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Novel ferrocenyl phosphonate derivatives. Inhibition of serine hydrolases by ferrocene azaphosphonates

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Full Paper
Received: 15 January 2010
Revised: 1 March 2010
Accepted: 18 April 2010
Published online in Wiley Online Library: 28 June 2010
( DOI 10.1002/aoc.1673
Novel ferrocenyl phosphonate derivatives.
Inhibition of serine hydrolases by ferrocene
Bogna Rudolfa∗ , Michèle Salmainb , Pierre Haquetteb, Marcin Stachowiczc
and Krzysztof Woźniakc
Owing to their unique properties, ferrocene compounds are gaining increasing interest for biological applications, particularly
as enzyme inhibitors. Phosphonate derivatives including a ferrocenyl moiety were synthesized by reaction of dimethyl- and
diphenylphosphite with ferrocenyl methyl maleimide. The ferrocenyl diphenyl phosphonate complex was characterized by
X-ray diffraction. The ability of these organometallic compounds to inhibit the enzymatic activity of the serine esterases acetyland butyrylcholinesterase was investigated. It appeared that the new ferrocenyl phosphonates inhibited both enzymes by
competitive, mixed or non competitve mechanisms with inhibition constants in the range 35–1000 µM. Both compounds also
behave as slow time-dependent inactivators of butyrylcholinesterase. The presence of the ferrocenyl entity seems then to have
c 2010 John Wiley & Sons, Ltd.
a dramatic effect on the biochemical behavior of the systems. Copyright Supporting information may be found in the online version of this article.
Keywords: ferrocene derivatives; phosphonates; biorganometallic chemistry; enzyme inhibition; cholinesterases
Appl. Organometal. Chem. 2010, 24, 721–726
Results and Discussion
Ferrocenyl dimethyl- and diphenyl phosphonate complexes 3a–b
were prepared by a method previously elaborated for their
metallocarbonyl analogs 1a–b.[7] The phospha-Michael addition
of dimethyl- or diphenyl-phosphite to maleimide 2 was carried
out in the presence of DBU to afford the ferrocenyl phosphonates
3a-b (Scheme 1).
The addition of phosphite to the carbon–carbon double
bond was confirmed by the lack of ethylenic protons signals
characteristic of the maleimide ferrocenyl derivative 2 in the
Correspondenceto:Bogna Rudolf,DepartmentofOrganicChemistry,University
of Łódź 91-403 Łódź, Tamka 12, Poland. E-mail:
a Department of Organic Chemistry, University of Łódź 91-403 Łódź, Tamka 12,
b Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire
Charles Friedel, CNRS UMR 7223, 11 rue Pierre et Marie Curie 75231 Paris cedex
05, France
c Chemistry Department, University of Warsaw, Pasteura 1, 02093 Warszawa,
c 2010 John Wiley & Sons, Ltd.
Copyright 721
Ferrocene and its derivatives are of increasing importance as
regards biological applications.[1] Incorporation of ferrocene units
into molecules of biological relevance introduces new useful
features and properties in these compounds.[2] Because of their
chemical stability and non-toxicity, ferrocenyl compounds are
already applied as electroactive labels of biologically active
compounds.[3] Other ferrocenyl derivatives display therapeutically
useful properties that may open new avenues in the treatment of
diseases like malaria[4] and cancer.[5]
Ferrocene compounds containing a phosphonate moiety are of
interest owing to their electrochemical properties and their ability
to strongly bind to metal oxide solid supports.[6] Interestingly, no
data have been reported so far on the biochemical activity of these
molecules, in particular their ability to inhibit serine hydrolases.
