Discovery of novel lipophilic inhibitors of OXA-10 enzyme (class D β-lactamase) by screening amino analogs and homologs of citrate and isocitrate

Article abstract of DOI:10.1016/j.bmcl.2009.04.149

Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete) deprotection. Aminoisocitrate was similarly obtained by

Full text of DOI:10.1016/j.bmcl.2009.04.149

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3593–3597
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel lipophilic inhibitors of OXA-10 enzyme (class D
b-lactamase) by screening amino analogs and homologs of citrate and isocitrate
Joséphine Beck a, Lionel Vercheval b, Carine Bebrone b, Adriana Herteg-Fernea b, Patricia Lassaux b,
Jacqueline Marchand-Brynaert a,
*
a Unité de Chimie Organique et Médicinale, Université Catholique de Louvain, Bâtiment Lavoisier, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium
b Centre d’Ingénierie des Protéines, Université de Liège, Bâtiment B6, Allée de la Chimie 17, B-4000 Sart-Tilman, Liège, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases
with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete) depro-
tection. Aminoisocitrate was similarly obtained by mono-alkylation with diethyl fumarate. Evaluation
against representative b-lactamases revealed that the free acid derivatives are modest inhibitors of class
A enzymes, whilst their benzyl esters showed a good inhibition of OXA-10 (class D enzyme). A docking
experiment featured hydrophobic interactions in the active site.
Received 17 March 2009
Revised 27 April 2009
Accepted 27 April 2009
Available online 6 May 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
2-Aminopropane-1,2,3-tricarboxylates
1-Aminopropane-1,2,3-tricarboxylates
3-Aminopropane-1,3,5-tricarboxylates
b-Lactamase inhibition
OXA-10 inhibitors
The extensive and uncontrolled use of b-lactam antibiotics has
resulted in the evolution of resistance in many strains of bacteria.1
The bacterial resistance is now a major medical concern and there
is a pressing need to discover new anti-infective agents, or to in-
crease the therapeutic efficacy of the existing drugs.2
longs to the latter approach and is based on a fortuitous
observation made by Fonze et al. during the crystallographic study
of Bacillus licheniformis BS3 b-lactamase, a class A enzyme.10
The crystallization of BS3 from a 100 mM citrate buffer revealed
the unexpected role of this hydroxy acid which is perfectly located
in the enzymic active site, as shown by the X-ray diffraction anal-
ysis. Similarly, isocitrate could be co-crystallized with BS3; in both
cases, one b-carboxylate group of (iso)citrate interacts with the
two catalytic serine residues, Ser70 and Ser130.11 Citrate and isoci-
trate are modest inhibitors of BS3 b-lactamase with Ki values at pH
The transpeptidases involved in the biosynthesis of peptidogly-
can, a major constituent of the bacterial cell wall, are serine en-
zymes which become inactive by binding penicillin and other b-
lactam antibiotics.3 The production of b-lactamases represents
the most widespread and often the most efficient mechanism de-
vised by bacteria to escape the lethal action of b-lactam antibiot-
ics.4 These defense enzymes have been grouped into four classes
on the basis of their primary structures and catalytic mechanisms.5
b-Lactamases of classes A, C and D are serine enzymes, whereas
class B b-lactamases are metalloproteins which require zinc(II)
ions for their activity. All b-lactamases catalyze very efficiently
the hydrolysis of the b-lactam function of antibiotics, rendering
them ineffective.6
Our research focuses on the synthesis and biochemical evalua-
tion of potential inhibitors of b-lactamases. Several strategies have
been considered to overcome the action of these b-lactam hydro-
lyzing enzymes.7 Beside modified penicillin/cephalosporin-derived
b-lactamase inhibitors,8 non traditional derivatives are also devel-
oped, which do not feature a b-lactam ring.9 The present work be-
5 of 490 lM and 2200 l
M respectively.11 These experimental data
prompted us to consider citrate and isocitrate as ‘hits’ for the con-
ception of new affinity inhibitors of class A b-lactamases. We
envisaged to increase the binding in the enzymic cavity by replac-
ing the alcohol function of (iso)citrate with an amine function on
which a variety of lateral chains could be fixed. Homologation, that
is, the lengthening of the acidic chains, was also considered in view
to possibly create novel interactions. The target molecules, de-
picted in Figure 1, are non natural amino acid derivatives related
H
H2N CO2H
HO2C CO2H
R N CO2H
H2N CO2H
CO2H
HO2C
R=H,acylchain
CO2H
HO2C
Aminocitrate derivatives
Aminocitrate homolog Aminoisocitrate
* Corresponding author. Tel.: +32 10 47 27 40; fax: +32 10 47 41 68.
