Lipase active-site-directed anchoring of organometallics: Metallopincer/protein hybrids

Article abstract of DOI:10.1002/chem.200500671

The work described herein presents a strategy for the regioselective introduction of organometallic complexes into the active site of the lipase cutinase. Nitrophenol phosphonate esters, well known for their lipase inhibitory activity, are used as anchor

Full text of DOI:10.1002/chem.200500671

FULL PAPER
DOI: 10.1002/chem.200500671
Lipase Active-Site-Directed Anchoring ofOrganometallics:
Metallopincer/Protein Hybrids
Cornelis A. Kruithof,[a] Miguel A. Casado,[a] Gabriela Guillena,[a] Maarten R. Egmond,[b]
Anca van der Kerk-van Hoof,[c] Albert J. R. Heck,[c] Robertus J. M. Klein Gebbink,[a] and
Gerard van Koten*[a]
Abstract: The work described herein
presents a strategy for the regioselec-
tive introduction of organometallic
complexes into the active site of the
lipase cutinase. Nitrophenol phospho-
nate esters, well known for their lipase
inhibitory activity, are used as anchor
functionalities and were found to be
ideal tools to develop a single-site-di-
rected immobilization method. A small
series of phosphonate esters, covalently
attached to ECE “pincer”-type d8-
organometallic phosphonate esters and
the new metal-complex/protein hybrids
were identified as containing exactly
one organometallic unit per protein.
The organometallic proteins were puri-
fied by membrane dialysis and ana-
lyzed by ESI-mass spectrometry. The
major advantages of this strategy are:
1) one transition metal can be intro-
duced regioselectively and, hence, the
metal environment can potentially be
fine-tuned; 2) purification procedures
are facile due to the use of pre-synthe-
sized metal complexes; and, most im-
portantly, 3) the covalent attachment
of robust organometallic pincer com-
plexes to an enzyme is achieved, which
will prevent metal leaching from these
hybrids. The approach presented
herein can be regarded as a tool in the
development of regio- and enantiose-
lective catalyst as well as analytical
probes for studying enzyme properties
(e.g., structure) and, hence, is a “proof-
of-principle design” study in enzyme
chemistry.
Keywords: inhibitors · lipases ·
metal complexes through
a propyl
metallopincer
organometallic
complexes
phosphonate
·
·
tether (ECE=[C6H3(CH2E)2-2,6]À; E=
NR2 or SR), were designed and synthe-
sized. Cutinase was treated with these
protein modifications
Introduction
ural fragments into proteins.[1] This strategy allows one to
combine the specific properties of proteins and enzymes
(substrate and stereoselectivity) with those of man-made
molecules. Inspired by the pioneering work of Kaiser[2] and
Whitesides,[3] this “hybrid” strategy has recently led to new
developments in the field of homogeneous catalysis.[4] Here,
this strategy relies on the idea of using proteins as scaffolds
for transition-metal catalysts and, as such, it represents a
promising method to transfer the properties of a protein
(water solubility, size, chiral environment) to a transition-
metal moiety. One of the first examples, developed by
Whitesides[3] in the 1970s and recently optimized by Ward
and co-workers,[4e] is based on biotin–avidin technology. In
this noncovalent anchoring approach, the hydrogenation of
N-protected dehydroamino acid derivatives by a biotinylat-
ed rhodium–diphosphine catalyst resulted in catalytic con-
versions with up to 96% ee.[4e]
Chemical modification of proteins is a fascinating approach
for altering protein function by the introduction of non-nat-
[a] C. A. Kruithof, Dr. M. A. Casado, Dr. G. Guillena,
Dr. R. J. M. Klein Gebbink, Prof. Dr. G. van Koten
Debye Institute, Organic Synthesis and Catalysis
Utrecht University, Padualaan 8, 3584 CH Utrecht (The Netherlands)
Fax : (+31)30-252-3615
E-mail: g.vankoten@chem.uu.nl
[b] Prof. Dr. M. R. Egmond
Bijvoet Center for Biomolecular Research
Membrane Enzymology, Utrecht University
Padualaan 8, 3584 CH Utrecht (The Netherlands)
[c] A. van der Kerk-van Hoof, Prof. Dr. A. J. R. Heck
Bijvoet Center for Biomolecular Research and
Utrecht Institute for Pharmaceutical Sciences
Regioselective anchoring of stable transition metal probes
to proteins may allow the controlled “tagging” of the pro-
tein of interest and can serve as biomarker[5] or as an addi-
tional tool in protein studies.[6] A classic example is the hy-
Biomolecular Mass Spectrometry, Utrecht University
Sorbonnelaan 16, 3584 CA Utrecht (The Netherlands)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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bridization of mercurybenzenesulfonyl fluoride derivatives
with the active site of proteases, for example, chymotryp-
sins.[7] These covalently bound labels were applied for the
multiple isomorphous replacement method, used by crystal-
lographers to accelerate phase determination in protein
structure elucidation.
established position.[10] Recently, Reetz and co-workers have
investigated a similar approach by immobilizing a dinitro-
phenol phosphonate diphosphine ligand as a potential pre-
cursor for the synthesis of hybrid catalysts.[4d,11] However, by
using this dinitrophenol phosphonate, the obtained hybrids
were unstable due to basic hydrolysis and the catalytic activ-
ity of the native lipase was restored in 24 h.
The protein we have chosen to use in our studies is the
enzyme cutinase, a 21 kDa lipase isolated from the fungi Fu-
sarium solani pisi.[12] Because this lipase does not contain a
“lid” covering the active site, it exhibits no interfacial activa-
tion, and, hence, the active site is accessible to hydrophilic
as well as hydrophobic substrates in aqueous media.[13]
Metallopincer complexes, containing a terdentate monoa-
nionic [C6H3(CH2E)2-2,6]À “pincer”-type ligand (ECE, E=
NR2, SR, or PR2), were selected as metal-containing build-
ing blocks. These organometallic complexes possess several
Evidently, controlled and site-directed protein modifica-
tion can become of great importance when constructing
transition metal complex/protein hybrid systems. Several re-
quirements to construct a structurally defined metal com-
plex/protein hybrid have to be considered. First of all, the
linkage of the metal complex to the protein has to be stable
under all future conditions (e.g., during purification and ap-
plication) and at the same time metal leaching from the
ligand has to be prevented. Furthermore, the protein needs
to have specific and accessible positions amenable toward
functionalization and the anchoring must preferably be in a
regioselective manner. These requirements intrinsically
demand three careful selections that have to be made: the
right protein, the proper anchor unit, and a chemically
stable metal complex.
interesting and useful characteristics, which include an ex-
[14]
À
ceedingly stable M C bond, and the potential applicabili-
ty in sensoring[15] and catalytic systems.[14,16] In recent years,
we have reported on the facile modification of the pincer
system with numerous functionalities.[17] Together with the
high chemical and thermal stability, these properties should
make the pincer unit a very suitable candidate for the con-
struction of metal complex/protein hybrids by means of an
active-site-directed approach.
