for the other
compounds mentioned above. The high resolution X-ray
crystal structures of hCA II in adduct with all these activators
were reported recently.8,11,12 These and other data13,14 on
compounds synthesized by considering histamine as lead
molecule, showed that the histamine/histidine based activa-
tors are highly efficient against many CA isoforms with
therapeutic applications.5,6 For example, β-alanyl-L-histidine
(L-carnosine) and many of its derivatives were shown earlier
to act as effective CAAs.14a We considered thus histamine,
histidine, and carnosine as lead molecules to design the
nanoscale enzyme activators reported in this article.
Similar to the strategy used to prepare NPs coated with sul-
fonamide CAIs, we employed lipoic acid 1 to derivatize L-His
methyl ester, L-carnosine methyl ester, or histamine, obtaining
thus the key intermediates 2-4 incorporating lipoyl moieties18
(Scheme 1). The conjugation of 1 with amines/amino acid
derivatives occurred by the classical BOP-DIEA chemistry,18
and the obtained conjugates 2-4 were subsequently treated with
Au(III) salts in the presence of reducing agents (sodium
borohydride) leading to the GNPs of types GNP2-GNP4,
coated with CAAs (L-His methyl ester, GNP2, L-carnosine,
GNP3, and histamine, GNP4, respectively) (Scheme 1). The
GNPs were characterized extensively (see Experimental Proto-
cols for details) by elemental analysis, EDX analysis, NMR
spectroscopy, MS, and TEM analysis. As for the corresponding
GNPs coated with sulfonamide CAIs, also GNP2-GNP4
reported here showed diameters in the range of 2.7-3.2 nm
(see Experimental Protocols). The ratio between the number of
gold atoms and the number of CAA moieties present in the NPs
reported here varied between 3:1 and 10.8:1 (see Experimental
protocols for details).
As seen from data of Table 1, where the content of gold atoms
and activator ligands (L) is presented for three different batches
of GNP2-GNP4, there was a good reproducibility for the
preparation of these nano-objects. Indeed, on the basis of the
elemental analysis (of gold, nitrogen and sulfur), it was observed
that the number of gold atoms varied between 1100 ( 5 for
GNP2, between 594 ( 4 for GNP3, and of 722 ( 6 for GNP4.
The number of activator molecules present in these nanoparti-
cles (L) were varying in the following way: 366 ( 6 for GNP2, 55
( 2 for GNP3, and 178 ( 3 for GNP4, respectively. The errors
in the concentrations measurements of the stock solution of the
activator (of 1 mM) were also estimated by measuring the gold
content of an aliquot and are presented in Table 1. These data
showed a good reproducibility of the measurements, with errors
in the range of 5-10% for the concentration estimation, which
are in the same range as those for the stopped flow measurements
used to assess the CA activating effects, reported later in this
work.
Figure 1. Binding of amine and amino acid CAAs to CA II. Super-
position of the L-His (gold, PDB 2ABE), D-His (sky blue, PDB 2EZ7),
L-Phe (magenta, PDB 2FMG), D-Phe (yellow, PDB 2FMZ), histamine
(pink, PDB 1AVN), and D-Trp (gray) adducts. The natural proton
shuttle, His64 (in red), the catalytic zinc ion (violet sphere), its three
histidine ligands (His94, 96, 119), and the ribbon representation of the
protein backbone (green) are also shown.
spatial learning, and memory, proves that this class of
relatively unexplored enzyme modulators may have pharma-
cological applications in conditions in which learning and
memory are impaired, such as for example Alzheimer’s
disease or aging. One must also mention that it was also
reported that the levels of CA are significantly diminished in the
brain of patients affected by Alzheimer’s disease,16 and these facts
strongly support the involvement of different CA isozymes in
cognitive functions.15-17
Continuing our interest in designing modulators of activity of
CAs with compounds/materials possessing a variety of struc-
tures, we report here the first nanoscale CAAs, obtained by
coating gold nanoparticles (GNPs) with derivatized amine and
amino acid activators, and investigate their biological activity
against several physiologically relevant CA isoforms.
