Non-tethered organometallic phosphonate inhibitors for lipase inhibition: Positioning of the metal center in the active site of cutinase

Article abstract of DOI:10.1002/ejic.200800654

Organometallic NCN-pincer complexes, bearing either a p-nitrophenyl phosphonate ester or a phosphonic acid group directly attached to the aromatic ring of the pincer complex, were synthesized. These compounds were tested as covalent inhibitors for the lipase cutinase. In a stoichiometric reaction of the NCN-pincer platinum phosphonate p-nitrophenyl ester 2 with cutinase, a 94% conversion to the protein-pincer metal complex hybrid was obtained in 48 h. The NCN-pincer metal phosphonic acid derivatives (3, 4) appeared to be inactive as cutinase inhibitors. In contrast to our previous work which entails propyl tethered phosphonate esters connected to pincer metal complexes, the presented strategy allows positioning of metal complexes inside the active site of lipases. This opens up the possibility for fine-tuning the chemical environment (second coordination sphere) around a synthetic metal center inside the pocket of an enzyme for diagnostic and catalytic purposes. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejic.200800654

FULL PAPER
DOI: 10.1002/ejic.200800654
Non-Tethered Organometallic Phosphonate Inhibitors for Lipase Inhibition:
Positioning of the Metal Center in the Active Site of Cutinase
Cornelis A. Kruithof,[a] Harmen P. Dijkstra,[a] Martin Lutz,[b] Anthony L. Spek,[b]
Maarten R. Egmond,[c] Robertus J. M. Klein Gebbink,*[a] and Gerard van Koten*[a]
Dedicated to Jan Reedijk on the occasion of his 65th birthday[‡]
Keywords: Protein modifications / Inhibitors / Phosphonates / ECE-Pincer complexes / Hydrolases
Organometallic NCN-pincer complexes, bearing either a p-
nitrophenyl phosphonate ester or a phosphonic acid group
directly attached to the aromatic ring of the pincer complex,
were synthesized. These compounds were tested as covalent
inhibitors for the lipase cutinase. In a stoichiometric reaction
of the NCN-pincer platinum phosphonate p-nitrophenyl es-
ter 2 with cutinase, a 94% conversion to the protein–pincer
metal complex hybrid was obtained in 48 h. The NCN-pincer
metal phosphonic acid derivatives (3, 4) appeared to be inac-
tive as cutinase inhibitors. In contrast to our previous work
which entails propyl tethered phosphonate esters connected
to pincer metal complexes, the presented strategy allows po-
sitioning of metal complexes inside the active site of lipases.
This opens up the possibility for fine-tuning the chemical en-
vironment (second coordination sphere) around a synthetic
metal center inside the pocket of an enzyme for diagnostic
and catalytic purposes.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
Introduction
from an inhibited hydrolase is extremely slow at normal pH
values (Ͻ pH ≈ 11), and for this reason the reaction is re-
garded as an irreversible process.[5]
Phosphonates bearing at least one good leaving group
(e.g. fluoride or p-nitrophenyl) are well-known for their
powerful inhibition activity of serine hydrolases, a family
of enzymes responsible for ester and amide hydrolysis in
nature.[1–4] Their inhibition efficiency originates from their
structural properties, which closely mimic the first transi-
tion state of ester and amide, hydrolysis reactions (Fig-
ure 1). Upon covalent binding of the phosphorus atom to
the nucleophilic Oγ atom of the catalytic serine residue, the
leaving group is released to afford a covalent protein–phos-
phonate adduct. Release of a second leaving group is pre-
vented because of the pentavalency of the phosphorus
atom. Therefore, cleavage of the phosphorylating agent
Figure 1. Left: transition state of ester hydrolysis by lipases; right:
the covalently attached phosphonate as the transition state ana-
logue.
[‡] In recognition of his outstanding contributions to the field of
coordination chemistry.
Recently, we reported the attachment of active-site-di-
[a] Chemical Biology & Organic Synthesis, Debye Institute for
Nanomaterials Science, Faculty of Science, Utrecht University, rected complexes (ASDCs) to the lipase cutinase, in which
Padualaan 8, 3584 CH Utrecht, The Netherlands
we implemented p-nitrophenylphosphonate esters as coval-
Fax: +31-30-2523615
ent inhibitors (1, Figure 2).[6] The phosphonate esters were
E-mail: g.vankoten@uu.nl
r.j.m.kleingebbink@uu.nl
linked by a propyl tether to terdentate pincer metal com-
plexes comprising the well-known ECE-pincer type ligand
{ECE = [2,6-(CH2E)2C6H3]–; E = e.g. PR2, SR, NR2}.[7]
Pincer metal complexes have proven to be versatile com-
pounds and have found many applications in the field of
homogeneous catalysis,[8] sensoring materials,[9] and supra-
[b] Membrane Enzymology, Faculty of Science, Utrecht University,
Padualaan 8, 3584 CH Utrecht, The Netherlands
[c] Crystal and Structural Chemistry, Faculty of Science, Utrecht
University,
Padualaan 8, 3584 CH Utrecht, The Netherlands
Supporting information for this article is available on the
WWW under http://www.eurjic.org or from the author.
Eur. J. Inorg. Chem. 2008, 4425–4432
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4425

R. J. M. Klein Gebbink, G. van Koten et al.
FULL PAPER
Figure 2. Phosphonate-appended NCN-pincer metal complexes.
molecular assemblies.[10] From molecular modeling studies desired product 2. The phosphonic acid pincer complexes 3
and preliminary X-ray crystal structures of some of these and 4 were prepared from previously reported complexes 9
protein–pincer metal hybrids,[11] we now know that the pro- and 10, respectively.[16] Treatment of a solution of either
pyl linker places the metal center at the periphery of the complex 9 or 10 with ten equivalents of bromotrimethylsil-
protein. Additionally, since the active site is well exposed at ane in dichloromethane, followed by addition of dry meth-
the protein surface, the metal pincer head group is located anol, generated the phosphonic acids (Scheme 2). Full con-
at the interface between the protein and the solvent.
version of the starting materials was confirmed by 31P{1H}
For potential applications of such ASDCs in the area of NMR (D2O) analysis, showing a singlet at δ = 14.38 and
protein diagnostics,[12] in non-natural (bio)catalysis,[13] or as 19.01 ppm for 3 and 4, respectively. Both complexes are
supramolecular building blocks using the unique properties water-soluble and nearly insoluble in dichloromethane and
of the pincer moiety, we have become interested in placing benzene.
the metal atom more deeply in the active site of cutinase.