In this study, we have employed p-nitrophenol (PnP)
phosphonates esters as anchoring units.[8] PnP phosphonate
esters are well known as lipase suicide inhibitors, which can
irreversibly bind to the active site of various lipases (the g-
OH group of the serine residue),[9] and thereby provide a
simple method to functionalize lipases selectively at a pre-
Three active-site-directed compounds (ASDC) were de-
veloped in which PnP phosphonate esters and metallopincer
moieties are combined. The design of these complexes (1–3)
is based on space-filling representations of metallopincer cu-
tinase hybrids (Figure 1).[18] Here we report on the synthesis
of these organometallic ASDCs, on their inhibition reaction
with wild type cutinase, and on the characterization of the
resulting organometallic cutinase hybrids.
Abstract in Dutch: Een eenvoudige methode voor het regio-
selectief verankeren van organometaalcomplexen aan het ac-
tieve centrum van het lipase cutinase is ontwikkeld. Hierbij is
gebruik gemaakt van nitrofenolfosfonaatesters als veranker-
ingsgroep. Deze geactiveerde fosfonaatesters zijn in staat om
de enzymatische katalytische activiteit van serine-hydrolasen
irreversibel te remmen en blijken daardoor ideale veranke-
ringsfunctionaliteiten te zijn. Door zogeheten pincer–metaal-
complexen te functionaliseren met een dergelijke fosfonaates-
ter is het mogelijk om het enzym cutinase te modificeren met
palladium en platina bevattende organometaalcomplexen. De
vorming van deze nieuwe hybride materialen zijn bestudeerd
met UV/Vis spectrometrie en de producten zijn gezuiverd
door middel van dialyse. De geïsoleerde hybriden zijn geka-
rakteriseerd met behulp van ESI massaspectrometrie. De
voordelen van deze strategie zijn: 1) de regioselectieve veran-
kering van een enkel overgangsmetaalcomplex aan een
enzym, wat de gecontroleerde modificatie van de omgeving
van het metaal mogelijk maakt, 2) zuiveringsprocedures zijn
eenvoudig doordat een kompleet metaalcomplex aan het
enzym gehecht wordt en bovenal, 3) de tridentate ligandmo-
dulus van het pincerligand zorgt ervoor dat het metaal niet
van het eiwit kan dissociren. Deze strategie kan beschouwd
worden als een protocol voor de ontwikkeling van regio- en
enantioselectieve katalysatoren, alsmede voor de ontwikkeling
van nieuwe analytische detectoren voor het bestuderen van ei-
witeigenschappen en -structuren.
Results and Discussion
Synthesis ofcomplexes 1–3 : The connection between the
pincer ligand and the phosphonate ester framework was
achieved by means of a Suzuki cross-coupling reaction
(Scheme 1).[4g] The allyl function of phosphonamidate 4[19]
was hydroborated in situ by using 9-borobicyclononane (9-
BBN) and reacted with either 2-bromo-1,3-bis[(dimethyl-
amino)methyl]-5-iodobenzene,
2-bromo-1,3-bis[(methyl-
thio)methyl]-5-iodobenzene or 2-bromo-1,3-bis[(phenyl-
thio)methyl]-5-iodobenzene[19] in the presence of [PdCl2-
(dppf)] as catalyst.[20]
À
The C C bond formation proceeded with high selectivity
at the iodo-position of the pincer units to yield 5, 6, and 7 in
high yields. Treatment of 6 and 7 with anhydrous HCl (1m
in Et2O) generated the corresponding chlorophosphonate
derivatives in situ, by replacement of the dimethylamino
functionality on the phosphorous atom.[21] These intermedi-
ates were immediately treated with p-nitrophenol furnishing
8 and 9. The palladium atom could readily be introduced
into SCS-pincer ligands 8 and 9. Oxidative addition by using
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Metallopincer/Protein Hybrids
FULL PAPER
Figure 1. Upper: organometallic ASDCs 1–3. Lower: close-up of space-filling presentations of the ASDCs hybridized with cutinase.[18]
inseparable mixture of phosphonic acid, phosphonic acid an-
hydride, and desired product, as well as some unidentified
compounds. This is most probably caused by the basic NMe2
functionalities of the pincer moiety. For this reason ligand 5
was first cyclometallated to give 10 by using [{Pt(tol-4)2-
(SEt2)}2][22] as the platinum source. This makes the pincerꢁs
NMe2 groups less basic and non-nucleophilic due to coordi-
nation of the nitrogen atoms to the platinum center. To
avoid halogen scrambling in the subsequent chlorination of
the phosphonate, the bromide ligand of the NCN platinum
moiety was first exchanged for chloride. This was achieved
by treatment of 10 with AgBF4 in wet acetone, followed by
addition of NaCl to obtain 11 as an off-white solid.[23] Anal-
ogous to 5 and 6, the P(O)NMe2 functionality of 9 was
treated with anhydrous HCl, affording the desired P(O)Cl-
derivative without decomposition of the NCN-pincer plati-
num complex. This clearly demonstrates the high stability of
À
the Pt C bond in organometallic NCN-pincer platinum
complexes. Reaction of the phosphonate chloride compound
with p-nitrophenol resulted in the desired complex
1
(Scheme 1). Unfortunately, the corresponding palladium
complex could not be prepared. This is due to the incompat-
ibility of the NCN pincer palladium chloride unit with the
anhydrous HCl conditions required to generate the P(O)Cl
functionality.
Anchoring ofthe metal complexes to cutinase : We made
use of PnP phosphonates for three main reasons: 1) they are
very active compounds which selectively react with the
active site, that is, serine residue 120 of cutinase; 2) the reac-
tion profile can be followed spectrometrically due to the
high UV-visible absorption of the p-nitrophenolate anion
(e=16230mÀ1 cmÀ1, 400 nm, pH 8);[24] and 3) the PnP phos-
phonates are more stable towards hydrolysis compared to
their chloro and dinitrophenol phosphonate counterparts.
The reactions of 1, 2, and 3 with the active site of cutinase
were monitored over time by measuring the increase of the
absorbance at 400 nm (pH 8.0, RT, 25 or 50 mm ASDC, and
25 mm cutinase). When two equivalents of 1 were used with
Scheme 1. Synthetic Scheme for the preparation of organometallic
ASDCs 1–3. Reagents and conditions: a) 9-BBN, THF, reflux, 3 h; b) I-4-
C6H2(CH2NMe2)2-2,6-Br,
I-4-C6H2(CH2SMe)2-2,6-Br
or
I-4-C6H2-
(CH2SPh)2-2,6-Br, DMF, [PdCl2(dppf)], K3PO4 (aq.), reflux 3 h; c) 1m
Et2O/HCl, RT, 2 h; d) p-nitrophenol, NEt3, C6H6, RT, 2 h; e) [Pd2-
(dba)3]·CHCl3, C6H6, RT, 16 h; f) [{Pt(tol-4)(SEt2)}2], C6H6, reflux, 2 h;
g) AgBF4, acetone, H2O, RT, 1 h; h) NaCl, CH2Cl2, RT, 1 h.