’ RESULTS AND DISCUSSION
Chemistry. CAAs bind at the entrance of the enzyme active
site cavity,8 not far away from His64 (Figure 1), the proton
shuttle residue of CAs.9,10 By means of its imidazole moiety and
due to its flexibility, this residue shuttles the protons from the
zinc bound water molecule to the reaction medium, facilitating
thus the formation of the nucleophilic, zinc hydroxide form of the
enzyme, which is the catalytically active one.8,11,12
Carbonic Anhydrase Activation. Activation of five physio-
logically relevant CA isoforms of human (h) origin, i.e., hCA I, II,
IV, VA, VII, and XIV, with L-histidine methyl ester, L-carnosine
methyl ester, histamine, lipoic acid conjugates 2-4, and nano-
particles GNP2-GNP4, are shown in Table 2 for the CO2
hydration reaction catalyzed by these enzymes.19 Control experi-
ments have also been performed with GNPs containing only
the lipoic acid moiety (without amine or amino acid derivatized
onto it) of type GNP5 (prepared in the same conditions as
As seen from data of Figure 1, CAAs such as histamine,8a L- or
D-histidine,12c and L- or D-phenylalanine12a among others, bind at
the entrance of the active site cavity, not far away from His64.
Their protonatable groups (imidazole, amino, or carboxylate
moieties) are able to participate in supplementary proton
shuttling processes, leading to an enhanced formation of the nucleo-
philic species and thus to an activation of the enzyme catalysis.
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Table 1. Statistical Analysis of the Gold Nanoparticles GNP2-GNP4 Composition and Concentration Determination, from
Three Different Batches Obtained by the Methods Reported in This Paper
Aua
ligandb
% N
concentrationc (mM)
found
NP
no. atoms
%
L moieties
% S
calcd
GNP2
GNP3
GNP4
1100 ( 5
594 ( 4
722 ( 7
62.31 ( 0.25
84.6 ( 0.41
72.69 ( 0.32
366 ( 6
55 ( 2
0.050 ( 0.001
0.022 ( 0.002
0.038 ( 0.002
0.067 ( 0.001
0.025 ( 0.004
0.058 ( 0.006
1.0
1.0
1.0
1.0 ( 0.04
1.0 ( 0.09
1.0 ( 0.08
178 ( 3
a The gold concentration was determined by atomic absorption after dissolution of the nanoparticles in a mixture of concentrated hydrochloric and nitric
acid. b By combustion. c The analytic, calculated concentration was of 1 mM based on the molecular weight of the NPs; the experimental (found) one was
assessed based on the gold concentrations, determined as above, for three different batches of each investigated NPs, GNP2-GNP4.
Scheme 1. Synthesis of Gold NPs (GNPs) GNP2-GNP4 Incorporating CAAs
GNP2-GNP4), as well as with a GNP coated with a sulfonam-
ide CAI, reported earlier,1 GNP6.
found in the brain among many other tissues/organs.20-23 It may
be observed that histamine has activation constants in the range
of 10 nM to 125 μM against these isoforms,20 with hCA VA and
XIV having the highest affinity and hCA II the lowest affinity for
this compound. L-His has an activation profile rather diverse, with
KAs in the range of 30 nM to 10.9 μM. The dipeptide L-carnosine
has rather similar CA activating properties with L-His, with KAs in
the range of 0.64-33.0 μM (Table 2). Derivatization of the last
two compounds by esterification of carboxyl moiety with metha-
nol, in the corresponding methyl esters, did not influence
significantly the activating properties of the esters compared to
the free carboxylic acids from which they were prepared
(Table 2). However, the attachment of the lipoyl moiety to these
amines/amino acid/dipeptide derivatives lead to compounds
2-4 with slightly enhanced CA activating properties compared
to the parent compound from which they were prepared. For
example, the lipoyl-L-His methyl ester 2 showed KAs in the range
of 20 nM to 9.7 μM; the lipoyl L-carnosine methyl ester 3 showed
KAs in the range of 0.96-30 μM, whereas lipoyl histamine 4 had
these parameters in the range of 13 nM to 116 μM, respectively,