For this reason, we have chosen a system in which a phos-
phonate moiety is directly attached to the aromatic ring of
the metal complex (see 2, Figure 2). This will place the
metal center well inside the active site of cutinase and po-
tentially other serine hydrolases (the superfamily to which
cutinase belongs).[14] Besides phosphonate esters, also phos-
phonic acid derivatives have been reported to be active in-
hibitors of serine hydrolases in various cases. However, they
have never been tested for lipases, e.g. cutinase.[15] From a
synthetic point of view, pincer-containing phosphonic acids
3 and 4 (Figure 2) are of interest, since the route to their
synthesis appeared to be considerably shorter than the syn-
thetic route to phosphonate compounds like 1 and 2.[16]
Herein, we report the synthesis of ASDCs 2, 3, and 4, as
well as their behavior in inhibition studies with cutinase.
Results and Discussion
Scheme 1. Synthesis of 2. Reagents and conditions: (a) tBuLi
(2 equiv.), Et2O, –100 °C. (b) –100 °C Ǟ room temp., 2 h. (c)
Synthesis of Complexes 2, 3, and 4
[Pt(tol-4)(SEt2)]2, C6H6, reflux, 1 h. (d) AgBF4, acetone, H2O,
room temp., 1 h. (e) excess NaCl, CH2Cl2, room temp., 2 h. (f) 1 
HCl/Et2O (5 equiv.), C6H6, room temp., 3 h. (g) p-nitrophenol,
NEt3, C6H6, room temp., 1 h.
Compound 2 was prepared in a five-step reaction se-
quence starting from 2-bromo-1,3-bis[(dimethylamino)-
methyl]-5-iodobenzene[17] and ethyl chloro-N,N-dimeth-
ylamino-phosphinate (5, Scheme 1). The formation of the
P–C bond was achieved by selective iodo-lithio exchange
using tBuLi,[18] followed by reaction of the lithiated inter-
mediate with 5 to obtain phosphinate–pincer adduct 6. Cy-
[19]
cloplatination of 6 was effected with [Pt(tol-4)(SEt2)]2 to
give organoplatinum complex 7. Next, a halide exchange
reaction using AgBF4 and NaCl was performed, to prevent
halogen scrambling in the subsequent reaction step,[20]
which resulted in complex 8. The p-nitrophenyl group was
introduced by first reacting 8 with dry HCl (1  in Et2O),
thereby replacing the NMe2 group on the phosphorus atom
Scheme 2. Synthesis of phosphonic acid pincer complexes. Rea-
gents and conditions: (a) 10 equiv. TMSBr, CH2Cl2, 4 h, room
temp., followed by dry MeOH.
Attempts to obtain single crystals suitable for X-ray dif-
by a chlorine atom. Subsequent substitution of the chloro fraction analysis for 2, 3, and 4 failed. We succeeded, how-
atom by a p-nitrophenyl moiety by treating the intermediate ever, in obtaining suitable crystals for 9 and 10 by slow
with p-nitrophenol in the presence of NEt3 provided the evaporation of a concentrated solution in dichloromethane.
4426
www.eurjic.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2008, 4425–4432

Non-Tethered Organometallic Phosphonate Inhibitors for Lipase Inhibition
Both complexes are isomorphous in the solid state (see Sup- releasing p-nitrophenolate, potentially becomes competitive
porting Information for crystallographic details). As a rep- and cannot be ignored anymore when monitoring p-nitro-
resentative picture, the molecular structure of 9 in the solid phenolate release as a measure of cutinase inhibition yields.
state is shown in Figure 3.
The P–O bond between the phosphonate moiety and the p-
nitrophenol group is rather labile and can lead to “sponta-
neous” hydrolysis under the applied reaction conditions
(pH 8.0). Therefore, for our inhibition process, we decided
to monitor the course of the reaction spectrophotometri-
cally by using a double beam UV/Vis spectrometer in which
a control mixture without cutinase was placed in the refer-
ence cuvette.
Figure 3. View of the molecular structure of 9 in the crystal. Dis-
placement ellipsoids are drawn at the 50% probability level. The
analogous Pt complex 10 is isomorphous in the solid state.
Inhibitory Activity of NCN-Pincer Phosphonate Ester 2
In order to quantitatively determine the inhibition rate
of cutinase with ASDCs 1 and 2, we typically monitored
the concomitant release of p-nitrophenolate during the inhi-
bition process with UV/Vis spectroscopy at 400 nm
(Scheme 3). Treatment of a buffered cutinase solution
(25 µ cutinase, buffer solution containing 0.1% Triton X-
100, 50 m Tris-HCl) at pH 8.0 (Scheme 3) with an excess
of ASDC 1 or 2 (10 equiv.) gave 100% inhibition of cu-
tinase in both cases (data not shown). However, this experi-
ment also revealed that the inhibition of cutinase with 2
was Ͼ500 times slower as compared to inhibition with 1.
Scheme 4. Base-catalyzed hydrolysis of 2 forming p-nitrophenolate
[pH 8.0, pKa (p-nitrophenol) = 7.15].
In order to get a more detailed picture of the inhibition
process with 2, only two equivalents of ASDC 2 in acetoni-
trile (5 m) were added to a buffered cutinase solution.
Monitoring the absorbance at 400 nm gave the reaction
profile as presented by curve A in Figure 4. ASDC 2 is used
as a racemate and, because cutinase is known to react with
enantiomeric preference (the phosphorus atom in 2 is a ste-
reogenic center),[21] addition of two equivalents of 2 was
necessary to ensure the presence of a stoichiometric amount
of the preferred, fast-reacting enantiomer with respect to
Scheme 3. ASDC reaction of 2 with cutinase.
Figure 4. Conversion of cutinase upon reaction with 2. Curve A:
release of p-nitrophenolate monitored by the increase in ab-
sorbance at 400 nm relative to blank. Decay curve B: decrease of
enzymatic activity monitored by using a spectrometric enzyme ac-
tivity assay containing p-nitrophenyl butyrate ester (0.25 m, pH
8.0). Reagents and conditions: 25 µ cutinase, 50 µ ASDC 2,
This is attributed to the C3-tether that is present in 1 and
not in 2, which gives rise to less steric hindrance during the
inhibition process in the case of 1. The consequence of this
much slower inhibition rate in the case of 2 is that also
the slow spontaneous hydrolysis of 2 (Scheme 4), a process buffer: 50 m Tris-HCl, 0.1% Triton X-100 at pH 8.0.