[Pd2(dba)3]·CHCl3 resulted in the desired complexes 2 and
3, respectively.[16b,c]
The same reaction sequence was also envisaged to synthe-
size platinum complex 1. However, reaction of NCN com-
pound 5 with anhydrous HCl resulted in the formation of an
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G. van Koten et al.
Figure 2. Conversion of cutinase upon reaction with 1. Release of nitro-
phenolate was monitored by following the absorbance increase at
400 nm. Conditions: 25 mm cutinase; 25 mm (stars) and 50 mm (diamonds)
of ASDC 1. Buffer: 50 mm Tris-HCl, 0.1% Triton X-100 at pH 8.0.
Figure 4. Reaction mixture of 2 with cutinase. Left: start of reaction.
Right: end of reaction containing the Cut-2 hybrid.
reasons we were unable to determine which enantiomer
reacts faster and which was left unreacted in solution.
The same reaction was performed with the SCS-pincer
complex 2 (with phenyl substituents on the sulfur donor
atom). In this reaction also a full conversion was observed
(25 mm cutinase:50 mm 2). Remarkably, the reaction proceed-
ed approximately 12 times slower than the corresponding re-
action with NCN ASDC 1 (5 min vs 60 min, Figure 3, un-
marked line).
This difference in reaction kinetics might be attributed to
the different solubility properties of 2 as compared to 1.
Whereas complex 1 was completely soluble in the buffer so-
lution, addition of 2 to the buffered cutinase solution result-
ed in a turbid solution, causing the negative absorption evi-
dent in Figure 3.[26] During the reaction the turbidity gradu-
ally disappeared, resulting in a clear yellowish solution
(Figure 4), which illustrates the solubility effect of metallo-
pincer cutinase hybrid formation on the metallopincer com-
plex.
The reaction between 2 and cutinase was also followed in
time by measuring the residual enzymatic catalytic activity
using a p-nitrophenyl butyrate based spectrometric assay
(decay line in Figure 3). The same reaction profile was
found confirming the results obtained by following the re-
lease of p-nitrophenolate by UV-visible spectroscopy.
When ASDC 3, bearing methyl substituents on the sulfur
donor atoms, was reacted with cutinase under the same re-
action conditions as for complexes 1 and 2 (i.e., in a 2:1
ASDC/cutinase ratio), the same total conversion time was
observed as for the reaction of 1 with cutinase (Figure 5,
stars). In this experiment 3 was completely soluble in the
buffer solution at the start of the reaction.
Figure 3. Conversion of cutinase upon reaction with 2. Unmarked line:
Release of nitrophenolate was monitored by following the absorbance in-
crease at 400 nm. Decay line: decrease of enzymatic activity monitored
by using a spectrometric assay containing p-nitrophenolbutyrate. Re-
agents and conditions: 25 mm cutinase, 50 mm ASDC 2, buffer: 50 mm
Tris-HCl, 0.1% Triton X-100 at pH 8.0.
respect to cutinase (Figure 2, upper line, diamonds), a rapid
time-dependent conversion of 1 to form the Cut-1 hybrid
was found to occur with a concomitant release of p-nitro-
phenolate anion. After five minutes, a full conversion of cu-
tinase to the hybrid was accomplished.
Earlier studies have shown that the reaction of cutinase
with phosphonates proceeds with high enantioselectivity.[25]
For this reason we also carried out an experiment by using a
stoichiometric amount of
1 with respect to cutinase
(Figure 2, bottom line, stars). In this experiment we ob-
served an initial and fast reaction in which 50% of the race-
mic mixture of 1 (one enantiomer) and 50% of the enzyme
are converted to the Cut-1 hybrid, followed by a slower re-
action of the remaining and less reactive enantiomer of 1.
These observations imply a 1:1 ASDC/cutinase stoichiome-
try and in addition, that a chiral preference of cutinase
exists for the organometallic ASDCs, much alike other
phosphonate inhibitors.[25] Unfortunately, we have not been
able to recover the slower reacting enantiomer of 1 after
completion of the 2:1 1/cutinase reaction. In addition, nei-
ther NMR shift reagents nor the use of chiral HPLC was
successful in discriminating the enantiomers of 1. For these
To examine the reaction rate effect during our previous
experiment in which we used ASCD 2, a final control ex-
periment was performed. Complexes 1 and 2 were incubated
with cutinase at concentrations at which clear solutions were
obtained (6 mm cutinase and 12 mm of ACSD). For these re-
actions, lower initial reaction rates were observed for each
reaction, but with the same rate ratio difference as in the
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Metallopincer/Protein Hybrids
FULL PAPER
mass spectra of metallopincer complexes. In the mass spec-
tra of hybrid materials no mass signals corresponding to
20604 Da were observed, indicating complete conversion of
wild-type cutinase to the hybrids.
The results of the MS measurements show the high integ-
rity of the metallopincer cutinase hybrids. Not only is a
100% conversion obtained, but also is a precise 1:1 metallo-
pincer/cutinase stoichiometry achieved. The metallopincer/
cutinase hybrids can thus be prepared in a single and selec-
tive step by specific modification of one amino acid residue
of the lipase and, hence, exactly one metal can be intro-
duced in the chiral cavity of cutinase.
Figure 5. Conversion of cutinase upon reaction with 1 (diamonds) and 3
(stars). Release of nitrophenolate was monitored by following the ab-
sorbance increase at 400 nm. Conditions: 25 mm cutinase, 50 mm ASDC,
buffer: 50 mm Tris-HCl, 0.1% Triton X-100 at pH 8.0.
Conclusion
This study demonstrates the use of irreversible enzyme in-
hibitors as anchoring moieties for the site-directed attach-
ment of organometallic complexes to enzymes. Suicide in-
hibitors are excellent tools for this purpose since they often
react chemo-, regio-, and enantioselectively, as well as stoi-
chiometrically with enzymes. Especially the last point is im-
portant in constructing a structurally well-defined hybrid.
The results from the ESI-mass analysis nicely confirm that
discrete 1:1 hybrids are obtained between cutinase and the
organometallic ASDCs 1–3 by using PnP phosphonate
esters as anchoring groups. Moreover, the ESI-mass results
also indicate that the metallopincer units of the ASDCs are
stable under the applied reaction conditions, 48 h in an
aqueous buffer solution at pH 8.0, and withstand aqueous
dialysis purification conditions (2”12 h). The stability of the
organometallic unit is evidently of great importance to
maintain its functionality and to prevent metal leaching.