Data of Table 2 show that both L-His and histamine, the two
lead compounds used here to design nanoscale enzyme activa-
tors, act as CAAs against the six physiologically important
isoforms, the cytosolic hCA I, II, and VI, the membrane-
associated one hCA IV, the mitochondrial one hCA VA, as well
as the transmembrane isoform hCA XIV, all of which are also
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Table 2. Activation of hCA Isozymes I, II, IV, VA, VII, and
XIV with L- and D-Histidine, at 25°C, for the CO2 Hydration
Reaction19
Table 3. Ex Vivo CA Activation Data after 30 and 60 min of
Incubation of Human Erythrocytes with Solutions Containing
0.1-5 μM Activators Investigated Here, Their Lipoic Acid
Conjugates and GNPsa
KAa (μM)
% CA activityb
hCA
Ib
hCA
IIb
hCA
IVc
hCA
VAd
hCA
VIIb
hCA
XIVd
activator
30 min
60 min
isozyme
L-Hise
L-His-OMec
2c
GNP2d
L-carnosine-OMec
3c
GNP3d
histaminec
4c
GNP4d
GNP5d
GNP6d
137 ( 3
141 ( 8
180 ( 5
131 ( 4
130 ( 6
171 ( 4
121 ( 3
125 ( 5
204 ( 12
89 ( 1
162 ( 9
166 ( 10
245 ( 9
155 ( 8
154 ( 7
263 ( 11
130 ( 5
135 ( 7
287 ( 9
87 ( 3
0.03
0.02
0.02
0.002
1.1
10.9
10.4
9.7
7.3
6.8
7.2
1.34
1.86
1.12
0.92 0.90
0.88 0.93
0.78 0.91
L-His-OMe
2
GNP2
0.008 0.001
0.002 0.003 0.001
L-carnosine
33
19
18
17
1.54
1.36
1.25
0.75 0.64
0.84 0.71
0.87 0.62
L-carnosine-OMe 10.9
32
30
3
0.96
GNP3
histaminef
4
0.009
0.007 0.002
0.002 0.001 0.001
2.1
125
25.3
24.1
0.010 37.5
0.009 35.0
0.010
0.013
0.77 116
11 ( 1
0
GNP4
GNP5
GNP6
0.005
0.002 0.001
inhibitiong
inhibitionh
0.001 0.003 0.007
a Data with GNP5 and GNP6 (CAI) are also provided for comparison.
b Mean ( standard error (n= 3); erythrocyte CA activity (hCA I þ hCA II)
in the absence of activator is taken as 100%. c At 5 μM. d At 0.1 μM.
a The activation constant (KA) for each isozyme was obtained by fitting
the observed catalytic enhancements as a function of the activator
concentration.11-14 Mean from at least three determinations by a
stopped-flow, CO2 hydrase method.19 Standard errors were in the range
of 5-10% of the reported values. b Human recombinant isozymes.
c Truncated human recombinant isozyme lacking the first 20 amino acid
residues.26 d Full length, human recombinant isoforms.20-22 e From ref 12c.
of a GNP derivatized with a sulfonamide CAI, such as GNP6, the
inhibitory activity is in the nanomolar range against all CA
isoforms (Table 2). These data represent a clear proof-of-concept
demonstration that it is possible to design not only nanoscale
enzyme inhibitors but also nanoscale enzyme activators, a field far
less investigated to date. As far as we know, this is the first report in
the literature of enzyme activation by NPs.
g
f From ref 20. The inhibition constants are in the range of 27-33 μM
against the investigated CA isoforms. h The inhibition constants are in the
range of 2.5-137 nM against the investigated CA isoforms.
Ex Vivo CA Activation. After incubation of normal blood red
cells (containing approximately 150 μM of hCA I and 20 μM of
hCA II)24-27 with micromolar concentrations of classical CAAs
(histamine, L-carnosine methyl ester, L-histidine methyl ester) or
new GNP activators synthesized in the present work (such as
GNP2, GNP3, GNP4), the total CA activity in homogenates of
treated cells is enhanced as compared to that of cells treated in a
blank experiment only with buffer (Table 3). Thus, small mole-
cule CAAs such as L-His-OMe and L-carnosine-OMe produces
only a weak-moderate activation of around 120-137% after half
an hour incubation and of around 130-162% of the basal CA
activity after one hour incubation with red cells. More or less the
same effects have been observed with the lipoic acid conjugates of
these small molecule CAAs mentioned above, of types 2-4,
which produced similar enhancements of the total CA activity
after 30 min or 1 h incubation with red blood cells (Table 3).