Eur. J. Inorg. Chem. 2008, 4425–4432
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
4427

R. J. M. Klein Gebbink, G. van Koten et al.
FULL PAPER
cutinase. Curve A in Figure 4 suggests that an incomplete B, respectively, Figure 4). Therefore, the different degrees of
reaction occurs and only 85% of the lipase is converted into spontaneous hydrolysis of the preferred enantiomer of 2 in
the hybrid Cut-2 after 40 hours. To confirm this result, we the inhibition cuvette vs. the reference cuvette fully account
performed an experiment in which the cutinase inhibition for the observed differences in cutinase inhibition rates as
reaction was followed by assaying the residual carboxylic determined by two independent experiments.[23]
ester hydrolysis activity of cutinase with p-nitrophenyl buty-
rate ester as a substrate.[6] At regular intervals, aliquots of
the inhibition reaction mixture were added to a buffer solu-
tion containing p-nitrophenyl butyrate ester. The rate of re-
lease of p-nitrophenol was monitored as a direct measure
for the concentration of residual free enzyme (Figure 4,
curve B). According to this experiment, a 94% cutinase in-
hibition (and not 85%, see curve A) was observed after
40 h. Between 40 and 48 h, no change in cutinase inhibition
was observed anymore, indicating that the reactive interme-
diate of 2 was completely converted.
Since curve A and B in Figure 4 give different inhibition
yields of cutinase, these results indicate indeed that, in par-
allel with the slow formation of the Cut-2 hybrid during the
inhibition experiment, a kinetically relevant side reaction
Figure 5. UV/Vis spectra of 2 (50 µ) in a buffer solution at pH
occurs in which also p-nitrophenol is released. Therefore,
we decided to monitor the reference cuvette with UV/Vis
spectroscopy (i.e. 50 µ of 2 in 0.1% Triton X-100, 50 m
Tris-HCl, pH 8.0), in order to determine whether this was
a result of spontaneous hydrolysis of 2 (Scheme 4) under
the applied reaction conditions (pH 8.0). At 20 min time
intervals UV/Vis spectra of this solution were measured for
a total period of 48 h. The combined spectra, presented in
Figure 5, show a decrease in absorbance between 272–
292 nm and an increase at 400 nm, the latter clearly show-
ing that in this time regime a substantial amount of p-nitro-
phenolate is released (note the presence of a single isosbes-
tic point at 332 nm). After 40 h, 15% (7.5 µ) of 2 is hy-
drolyzed in the reference cuvette (see inset of Figure 5). This
means that in our inhibition cuvette two competitive reac-
tions take place, both of which release p-nitrophenolate: (1)
cutinase inhibition with 2 (Scheme 3) and (2) spontaneous
hydrolysis of 2 under the reaction conditions (Scheme 4).
Control experiments showed that, during this time frame
(40 h), the catalytic lipase activity in the hydrolysis reaction
of p-nitrophenyl butyrate ester by cutinase is not affected,
ruling out the possibility that the observed discrepancy be-
tween curves A and B is caused by lipase deactivation or
degradation. Therefore, from curve B (Figure 4) it can be
concluded that 6% hydrolysis of the reactive enantiomer of
8.0 (0.1% Triton X-100, 50 m Tris-HCl) recorded over 48 h at
20 min intervals. The inset shows the hydrolysis of 2 (50 µ) re-
corded at 400 nm under the same conditions (kh = 1.13ϫ10–6 s–1/
6.8ϫ10–5 min–1).[22]
Analysis of the Cut-2 Hybrid by ESI-MS
In order to synthesize a pure Cut-2 hybrid sample for
ESI-MS analysis, an additional experiment was conducted
in which cutinase (25 µ) was allowed to react with a 10-
fold excess of 2 (250 µ). Within 5 h, full conversion to the
Cut-2 hybrid was obtained. This was confirmed by a con-
trol experiment using the p-nitrophenyl butyrate ester lipase
activity assay. No residual ester hydrolysis activity of cu-
tinase was found, proving that cutinase was indeed com-
2
[22] has occurred during the inhibition process as only 94%
cutinase inhibition was found. It should be noted that the
cutinase inhibition does not change anymore after 40 h,
meaning that the inhibition rate of the unreactive enanti-
omer of 2 with cutinase is too low to be measured and can
therefore be ignored here (85% of this enantiomer is still
present after 40 h). Since in the reference cuvette 15% hy-
drolysis of the reactive enantiomer occurred, inhibition
curve A was overcompensated by 9% (the difference in
spontaneous hydrolysis of 2 in the inhibition cuvette and
the reference cuvette) at the 40-h time point. This is exactly
the observed difference in cutinase inhibition as determined
Figure 6. ESI-mass spectrum of the cutinase–NCN-pincer–PtCl
by the two analysis techniques (85% vs. 94%, curves A and complex hybrid Cut-2.
4428
www.eurjic.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2008, 4425–4432

Non-Tethered Organometallic Phosphonate Inhibitors for Lipase Inhibition
pletely inhibited and that all the active sites were modified
Conclusions
by the organometallic pincer complex. After dialysis of the
mixture, a pure Cut-2 sample was obtained, which illus-
trates the simplicity of this modification strategy. The ESI-
mass spectrum showed the signal of Cut-2 minus the chlo-
rine atom (Mw: 21077.5 Da, Figure 6), a common observa-
tion in the case of pincer complexes. Importantly, no peak
at the m/z of free cutinase was found (Mw: 20619). This
provides an unambiguous proof for the quantitative forma-
tion of the cutinase–NCN-pincer–PtCl complex hybrid
Cut-2, and illustrates the stability of the SerO–P bond
towards hydrolysis.[5]
A new ASDC (2), having a phosphonate p-nitrophenyl
ester directly attached to an aromatic organometallic pincer
moiety, is reported. This probe shows good inhibitory ac-
tivity towards cutinase, affording an organometallic–cu-
tinase hybrid in which the organometallic head group is
now positioned in the chiral surroundings of the active site
of cutinase. This in contrast with the previously reported
ASDC 1, which has the metal center positioned at the pe-
riphery of the of Cut-1 lipase hybrid as determined by X-
ray crystallographic studies.[11] The inhibition rate of 2 with
cutinase was found to be Ͼ500 times slower than that of 1,
which is attributed to increased steric hindrance in 2. Con-
sequently, in the case of very slow inhibition rates (as with
2), one cannot simply rely on monitoring the increase in
color intensity in solution as a measure of protein inhibi-
tion, since slow side reactions (hydrolysis of 2) now become
significant. In such cases, appropriate control experiments
(enzyme activity tests) are needed to confirm the inhibition
yield. The isolation of pure Cut-2 hybrid was confirmed
by ESI-MS spectrometry, showing the absence of wild-type
cutinase in the sample: only the signal corresponding to
Cut-2 was observed. NCN-pincer metal phosphonic acids
on the other hand were found to be inactive as inhibitors
towards cutinase. This latter result was attributed to unfa-
vorable polar–apolar repulsion interactions, showing the
importance of the p-nitrophenol leaving group in the use of
ASDCs for lipase modification.