Noteworthy is that, due to the covalent bond formed during
the hybridization reaction of the ASDCs with the lipase, dis-
sociation of the phosphonate from the lipase will not occur
(pH<11). This feature permits the use of polar and apolar
solvents in future applications. The terdentate pincer ligand
allows the introduction of a variety of transition metals and
in this respect provides a unique method to merge organo-
metallics with the properties found in biology (selectivity,
solubility, and size) by using the hybridization protocol out-
lined in this study. One other major advantage of this ap-
proach is the positional precision with which the transition
metal unit is introduced in the active site of the enzyme.
This positioning promises a fine-tuning of the organometal-
lic/enzyme hybrids on the atomic level. Our current research
efforts indeed focus on the hybridization of a variety of
transition-metal complexes with cutinase and related pro-
teases, and on the use of such hybrids in aqueous homoge-
neous catalysis.
previous experiments with higher reactant concentrations.
This observation strongly suggests that steric factors deter-
mine the rate of inhibition to a greater extent than the solu-
bility of the ASDCs.
Isolation, purification and characterization of cutinase met-
allopincer hybrids: A series of ESI-MS samples were pre-
pared by reacting cutinase with two equivalents of 1, 2, or 3
over a period of 48 h (Tris buffer, pH 8.0). Prior to purifica-
tion and analysis of the product, the residual enzymatic cata-
lytic activity was determined by using a p-nitrophenyl buty-
rate based spectrometric assay. No release of p-nitropheno-
late was observed in all reactions after 48 h of incubation of
cutinase; a result that indicates that no esterase activity is
left and no wild-type cutinase is present in solution. The
crude materials were then purified by dialysis[27] (2”12 h)
[28]
and analyzed by ESImass spectrometry.
The organome-
tallic complexes are permeable through the dialysis mem-
brane in the set-up used for sample preparation, whereas
the native enzyme and the hybrids are not. The dialysis set-
up does not seem to degrade the organometallic moieties
and, therefore, is ideal for the purification of the metallo-
pincer/protein hybrids.
The native enzyme was used as calibration material for
the ESIanalysis and a mass of 20603.9 Æ0.2 Da was found
(calculated: 20604.1 Da; Table 1). All cutinase metallopinc-
Table 1. ESI-MS results of ASDC-cutinase hybrids.[28]
Compound
Calculated [Da]
Calculated
Found [Da]
Àhalide [Da]
cutinase
Cut-1
Cut-2
20603.1
21158.0
21244.0
21119.8
–
20603.9Æ0.2
21120.9Æ0.4
21163.2Æ0.4
21039.1Æ0.7
21122.5[a]
21164.1[b]
21039.9[b]
Cut-3
[a] [M+ÀCl]. [b] [M+ÀBr].
er hybrids showed a somewhat lower mass in the ESImass
spectra than the calculated value. This difference originates
from the release of a halide ion from the metal center
during the measurement; a result typically found in ESI
Experimental Section
General comments: All organic and organometallic reactions were con-
ducted under a dry dinitrogen atmosphere by using standard Schlenk
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6873

G. van Koten et al.
techniques. Organic solvents were dried over appropriate materials and
distilled prior to use. All reagents were obtained commercially and used
without further purification unless otherwise stated. Triton X-100 was
purchased from Serva and tris(hydroxymethyl)aminomethane from J. T.
Baker. Buffer solutions were prepared using Milli-Q grade water. Purifi-
cation of water (18.2MWcm) was performed with the Milli-Q Synthesis
system (Millipore, Quantum Ultrapure). 1H and 13C{1H} NMR spectra
were recorded at 298 K on a Varian Inova 300 spectrometer at 300 and
75 MHz, respectively, and 31P{1H} NMR spectra were recorded at 298 K
on a Bruker AC200 at 81 MHz. All NMR chemical shifts are in ppm ref-
erenced to residual solvents (31P{1H} NMR shifts to H3PO4). The
MALDI-TOF mass spectra were acquired using a Voyager-DE Biospectr-
ometry Workstation (PerSeptive Biosystems Inc, Framingham, MA,
USA) mass spectrometer. Sample solutions with an approximate concen-
tration of 20–30 mgmLÀ1 in CH2Cl2 or THF were prepared. The matrix
was 5-chlorosalicylic acid (CSA) with an approximate concentration of
20–30 mgmLÀ1. A 0.2 mL of the sample and 0.2 mL of the matrix solution
were combined and placed on a golden MALDItarget plate and ana-
lyzed after evaporation of the solvents. [{Pt(tol-4)2(SEt2)}2],[22] [Pd2-
(40 mL). The organic phase was separated and the aqueous phase ex-
tracted with Et2O (2”50 mL). The organic fractions were combined,
washed with 1n NaOH (2”50 mL) and brine, dried over MgSO4, and
concentrated in vacuo to obtain a dark brown oil. The crude was dis-
solved in Et2O (5 mL) and purified by column chromatography (silica,
eluting with Et2O, then CH2Cl2 and finally with MeOH/5% NEt3) to
obtain the product as a yellowish oil (3.20 g, 90%). 1H NMR (300 MHz,
CDCl3): d=7.15 (s, 2H; ArH), 3.90 (m, 2H; CH2O), 3.52 (s, 4H; CH2N),
2.63 (d, 3J(H,P)=8.79 Hz, 6H; PNMe2 and 2H; CH2Ar), 2.29 (s, 12H;
NMe2), 1.80 (m, 2H; PCH2), 1.75 (m, 2H; PCH2CH2), 1.26 ppm (t,
3J(H,H)=6.96 Hz, CH3; 3H); 31P NMR (81 MHz, CDCl3): d=37.25 ppm
(s); 13C NMR (75 MHz, CDCl3): d=140.43, 139.05, 129.74, 124.27 (s;
ArC), 63.85 (s; CH2N), 59.06 (d, 2J(C,P)=6.71 Hz; CH2O), 45.40 (s;
NMe2), 36.09 (d, 2J(C,P)=15.26 Hz; CH2CH2P), 35.73 (d, 2J(C,P)=
4.27 Hz; PNMe2), 26.41 (d, 1J(C,P)=131.23 Hz; CH2P), 24.27 (d,
3J(C,P)=3.62 Hz; ArCH2), 16.23 ppm (d, 3J(C,P)=6.72 Hz; CH3); ele-
mental analysis calcd (%) for C19H35BrN3O2P (448.38): C 50.89, H 7.87,
N 9.37; found: C 50.96, H 7.97, N 9.31.