We are interested in the synthesis of phosphonate derivatives containing a metallocarbonyl entity and recently reported
the synthesis of the half-sandwich iron phosphonate complexes
1a–b (Fig. 1) derived from (η5 -C5 H5 )Fe(CO)2 (η1 -N-maleimidato).[7]
Organophosphorus compounds are able to inhibit serine hydrolases by phosphorylation of the active site serine residue. For
example, the diphenyl phosphonate 1b was shown to inhibit the
serine hydrolase butyrylcholinesterase (BChE) in a reversible manner according to a competitive mechanism and inactivated this
enzyme in a time-dependent fashion by phosphonylation of its
active site serine residue. Likewise, NCN pincer complexes bearing
a phosphonate group were able to phosphonylate the active site
serine residue of the lipase cutinase.[8]
In this paper, we describe the synthesis of two novel
phosphonate derivatives containing a ferrocene moiety 3a–b
derived from the maleimide complex 2. The molecular structure
of compound 3b was established by X-ray crystallography. We
studied the inhibitory activity of 3a–b towards two clinically
important serine hydrolases, namely acetylcholinesterase (AChE)
and BChE. It appeared that the diphenyl phosphonate ester 3b
behaved as a competitive inhibitor of BChE in a short timescale
whereas both compounds very slowly inactivated this enzyme in
a time-dependent fashion.
B. Rudolf et al.
R1 P OR3
3 R1 P OR + R O
O Enz
spontaneous hydrolysis
Enz-OH+ R1 P OR
Figure 1. Structure of 1a–b.
Scheme 2. Mechanism of inhibition of serine hydrolases by phosphonates.
+H P
OR (i)
3a- b
a R = CH3 ; b R = Ph
Scheme 1. Reagents and conditions: (i) 2 (0.33 mmol), dimethyl- or
diphenylphosphite (0.31 mmol), DBU (0,13 mmol), CH2 Cl2 , 18 h, r.t.,
was almost flat as the torsion between the four carbons of this
ring was equal to 12.5◦ . The configuration at the chiral carbon
of the succinimide ring was R. Intermolecular interactions were
observed between the two oxygens of the succinimide ring and
the unsubstituted Cp ring of two neighbouring molecules on the
one hand and one of the phenyl groups of two neighbouring
molecules (2.41 ≤ dO-H ≤ 2.65 Å) on the other hand.
Biochemical Assays
NMR spectrum of 3a–b. Complex patterns observed at
2.9–3.4 ppm suggest the formation of two enantiomers owing
to the presence of a stereogenic center on the succinimide ring.
The 31 P NMR spectrum of 3a–b displayed a singlet in the region
between 10 and 25 ppm, typical of phosphonates. Their IR spectra
display an imide carbonyl stretching band at ∼1700 cm−1 and a
band around 1250 cm−1 , assignable to the stretching vibration of
the P O bond.
Compound 3b crystallized in the monoclinic C2/c space group
with eight molecules of 3b and four molecules of disordered
dichloromethane (Fig. 2) per unit cell. The bulky diphenyl
phosphonate substituent was attached to the succinimide ring
in a direction opposite to the Fc unit. The best planes formed
by the substituted Cp ring of the Fc unit formed an angle of
111.5◦ with the best plane of the succinimide ring. This ring
Several very important enzymes pertain to the serine hydrolases
class, namely proteases such as trypsin, lipases and esterases
such as acetylcholinesterase and butyrylcholinesterase. All these
enzymes contain an essential serine residue in the catalytic
site together with two other residues forming altogether the
catalytic triad that is involved in the catalytic cycle. Phosphonate
derivatives are known to react with the active site serine
residue of these enzymes, affording the phosphorylated enzymes
as illustrated in Scheme 2. The phosphorylated enzyme may
further move towards the regenerated form or alternatively
towards an ‘aged’ form corresponding to irreversible inhibition.
Therefore organophosphates are considered as potent inhibitors
of serine hydrolases.[9 – 11] This process has also been exploited
for the detection of organophosphorous pesticides by enzyme
Figure 2. Molecular structure of 3b. Thermal ellipsoids were drawn at the 50% probability level. Some bond lengths (Å) and angles (deg): average
(Fe1–C1–5) = 2.044(3); average (Fe1–C6–10) = 2.042(7), N12–C11 = 1.466(2), P16–C15 = 1.7915(18), P16–O3 = 1.4565(15), P16–O24 = 1.5762(13),
O24–P16–O17 = 102.16(7).
c 2010 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2010, 24, 721–726
Novel ferrocenyl phosphonate derivatives
Table 1. Inhibition constants KI and K I and IC50 for the inhibition of
AChE and BChE by complexes 1a, 1b, 3a and 3b
Compound Enzyme
IC50 ,
Inhibition constants,
No inhibition
No inhibition
No inhibition
n.d., Not determined.