However, some of the new GNPs tested ex vivo (which showed
strong in vitro CA activity enhancements, Table 3) of types
GNP2-GNP4, produced much higher activations of 171-
204% after half an hour incubation and of 245-287% after one
hour incubation (Table 3). As CA is one of the essential buffer
systems in biological systems through its production of bicar-
bonate and protons, a triplicate production of such anions under
the effect of an activator as those described here, may be highly
relevant. For example, in the CA deficiency syndrome,25 no CA II
is present at all in the body of the affected patients, which has
dramatic clinical consequences, such as mental retardation,
osteopetrosis, renal calcification, and other life threatening
conditions due to the lack of the activity of just one CA isoform.
More recently, Birk’s and our group28 reported a single point
mutation in the CA XII gene which leads to a protein with 71%
against the six investigated CA isoforms (Table 2). The GNPs
incorporating either histamine, L-His methyl ester, or L-carnosine
methyl ester moieties, GNP2-GNP4, were on the other hand
highly efficient CAAs against all isoforms, with activation con-
stants in the low nanomolar range of 1-9 nM (Table 2). The
increase of activating power of the GNPs as compared to the
original lead is sometimes dramatic, with factors as high as 62500
for hCA II with GNP4 and histamine (containing the same CAA
moiety), of 12500 for hCA VII (again comparing histamine and
the corresponding GNP4), etc. This highly increased activating
effects observed with the NPs investigated here may be ac-
counted for on the cooperativity effect triggered by the nano-
object. Indeed, as there is a rather large number (between 55 and
366) of moieties of the ligand (L) possessing itself activation
effects within the reported NP, it is to be expected that these
moieties may better act in transferring protons from the enzyme
active site to the environment with the assistance of all these
multiple proton-shuttling groups present in the nano-object.
Furthermore, in the enzyme-activator complex (the case in
which the activator is a nanoparticle is discussed here) containing
all these multiple proton shuttling moieties, the proton transfer
processes become intramolecular,8 being thus more rapid com-
pared to the intermolecular ones, and this may be an additional
effect explaining the high efficiency of these NPs as CAAs.
On the other hand, the GNPs coated only with lipoic acid,
GNP5, show inhibitory activity against all CA isoforms, similar to
the GNPs (Au@) without any derivatization, investigated earlier.1
Indeed, inhibition constants in the micromolar range have been
measured against all isoforms in the presence of GNP5. In the case
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catalytic activity of the wild type one. However, also in this case
there were relevant clinical features associated with this geno-
type, such as hyperchlorohydrosis, mental retardation, delayed
development in childhood, etc. Such data show that even a small
loss of catalytic activity of one CA isoform may have dramatic
clinical consequences. Correction of such a loss with potent
activators as those described here may thus have clinical benefit.
As control, we have also performed experiments in which red
blood cells have been incubated with the NPs coated only with
lipoic acid (GNP5) or with a sulfonamide conjugate of lipoic acid
(GNP6). Data of Table 3 show that GNP5 produced a weak
inhibitory activity on the total blood CA activity (probably due to
the gold nanoparticle itself, which is weakly inhibitory against
these enzymes).1 On the other hand, the sulfonamide coated
nanoparticles GNP6 led to a powerful inhibition of the CA
activity in red blood cells already after 30 min incubation (an
activity of 11% has been measured), whereas a longer incubation,
of 1 h, led to the total inhibition of CAs present in red blood cells,
as reported earlier for this type of nano-objects.1 These are clear-
cut experiments proving that some of the compounds reported
here might act as effective in vivo CA activators and might thus
constitute interesting candidates for animal studies regarding
their involvement in cognitive processes.
fuming hydrochloric and nitric acids (3:1, v/v). Purity of the obtained
compounds was assessed by HPLC and elemental analysis (combustion, C,
H, N) and was >95% for all of the new derivatives reported here.