E-mail address: jacqueline.marchand@uclouvain.be (J. Marchand-Brynaert).
Figure 1. Target molecules (acids and corresponding esters).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.149

3594
J. Beck et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3593–3597
to glutamate and aspartate. Such compounds have traditionally
been developed for drug discovery in the CNS (central nervous sys-
tem) and anticancer research fields.12 To our knowledge, analogs
and homologs of (iso)citrate were not considered as potential
inhibitors of b-lactamases before our work.13
methyl or tris-ethyl precursors 3a–b. A practical protocol using
6 N HCl at reflux was previously set up.11
The synthesis of products possessing longer acidic chains was
based on the bis-alkylation of Schiff base by Michael addition.18
Thus, imine 1b was treated with potassium carbonate and ethyl
acrylate in excess to furnish compound 7 with 90% crude yield
(Scheme 2). Imine hydrolysis led to the formation of pyrrolidinone
8, isolated with 50% yield after purification by chromatography.19
This was treated with 6 N HCl at reflux in view of producing the
target-molecule 9. In fact, we recovered a mixture of aminocitrate
homolog 9 and unprotected pyrrolidinone 10, in a 80:20 ratio from
1H NMR analysis. Treatment with Dowex-H+ resin did not improve
the amount of 9.
Aminocitrate (and homolog) derivatives are
bis-substituted -amino acids, for which at least three synthetic
a,
a0-symmetrically
a
strategies could be applied, namely the bis-alkylation of anions de-
rived from oxazolin-5-ones,14 nitroacetates15 and glycinate Schiff
bases.16 We found that the last method was the most practical
and versatile one, allowing a rapid access to the library of com-
pounds of interest.
The Schiff bases 1, prepared as usual by condensation of glycine
esters with m-chlorobenzaldehyde, were treated with potassium
carbonate, triethylammonium bromide (phase transfer catalyst)16
and (m)ethyl bromoacetate (method A) or with LDA at low temper-
ature and benzyl bromoacetate (method B),11 to furnish the crude
imines 2 (Scheme 1). Yields are quantitative for 2a–c, but com-
pounds 2d–e (R = Bn) were contaminated with 10–20% of mono-
alkylated products (from 1H NMR analysis). The amines 3, obtained
after acidic hydrolysis,17 were acylated with different acid chlo-
rides in the presence of pyridine. Amides 4a–e were recovered in
about 60% overall yields after chromatographic purifications.
Deprotection of the methyl esters 4a–c posed unexpected prob-
lems: numerous acidic and basic conditions were tested, but com-
petitive hydrolysis of the amide bond could not be avoided and the
use of nucleophilic conditions was ineffective. Fortunately, hydrog-
enolysis of benzyl esters 4d–e led quantitatively to the amides 5d–
e featuring two and three free carboxyl functions, respectively. Par-
tially deprotected amine derivatives 6b–c were also prepared by
hydrogenolysis in aqueous HCl of the corresponding benzyl esters
3c–d and isolated as hydrochloride salts. The fully deprotected
aminocitrate 6a could be obtained, either by hydrogenolysis of
the tris-benzyl precursor 3e, or by acidic hydrolysis of the tris-
Finally, we prepared aminoisocitrate 15 (also named
a-amino
tricarballylic acid20) by the same strategy, as a racemic mixture
of diastereoisomers (Scheme 3). The mono-alkylation of glycine
Schiff bases requires the use of a sterically hindered derivative of
benzophenone.21 Thus, imine 11 was reacted with sodium ethox-
ide as catalyst and diethylfumarate in ethanol to obtain the
mono-alkylated product 12 with 95% crude yield and a diastereo-
isomeric ratio of 27/73 determined by 1H NMR analysis. The acidic
hydrolysis of 12 led to a mixture of products, aminoisocitrate 13
and pyrrolidinone 14 (ratio 85/15), with 66% yield after chroma-
tography. Tris-ethyl aminoisocitrate 1322 was isolated as a 46/54
mixture of diastereoisomers, whereas ethyl 3-ethoxycarbonylpyr-
roglutamate 1423, recovered in the last chromatographic fractions,
was mainly one diastereoisomer. The previous mixture was hydro-
lyzed with 6 N HCl to give the racemic aminoisocitric acid 15
(50:50 ratio of diastereoisomers) with 80% yield.