Preparation ofethyl P-{3-{4-bromo-1,3-bis[(phenylthio)methyl]phenyl}-
propyl}-N,N-dimethylaminophosphinate (6): A similar procedure as for 5
was applied. Compound 4 (0.50 g, 2.85 mmol), THF (10 mL), 9-BBN
(0.41m, 15.3 mL, 6.27 mmol). K3PO4 (3m, 2.0 mL, 6 mmol), [PdCl2(dppf)]
(0.18 g, 0.23 mmol), 2-bromo-1,3-bis[(phenylthio)methyl]-5-iodoben-
zene[19] (1.50 g, 2.85 mmol), DMF (20 mL). The product was purified by
column chromatography eluting with Et2O and acetone. After concentra-
tion of the product containing fractions (acetone), a yellow oil was ob-
tained. Yield: 1.31 (80%); 1H NMR (300 MHz, CDCl3): d=7.34–7.18
(brm, 10H; SArH), 6.89 (s, 2H; ArH), 4.22 (s, 4H; CH2S), 3.90 (m, 2H;
CH2O), 2.65 (m, 2H; CH2Ar), 2.63 (d, 3J(H,P)=8.79 Hz, PNMe2; 6H),
2.29 (s, 12H; NMe2), 1.80 (m, 2H; PCH2), 1.75 (m, 2H; PCH2CH2),
1.26 ppm (t, 3J(H,H)=6.96 Hz, CH3; 3H); 31P NMR (81 MHz, CDCl3):
d=37.09 ppm (s); 13C NMR (75 MHz, CDCl3): d=142.03, 137.99, 136.48,
130.19, 129.06, 128.16, 127.35, 126.62, 59.37 (d, 2J(C,P)=6.72 Hz; CH2O),
40.88 (s; CH2S), 36.36 (d, 2J(C,P)=3.62 Hz; PCH2CH2), 36.01 (d,
3J(C,P)=16.53 Hz; PN(CH3)2), 24.86 (d, 1J(C,P)=131.24 Hz; PCH2),
23.78 (d, 3J(C,P)=3.70 Hz; CH2Ar), 16.56 ppm (d, 3J(C,P)=6.72 Hz;
CH3); elemental analysis cald (%) for C27H33BrNO2PS2 (578.56): C 56.05,
H 5.75, N 2.42, S 11.08; found: C 56.18, H 5.83, N 2.37, S 11.16.
[29]
(dba)3]·CHCl3 and 2-bromo-1,3-bis[(dimethylamino)methyl]-5-iodoben-
zene[30] were prepared according to described procedures. Microcoanaly-
ses were performed by Kolbe, Mikroanalystisches Laboratorium (Müll-
heim a/d Ruhr, Germany). UV-visible experiments were performed at
room temperature using a Carey-100 spectrometer. Electrospray ioniza-
tion mass spectra of the wild-type cutinase and modified cutinase were
recorded on a Finnigan LC-Q ion-trap mass spectrometer. All samples
were introduced using a nanoflow electrospray source (Protana, Odense,
Denmark).
Inhibition experiments: A solution of 1, 2, or 3 in CH3CN (5–10 mL;
50 mmol, 10 mL µ 5 mm) was added to a buffer solution (0.1% Triton X-
100, 50 mm Tris-HCl, pH 8.0) containing wild-type cutinase (25 mm,
25 mmol). The reaction was followed by UV-visible spectroscopy until the
reaction was complete, which was confirmed by a plateauing of the inten-
sity of the UV absorption at 400 nm.
Hydrolysis activity ofcutinase : The catalytic activity of cutinase was de-
termined spectrometrically on 0.25 mm p-nitrophenol butyrate in the
presence of 100 mm Triton X-100 and 10 mm Tris-HCl at pH 8.0. Aliquots
of 5–10 mL were taken and added to the assay. Activities were calculated
from the increase of absorbance at 400 nm.
Preparation ofethyl P-{3-{4-bromo-1,3-bis[(methylthio)methyl]phenyl}-
propyl}-N,N-dimethylaminophosphinate (7): A similar procedure as for 5
was applied. Compound 4 (0.88 g, 5.00 mmol), THF (20 mL), 9-BBN
(1.34 g, 10.10 mmol, 0.5m in THF), K3PO4 (3m, 5.5 mL, 17.50 mmol), 2-
bromo-1,3-bis[(methylthio)methyl]-5-iodobenzene[19] (2.01 g, 5.00 mmol),
[PdCl2(dppf)] (0.33 g, 0.43 mmol), DMF (15 mL). The crude was dis-
solved in 5 mL Et2O and purified by column chromatography (silica, elut-
ing with CH2Cl2/acetone 3:1) to obtain the product as a yellowish oil
2.02 g (88%). 1H NMR (300 MHz, CDCl3): d=7.06 (s, 2H; ArH), 4.01
(m, 1H; CH2O), 3.88 (m, 1H; CH2O), 3.83 (s, 4H; CH2S), 2.66 (d,
3J(H,P)=9.00 Hz, 6H; NMe2 and 2H; CH2Ar), 2.07 (s, 6H; SMe), 1.80
(brm, 4H; CH2), 1.28 ppm (t, 3J(H,H)=7.20 Hz, 3H; CH3); 31P NMR
(81 MHz, CDCl3): d=37.07 ppm (s); 13C NMR (75 MHz, CDCl3): d=
140.40, 138.68, 129.83, 124.30 (s; ArC), 59.41 (d, 2J(C,P)=6.68 Hz,
CH2O; 2H), 39.56 (s; CH2S), 36.37 (d, 2J(C,P)=16.43 Hz; PCH2CH2),
36.11 (d, 2J(C,P)=3.68 Hz; NMe2), 24.87 (d, 1J(C,P)=132.83 Hz; PCH2),
24.03 (d, 3J(C,P)=3.62 Hz; CH2Ar), 16.54 (d, 3J(C,P)=6.68 Hz;
CH3CH2O), 15.64 ppm (s; SMe); elemental analysis cald (%) for
C15H24BrNOPS2 (454.43): C 44.93, H 6.43, N 3.08, S 14.11; found: C
45.10, H 6.51, N 3.03, S 14.06.
Chemical stability experiments: The chemical stability of ACDC 1 in the
buffer solution (0.1% Triton X-100, 50 mm Tris-HCl, pH 8.0) was exam-
ined over 48 h using UV-visible spectroscopy (400 nm). The compound
did not show any hydrolysis of PnP phosphonate ester functionality
during this time. Naturally, the stability of cutinase was also tested under
the same conditions. Aliquots were taken and added to a spectrometric
assay containing 0.25 mm p-nitrophenyl butyrate. No decrease of ester hy-
drolysis activity, and thus no catalytic deactivation, was observed during
a 3 day period.
Preparation ofsamples of r mass spectrometry analysis : Cutinase (25 mm)
in a 10 mm Tris-HCl buffer solution (pH 8.0) was modified during 48 h by
adding an excess of 1, 2, or 3. Completion of the reaction was confirmed
by determining the residual catalytic hydrolyses activity of cutinase. The
clear yellow solutions were subsequently dialyzed for 2”12 h using the
same buffer as dialysis solvent. Prior to ESImass measurements the solu-
tions were diluted to 2–5 mm with CH3CN.
Preparation ofethyl P-{3-{4-bromo-1,3-bis[(dimethylamino)methyl]phe-
nyl}propyl}-N,N-dimethylaminophosphinate (5):[4g]
A solution of allyl
ethyl N,N-dimethylaminophosphinate 4[19] (1.53 g, 8.65 mmol) in THF
(15 mL) was treated with 9-BBN (34.4 mL a 0.5m in THF, 17.20 mmol).