Methyl 2-(5-(1,2-Dithiolan-3-yl)pentanamido)-3-(1H-imi-
dazol-4-yl)propanoate 2. To a solution of 300 mg of lipoic acid 1
(1.45, 1 equiv) in 5 mL of dimethylacetamide was added 342 mg (1.45
mmol, 1 equiv) of L-histidine methyl ester dihydrochloride, 771 mg (1.74
mmol, 1.2 equiv) of BOP (benzotriazole-1-yl-oxy-tris-(dimethylamino)-
phosphonium hexafluorophosphate), and 1 mL (5.81 mmol, 4 equiv) of
DIEA. The mixture was stirred overnight at room temperature and then
diluted with water and extracted twice by ethyl acetate. The organic layer
was dried over anhydrous sodium sulfate and filtered. The filtrate was con-
centrated under vacuum. The residue was purified on silica gel using a mix-
ture methylene chloride/methanol (90-10, v/v) as eluent; yield 65%; mp
77 °C. Rf = 0.4 (methylene chloride/methanol (90-10, v/v)). 1H NMR
(DMSO-d6, 400 MHz) δ 8.21 (d, 1H, J = 7.6 Hz), 7.71 (s, 1H), 6.86 (s,
1H), 4.48 (td, 1H, J = 8.2 Hz, J = 5.7 Hz), 3.66-3.51 (m, 5H), 3.22-3.13
(m, 1H), 3.13-3.07 (m, 1H), 2.93 (dd, 1H, J = 14.7 Hz, J = 5.5 Hz), 2.84
(dd, 1H, J = 4.7 Hz, J = 8.7 Hz), 2.40 (dq, 1H, J = 12.2 Hz, J = 6.0 Hz), 2.08
(t, 2H, J = 7.2 Hz), 1.92-1.76 (m, 1H), 1.69-1.57 (m, 1H), 1.57-1.38
(m, 3H), 1.34-1.17 (m, 5H). 13C NMR (DMSO-d6, 101 MHz) δ 172.16,
172.12, 134.77, 132.70, 116.71, 56.12, 56.10, 53.57, 52.13, 51.81, 38.10,
34.80, 34.09, 28.62, 28.12, 28.09, 24.90, 24.89. MS ESIþ m/z 358.20 [M þ
H]þ, 480.18 [M þ Na]þ. Elemental analysis, found: C, 50.43; H, 6.59; N,
11.60%. C15H23O3N3S2 requires: C, 50.20; H, 6.47; N, 11.73%.
Synthesis of Compound 2 Coated Gold Nanoparticles GNP 2. To a
solution of 115 mg of NaBH4 and 9 mg of compound 2 in 10 mL of
DMSO was added a solution of HAuCl4, 4H2O (80 mg) in 10 mL of
DMSO. The reaction mixture turned deep brown immediately. The
reaction mixture was stirred at room temperature for 24 h. Then 40 mL
of CH3CN was added to give a black precipitate which was collected by
centrifugation, washed two times with 60 mL of acetonitrile and 60 mL
of ethanol, and dried under vacuum. EDX and elemental analysis were in
agreement with Au1100(C15H23O3N3S2)366
’ CONCLUSIONS
By attaching lipoic acid moieties to amine or amino acid type
CAAs, we prepared lipoic acid conjugates of histamine, L-histidine
methyl ester, and L-carnosine methyl ester. Gold nanoparticles were
then prepared, coated with these CAAs, which showed low nano-
molar activation (KAs of 1-9 nM) of relevant cytosolic, membrane-
bound, mitochondrial and transmembrane CA isoforms, such as CA
I, II, IV, VA, VII, and XIV. There was a dramatic enhancement of the
CA activating properties of the NPs compared to the low molecular
activators with comparable structure. The NPs coated with these
compounds also effectively activated CA ex vivo, in whole blood
experiments, with an increase of 200-280% of the CA activity. This
is the first example of enzyme activation with nanoparticles and may
lead to biomedical applications.
TEM Analysis.
Average size of gold nanoparticles GNP2 = 3.27 nm.
’ EXPERIMENTAL PROTOCOLS
General. All reagents and solvents were of commercial quality and used
without further purification unless otherwise specified. All reactions were
carried out under an inert atmosphere of nitrogen. TLC analyses were
performed on silica gel 60 F254 plates (Merck Art. 1.05554). Spots were
visualized under 254 nm UV illumination or by ninhydrin solution spraying.
Melting points were determined o on a B€uchi melting point 510 apparatus
and are uncorrected. 1H and 13C NMR spectra were recorded on Bruker
DRX-400 spectrometer using DMSO-d6 as solvent and tetramethylsilane as
1
internal standard. For H NMR spectra, chemical shifts are expressed in
(2S)-Methyl 2-(3-(5-(1,2-dithiolan-3-yl)pentanamido)propanamido)-
3-(4H-imidazol-5-yl)propanoate 3. L-Carnosine methyl ester hydrochlor-
ide (413 mg, 1.72 mmol, 1.2 equiv) , 771 mg (1.74 mmol, 1.2 equiv) of
BOP, and 1 mL (5.81 mmol, 4 equiv) of DIEA were added to a solution of
300 mg of lipoic acid 1 (1.45, 1 equiv) in 5 mL of dimethylacetamide. The
mixture was stirred overnight at room temperature and then diluted with
water and extracted twice with ethyl acetate. The organic layers were dried
over anhydrous sodium sulfate, filtered, and concentrated under vacuum
The residue was purified on silica gel using a mixture methylene chloride.
methanol (90-10, v/v) as eluent.