Appl. Organometal. Chem. 2010, 24, 721–726
c 2010 John Wiley & Sons, Ltd.
Cholinesterase inhibitors have been used as drugs administered
to slow the progression of Alzheimer’s disease, which is associated
with a decrease in AChE activity while the activity of BChE
increases. In this context, the discovery of BChE-selective inhibitors
would be appreciable.[13] Law reported that simple dialkyl phenyl
phosphates were able to inhibit AChE and/or BChE in a reversible
manner by a competitive or partially competitive mechanism.[14]
Recently we found out that the metallocarbonyl phosphonate 1b
acted as a competitive inhibitor of BChE and was also able to
inactivate both BChE and α-chymotrypsin in a time-dependent
fashion.[7] Thus, we thought it was interesting to investigate
whether the ferrocenyl phosphonate diester complexes 3a–b
were also able to inhibit the cholinesterases AChE and BChE and
what was their mechanism of inhibition.
The enzymes (AChE from electrical eel and BChE from horse
serum) were from commercial sources and used as received.
Several types of experiments were carried out in order to delineate
the reversible and irreversible inhibiting properties of these
compounds and determine kinetic constants. Table 1 summarizes
the results of the inhibition experiments for the organometallic
complexes 1a–b and 3a–b towards AChE and BChE.
One immediately notices a very different behavior for the halfsandwich 1a–b and sandwich complexes 3a–b towards both
enzymes. Globally, the ferrocenyl derivatives were much more
efficient inhibitors than the metallocarbonyl derivatives whatever
the enzyme was. For instance, compound 1a had no effect on
both AChE and BChE whereas compound 3a behaved as a mixed
inhibitor of both AChE and BChE. However, let us notice that 3a
did not exhibit any selectivity between both enzymes, making
it less interesting as a potential drug. Conversely, the ferrocenyl
derivative 3b was 10 times more efficient on BChE than on
AChE with an inhibition constant as low as 35 µM; this is an
improvement by a factor of 5 with respect to compound 1b.
Very few other ferrocenyl derivatives are actually known to inhibit
cholinesterases,[15] making this compound interesting for further
Although both enzymes share a high degree of homology, they
display subtle differences in their active site. This site consists of a
20 Å length hydrophobic gorge at the bottom of which are located
the esterasic subsites including the catalytic triad Ser, His and Glu
and the anionic subsite accommodating the cationic (choline)
part of the substrate.[16] The latter subsite is known to the larger
in BChE because of the replacement of two aromatic residues
in AChE by aliphatic residues.[17] Both enzymes also display a
peripheral anionic site (containing aromatic residues) involved
in the accessibility of small molecules to the active site.[16] The
gorge of AChE is lined with 14 aromatic residues while six of these
residues are aliphatic in the case of BChE, making the volume of
the active site of BChE larger by 200 Å 3 (180 and 362 Å 3 for AChE
and BChE, respectively).[17] Previous studies on cholinesterase
inhibitors enlightened the importance of the dimension and
microenvironment of the gorge as regards the selectivity and
mode of inhibition of ligands.[14,17] For example, propidium is
an uncompetitive inhibitor of AChE and X-ray crystallographic
studies actually showed that it bound to the peripheral anionic
site,[18] while it is a competitive inhibitor of BChE. Similarly, tacrine
is a noncompetitive inhibitor of AChE but a mixed inhibitor of
BChE.[17] By analogy with these results, the relative sizes of the
active site of AChE and BChE probably dictated the ability of 3a
and 3b to inhibit these enzymes and the mechanism by which
they operated. The presence of two bulky phenyl substituents
may prevent 3b (estimated volume 323 Å 3 ) enteing deeply in the
gorge of AChE because of its narrowness, allowing the substrate
to enter and be hydrolyzed. Conversely, 3b may be allowed to
penetrate more deeply in the gorge of BChE and thus competes
with butyrylthiocholine for binding. Compound 3a, being less
voluminous, (240 Å 3 ) may enter more freely into both enzymes’
active sites.