Inhibitory Activity of ASDCs 3 and 4
To determine whether phosphonic acids 3 and 4 are able
to interact with the active site of cutinase, we performed a
similar incubation experiment as described for 2. Cutinase
(25 µ, in 50 m Tris-HCl, 0.1% Triton X-100 at pH 8.0)
was exposed to either 1 or 100 equiv. of 3 or 4. After incub-
ating the solution for 48 h, the residual hydrolytic activity
of cutinase was tested with the standard activity assay com-
prising p-nitrophenyl butyrate ester in a Tris-HCl buffer at
pH 8.0. Any decrease in hydrolytic activity would imply
blocking of the active site of cutinase. However, with 3 and
4 no decrease in hydrolysis activity of cutinase towards the
p-nitrophenol butyrate ester was observed, not even when
100 equiv. of either 3 or 4 were used. Thus, although others
have reported similar arylphosphonic acid structures to be
active towards serine hydrolases, no formation of a Cut-
3 or Cut-4 hybrid seems to occur. Becker and co-workers
reported that hydrophobic interactions of side chains of the
inhibitor with a lipase have a minor influence on the inhibi-
tory power of phosphonates.[24] In the case of cutinase, the
active site is accessible for water-soluble as well as water-
insoluble esters. This is due to the absence of a so-called
lid, covering the active site, and for this reason cutinase
does not show any interfacial activation towards hydro-
phobic surfaces such as lipid micelles as common lipases
do. A possible explanation for the lack of inhibitory activity
by 3 and 4 could be that these complexes exist in the basic
buffer solution as ionic complexes, which probably interact
with the hydrophobic surface of cutinase in a repelling man-
ner, thereby preventing them from entering the active site.
Related to this, Freedman et al. reported the inhibitory ac-
tivity of related phenylphosphonic and phenylphosphinic
Due to the specificity of p-nitrophenyl phosphonate es-
ters for serine hydrolases,[14] this methodology should pro-
vide relatively easy access to a whole range of organometal-
lic–enzyme hybrids. Therefore, the presented protocol pro-
vides a straightforward and unique approach for further
fine-tuning of the organometallic–enzyme hybrid for diag-
nostic (transition-metal bioprobes) and catalytic (artificial
metalloenzymes) applications.
Experimental Section
General Remarks: All organic and organometallic reactions were
conducted under a dry dinitrogen atmosphere by using standard
Schlenk techniques. Organic solvents were dried with appropriate
materials and distilled prior to use. All reagents were obtained com-
mercially and used without further purification unless stated other-
wise. Triton X-100 was purchased from Serva and tris(hydroxy-
methyl)aminomethane from J. T. Baker. Buffer solutions were pre-
acids toward acetylcholine esterase, for which strong inhibi- pared with Milli-Q grade water. Purification of water (18.2 Mécm)
tion activities were found in some examples.[15] The enzyme
was performed with the Milli-Q Synthesis system (Millipore; Quan-
tum Ultrapure). 1H and 13C{1H} NMR spectra were recorded at
has an active site similar to that of cutinase (i.e. an serine
298 K with a Varian Inova 300 spectrometer at 300 and 75 MHz,
hydrolase with a Glu, His, Ser catalytic triad), however, the
respectively, and 31P{1H} NMR spectra were recorded at 298 K
catalytic cavity is more exposed and larger. In the case of
with a Bruker AC200 instrument at 81 MHz. All NMR chemical
acetylcholine esterase, phosphonic acid inhibitors are able
shifts are in ppm and are referenced to residual solvents (31P{1H}
to interact with the active site because there is less interac-
NMR shifts to H3PO4). The MALDI-TOF mass spectra were ac-
tion with the protein surface around the active site. Because
quired by using a Voyager-DE Biospectrometry Workstation (Per-
of its narrow catalytic pocket, this is apparently not the case
for cutinase, which provides an alternative explanation for
the lack of inhibition power of 3 and 4.
Septive Biosystems Inc., Framingham, Ma, USA) mass spectrome-
ter. Sample solutions with an approximate concentration of 20–
30 mg/mL in CH2Cl2 or thf were prepared. The matrix was 5-chlo-
Eur. J. Inorg. Chem. 2008, 4425–4432
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
4429

R. J. M. Klein Gebbink, G. van Koten et al.
FULL PAPER
1
rosalicylic acid (csa) with an approximate concentration of 20–
30 mg/mL. The sample (0.2 µL) and the matrix solution (0.2 µL)
were combined and placed on a golden MALDI target plate and
analyzed after evaporation of the solvents. [Pt(tol-4)2(SEt2)]2,[19] 2-
bromo-1,3-bis[(dimethylamino)methyl]-5-iodobenzene,[17] 9,[16] and
10[16] were prepared according to described procedures. Micro-
analyses were performed by Kolbe, Mikroanalytisches Laborator-
ium (Mülheim a/d Ruhr, Germany). UV/Vis spectroscopic experi-
ments were performed at room temperature by using a Carey-100
spectrometer. Electrospray ionization (ESI) mass spectra of the
wild-type cutinase and modified cutinase were recorded with a Fin-
nigan LC-Q ion trap mass spectrometer. All samples were intro-
duced by using a nanoflow electrospray source (Protana, Odense,
Denmark).
product as colorless oil. Yield: 11.84 g (75%). H NMR (CDCl3):
3
δ = 4.19 (m, CH2O, 2 H), 2.67 [d, JH,P = 13.92 Hz, 6 H, N-
(CH3)2], 1.35 [t, JC,P = 5.99 Hz, 3 H, CH3CH2O] ppm. 31P NMR
3
2
(CDCl3): δ = 18.84 (s) ppm. 13C NMR (CDCl3): δ = 64.5 (d, JC,P
= 6.0 Hz, CH2O), 36.9 [d, 2JC,P = 2.8 Hz, PN(CH3)2], 16.1 (d, 2JC,P
= 7.8 Hz, CH3CH2O) ppm. C4H11ClNO2P (171.56): calcd. C 28.00,
H 6.46, N 8.16; found C 27.88, H 6.56, N 8.28. GC-MS: calcd.
171.56; found 171.
Ethyl
P-{4-bromo-1,3-bis[(dimethylamino)methyl]phenyl}-N,N-di-
methylaminophosphinate (6): A solution of 2-bromo-1,3-bis[(dime-
thylamino)methyl]-5-iodobenzene (1.48, 3.73 mmol) in Et2O
(30 mL) was cooled to –100 °C and tBuLi (2 equiv., 4.97 mL,
7.45 mmol, 1.5  in pentane) was added dropwise. Stirring was con-
tinued for 10 min at this temperature and the reaction mixture was
subsequently quenched with 5 (0.63 g, 3.73 mmol) in Et2O (10 mL).