Protocols and spectroscopic data for compounds 1-6 (Scheme
1), 7–10 (Scheme 2) and 12-15 (Scheme 3) are given as Supple-
mentary data.
All the amino(iso)citrate derivatives (esters and acids) were
evaluated for their activity against a series of b-lactamases of class
A (TEM-1,24 BS3,10 NMCA25), class C (P9926), class D (OXA-1027),
and class B (BcII28, VIM-429). Most of them are clinically reprensta-
tive. Class A enzymes are primarily penicillinases, susceptible to
inhibition by the presently marketed b-lactamase inhibitors,
namely potassium clavulanate, sulbactam and tazobactam, which
Cl
Cl
i
N
N
CO2R
CO2R
CO2R'
H
H
R'O2C
H
ClPh
H
O
N
i
ii
N
CO2Et
CO2Et
CO2Et
CO2Et
1b
1a
:R=Me
1b:R=Et
1c
2a:R=R'=Me
2b:R=R'=Et
:R=Bn
8
2c
:R=Me,R'=Bn
EtO2C
7
2d:R=Bn,R'=Me
2e:R=R'=Bn
H
ClH3N CO2H
HO2C CO2H
O
N
R''
iii
H
CO2H
CO2H
iii
ii
H2N CO2R
R'O2C CO2R'
3a
N
CO2R
CO2R'
O
R'O2C
10
9
4a
:R=R'=Me
3b
:R=R'=Me,R''=PhO
Scheme 2. Synthesis of aminocitrate homolog. Reagents and conditions: (i) K2CO3,
CH2@CHCO2Et, EtOH, rt, 16 h; (ii) 1 N HCl, CH3CN, rt, 30 min, then basic work-up;
(iii) 6 N HCl, 120 °C, 24 h.
4b:R=R'=Me,R''=Ph
:R=R'=Et
3c:R=Me,R'=Bn
4c:R=R'=Me,R''=2-thienyl
3d
3e
4d
:R=Me,R'=Bn,R''=PhO
:R=Bn,R'=Me
:R=R'=Bn
4e:R=R'=Bn,R''=PhO
v
iv
Ph
Ph
Ph
Ph
12
i
ii
N
CO2Et
N
CO2Et
CO2Et
R''
H
N
11
ClH3N CO2R
R'O2C CO2R'
6a
CO2R
CO2H
CO2Et
O
HO2C
H
N
H2N CO2Et
O
ClH3N CO2H
iii
5d
5e
CO2Et
:R=Me,R''=PhO
:R=H,R''=PhO
:R=R'=H
CO2Et
CO2Et
CO2H
CO2H
15
6b:R=Me,R'=H
6c:R=H,R'=Me
CO2Et
13
14
Scheme 1. Synthesis of aminocitrate derivatives. Reagents and conditions: (i)
K2CO3, Bu4NBr, excess BrCH2CO2R0, CH3CN, 50 °C or LDA, excess BrCH2CO2R0, THF,
ꢀ78 °C to 20 °C; (ii) 1 N HCl, CH3CN, rt, 30 min, then basic work-up; (iv) pyridine,
R00CH2COCl, rt, 5 h; (iv) H2, Pd/C, EtOAc, rt, 4 h; (v) H2, Pd/C, HCl concd, EtOAc, rt, 5 h.
Scheme 3. Synthesis of aminoisocitrate. Reagents and conditions: (i) NaOEt, diethyl
fumarate, EtOH, rt, 1 h; (ii) HCl 1 N, CH3CN, rt, 30 min, then basic work-up; (iii) 6 N
HCl, 120 °C, 24 h.

J. Beck et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3593–3597
3595
are efficient only against a few class D b-lactamases. Some class D
enzymes present rather a carbapenemase activity. Class C enzymes
are primarily cephalosporinases, resistant to inhibition by the pre-
vious agents. There is no clinically useful inhibitor for class B en-
zymes that emerge now as worldwide source of carbapenem
resistance.