Although neither 3a nor 3b contained good leaving groups to
behave as irreversible inhibitors, their ability to behave as timedependent inactivators of AChE and BChE was further examined as
1b was previously shown to inactivate BChE with a second-order
rate constant k2nd of 8.6 M−1 min−1 . For this purpose, the enzymes
were incubated with the complexes in large excess. Aliquots were
removed periodically and the enzyme activity of the mixtures
measured with appropriate substrates according to the Kitz and
Wilson procedure.[19] No time-dependent inactivation of AChE was
observed over a period of 4000 min, while slow inactivation of BChE
was observed with the two complexes. The rates of inactivation
followed a pseudo-first-order law (Fig. 3). This allowed calculation
of k2nd of 0.6 and 1 M−1 min−1 for the dimethyl complex 3a and
the diphenyl complex 3b, respectively. Thus, both compounds
inactivated BChE much more slowly than 1b. Inactivation of serine
hydrolases by organophosphates occurs via the nucleophilic
addition of the active site serine to the phosphorous atom to
form a pentacoordinate intermediate from which is released
an alcoholate anion to give the phosphor(n)ylated enzyme
(Scheme 2). The rate of nucleophilic addition is obviously related
to the positioning of the inhibitor within the enzyme’s active
site. The very slow rates observed here may be related to the
unfavorable positioning of the phosphonate group relatively to
the serine residue that makes the nucleophilic attack difficult.
MeO− being a poorer leaving group than PhO− , it may explain
why 3a inactivated BChE more slowly than 3b. It can be concluded
that these phosphonate compounds behave almost exclusively as
reversible inhibitors of AChE and BChE.
In conclusion, two ferrocenyl complexes containing a phosphonate moiety were prepared by phospha-Michael addition of
diphenyl- or dimethyl phosphite to ferrocenyl methylmaleimide
and characterized by classical spectroscopic methods including
X-ray crystallography for one of them. Biochemical studies carried
out on serine hydrolases showed that these compounds were able
to inhibit the catalytic activity of acetyl- and butyrylcholinesterase
in a reversible manner. Very slow time-dependent inactivation of
BChE by the two derivatives was also observed. The ferrocenyl
diphenyl complex appeared to be the most active compound of
B. Rudolf et al.
3b: yield 140 mg (85%). 1 H NMR (600 MHz, CDCl3 , δ in ppm):
2.85–3.25 (m, 2H), 3.45–3.65 (m, 1H), 4.11 (t, J = 1.7 2H), 4.17
(s, 5H), 4.34 (t, J = 1.7 2H), 4.51 (s, 2H), 7.04–7.34 (m, 10H). 13 C
NMR (150 MHz, CDCl3 , δ in ppm) 30.35 (d, JCP = 3.0 Hz), 38.84 (d,
JCP = 144.0 Hz), 38.96, 68.50, 68.68, 69.66 (d, JCP = 9, 0 Hz), 81,10,
120,43 (d, JCP = 4, 5 Hz), 120,72 (d, JCP = 4, 5 Hz), 125,69, 125,84,
129.84, 129.95, 149.74 (d, JCP = 9, 0 Hz), 170.49 (d, JCP = 6, 0 Hz),
173.77 (d, JCP = 6, 0 Hz); 31 P NMR (243 MHz, CDCl3 , δ in ppm):
12.24. IR (CHCl3 ν in cm−1 ) 1707 (C O imide), 1280 (P O). Anal.
calcd for C27 H24 FeNO5 P + 1/3 CH2 Cl2 : C, 58.87; H, 4.46; N, 2.51;
found C, 58.85; H, 4.57; N, 2.61.