The reaction mixture was stirred for 2 h at room temperature and
then water (40 mL) was added. The aqueous phase was separated
and the organic phase was washed with NaOH (2ϫ30 mL) and
brine. Afterwards it was dried with MgSO4. The mixture was fil-
tered and the filtrate was concentrated in vacuo to afford the prod-
uct as yellowish oil. Yield: 1.24 g (82%). 1H NMR (CDCl3): δ =
Inhibitory Activity of 2: To a buffer solution (0.1% Triton X-100,
50 m Tris-HCl, pH 8.0) containing wild-type cutinase (25 µ,
25 nmol, 0.52 mg) was added a solution of 2 in CH3CN (10 µL,
5 m, 50 µmol). The reaction was followed by UV/Vis spectroscopy
until it was complete, which was confirmed by a plateau that ap-
peared in the UV absorption curve at 400 nm. The conversion rate
of the reaction was calculated from the increase in absorbance gen-
erated by the p-nitrophenolate anion by using an experimentally
determined molar extinction coefficient of 14580 –1 cm–1 in this
buffer.
3
7.64 (d, JH,P = 12.45 Hz, 2 H, ArH), 4.10 (m, CH2O, 2 H), 3.54
3
(s, ArCH2N, 4 H), 2.66 [d, JH,P = 9.89 Hz, 6 H, PN(CH3)2], 2.28
[s, N(CH3)2, 12 H], 1.37 (t, 3JH,H = 6.96 Hz, 3 H, CH3CH2O) ppm.
31P NMR (CDCl3): δ = 24.33 (s) ppm. 13C NMR (CDCl3): δ =
Inhibitory Activity Studies with 3 and 4: To a buffer solution (0.1%
Triton X-100, 50 m Tris-HCl, pH 8.0) containing wild-type cu-
tinase (25 µ, 25 nmol, 0.52 mg) was added a solution of 7 or 8 in
water (50 µL, 50 m, 2.5 mmol). The samples were incubated for
48 h at room temperature. Afterwards, the free enzyme concentra-
tion was determined by examining the residual catalytic activity
using the UV/Vis spectroscopic assay containing the substrate p-
nitrophenol butyrate ester as described below. In a control experi-
ment, cutinase without a phosphonic acid complex in the buffer
solution was also tested for ester hydrolysis activity.
3
2
139.5 (d, JC,P = 14.0 Hz, ArC), 132.2 (d, JC,P = 11.0 Hz, ArC),
4
1
131.40 (d, JC,P = 4.0 Hz, ArC), 129.0 (d, JC,P = 173.8 Hz, ArC),
2
64.1 (s, ArCH2N), 60.7 (d, JC,P = 5.4 Hz, CH2O), 45.8 [s, N-
(CH3)2], 36.6 [d, 2JC,P = 4.2 Hz, PN(CH3)2], 16.6 (d, 3JC,P = 6.1 Hz,
CH3CH2O) ppm. C16H29BrN3O2P (406.30): calcd. C 47.30, H 7.19,
N 10.34; found C 47.21, H 7.25, N 10.12.
Ethyl P-{4-Bromoplatinum-1,3-bis[(dimethylamino)methyl]phenyl}-
N,N-dimethylaminophosphinate (7): To a solution of 6 (0.36 g,
0.88 mmol) in benzene (10 mL) was added solid [Pt(tol-4)(SEt2)]2
(0.37 g, 0.40 mmol) and the resulting mixture was subsequently
heated to reflux for 1 h. All volatiles were removed in vacuo and
the remaining dark brown oil was dissolved in CH2Cl2 (10 mL) and
filtered through a short path of Celite. The filtrate was concentrated
and washed with pentane (3ϫ10 mL, stirring for 30 min) and Et2O
(2ϫ10 mL, stirring for 30 min). The resulting solid was dried in
vacuo to obtain the product as an off-white solid. Yield: 0.38 g
Ester Hydrolysis Activity of Cutinase: The catalytic activity of cu-
tinase was determined by UV/Vis spectroscopy on p-nitrophenol
butyrate ester (0.25 m) in the presence of Triton X-100 (100 m)
and Tris-HCl (10 m) at pH 8.0. Aliquots (5 µL) were taken and
added to the assay. Activities were calculated from the increase in
absorbance at 400 nm.
Hydrolysis of 2: The hydrolysis reaction of the phosphonate ester
in ASDC 2, caused by reaction of 2 with the basic buffer solution
(0.1% Triton X-100, 50 m Tris-HCl, pH 8.0) and thereby releasing
p-nitrophenol, was examined over 48 h by recording UV/Vis spec-
tra at 400 nm at 20 min time intervals.
3
(65%). 1H NMR (CDCl3): δ = 7.06 (d, JH,P = 12.82 Hz, 2 H,
ArH), 3.96 (s, 3JH,Pt = 46.88 Hz, 4 H, CH2N), 3.92 (m, 2 H, CH2O),
3.03 [s, 3JH,Pt = 38.09 Hz, 12 H, N(CH3)2], 2.61 [d, 3JH,P = 8.06 Hz,
3
6 H, PN(CH3)2], 1.21 (t, JH,H = 6.96 Hz, 3 H, CH3CH2O) ppm.
31P NMR (CDCl3): δ = 27.63 (s) ppm. 13C NMR (CDCl3): δ =
4
3
152.3 (d, JC,P = 2.3 Hz, ArC), 143.8 (d, JC,P = 15.9 Hz, ArC),
Preparation of Samples for Mass Spectrometry Analysis: Cutinase
(25 µ) in a Tris-HCl buffer solution (10 m, pH 8.0) was incu-
bated for 48 h with excess of 2 (10 equiv.). Completion of the reac-
tion was confirmed by determining the residual catalytic hydrolysis
activity of cutinase. The clear yellow solutions were subsequently
dialyzed for 2ϫ12 h with the same buffer. Prior to measurements
of ESI mass spectra, the solutions were diluted to 2–5 µ with
CH3CN.
1
2
124.8 (d, JC,P = 177.1 Hz, ArC), 122.4 (d, JC,P = 10.9 Hz, ArC),
77.2 (s, 3JC,Pt = 31.7 Hz, ArCH2N), 59.9 (d, 2JC,P = 5.5 Hz, CH2O),
53.9 [s, N(CH3)2], 36.3 [d, 2JC,P = 3.7 Hz, PN(CH3)2], 16.5 (d, 3JC,P
= 6.7 Hz, CH3CH2O) ppm. C16H29BrN3O2PPt (601.38): calcd. C
31.96, H 4.86, N 6.99; found C 31.85, H 4.67, N, 6.78.