δ (ppm) downfield from tetramethylsilane, and coupling constants (J) are
expressed in hertz. Electron Ionization mass spectra were recorded in posi-
tive or negative mode on a Water MicroMass ZQ. Transmission electron
microscopy (TEM) observations were carried out at 100 kV (JEOL 1200
EXII). Samples for TEM measurements were prepared by dissolving gold
NPs suspension in ethanol and placing a drop of the obtained mixture onto a
carbon coated copper grip, followed by natural evaporation of the solvent at
room temperature. An estimation of the Au/S ratio was performed by using
an environmental secondary electron microscope FEI Quanta 200 FEG
coupled with an electron dispersive spectroscope Oxford INCA detector.
Elemental analysis has been performed by combustion for N and S, and by
atomic absorption for Au, after dissolution of the samples in a mixture of
Yiel: 75%; mp 56 °C. Rf = 0.29 (methylene chloride/methanol (90-
10, v/v)). 1H NMR (DMSO-d6, 400 MHz) δ 8.31 (d, 1H, J = 7.6 Hz),
7.81(s, 1H), 7.82-7.79 (t, 1H), 4.49 (td, 1H, J = 8.0 Hz, J = 5.8 Hz),
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3.65-3.54 (m, 4H), 3.24-3.14 (m, 3H), 3.14-3.08 (m, 1H), 2.96 (dd,
1H, J = 14.7 Hz, J = 5.6 Hz), 2.87 (dd, 1H, J = 14.7 Hz, J = 8.4 Hz), 2.40
(td, 1H, J = 12.6 Hz, J = 6.4 Hz), 2.25 (t, 2H, J = 7.3 Hz), 2.03 (t, 2H, J =
7.4 Hz), 1.85 (m, 1H), 1.71-1.59 (m, 1H), 1.59-1.40 (m, 3H),
1.38-1.28 (m, 2H), 1.28-1.21 (m, 2H). 13C NMR (DMSO-d6, 101
MHz) δ 171.98, 171.89, 170.49, 134.68, 132.35, 116.59, 56.07, 54.86,
52.08, 51.84, 39.87, 38.04, 35.09, 35.04, 34.07, 28.34, 28.27, 24.95. MS
ESIþ m/z 429.20 [M þ H]þ, 451.19 [M þ Na]þ, ESI- m/z 387.18
[M - H]-. Elemental analysis, found: C, 55.62; H, 7.12; N, 14.31%.
C18H28O4N4S2 requires: C, 55.83; H, 7.28; N, 14.47%.
Synthesis of CAA-Carnosine Coated Gold Nanoparticles GNP 3. To
a solution of 115 mg of NaBH4 and 14 mg of compound 3 in 10 mL of
DMSO was added a solution of HAuCl4,4H2O (80 mg) in 10 mL of
DMSO. The reaction mixture turned deep brown immediately. The
reaction mixture was stirred at room temperature for 24 h. Then 40 mL
of CH3CN was added to give a black precipitate which was collected by
centrifugation, washed two times with 60 mL of acetonitrile and 60 mL
of ethanol, and dried under vacuum. EDX and Elemental analysis were in
agreement with [Au594(C18H28O4N4S2)55]
Hz), 3.24(dd, 3H, J=13.3Hz, J = 7.3 Hz), 3.21 - 3.06 (m, 3H), 2.60 (t, 2H,
J=5.9Hz),2.40(td,1H,J=12.4Hz,J= 6.2 Hz), 2.04 (t, 2H, J= 7.3 Hz), 1.85
(dq, 1H, J = 13.5 Hz, J = 6.8 Hz), 1.65 (m, 1H), 1.59-1.42 (m, 3H),
1.38-1.26 (m, 2H). 13C NMR (DMSO-d6, 101 MHz) δ 171.81, 134.55,
56.09, 38.58, 38.06, 35.18, 34.08, 28.26, 26.96, 25.01. MS ESIþ m/z 300.20
[Mþ H]þ, 322.30[Mþ Na]þ. Elementalanalysis, found:C, 52.13;H, 6.79;
N, 13.67%. C13H21ON3S2 requires: C, 52.00; H, 7.05; N, 14.00%.