Structure Determination
Figure 3. Time-dependent inactivation of BChE by 3a () and 3b () at 0.5
and 0.1 mM, respectively.
the series including the metallocarbonyl complex analogues with
an IC50 of 10 µM and a competitive inhibition constant of 35 µM.
It also allowed efficient discrimination between AChE and BChE. It
turns out that the nature of the substituents on the phosphorus
atom has a marked influence on the affinity constant and the
reactivity of these molecules.
1,8-Diazabicyclo-(5,4,0)undec-7-ene (DBU) was purchased from
Aldrich. Dimethyl- and diphenylphosphite were obtained from
Fluka. The starting ferrocenyl derivative 2 containing the
maleimide moiety was synthesized according to a previously
reported method.[20] 1 H, 31 P, 13 C NMR spectra were recorded in
CDCl3 on Varian Gemini 200BB (200 MHz for 1 H) and Brucker
Avance III (600 MHz for 1 H), referenced to internal TMS. IR spectra
were recorded as KBr pellets on an FT-IR Nexus (Thermo Nicolet)
spectrometer. All solvents were purified according to standard
procedures. Chromatographic separations were performed on Silica gel Merck 60 (230–400 mesh ASTM). All reactions were carried
out under argon.
Ferrocenyl complexes (3a–b)
An argon-saturated solution of ferrocenyl methylmaleimide
2 (100 mg, 0.33 mmol) in dichloromethane was treated with
dimethyl- (28.5 µl, 0.31 mmol) or diphenyl- (60 µl, 0.31 mmol)
phosphite containing DBU (20 mg, 0,13 mmol). The reaction
mixture was stirred overnight at room temperature. The mixture
was purified by column chromatography on silica gel. An
orange band containing the starting material 2 was eluted by
dichloromethane, followed by second light orange band eluted
by dichloromethane–ethyl acetate 2 : 1. Products 3a–b were
crystallized from dichloromethane–heptane.
3a: yield 61 mg (49%). 1 H NMR (200 MHz, CDCl3 , δ in ppm):
2.81–3.33 (m, 3H, succinimide), 3.78 (dd, J = 2.4, 11, 6H), 4.12 (s,
2H), 4.20 (s, 5H), 4.33 (s, 2H), 4.44 (s, 2H). 13 C NMR (50 MHz, CDCl3 ,
δ in ppm) 30.43 (d, JCP = 3.5 Hz), 38.75, 38.87 (d, JCP = 142.5 Hz),
54.03 (d, JCP = 6.6 Hz), 54.44 (d, JCP = 6.6 Hz), 68.40, 68.69, 69.53
(d, JCP = 2.5 Hz), 81.26, 171.98, 174.20; 31 P NMR (81 MHz, CDCl3 ,
δ in ppm): 22.19. IR (CHCl3 , ν in cm−1 ) 1705 (C O imide), 1258
(P O). Anal. calcd for C17 H20 FeNO5 P: C, 50.39; H, 4.98; N, 3.46;
found C, 50.25; H, 5.00; N, 3.21.
The single-crystal X-ray diffraction experiment was carried out from
an orange crystal of 3b (grown from CH2 Cl2 –heptane mixture)
with a Bruker Kappa ApexII Ultra controlled by ApexII software,
equipped with Mo Kα rotating anode X-ray source (λ = 0.71073 Å,
50.0 kV, 22.0 mA) monochromatized by multi-layer optics and an
ApexII CCD detector. The temperature was maintained at 100 K
using the Oxford Cryostream cooling device. The structure was
solved by direct methods using the SHELXS-97 program and
refined with the SHELXL-97.[21] Multi-scan absorption corrections
were applied in the scaling procedure.