The enzymes were incubated with the tested compounds dur-
ing 30 min at 37 °C, at pH 7 or pH 5. Then nitrocefin (a chromo-
genic substrate) was added and the hydrolysis rate of this
substrate was followed by spectrophotometry at 482 nm. In this
way, the residual activity of the b-lactamases could be deter-
mined.30,31 The results shown in Table 1 are expressed as percent-
ages of b-lactamase initial activity. The compounds were tested at
Our initial hypothesis had been to improve the activity of (iso)-
citrates versus class A b-lactamases by replacing the hydroxyl
function with an amine/amide substituent, and/or by the homolo-
gation of two acid chains. Aminocitrate 6a33 was indeed slightly
more active than citrate and its co-crystallization with BS3 b-lacta-
mase showed, in a preliminary communication, that this inhibitor
adopts a different position in the active site, the amine group inter-
acting directly with the Ser70 and Ser130 catalytic residues.11 The
corresponding amide 5e, featuring the side chain of penicillin V,
was also an inhibitor of BS3. Lastly, the homolog 933 of aminoci-
trate inhibited TEM-1, another class A b-lactamase. Compounds
6a, 5e and 9 are tricarboxylic acid derivatives, like citrate. This
structural characteristic seems however not to be essential for
inhibiting class A enzymes, since 3b, 3c (aminocitrate triesters)
and 5d (amidocitrate monoester) are also active compounds,
against NMCA. Unfortunately, all attempts to co-crystallize the
previous inhibitors with class A enzymes failed; thus structural
data of the enzyme-inhibitor complexes are not available.
Interestingly, we have discovered novel inhibitors of OXA-10, a
class D b-lactamase for which only few inhibitors were previously
described in the literature.34–37 Tribenzyl aminocitrate 3e, and
dibenzyl amidocitrates 4d and 4e were efficient against OXA-10
(the enzyme has a residual activity inferior to 50% in the presence
of these compounds), whereas corresponding derivatives 3 and 4
bearing smaller ester groups were inactive (for instance 3b, 3c,
3d, 5d).
a concentration of 100 lM (unless otherwise mentioned); low%
values indicate a very active compound.
None of the tested compounds inhibited BcII and VIM-4 (class B,
zinc b-lactamases) nor P99 (Class C), except 6c (pH 5). The activity
of (iso)citrate-type derivatives against class A b-lactamases (TEM-
1, BS3, NMCA) has been confirmed11 and promising results were
recorded for the inhibition of class D b-lactamase OXA-10.
Among the aminocitrate esters (3), 3b (entry 1) inhibited TEM-
1 and NMCA, 3c (entry 2) inhibited NMCA and 3e (entry 4) was a
good inhibitor of OXA-10, whereas the less lipophilic ester 3d
(entry 3) was not active against OXA-10. Partially or totally
deprotected aminocitrates 6a (entry 10), 6b (entry 11) and 6c
(entry 12) were poorly active or inactive. Among amide deriva-
tives (4) fully protected as tris-esters, 4d (entry 6) and 4e (entry
7), but not 4b (entry 5), inactivated OXA-10, confirming the
importance of the lipophilic character of inhibitors to interact
with this b-lactamase. As a matter of fact, partially or totally
deprotected amides 5d (entry 8) and 5e (entry 9) were not active
against OXA-10. However, bis-acid 5d and tris-acid 5e inhibited
NMCA and BS3 respectively. Pyrrolidinones 8 (entry 13) and 14
(entry 16) were inactive. Aminocitrate homolog 9 (entry 14)
was an inhibitor of TEM-1, whereas amino(iso)citrates 13 (entry
15) and 15 (entry 17) were devoid of significant activities. The
selectivity of the tris-carboxyl derivatives 6a and 9 for class A
b-lactamases versus DD-peptidases was controlled by measuring
their capacity to inhibit R39 enzyme,32 a low molecular weight
DD-transpeptidase/carboxypeptidase. No significant activity could
be detected (data not shown).
Thus, from our initial screening (Table 1), three compounds
emerged, namely 3e, 4e and 4d. They were further investigated
for determining kinetic parameters. Compound 3e is an inactivator
of OXA-10, as shown by the inactivation graph (ki vs [I]) of Figure 2,
with a Ki value of 95 25
lM (pH 7). On the other hand, compound
4e behaves as a competitive inhibitor with a Ki value of 20
4 lM
(pH 7). The Hanes linearization has been used to assess the inhibi-
tion type (see Supplementary data).38 The last compound, 4d, is
less efficient than 3e and 4e for inhibiting the b-lactamase.