Synthesis of Histamine Coated Gold Nanoparticles GNP4. To a
solution of 115 mg of NaBH4 and 9 mg of compound 4 in 10 mL of
DMSO was added a solution of HAuCl4 and 4H2O (80 mg) in 10 mL of
DMSO. The reaction mixture turned deep brown immediately. The
reaction mixture was stirred at room temperature for 24 h. Then 40 mL
of CH3CN was added to give a black precipitate which was collected by
centrifugation, washed two times with 60 mL of acetonitrile and 60 mL
of ethanol, and dried under vacuum. EDX and elemental analysis were in
agreement with [Au722(C13H21ON3S2)178].
TEM Analysis.
Average size of gold nanoparticles GNP4 = 2.86 nm.
CA Assay. A stopped-flow method19 has been used for assaying the
CA catalyzed CO2 hydration activity with Phenol Red as indicator,
working at the absorbance maximum of 557 nm, following the initial
rates of the CA-catalyzed CO2 hydration reaction for 10-100 s. For
each activator at least six traces of the initial 5-10% 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 activator (0.01 mM) were prepared in
distilled-deionized water with 5% DMSO, and dilutions up to 0.01 nM
were done thereafter with distilled-deionized water. The nanoparticles
were totally soluble in this solvent mixture. Activator (concentration
range 0.01 μM to 0.01 nM) and enzyme solutions ([E] = 10 nM) were
preincubated together for 15 min to 2 h at room temperature prior
to assay in order to allow for the formation of the E-A complex.
The activation constant (KA), defined similarly with the inhibition
constant KI,8,11-14 can be obtained by considering the classical Michae-
lis-Menten equation (eq 1), which has been fitted by nonlinear least-
squares by using PRISM 3:
TEM Analysis.
Average size of gold nanoparticles GNP3 = 2.68 nm.
N-(2-(1H-Imidazol-4-yl)ethyl)-5-(1,2-dithiolan-3-yl)pentanamide 4. A
solution of 250 mg of lipoate-NHS ester (0.82 mmol, 1.1 equiv) (prepared
as described by Liu et al.26) in 4 mL of DMF was added dropwise over 10 min
to a solution of 83 mg of histamine (0.75 mmol, 1equiv) and 63 mg of sodium
bicarbonate (0.75 mmol, 1 equiv) in DMF/water (6 mL, 1:1 v/v) at 0 °C.
The solution was stirred under reflux overnight. The mixture was then
extracted with chloroform (3 ꢀ 10 mL). The combined organic extracts were
washed with water (3 ꢀ 10 mL), dried over anhydrous Na2SO4, filtered, and
concentrated under vacuum. The crude product was purified on silica gel
using a mixture methylene chloride. methanol (90-10, v/v) as eluent to give
the final product as a yellow solid; yield 60%; mp 98 °C; Rf =0.32(methylene
chloride/methanol (90-10, v/v)). 1H NMR (DMSO-d6, 400 MHz) δ 7.86
(t, 1H,J=5.4Hz),7.52(s, 1H), 6.77(s,1H),3.60(dt, 2H,J=14.8Hz,J=6.2
v ¼vmax=f1þKM=½Sꢁð1þ½Aꢁf =KAÞg
ð1Þ
where [A]f is the free concentration of activator.