Ethyl P-{4-Chloroplatinum-1,3-bis[(dimethylamino)methyl]phenyl}-
N,N-dimethylaminophosphinate (8):
A solution of 7 (0.77 g,
Ethyl Chloro-N,N-dimethylaminophosphinate (5): Dimethylamine
(8.29 g, 12.75 mL, 182.12 mmol) in Et2O (20 mL) was added drop-
wise to a solution of dichloroethylphosphonate in Et2O (30 mL)
at 0 °C. The mixture was stirred for 2 h at room temperature, the
precipitated salt was filtered off, and the residue was washed with
Et2O (2ϫ20 mL). The combined filtrate and washings were con-
centrated to dryness to afford an orange oil. The crude product
was distilled under reduced pressure (54 °C/ 12 Torr) to obtain the
1.28 mmol) in wet acetone (20 mL) was treated with AgBF4 (0.25 g,
1.28 mmol) and stirred for 30 min at room temperature. The reac-
tion mixture was filtered through a short pad of Celite and NaCl
(0.75 g, 12.80 mmol) was added to the filtrate. After stirring at
room temperature for 2 h, the solvent was removed and the crude
product was dissolved in CH2Cl2 (20 mL). Water (20 mL) was
added and the CH2Cl2 phase was separated. The aqueous phase
was extracted with CH2Cl2 (2ϫ20 mL) and the combined organic
4430
www.eurjic.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2008, 4425–4432

Non-Tethered Organometallic Phosphonate Inhibitors for Lipase Inhibition
fractions were dried with MgSO4. The mixture was filtered, the
filtrate concentrated to 2 mL and pentane was added to precipitate
the product as an off-white solid. Yield: 0.64 g (92%). 1H NMR
4-Bromoplatinum{3,5-bis[(dimethylamino)methyl]phenyl}phosphonic
Acid (4): The same procedure as that for 3 was applied: 10 (100 mg,
0.17 mmol), bromotrimethylsilane (252 mg, 218 µL, 1.66 mmol) in
3
3
(CDCl3): δ = 7.06 (d, JH,P = 12.82 Hz, 2 H, ArH), 3.96 (s, JH,Pt CH2Cl2 (5 mL), MeOH (8 mL), and dichloromethane (2 mL) were
3
3
= 46.88 Hz, 4 H, CH2N), 3.92 (m, CH2O, 2 H), 3.03 [s, JH,Pt
=
used. Yield: 85 mg (94%). 1H NMR (D2O): δ = 7.13 (d, JH,P
=
3
3
38.09 Hz, 12 H, N(CH3)2], 2.61 [d, JHP = 8.06 Hz, 6 H, PN- 13.2 Hz, 2 H, ArH), 4.04 (s, JH,Pt = 43.9 Hz, 4 H, CH2), 2.89 (s,
(CH3)2], 1.21 (t, 3JH,H = 6.96 Hz, 3 H, CH3CH2O) ppm. 31P NMR 3JH,Pt = 36.2 Hz, 12 H, NMe2) ppm. 31P NMR (D2O): δ = 19.01
(CDCl3): δ = 27.63 (s) ppm. 13C NMR (CDCl3): δ = 152.9 (d, 4JC,P (s) ppm. 13C NMR (D2O): δ = 161.5 (s, ArC), 149.1 (d, JC,P
=
=
3
3
1
1
2
= 2.3 Hz, ArC), 144.0 (d, JC,P = 15.2 Hz), 125.0 (d, JC,P
=
16.8 Hz, ArC), 125.4 (d, JC,P = 176.9 Hz, ArC), 121.8 (d, JC,P
1
2
3
175.0 Hz, ArC), 125.0 (d, JC,P = 175.0 Hz, ArC), 122.46 (d, JC,P
= 10.4, JC,Pt = 17.3 Hz, ArC), 77.4 (s, ArCH2N), 60.4 (d, JC,P
6.1 Hz, CH2O), 55.3 [s, N(CH3)2], 36.7 [d, JC,P = 4.2 Hz,
11.6 Hz, ArC), 76.3 (s, JC,Pt = 41.6 Hz, CH2), 54.4 [s, N(CH3)2]
3
2
=
ppm.
2
Supporting Information (see footnote on the first page of this arti-
cle): Crystallographic data of the crystal structures complexes 9 and
10.
3
PN(CH3)2], 16.6 (d, JC,P
=
6.7 Hz, CH3CH2O) ppm.
C16H29ClN3O2PPt (556.93): calcd. C 34.51, H 5.25, N 7.54; found
C 34.62, H 5.28, N 7.48.
Ethyl 4-Nitrophenyl P-{4-Chloroplatinum-3,5-bis[(dimethylamino)-
methyl]phenyl}phosphonate (2): A solution of compound 8 (200 mg,
3.59 mmol) in benzene (20 mL) was treated dropwise with anhy-
drous HCl (1  in Et2O, 1.8 mL, 1.8 mmol, 5 equiv.) and afterwards
the mixture was stirred for 3 h. The formed turbid mixture was
filtered under a N2 atmosphere, and a clear yellow solution was
collected as filtrate. The filtrate was concentrated in vacuo to a
yellowish solid. The solid residue was redissolved in benzene
(20 mL) and dry NEt3 (36.3 mg, 50 µL, 3.59 mmol) was added to
the mixture, followed by the dropwise addition of a solution of p-
nitrophenol (50 mg, 3.59 mmol) in benzene (20 mL). Slowly, a tur-
bid mixture was formed while the reaction mixture was stirred for
one hour at room temperature. After filtration of the reaction mix-
ture under a N2 atmosphere, a saturated NaHCO3 solution (20 mL)
was added. The organic phase was separated and washed with a
saturated NaHCO3 solution (2ϫ20 mL) followed by water
(20 mL). The product-containing organic phase was dried with
MgSO4, filtered, and concentrated to a pale yellow solid. Yield:
Acknowledgments
The authors kindly wish to thank C. Versluis and Prof. A. J. R.
Heck of the Biomolecular Mass Spectrometry group in the Depart-
ment of Pharmaceutical Sciences at Utrecht University for measur-
ing the ESI-MS spectra of various protein structures. This work
was supported by the Council for Chemical Sciences of the Dutch
organization for Scientific Research (CW-NWO) . The Unilever Re-
search Laboratory at Vlaardingen in The Netherlands, is kindly
acknowledged for the enzyme supply.
[1] a) J. Kraut, Ann. Rev. Biochem. 1977, 46, 331–358; b) P. E. Kol-
attukudy in Lipases, Elsevier, Amsterdam, 1984, 471–501; c) S.
Ransac, Y. Gargouri, F. Marguet, G. Buono, C. Beglinger, P.
Hildebrand, H. Lengsfeld, P. Hadvary, R. Verger, Methods En-
zymol. 1997, 286, 190–231.