There is a hydrophobic pocket in the active site of OXA-10 in
which the molecules equipped with lipophilic benzyl groups may
interact. The residues located in this hydrophobic core are essential
for the catalytic efficiency of class D b-lactamases. In order to pro-
pose a mode of interaction of this series of compounds, manual
docking of molecule 4e in the active site has been carried out on
Table 1
Inhibition of b-lactamases by aminocitrate derivatives at pH 7 (pH 5)
Entry
Compounda
TEM-1
BS3
NMCA
P99
OXA-10
BcII
VIM-4e
1
2
3
4
5
3b11
3c
3d
3e
4b
71b (X)
81b (X)
X
100 (X)
X
82 (X)
100 (X)
X
100 (X)
X
60 (X)
63 (X)
X
X
X
87 (X)
100 (X)
X
X
X
98 (X)
92 (93)
100 (93)
X (100)
X (90)
100 (X)
X
X
X
X
X
X
X
100 (X)
100 (X)
10 (X)
X
6
4d
X
X
X
X
10 (X)
7
8
9
4e
5d
X
X
X
X
10 (X)
X
X
98d (X)
96 (100)
95d (X)
99 (92)
99 (87)
94 (80)
88 (69)
94 (80)
100 (86)
100 (90)
96 (X)
80 (70)
88 (X)
96 (97)
95 (100)
85 (96)
100 (100)
100 (100)
100 (100)
85 (97)
66 (X)
96 (85)
94 (X)
94 (96)
95 (99)
100 (100)
100 (97)
100 (98)
100 (93)
100 (86)
85 (X)
97 (100)
81 (X)
100 (93)
100 (73)
100 (100)
100 (81)
100 (99)
100 (100)
100 (94)
100 (X)
84 (82)
100 (X)
96 (83)
100 (79)
97 (98)
100 (100)
82 (88)
91 (87)
92 (85)
86 (100)
X (100)
83 (X)
100 (90)
100 (100)
X (100)
100 (93)
X (79)
100
100
100
100
100
96
96
100
94
94
5e
10
11
12
13
14
15
16
17
6a11
6b
6c
8
9 + 10
13 + 14
14
X (100)
15
X (100)c
Results expressed as percentages of initial activities.
a
Compounds were tested at pH 7 in 50 mM phosphate buffer and at pH 5 in 50 mM acetate buffer at a concentration of 100
l
M, otherwise mentioned.
b
c
0.2 mM.
1 mM.
5 mM
d
e
It is not possible to test the activity of VIM-4 at pH 5; X = not tested. The standard deviation for the percentages of initial activities is below 5%.

3596
J. Beck et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3593–3597
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
biochemical evaluation against R39, Eric Sauvage and Georges Dive
for the docking experiments. This work was supported by the Bel-
gian Program on Interuniversity Poles of Attraction (PAI 5/33 and
PAI 6/19), the Fonds de la Recherche Scientifique (FRS-FNRS) and
the Université catholique de Louvain. C.B. is a FRS-FNRS (Belgium)
post-doctoral researcher. J.M.-B. is senior research associate of the
FRS-FNRS (Belgium).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.149.
References and notes
0
100
200
[I] (µM)
300
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l
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Figure 3. Docking of 4e in OXA-10 enzyme. The hydrophobic residues in the active
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the basis of the known position of the amide chain in the complex
of benzylpenicillin with OXA-10.37 As shown in Figure 3, the aro-
matic rings are well located in the hydrophobic pocket.
17. Acidic hydrolysis was followed by a basic work-up for recovering the free
amines 3. Crude amines 3a–b were used in the acylation step. Amines 3c–e
could be purified by column chromatography on silica gel. Thus, pure amines
were used for the acylation step. However, the overall yields of amides 4a–e
from Schiff bases 1a–c remained in the range of 60% (see Supplementary data).
18. Jiang, Y.; Wu, S.; Chen, D.; Ma, Y.; Liu, G. Tetrahedron 1988, 44, 5343.
19. The same cyclized product 8 has been described as the result of the following
sequence of reactions: (i) bis-alkylation of ethyl nitroacetate with ethyl
acrylate in the presence of diisopropylamine; (ii) reduction of the nitro group
by hydrogenation on Raney Ni. (a) Kornblum, N.; Eicher, J. H. J. Am. Chem. Soc.
1956, 78, 1494; (b) Smirnova, A. A.; Perekalin, V. V.; Shcherbakov, V. A. Zh. Org.
Khim. 1968, 4, 2245.
