Site-selective ser-hydrolase labelling with a luminescent organometallic NCN-platinum complex

Article abstract of DOI:10.1002/ejic.200900980

The synthesis, spectroscopic properties and protein-binding studies of a novel luminescent organometallic NCN-platinum complex are described. The luminescent organometallic complex was linked to a serine hydrolase reactive phosphonate group by means of cl

Full text of DOI:10.1002/ejic.200900980

FULL PAPER
DOI: 10.1002/ejic.200900980
Site-Selective Ser-Hydrolase Labelling with a Luminescent Organometallic
NCN–Platinum Complex
Birgit Wieczorek,[a][‡] Bart Lemcke,[a] Harm P. Dijkstra,[a] Maarten R. Egmond,[b]
Robertus J. M. Klein Gebbink,*[a] and Gerard van Koten*[a]
Keywords: Luminescence / Hydrolases / Metalloenzymes / Protein labeling / Platinum
The synthesis, spectroscopic properties and protein-binding
studies of a novel luminescent organometallic NCN–plati-
num complex are described. The luminescent organometallic
complex was linked to a serine hydrolase reactive phos-
phonate group by means of click chemistry, and was ex-
ploited in serine hydrolase specific-binding studies using gel
electrophoresis. The NCN–platinum protein label was found
to be a robust dye with absorbance and emission maxima
between 374–380 and 482–493 nm, respectively, and quan-
tum yields between 0.04 and 0.15.
enables chemists to more easily alter their spectroscopic and
protein-binding properties through chemical synthesis.[13,14]
In the emerging field of activity-based protein profiling,
novel small molecular probes are highly desirable (e.g., for
the targeting of cancer-inducing mutated proteins).[15]
When fluorescent dyes with different emission wavelengths
are combined with various protein-reactive functional
groups, a wide spectrum of the proteome can potentially be
addressed and studied with these fluorescent probes. For
instance, Cravatt and co-workers have probed several dif-
ferent enzyme classes like aspartyl proteases, deubiquitinat-
ing enzymes, glycosidases and serine hydrolases through the
design of a variety of enzyme-targeted probes based on
their active-site reactivity and substrate selectivity.[16–18]
Currently, our group is working on the development of
protein reactive probes as well, with the aim of modifying
serine hydrolases and lipases for protein diagnosis purposes
and/or for the construction of novel semisynthetic en-
zymes.[19–24] By doing so, various phosphonate inhibitors
substituted by different organic[22,23] and organometallic
moieties[19–21,24] have been used to target different lipases
(Figure 1). By substituting organometallic ECE-pincer–
metal complexes (in which E is an electron-donating sub-
stituent such as nitrogen or sulfur) with a protein-reactive
phosphonate group, the special properties of the metal cen-
tre can be introduced into a protein. By doing so, novel,
unnatural features can be added to the protein, which can
be exploited in coordination studies involving the metal
centre or in the phasing of protein diffraction data.[21]
Phosphonates have long been known to be substrate ana-
logues for serine hydrolases, and even phosphonates with
various organic fluorescent reporter tags have been studied
by different groups.[17,22,29–31] So far, phosphonates substi-
tuted by organic dyes [e.g., fluorescent dansyl (Figure 1),[22]
rhodamine[15] and p-nitrobenzofurazan[32] groups] have
Introduction
Luminescent probes are playing an important role in the
study and understanding of biomolecular processes, such as
protein functioning within cells,[1] membrane dynamics[2]
and tumour cell surface targeting.[3] Various luminescence
techniques [e.g., Förster resonance energy transfer
(FRET)[4,5] and single molecule spectroscopy][5,6] have be-
come increasingly important tools in the study of these
phenomena. The design and development of novel lumines-
cent probes is crucial for the advancement of this field.
Excellent examples that illustrate the power of lumines-
cence in biology are the use of the green,[7] yellow or cyan
fluorescent proteins (GFP, YFP, CFP) in the labelling of
reporter molecules in cells to study intracellular dy-
namics.[8–10] Also various commercially available synthetic
fluorescent small-molecule organic dyes,[11,12] with different