The prepared mixture was stirred for 3 h at reflux temperature. The col-
orless solution was allowed to cool to room temperature and a degassed
aqueous solution of K3PO4 (3m, 7.2 mL, 21.60 mmol) was added, fol-
lowed by a solution of 2-bromo-1,3-bis[(dimethylamino)methyl]-5-iodo-
benzene (3.26 g, 8.21 mmol) and [PdCl2(dppf)] (0.5 g, 0.68 mmol) in
DMF (15 mL). The solution immediately turned dark red after addition
of the palladium catalyst. The mixture was heated to reflux for 3 h and
during this period a clear orange solution was formed. THF was evapo-
rated in vacuo and the mixture diluted with Et2O (60 mL) and water
Preparation ofethyl 4-nitrophenyl P-{3-{4-bromo-1,3-bis[(phenylthio)me-
thyl]phenyl}propyl}phosphonate (8): Compound
6 (0.49 g, 0.85 mmol)
was dissolved in dry benzene (10 mL) and treated with a solution of an-
hydrous HCl (4.25 mL, 4.25 mmol, 1m in Et2O). After stirring for 2 h at
room temperature under a nitrogen atmosphere, more benzene (10 mL)
was added and the yellow suspension was filtered. A solution of p-nitro-
phenol (0.12 g, 0.85 mmol) and NEt3 (0.6 mL, 4.24 mmol) in benzene
(20 mL) was added dropwise to the filtrate and stirring was continued for
2 h. All volatiles were removed in vacuo and Et2O (30 mL) was added
followed by a saturated solution of K2CO3 (30 mL). The organic phase
6874
www.chemeurj.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2005, 11, 6869 – 6877

Metallopincer/Protein Hybrids
FULL PAPER
was separated and washed with a saturated solution of K2CO3 (30 mL),
water (30 mL), and brine (30 mL). The solution was dried on MgSO4 and
concentrated to give a pale yellow solid. Yield: 0.35 g (62%); 1H NMR
(300 MHz, CDCl3): d=8.21 (d, 3J(H,H)=8.70 Hz, 2H; ArH), 7.35–7.17
(brm; 10H; SArH), 6.89 (s, 2H; ArH), 4.22 (s, 4H; CH2S), 4.16 (m, 2H;
CH2O), 2.51 (t, 3J(H,H)=6.30 Hz, 2H; CH2Ar), 1.81 (brm, 4H; PCH2,
PCH2CH2), 1.30 ppm (t, 3J(H,H)=6.90 Hz, CH3; 3H); 31P NMR
(81 MHz, CDCl3): d=30.56 ppm (s); 13C NMR (75 MHz, CDCl3): d=
155.82 (d, 2J(C,P)=8.48 Hz; ArC), 144.78, 139.58, 138.03, 135.83, 131.18,
130.02, 129.14, 127.12, 126.30, 124.49 (s; ArC), 121.19 (d, ArC, 3J(C,P)=
4.28 Hz), 63.25 (d, 2J(C,P)=7.32 Hz; CH2O), 40.82 (s; CH2S), 35.54 (d,
2J(C,P)=17.06 Hz; PCH2CH2), 25.47 (d, 1J(C,P)=140.97 Hz; PCH2),
23.77 (d, 3J(C,P)=4.91 Hz; CH2Ar), 16.64 ppm (d, 3J(C,P)=5.51 Hz;
CH3CH2O); elemental analysis cald (%) for C31H31BrNO5PS2 (672.59): C
55.36, H 4.65, N 2.08, S 9.53; found: C 55.36, H 4.77, N 2.02, S 9.58.
CH2Ar), 2.43 (d, 3J(H,P)=8.79 Hz, 6H; PNMe2), 2.03 (m, 2H; PCH2),
1.71 (m, 2H; PCH2CH2), 1.06 ppm (t, 3J(H,H)=7.01 Hz, 3H; CH3);
31P NMR (81 MHz, C6D6): d=36.13 ppm (s); 13C NMR (75 MHz, C6D6):
d=144.62, 143.79, 136.21 (s; ArC), 119.55 (s, 3J(C,Pt)=31.74 Hz; ArC),
77.69 (s, 3J(C,Pt)=31.13 Hz; CH2N), 58.89 (d, 2J(C,P)=6.71 Hz; CH2O),
53.98 (s; NMe2), 37.48 (d, 2J(C,P)=15.32 Hz; CH2CH2P), 35.83 (d,
1
2J(C,P)=3.70 Hz; PNMe2), 24.97 (d, J(C,P)=130.64 Hz; CH2P), 24.87 (s,
3J(C,P)=3.70 Hz; ArCH2), 16.38 ppm (d; CH3); elemental analysis cald
(%) for C19H35ClN3O2PPt (599.00): C 38.10, H 5.92, N 7.02; found: C
37.95, H 5.76, N 6.88.
Preparation ofethyl 4-nitrophenyl P-{3-{4-(chloroplatino)-1,3-bis[(dime-
thylamino)methyl]phenyl}propyl}phosphonate (1): A similar procedure
as for 8 and 9 was applied. Compound 11 (246.1 mg, 0.41 mmol), anhy-
drous HCl (2.05 mL, 2.05 mmol, 1.0m in Et2O); triethylamine (0.3 mL),
p-nitrophenol (57.2 mg, 0.41 mmol) in benzene (15 mL). The product was
obtained as a yellowish solid (199 mg, 66%). 1H NMR (300 MHz, C6D6):
Preparation ofethyl 4-nitrophenyl P-{3-{4-bromo-1,3-bis[(methylthio)me-
thyl]phenyl}propyl}phosphonate (9): A similar procedure as for 8 was ap-
3
3
d=7.76 (d, J(H,H)=7.63 Hz, 2H; ArH), 7.05 (d, J(H,H)=8.24 Hz, 2H;
ArH), 6.45 (s, 2H; ArH), 3.88 (m, 2H; CH2O), 3.35 (s, 3J(H,Pt)=
43.94 Hz, 4H; CH2N), 2.73 (s, 3J(H,Pt)=32.00 Hz, 12H; NMe2), 2.51 (t,
3J(H,H)=7.63, 2H; ArCH2), 2.02 (m, 2H; PCH2CH2), 1.79 (m, 2H;
CH2P), 0.95 ppm (t, 3J(H,H)=7.02 Hz, 3H; CH3,); 31P NMR (81 MHz,
C6D6): d=30.52 ppm (s); 13C NMR (75 MHz, C6D6): d=155.85 (d,
2J(C,P)=7.92 Hz; ArC), 144.65, 144.00, 135.25, 125.51 (s; ArC), 120.81
(d, 3J(C,P)=4.88; ArC), 119.41 (s; ArC), 77.68 (s; CH2N), 62.52 (d,
2J(C,P)=6.71 Hz; CH2O), 53.99 (s; NMe2), 37.00 (d, 2J(C,P)=16.48 Hz;
CH2CH2P), 25.70 (s, 1J(C,P)=141.00 Hz; PCH2), 24.66 (d, 3J(C,P)=
4.88 Hz; ArCH2), 16.23 ppm (d, 3J(C,P)=6.10 Hz; CH3); MS (MALDI-
TOF, CSA): m/z: 656.3 [M+ÀCl], 462.2 [M+ÀClÀPt], 374.6 [M+
ÀClÀPtÀNO2ÀOEt], 343.1 [M+ÀClÀPtÀNO2ÀOEtÀ2Me]; elemental
analysis cald (%) for C23H33ClN3O5PPt (693.03): C 39.86, H 4.80, N 6.06;
found: C 40.05, H 4.72, N 5.93.