This model remains to be confirmed by co-crystallization exper-
iments. Nevertheless, the 2-aminopropane-1,3-di(benzyloxycar-
bonyl)-2-carboxylic acid motif represents a novel hydrophobic
scaffold for the inhibition of class D b-lactamases. Usually, the
non b-lactamic inhibitors of serine b-lactamases are designed to
covalently interact with the active serine residue, as exemplified
by Pratt and co-workers with
a
-ketoheterocycles,39 phenacetu-
rates40 and diaroyl phosphates.35 Recently, They have described
amido ketophosph(on)ates acting as micromolar inhibitors of class
D b-lactamases and suggested that these inhibitors interact at the
active site in the carbonyl form rather than by formation of tetra-
hedral adducts.36 Our work discloses original inhibitors of class D
b-lactamases which mechanism is most probably based on non
covalent interactions. Mechanistic studies are in progress.
20. (a) Greenstein, J. P.; Izumiya, N.; Winitz, M.; Birnbaum, S. M. J. Am. Chem. Soc.
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Acta Crystallogr., Sect. D 1995, D51, 682.
25. Swaren, P.; Maveyraud, L.; Raquet, X.; Cabantous, S.; Duez, C.; Pedelacq, J.-D.;
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Acknowledgments
The biochemical evaluations against b-lactamases have been
performed in the laboratories of Professors B. Joris and M. Galleni
(University of Liège, Belgium). We thank Astrid Zervosen for the

J. Beck et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3593–3597
3597
28. (a) Carfi, A.; Duee, E.; Galleni, M.; Frère, J.-M.; Dideberg, O. Acta Crystallogr.,
31. The catalytic parameters (kcat
,
Km,
kcat/Km
)
of tested b-lactamases versus
Sect. D 1998, D54, 313; (b) Badarau, A.; Page Michael, I. Biochemistry 2006, 45,
10654.
29. Pournaras, S.; Tsakris, A.; Maniati, M.; Tzouvelekis Leonidas, S.; Maniatis
Antonios, N. Antimicrob. Agents Chemother. 2002, 46, 4026.
nitrocefin are given as Supplementary data.
32. Sauvage, E.; Herman, R.; Petrella, S.; Duez, C.; Bouillenne, F.; Frère, J.-M.;
Charlier, P. J. Biol. Chem. 2005, 280, 31249.
33. Plot v0/vi (ratios of hydrolysis in the absence and in the presence of inhibitors)
30. Protocol of biochemical evaluation: The enzymes were produced and purified
versus inhibitor concentration gave the inhibition constant Ki. For 6a,11
as previously described.10,24–29 The enzymes (1-100 nM) were incubated with
Ki = 250
lM (BS3, pH 5) and 150 lM (TEM-1, pH 5); for 9 + 10, Ki = 105 lM
the tested compounds (100
substrate31 (nitrocefin, 100
buffer (50 mM, pH 5). In the case of metallo-enzymes (class B), HEPES buffer
(10 mM, pH 7) added with ZnCl2 (50 M) was used. Tested compounds were
dissolved in DMSO at 10–100 mM and then diluted with the buffer (final
concentration of DMSO in the testing = 2%); DMSO = 2% had no effect on the
enzyme activity. The hydrolysis rate of nitrocefin was followed by
spectrophotometry at 482 nm with UVIKON 860, 940 and XL apparatus
connected to a computer via a RS232 line. The residual activity was obtained
by comparison with the variation of the absorbance of the reference (sample
without inhibitor) and indicated in Table 1. Results are expressed as % of initial
activities. The standard deviation is below 5%. All experiments were performed
three times.
l
M, otherwise mentioned) and the chromogenic
(TEM-1, pH 5).
lM) in phosphate buffer (50 mM, pH 7) or acetate
34. Adediran, S. A.; Nukaga, M.; Baurin, S.; Frère, J. M.; Pratt, R. F. Antimicrob. Agents
Chemother. 2005, 49, 4410.
35. Majumdar, S.; Adediran, S. A.; Nukaga, M.; Pratt, R. F. Biochemistry 2005, 44,
16121.
36. Perumal, S. K.; Pratt, R. F. J. Org. Chem. 2006, 71, 4778.
37. Maveyraud, L.; Golemi-Kotra, D.; Ishiwata, A.; Meroueh, O.; Mobashery, S.;
Samama, J.-P. J. Am. Chem. Soc. 2002, 124, 2461.
38. Cornish-Bowden, A. Fundamental of Enzyme Kinetics; Portland Press Ltd:
London, UK, 2001.
l
39. Kumar, S.; Pearson, A. L.; Pratt, R. F. Bioorg. Med. Chem. 2001, 9, 2035.
40. Adediran, S. A.; Cabaret, D.; Flavell, R. R.; Sammons, J. A.; Wakselman, M.; Pratt,
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