Working at substrate concentrations considerably lower than KM ([S]
, KM), and considering that [A]f can be represented in the form of the
total concentration of the enzyme ([E]t) and activator ([A]t), the
obtained competitive steady-state equation for determining the activa-
tion constant is given by eq 2:11-14
2
v ¼v0 KA=fKAþð½Aꢁt-0:5fð½Aꢁtþ½EꢁtþKAÞ-ð½Aꢁtþ½EꢁtþKAÞ
3
1 = 2
-4½Aꢁt 3 ½EꢁtÞ
gg
ð2Þ
where v0 represents the initial velocity of the enzyme-catalyzed reaction
in the absence of activator.11-14 All CA isozymes used in the experi-
ments were purified recombinant proteins obtained as reported by our
group.1,12,14
Ex Vivo CA Activation. An amount of 2 mL of freshly isolated
human blood was thoroughly washed several times with 5 mL of
Tris buffer (pH 7.40, 5 mM) and centrifuged for 10 min. The
obtained erythrocytes were then treated with 2 mL of a 0.1-5 μM
solution of CA activator. Incubation has been done at 37 °C with
gentle stirring for periods of 30-60 min. After that time, the red cells
were centrifuged again for 10 min, the supernatant discarded, and the
cells washed three times with 5 mL of the above-mentioned buffer in
order to eliminate all unbound activator. The cells were then lysed
in 5 mL of distilled water, centrifuged for eliminating membranes
and other insoluble materials, and CA activity was assayed as
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Journal of Medicinal Chemistry
ARTICLE
described above.19 Blank experiments were done in which no activator has
been added to the blood red cells treated as described above, and CA
activity determined in such conditions has been taken as 100%.27
(6) (a) Domsic, J. F.; Avvaru, B. S.; Kim, C. U.; Gruner, S. M.;
Agbandje-McKenna, M.; Silverman, D. N.; McKenna, R. Entrapment of
carbon dioxide in the active site of carbonic anhydrase II. J. Biol. Chem.
2008, 283, 30766–307681. (b) Fisher, S. Z.; Maupin, C. M.; Budayova-
Spano, M.; Govindasamy, L.; Tu, C. K.; Agbandje-McKenna, M.;
Silverman, D. N.; Voth, G. A.; McKenna, R. Atomic crystal and
molecular dynamics simulation structures of human carbonic anhydrase
II: insights into the proton transfer mechanism. Biochemistry 2007, 42,
2930–2937. (c) Winum, J. Y.; Rami, M.; Scozzafava, A.; Montero, J. L.;
Supuran, C. Carbonic Anhydrase IX: a new druggable target for the
design of antitumor agents. Med. Res. Rev. 2008, 28, 445–463. (d)
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tors. Med. Res. Rev. 2003, 23, 146–189.
’ AUTHOR INFORMATION
Corresponding Author
*For J.-Y.W.: E-mail, jean-yves.winum@univ-montp2.fr. For
C.T.S.: phone, 39-055-4573005; fax, 39-055-4573385; E-mail,
claudiu.supuran@unifi.it.
’ ACKNOWLEDGMENT
(7) Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.;
Scozzafava, A.; Quinn, R. J.; Supuran, C. T. Non-zinc mediated inhibi-
tion of carbonic anhydrases: coumarins are a new class of suicide
inhibitors. J. Am. Chem. Soc. 2009, 131, 3057–3062.
This research was financed in part by a grant of the 7th
Framework Programme of the European Union (Metoxia project, to
C.T.S. and A.S.) and by the grant CNRS MIE-2007 (Programme
“Maladies Infectieuses Emergentes”Centre National de la Recherche
Scientifique, France, to J.-Y.W).
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G.; Supuran, C. T. Carbonic anhydrase activators: X-ray crystallographic
and spectroscopic investigations for the interaction of isozymes I and II
with histamine. Biochemistry 1997, 36, 10384–10392. (b) Temperini, C.;
Innocenti, A.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase activators.
Kinetic and X-ray crystallographic study for the interaction of D- and
L-tryptophan with the mammalian isoforms I-XIV. Bioorg. Med. Chem.
2008, 16, 8373–8378. (b) Temperini, C.; Scozzafava, A.; Puccetti, L.;
Supuran, C. T. Carbonic anhydrase activators: X-ray crystal structure of the
adduct of human isozyme II with L-histidine as a platform for the design of
stronger activators. Biorg. Med. Chem. Lett. 2005, 15, 5136–5141.
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Lindskog, S. Role of histidine 64 in the catalytic mechanism of human
carbonic anhydrase II studied with a site-specific mutant. Biochemistry
1989, 28, 7913–7918. (b) Behravan, G.; Jonasson, P.; Jonsson, B. H.;
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’ ABBREVIATIONS USED
BOP, benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexa-fluorophosphate; CA, carbonic anhydrase; CAA, CA activator;
CAI, CA inhibitor; DIEA, diisopropylethylamine; EDX, energy dis-
persive X-ray analysis; GNP, gold nanoparticle; MRI, magnetic
resonance imaging; NP, nanoparticle; TEM, transmission electron
microscopy
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