The refinement was based on F2 for all reflections except those
with negative intensities. Weighted R factors, wR and all goodnessof-fit S values were based on F2 , whereas conventional R factors
were based on the amplitudes, with F set to zero for negative
F 2 . The F0 2 > 2σ (F0 2 ) criterion was applied only for R factors
calculation was not relevant to the choice of reflections for the
refinement. The R factors based on F2 are for all structures about
twice as large as those based on F. The hydrogen atoms were
located in idealized geometrical positions, except hydrogens in
solvent molecule. Scattering factors were taken from Tables
and of the International Crystallographic Tables, Vol. C.[22]
A 15% disorder for Cl1 atom in solvent molecule present was
Crystal data for 3b: C55 H49 Cl2 Fe2 N2 O10 P2 , M = 1142.50,
monoclinic, C2/c, a = 24.7102(8), b = 7.8672(3), c = 25.9289(9) Å,
β = 99.035(2), V = 4978.0(3) Å 3 , Z = 4, Dcalc = 1.524 g cm−3 ,
µ = 0.818 mm−1 , θmax = 28.47◦ , no. of reflections = 22398, no. of
unique reflections with I > 2σ (I) = 6198, no. parameters = 348,
R1 = 0.0368, wR2 = 0.0813.
Enzyme Assays
Butyrylcholinesterase (E.C., horse serum, Fluka) activity
measurements were performed on the basis of the Ellman’s
thiol assay according to Doctor et al.[23] with slight modification.
A stock solution of BChE (10 mg/ml) was prepared in 50 mM
phosphate buffer pH 8.0. The solution was diluted to 40 µg/ml
in the same buffer. The substrate mixture was prepared by
mixing butyrylthiocholine iodide (BTCh, Fluka; 50 mM in H2 O, 50 µl)
and 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB, Lancaster; 1 mM in
10 mM Tris–HCl, 0.01% gelatine pH 7.6, 5 µl) and incubated at
30 ◦ C. A 200 µl aliquot of substrate mixture was dispersed in wells
of microplate (Grenier). The enzymatic reaction was initiated by
dispensing 10 µl of BChE solution (40 µg/ml) into the wells. The
optical density at 415 nm was monitored during 4 min with a
microplate reader (model 680, Biorad) thermostated at 30 ◦ C. In
these conditions, BChE has a specific activity of 6 u/mg of solid.
Acetylcholinesterase (from electric eel, type VI-S, EC, Sigma)
c 2010 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2010, 24, 721–726
Novel ferrocenyl phosphonate derivatives
The Michaelis–Menten equation reflecting both interactions is:
EI + S
E + P 1 + P2
where the modifying factors α and α are:
Scheme 3. Reversible inhibition. Competitive type: K I = 0; mixed type:
KI = K I ; non-competitive type: KI = K I .
activity measurements were performed as above except that BTCh
was replaced by acetylthiocholine (ATCh, Sigma) and the enzyme
solution was diluted to 0.8 µg/ml. In these conditions, AChE has a
specific activity of 166 u/mg of protein.
Inhibition Assays of BChE
IC50 of 3a
Various amounts of a 5 mM solution of 3a in MeOH (0 to 20 µl)
were pipetted in a series of glass tubes and the volume was made
up to 20 µl by addition of MeOH. Then 200 µl of 0.5 mM BTCh
and 1 mM DTNB in 10 mM TRIS HCl pH 7.6 were added. Catalytic
activity was measured by monitoring of OD415 nm during 1 min
at RT after addition of 10 µl of a 40 µg/ml solution of BChE in
100 mM phosphate pH 8.0 with a UV–vis spectrometer. The IC50
was calculated from linear regression of plot of logit (% residual
activity) vs log([3a]) [logit(% residual activity) = log(% residual
activity)/(100 − % residual activity)].
IC50 of 3b
Various solutions of 3b in MeOH (0 to 0.08 mM, 10 µl) were
pipetted in wells of a microtiter plate. Then BChE (40 µg/ml,
100 µl) was added to the wells. Ten microliters of these mixtures
was transferred into the adjoining wells and the mixture of 0.5 mM
BTCh and 1 mM DTNB in 10 mM TRIS HCl pH 7.6 (200 µl) was
added. Catalytic activity was measured by monitoring of OD415 nm
during 4 min at 30 ◦ C with a microtiter plate reader. The IC50
was calculated from linear regression of plot of logit(% residual
activity) vs log([3b]) [logit(% residual activity) = log(% residual
activity)/(100 − % residual activity)].