[2] a) O. V. Oskolkova, R. Saf, E. Zenzmaier, A. Hermetter, Chem.
Phys. Lipids 2003, 125, 103–114; b) H. Schmidinger, R. Birner-
Gruenberger, G. Riesenhuber, R. Saf, H. Susani-Etzerodt, A.
Hermetter, ChemBioChem 2005, 6, 1776–1781; c) G. Zan-
donella, P. Stadler, L. Haalck, F. Spener, F. Paltauf, A. Her-
metter, Eur. J. Biochem. 1999, 262, 63–69.
[3] C. M. Salisbury, J. Ellman, ChemBioChem 2006, 7, 1034–1037.
[4] a) N. Jessani, J. A. Young, S. L. Diaz, M. P. Patricelli, A. Varki,
B. F. Cravatt, Angew. Chem. Int. Ed. 2005, 44, 2400–2403; b)
A. E. Speers, B. F. Cravatt, ChemBioChem 2004, 5, 41–47; c)
H. Schmidinger, H. Susani-Etzerodt, R. Birner-Gruenberger,
A. Hermetter, ChemBioChem 2006, 7, 527–534.
[5] It should be noted that the hydrolytic sensitivity of the SerO–
P bond is dependent upon the nature of R1 (Figure 1). If for
example R1 = p-nitrophenyl, the SerO–P bond is easily hy-
drolyzed, thereby releasing the phosphonate group from the
protein again (see ref.[7]). For the phosphonate inhibitors de-
scribed in this paper with R1 = Et, no observable hydrolysis
occurred for at least two weeks.
[6] a) C. A. Kruithof, G. Guillena, M. R. Egmond, G. van Koten
in Book of Abstracts, First International Symposium on Bioor-
ganometallic Chemistry, 18–20 July 2002, Paris, p. 104; b) C. A.
Kruithof, M. A. Casado, G. Guillena, M. R. Egmond, A.
van der Kerk-van Hoof, A. J. R. Heck, R. J. M. Klein Geb-
bink, G. van Koten, Chem. Eur. J. 2005, 11, 6869–6877.
[7] It should be stated that Reetz and co-workers were the first to
publish the concept of site-specific introduction of metal–li-
gand systems into lipases via phosphonate inhibitors. Their ex-
amples, however, did not result in irreversible protein modifica-
tion because of fast hydrolysis of the SerO–P bond (see also
ref.[5]): see M. T. Reetz, M. Rentzsch, A. Pletsch, M. Maywald,
Chimia 2002, 56, 721–723.
1
3
187 mg (80%). H NMR (C6D6): δ = 7.74 (d, JH,H = 9.16 Hz, 2
3
3
H, ArH), 7.42 (d, JH,P = 13.73 Hz, 2 H, ArH), 7.22 (d, JH,H
=
3
3
9.16 Hz, 2 H, ArH), 4.12 (dt, JH,H = 7.02, JH,P = 15.57 Hz, 2 H,
CH2O), 3.19 (s, JH,Pt = 19.53 Hz, 4 H, ArCH2), 2.59 [s, JH,Pt
3
3
=
3
18.31 Hz, 12 H, N(CH3)2], 1.10 (t, JH,H = 7.02 Hz, 3 H,
CH3CH2O) ppm. 31P NMR (C6D6): δ = 20.6 (s) ppm. 13C NMR
4
3
(C6D6): δ = 156.1 (d, JC,P = 6.1 Hz, ArC), 144.6 (d, JC,P
=
17.7 Hz, ArC), 144.6 (s, ArC), 127.1, 125.5 (s, ArC), 123.0 (d, 2JC,P
3
1
= 11.6 Hz, ArC), 120.8 (d, JC,P = 4.9 Hz, ArC), 120.6 (d, JC,P
194.1 Hz, ArC), 76.9 (s, JC,Pt = 31.1 Hz, ArCH2N), 62.7 (d, JC,P
= 5.5 Hz, CH2O), 53.8 [s, N(CH3)2], 16.3 (d, JC,P = 6.1 Hz,
=
3
2
3
CH3CH2O) ppm. MS (MALDI-TOF, CSA): m/z = 614.2 [M – Cl]+,
420.1 [M – Cl – Pt]+, 378.3 [M – Cl – Pt – OEt]+. C20H27ClN3O5PPt
(650.95): calcd. C 36.90, H 4.18, N 6.46; found C 37.01, H 4.26, N
6.37.
4-Bromopalladium{3,5-bis[(dimethylamino)methyl]phenyl}phosphonic
Acid (3): A solution of 9 (121 mg, 0.24 mmol) in CH2Cl2 (6 mL)
was treated with of bromotrimethylsilane (362 mg, 312.2 µL,
2.36 mmol, 10 equiv.) and subsequently stirred for 4 h at room tem-
perature. Dry MeOH (10 mL) was added and the resulting yellow
solution was stirred for 1 h. All volatiles were evaporated in vacuo
and the remaining residue was washed with dichloromethane
(2 mL). The crude product was dissolved in a minimum amount of
dry MeOH. Slow addition of diethyl ether precipitated the product
as an off-white solid. Yield: 96 mg (89%). 1H NMR (D2O): δ =
3
7.05 (d, JH,P = 12.6 Hz, 2 H, ArH), 3.96 (s, CH2, 4 H), 2.68 (s,
NMe2, 12 H) ppm. 31P NMR (D2O): δ = 14.83 (s) ppm. 13C NMR
(D2O): δ = 156.4 (s, ArC), 145.9 (d, 3JC,P = 15.4 Hz), 132.8 (d, 1JC,P
[8] a) J. Kjellgren, H. Sundén, K. J. Szabó, J. Am. Chem. Soc.
2005, 127, 1787–1796; b) J. T. Singleton, Tetrahedron 2003, 59,
1837–1857; c) H. P. Dijkstra, M. Q. Slagt, A. McDonald, C. A.
2
= 176.2 Hz, ArC), 122.1 (d, JC,P = 10.4 Hz, ArC), 73.4 (s, CH2),
52.2 [s, N(CH3)2] ppm.
Eur. J. Inorg. Chem. 2008, 4425–4432
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
4431

R. J. M. Klein Gebbink, G. van Koten et al.
FULL PAPER
Kruithof, R. Kreiter, A. M. Mills, M. Lutz, A. L. Spek, W.
Klopper, G. P. M. van Klink, G. van Koten, Eur. J. Inorg.
Chem. 2003, 830–838; d) H. P. Dijkstra, C. A. Kruithof, N.
Ronde, R. van de Coevering, D. J. Ramón, D. Vogt, G. P. M.
van Klink, G. van Koten, J. Org. Chem. 2003, 68, 675–685.