plied. Compound
7 (0.41 mg, 0.90 mmol), anhydrous HCl (4.50 mL,
4.50 mmol, 1.0m in Et2O); triethylamine (0.14 mL), p-nitrophenol
(125.5 mg, 0.90 mmol) in benzene (15 mL). The product was obtained as
a yellowish solid (0.39 g, 78%). 1H NMR (300 MHz, CDCl3): d=8.16 (d,
3
3J(H,H)=7.20 Hz, ArH; 2H), 7.31 (d, J(H,H)=8.10 Hz, ArH; 2H), 7.03
(s, 2H; ArH), 4.20–4.10 (brm, 2H; CH2O), 3.76 (s, 4H; CH2), 2.66 (t,
3J(H,H)=6.90 Hz, 2H; PCH2CH2), 2.02–1.86 (m, 4H; CH2Ar, PCH2),
2.01 (s, 6H; SMe), 1.26 ppm (t, 3J(H,H)=7.20 Hz, 3H; CH3); 31P NMR
(81 MHz, CDCl3): d = 30.50 ppm (s); 13C NMR (75 MHz, CDCl3): d=
155.84 (d, 2J(C,P)=7.93 Hz; ArC), 144.72, 139.60, 138.87, 129.70, 125.87,
124.58 (s; ArC), 121.21 (d, 3J(C,P)=4.30 Hz; ArC), 63.56 (d, 2J(C,P)=
7.31 Hz; CH2O), 39.50 (s; CH2S), 35.60 (d, 2J(C,P)=17.67 Hz;
PCH2CH2), 25.58 (d, 1J(C,P)=140.10 Hz; PCH2), 24.11 (d, 3J(C,P)=
4.80 Hz; CH2Ar), 16.60 ppm (d, 3J(C,P)=5.50 Hz; CH3); elemental anal-
ysis cald (%) for C21H27BrNO5PS2 (548,45): C 45.99, H 4.96, N 2.55, S
11.69; found: C 46.15, H 5.08, N 2.48, S 11.52.
Preparation ofethyl 4-nitrophenyl P-{3-{4-(bromopallado)-1,3-bis[(phe-
nylthio)methyl]phenyl}propyl}phosphonate (2): Solid [Pd2(dba)3]·CHCl3
(0.17 g, 0.16 mmol) was added to a solution of 8 (0.22 g, 0.32 mmol) in
benzene (30 mL) and stirred for 16 h at room temperature. All volatiles
were removed in vacuo and the residue dissolved in THF (30 mL). Then
Et3N (0.5 mL) was added and the mixture was stirred for 2 h. The color
of the mixture turned from purple-black to dark green indicating the for-
mation of Pd black. The Pd black was then removed by filtration over a
short path of Celite leaving the filtrate as a clear yellow solution. The fil-
trate was concentrated under reduced pressure to obtain the crude prod-
uct as a yellow solid. Pure product was obtained by purification using
column chromatography, eluting with Et2O to remove dba, and acetone
to obtain the product as a pale yellow solid. Yield: 0.16 g (63%);
1H NMR (300 MHz, CDCl3): d=8.23 (d, 3J(H,H)=9.30 Hz, 2H; ArH),
7.83 and 7.36 (m, 12H; ArH), 6.82 (s, 2H; ArH), 4.60 (brs, 4H; CH2S),
4.16 (m, 2H; CH2O), 2.62 (t, 3J(H,H)=6.90 Hz, 2H; CH2Ar,), 1.96 (m,
4H; PCH2 and PCH2CH2), 1.30 ppm (t, 3J(H,H)=6.90 Hz, 3H; CH3);
31P NMR (81 MHz, CDCl3): d=30.74 ppm (s); 13C NMR (75 MHz,
CDCl3): d=155.84 (d, 2J(C,P)=8.53 Hz; ArC), 149.75, 144.79, 137.76,
132.63, 131.90, 130.12, 129.85, 125.91, 122.40 (s; ArC), 121.17 (d,
3J(C,P)=4.91 Hz; ArC), 63.27 (d, 2J(C,P)=8.53 Hz; CH2O), 52.99 (s;
CH2S), 36.07 (d, 2J(C,P)=17.06 Hz; PCH2CH2), 25.80 (d, 1J(C,P)=
141.65 Hz; PCH2), 24.11 (d, 3J(C,P)=4.83 Hz; CH2Ar), 16.63 ppm (d,
Preparation ofethyl
N,N-dimethylamino-P-{3-{4-(bromoplatino)-1,3-
bis[(dimethylamino)methyl]phenyl}propyl}phosphinate (10):[4g] Solid [{Pt-
(tol-4)2(SEt2)}2] (1.06 g, 1.14 mmol) was added to a solution of 5 (1.02 g,
2.27 mmol) in dry toluene (45 mL) and heated to reflux for 2 h. The sol-
vent was removed in vacuo and the remaining dark brown oil dissolved
in CH2Cl2 (15 mL) and filtered through a short path of Celite. The filtrate
was concentrated under reduced pressure leaving a brown oil, which was
subsequently washed with pentane (3”30 mL, stirring 30 min) and Et2O
(2”30 mL, stirring 30 min). The resulting mixture was dried in vacuo to
obtain the product as an off-white solid (1.11 g, 78%). 1H NMR
(300 MHz, C6D6): d=6.52 (s, 2H; ArH), 3.91 (m, 2H; CH2O), 3.33 (s,
3J(H,Pt)=44.56 Hz, 4H; CH2N), 2.77 (s, 3J(H,Pt)=37.54 Hz, 12H;
NMe2), 2.60 (t, 3J(H,H)=7.32 Hz, 4H; CH2Ar), 2.43 (d, 3J(H,P)=
8.85 Hz, 6H; PNMe2), 2.02 (m, 2H; CH2), 1.71 (m, 2H; PCH2CH2),
1.06 ppm (t, 3J(H,H)=7.02 Hz, 3H; CH3,); 31P NMR (81 MHz, C6D6):
d=36.26 ppm (s); 13C NMR (75 MHz, C6D6): d=145.52, 143.77, 136.38,
134.52, 119.54 (s; ArC), 77.13 (s, 2J(C,Pt)=32.96 Hz; CH2N), 58.82 (s;
CH2O), 54.62 (s; NMe2), 37.44 (d, 2J(C,P)=14.65 Hz; CH2CH2P), 35.81
(d, 2J(C,P)=3.70 Hz; PNMe2), 25.33 (d, 1J(C,P)=130.64 Hz; CH2P),
24.27 (d; ArCH2), 16.23 ppm (d, 3J(C,P)=6.72 Hz; CH3); MS (MALDI-
TOF, CSA): m/z: 561.1 [M+ÀBr], 467.2 [M+ÀBrÀPt]; elemental analysis
cald (%) for C19H35BrN3O2PPt (643.46): C 35.47, H 5.48, N 6.53; found:
C 35.48, H 5.41, N 6.38.