Inhibition constants determination
α =1+
and α = 1 + KI
KM , V, (1/α )V and (α/α )KM were estimated by nonlinear
regression analysis of rate vs substrate concentration plots in
the absence or presence of inhibitor with Kaleidagraph.
Inhibition assay of AChE
Various volumes of a 6 mM aqueous solution of ATCh (1–10 µl)
were pipetted in a series of glass tubes and the volume was
brought up to 250 µl by addition of 1 mM DTNB in 10 mM TRIS.HCl
pH 7.6. Then 20 µl of a methanolic solution of complex was added
(1 mM for 3b or 5 mM for 3a). Alternatively, 20 µl of methanol was
added for Km and V determination. Catalytic activity was measured
by monitoring of OD415 nm during 1 min at RT after addition of 30 µl
of a 0.8 µg/ml solution of AChE in 100 mM phosphate pH 8.0. KM ,
V, (1/α )V and (α/α )KM were estimated by nonlinear regression
analysis of rate vs substrate concentration plots in the absence or
presence of inhibitor with Kaleidagraph.
Time-dependent Inactivation of AChE and BChE
BChE (40 µg/ml, 50 mM phosphate buffer pH 8, 100 µl) was mixed
with inhibitor (1 mM for 3b or 5 mM for 3a, in MeOH, 10 µl).
A control experiment was performed where the solution of
complex was replaced by MeOH. Aliquots (10 µl) were withdrawn
at recorded times and the enzymatic activity was measured under
standard conditions (see above). The logarithm of the percentage
residual activity was plotted as a function of time to calculate the
pseudo-first-order rate constant kobs and deduce the second order
rate constant k2nd . The same procedure was applied with AChE
(0.8 µg/ml).
Supporting Information
Crystallographic data (excluding structure factors) for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication numbers
CCDC 756101. Copies of the data can be obtained, free of charge,
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK [fax: +44 (0)1223–336033 or e-mail:].
Supporting information can also be found in the online version of
this article
The French Ministry of Foreign Affairs and the Polish Ministry
of Science and Education are gratefully acknowledged for scientific exchanges through the ‘POLONIUM’ programme. The
Structural Research Laboratory of the Chemistry Department,
Warsaw University, Poland, gratefully acknowledges financial support from the European Regional Development Fund in the
Sectorial Operational Programme ‘Improvement of the Competitiveness of Enterprises, years 2004–2006’ (project no. WKP
1/1.4.3./1/2004/72/72/165/2005/U) and from the Foundation for
Polish Science for K.W. (Mistrz Professorship).
c 2010 John Wiley & Sons, Ltd.
Various volumes of a 12.5 mM aqueous solution of BTCh (1–10 µl)
were pipetted in a series of glass tubes and the volume was
brought up to 200 µl by addition of 1 mM DTNB in 10 mM TRIS.HCl
pH 7.6. Then 20 µl of a methanolic solution of complex was added
(1 mM for 3b or 5 mM for 3a). Alternatively, 20 µl of methanol was
added for Km and V determination. Catalytic activity was measured
by monitoring of OD415 nm during 1 min at RT after addition of 10 µl
of a 40 µg/ml solution of BChE in 100 mM phosphate pH 8.0. The
interaction between the enzymes and the inhibitors is described
in Scheme 3, where E is the enzyme, S is the substrate, I is the
inhibitor, ES is the enzyme–substrate complex and P1 and P2 are
the products. KI and K I are the inhibition constants reflecting the
interaction of inhibitor with free enzyme and enzyme–substrate
complex, respectively.
Appl. Organometal. Chem. 2010, 24, 721–726
(1/α ) × V × [S]
V × [S]
α × KM + α × [S]
(α/α ) × KM + [S]
B. Rudolf et al.
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serine, azaphosphonates, inhibition, phosphonate, hydrolases, novem, derivatives, ferrocenyl
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