[9] a) M. Albrecht, M. Lutz, A. L. Spek, G. van Koten, Nature
2000, 406, 970–974; b) G. Guillena, K. M. Halkes, G. Rodríg-
uez, G. D. Batema, G. van Koten, J. P. Kamerling, Org. Lett.
[18] M. Albrecht, G. Rodríguez, J. Schoenmaker, G. van Koten,
Org. Lett. 2000, 2, 3461–3464.
[19] a) A. J. Canty, S. D. Fritsche, H. Jin, J. Patel, B. W. Skelton,
A. H. White, Organometallics 1997, 16, 2175–2182; b) B. R.
Steele, K. Vrieze, Trans. Met. Chem. 1977, 2, 140–144.
[20] D. M. Grove, G. van Koten, J. N. Louwen, L. G. Noltes, A. L.
Spek, H. J. C. J. Ubbels, J. Am. Chem. Soc. 1982, 104, 6609–
6616.
2003, 5, 2021–2024; c) D. Becatti, K. M. Halkes, G. Guillena, [21] a) M. L. M. Mannesse, G. H. de Haas, H. T. W. M.
A. Carvalho de Souza, G. van Koten, J. P. Kamerling, ChemBi-
oChem 2005, 6, 1196–1203.
van der Heijden, M. R. Egmond, H. M. Verheij, Biochem. Soc.
Trans. 1997, 25, 165–170; b) S. Longhi, M. L. M. Mannesse,
H. M. Verheij, G. H. de Haas, M. R. Egmond, E. Knoop-
Mouthuy, C. Cambillau, Protein Sci. 1997, 6, 275–286; c) J.-F.
Cavalier, G. Buono, Acc. Chem. Res. 2000, 33, 579–588. Prelim-
inary results of an X-ray structure of the NCN-pincer Pt com-
plex phosphonate inhibitor 1 attached to cutinase N172K re-
veals the presence of the S enantiomer in the active site. The
commonly accepted mechanism for the inhibition reaction is
the SN2 mechanism, which implicates that the reaction of 1
with cutinase has proceeded with the R enantiomer as the most
reactive isomer.
[10] a) W. W. Gerhardt, A. J. Zucchero, J. N. Wilson, C. R. South,
U. H. F. Bunz, M. Weck, Chem. Commun. 2006, 2141–2143; b)
S.-E. Stiriba, M. Q. Slagt, H. Kautz, R. J. M. Klein Gebbink,
R. Thomann, H. Frey, G. van Koten, Chem. Eur. J. 2004, 10,
1267–1273; c) H. J. van Manen, K. Nakashima, S. Shinkai, H.
Kooijman, A. L. Spek, F. van Veggel, D. N. Reinhoudt, Eur. J.
Inorg. Chem. 2000, 2533–2540; d) W. T. S. Huck, L. J. Prins,
R. H. Fokkens, N. M. M. Nibbering, F. C. J. M. van Veggel,
D. N. Reinhoudt, J. Am. Chem. Soc. 1998, 120, 6240–6246.
[11] L. Rutten, B. Wieczorek, J.-P. B. A. Mannie, H. P. Dijkstra,
C. A. Kruithof, M. R. Egmond, M. Lutz, A. L. Spek, R. J. M.
Klein Gebbink, G. van Koten, P. Gros, manuscript in prepara-
tion.
[12] a) K. K.-W. Lo, W.-K. Hui, C.-K. Chung, K. H.-K. Tsang,
D. C.-M. Ng, N. Zhu, K.-K. Cheung, Coord. Chem. Rev. 2005,
249, 1434–1450; b) K. K.-W. Lo, W.-K. Hui, C.-K. Chung,
K. H.-K. Tsang, T. K.-M. Lee, C.-K. Li, J. S.-Y. Lau, D. C.-M.
Ng, Coord. Chem. Rev. 2006, 250, 1724–1736; c) M. Salmain,
G. Jaouen, C. R. Chimie 2003, 6, 249–258.
[13] a) C. Letondor, T. R. Ward, ChemBioChem 2006, 7, 1845–1852;
b) T. Reetz, J. J.-P. Peyralans, A. Maichele, Y. Gu, M. Maywald,
Chem. Commun. 2006, 4318–4320; c) D. Qi, C.-M. Tann, D.
Haring, M. D. Distefano, Chem. Rev. 2001, 101, 3081–3111.
[14] Recently, we showed that this type of reactive phosphonate es-
ter selectively inhibits serine hydrolases, see: H. P. Dijkstra, H.
Sprong, B. N. H. Aerts, C. A. Kruithof, M. R. Egmond,
R. J. M. Klein Gebbink, Org. Biolmol. Chem. 2008, 6, 523–531.
[15] L. D. Freedman, H. Tauber, G. O. Doak, H. J. Magnuson, J.
Am. Chem. Soc. 1953, 75, 1379–1381.
[22] Please note that the nonreactive enantiomer of 2 hydrolyzes to
the same extent in both cuvettes and for this reason it does
not account for changes in the absorbance level; the hydrolysis
progresses equally in both cuvettes.
[23] a) The 85% inhibition of cutinase by the reactive enantiomer
of 2 as determined from curve A (Figure 4), can also be ex-
plained by only considering the two competing reactions
(Scheme 3 and Scheme 4). Whereas in the inhibition cuvette
100% p-nitrophenolate release from 2 is a combined result of
reactions 1 and 2, in the reference cuvette 15% p-nitrophenol-
ate release occurs as a results of reaction 2 only. Ideally, in the
inhibition cuvette 100% p-nitrophenolate release would result
solely from reaction 1 (thus assuming reaction 2 does not oc-
cur), leading to the same increase in absorption as observed
now (curve A). Obviously, this means that the 15% release in
the reference cuvette is an overcompensation in the UV/Vis ab-
sorption, thus fully explaining the observed 85% inhibition rate
of curve A. It should be noted that following this line of analy-
sis does not tell anything about the amount of spontaneous
hydrolysis of 2 in the inhibition cuvette. b) Rate was calculated
by determining the slope of the ln([PnP]/[PnP]0) vs. time plot.
R2 = 0.9992.
[16] M. Q. Slagt, G. Rodríguez, M. P. Grutters, R. J. M. Klein Geb-
bink, W. Klopper, L. W. Jenneskens, M. Lutz, A. L. Spek, G.
van Koten, Chem. Eur. J. 2004, 10, 1331–1344.
[17] G. Rodríguez, M. Albrecht, J. Schoenmaker, A. Ford, M. Lutz,
A. L. Spek, G. van Koten, J. Am. Chem. Soc. 2002, 124, 5127–
5138.
[24] E. L. Becker, Biochim. Biophys. Acta 1967, 147, 289–296.
Received: June 30, 2008
Published Online: August 25, 2008
4432
www.eurjic.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2008, 4425–4432