3J(C,P)=6.04 Hz
CH3);
elemental
analysis
cald
(%)
for
C31H31BrNO5PPdS2 (779.01): C 47.80, H 4.01, N 1.80, S 8.23; found: C
47.98, H 4.07, N 1.70, S 8.29; MS (MALDI-TOF, CSA): m/z: 700.6 [M+
ÀBr].
Preparation ofethyl
N,N-dimethylamino-P-{3-{4-(chloroplatino)-1,3-
bis[(dimethylamino)methyl]phenyl}propyl}phosphinate (11): Compound
10 (0.76 g, 1.20 mmol) in wet acetone (10 mL) was treated with AgBF4
(0.25 g, 1.26 mmol) for 1 h. The reaction mixture was filtered over Celite
and NaCl (0.74 g, 12.60 mmol) was added to the filtrate. The solvent was
removed in vacuo after 1 h stirring of the reaction mixture, and subse-
quently water (20 mL) was added. The product was extracted with
CH2Cl2 (4”20 mL), the combined fractions dried on MgSO4 and concen-
trated under reduced pressure leaving an off-white solid (0.61 g, 85%).
1H NMR (300 MHz, C6D6): d=6.54 (s, 2H; ArH), 3.93 (m, 1H; CH2O),
3.69 (m, 1H; CH2O), 3.38 (s, 3J(H,Pt)=44.69 Hz, 4H; CH2N), 2.75 (s,
3J(H,Pt)=38.46 Hz, 12H; NMe2), 2.60 (t, 3J(H,H)=7.33 Hz, 2H;
Preparation ofethyl 4-nitrophenyl P-{3-{4-(bromopallado)-1,3-bis[(me-
thylthio)methyl]phenyl}propyl}phosphonate (3): A similar procedure as
for 2 was applied. Compound 9 (0.40 g, 0.73 mmol), [Pd2(dba)3]·CHCl3
(0.38 g, 0.36 mmol). The product was purified by column chromatography
(silica, eluting with Et2O (dba) and acetone (product)) to leave the prod-
uct as a yellow solid after removal of the eluent in vacuo. Yield: 0.3 g
(71%); 1H NMR (300 MHz, CDCl3): d=8.11 (d, 3J(H,H)=9.32 Hz, 2H;
ArH), 7.28 (d, 3J(H,H)=9.60 Hz, 2H; ArH), 6.73 (s, 2H; ArH), 4.30
(brs, 2H; CH2S), 4.20–3.99 (brm, 6H; CH2S, CH2O), 2.71 (s, 6H; SMe),
2.52 (t, 3J(H,H)=6.60 Hz, 2H; CH2Ar), 1.85 (m, 4H; PCH2, PCH2CH2),
Chem. Eur. J. 2005, 11, 6869 – 6877
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6875

G. van Koten et al.
1.21 ppm (t, 3J(H,H)=6.90 Hz, 3H; CH3); 31P NMR (81 MHz, CDCl3):
d=30.74 ppm (s); 13C NMR (75 MHz, CDCl3): d=160.11 (s; ArC),
155.84 (d, 2J(C,P)=8.48 Hz; ArC), 148.90, 144.58, 137.78, 125.84, 122.80
(s; ArC), 121.21 (d, 3J(C,P)=4.20 Hz; ArC), 63.21 (d, 2J(C,P)=6.68 Hz;
CH2O), 49.76 (s; CH2S), 35.93 (d, 2J(C,P)=17.63 Hz; PCH2CH2), 25.58
(d, 1J(C,P)=140.10 Hz; PCH2), 24.11 (d, 3J(C,P)=4.80 Hz; CH2Ar),
16.62 ppm (d, 3J(C,P)=6.08 Hz; CH3); MS (MALDI-TOF, CSA):
[8] C. A. Kruithof, G. Guillena, M. R. Egmond, G. van Koten, Book of
Abstracts, First International Symposium on Bioorganometallic
Chemistry, 18–20 July 2002, Paris, p. 104.
[9] a) F. Bjçrkling, A. Dahl, S. Patkar, M. Zundel, Bioorg. Med. Chem.
1994, 2, 697–705; b) J. C. Powers, J. L. Asgian, ꢂ. D. Ekici, K. E.
James, Chem. Rev. 2002, 102, 4639–4750; c) N. Jessani, J. A. Young,
S. L. Diaz, M. P. Patricelli, A. Varki, B. F. Cravatt, Angew. Chem.
2005, 117, 2452–2455; Angew. Chem. Int. Ed. 2005, 44, 2400–2403.
[10] a) C. D. Hodneland, Y.-S. Lee, D.-H. Min, M. MrKsich, Proc. Natl.
Acad. Sci. USA 2002, 99, 5048–5052; b) G. Zandonella, P. Stadler,
L. Haalck, F. Spener, F. Paltauf, A. Hermetter, Eur. J. Biochem.
1999, 262, 63–69.
m/z:
575.0
[M+ÀBr],
452
[M+ÀBrÀPdÀMe],
303.7
[M+
ÀBrÀPdÀ2MeÀNO2C6H4O]; elemental analysis cald (%) for
C21H27BrNO5PPdS2 (654,87): C 38.52, H 4.16, N 2.14, S 9.79; found: C
38.65, H 4.22, N 1.98, S 9.65.
[11] So far, no experimental details concerning the formation of such a
metal complex have been presented, see reference [4d].
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G.G. acknowledges the European Commission for a TMR grant (HPMF-
CT-2000-00472). This work was supported by the Council for Chemical
Sciences from the Dutch organization for Scientific Research (CW-
NWO). Unilever Research Laboratory, Vlaardingen, is kindly acknowl-
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Prof. Robert A. Gossage for their contribution in preparing this manu-
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360 slower. This result also indicates that the choice of tether length
can be estimated according to these representations of the hybrids.
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Chem. Eur. J. 2005, 11, 6869 – 6877

Metallopincer/Protein Hybrids
FULL PAPER
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Received: June 10, 2005
Published online: October 13, 2005
Chem. Eur. J. 2005, 11, 6869 – 6877
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6877