emission wavelengths and various protein reactive groups
(e.g., maleimide functional groups for cysteine labelling) are
increasingly used in proteomics to screen for different types
of proteins and monitor their activity profiles.[13,14] These
dyes are generally smaller in size and possess a lower molec-
ular weight than the protein-based fluorescent labels, which
[a] Organic Chemistry & Catalysis, Debye Institute for Nano-
materials Science,
Faculty of Science, Utrecht University,
Padualaan 8, 3584 CH Utrecht, The Netherlands
Fax: +31-30-2523615
E-mail: r.j.m.kleingebbink@uu.nl
g.vankoten@uu.nl
[b] Membrane Enzymology, Bijvoet Centre for Biomolecular
Research,
Faculty of Science, Utrecht University,
Padualaan 8, 3584 CH Utrecht, The Netherlands
[‡] Current address: Novo Nordisk A/S, Diabetes Protein & Pep-
tide Chemistry,
Novo Nordisk Park, 2760 Måløv, Denmark
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.200900980.
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R. J. M. Klein Gebbink, G. van Koten et al.
FULL PAPER
Figure 1. (a) General inhibition reaction of serine hydrolases with phosphonates (PNP = p-nitrophenolate anion). (b) Structures of
organometallic ECE-pincer–metal phosphonates (left),[20,21] of an organic luminescent dansyl phosphonate inhibitor (middle)[22] and of
the organometallic luminescent target inhibitor 6 (right).
been used in fluorescent labelling studies. Due to the excit-
Contrary to common organic fluorescent probes, which
ing spectroscopic properties of certain NCN–metal com- generally emit from a singlet excited state, these complexes
plexes,[19,20,25–28] we became interested in applying these in emit from a low-lying triplet excited state due to the influ-
the labelling of proteins.
ence of the heavy metal.[27,36,60] In general, the absorbance
With the organometallic ECE-pincer–metal phosphonate and the emission maxima display a large Stokes shift, which
inhibitors[19–21] developed by our group, we have been ex- is different from common organic fluorescent dyes.[27,37,42]
ploiting the special properties of the heavy-metal atom These special features of organometallic luminescent com-
probe as, for example, coordination target for phosphanes plexes can potentially be used in novel spectroscopic tech-
and as phasing tool for the elucidation of protein crystal niques [e.g., luminescence resonance energy transfer
structures.[21,33]
(LRET) and anisotropy studies on dynamic protein interac-
Interestingly, many different coordination complexes tions[37,52] or in time-resolved spectroscopy].[27] Addition-
with exciting luminescent properties are known from the ally, a rich and diverse chemistry is known for similar NCN
literature,[34–36] but only very few of them have been used transition-metal complexes, including chemical sensing,
in the labelling of biomolecules,[37–54] which is mainly supramolecular coordination chemistry and catalysis.[58]
caused by the instability (e.g., water or redox sensitivity) of
Here, we report on the synthesis of the novel luminescent
these complexes under biological conditions. Inspired by NCN-pincer–platinum probe 6 and its photophysical prop-
the high quantum yields of various organoplatinum com- erties in both organic and biological media. Furthermore,
plexes, as demonstrated by the Williams[25–27,55] and other we illustrate how this probe can be used for the efficient,
groups[56] and by our own results on the design and applica- diagnostic labelling of several proteins.
tions of novel organometallic phosphonate inhibitors, we
embarked on the modification of one of these highly lumi-
nescent NCN–platinum complexes[57] (complex 1,
Scheme 1) to develop a novel luminescent organometallic
Results and Discussion
NCN–platinum phosphonate inhibitor (6, Figure 1,
Scheme 1). Organometallic pincer complexes possess a co-
Synthesis of the Luminescent Pincer Platinum Phosphonate
Complex
valent metal–carbon bond, which is completed by two or-
tho-chelated metal–heteroatom interactions.[19–21,27,28,57–59]
The synthesis of the luminescent NCN–platinum inhibi-
This combination of metal–ligand interactions leads to a tor 6 was pursued by means of two different routes (a) and
high stability in aqueous and biological media, which in (b), both by means of a CuI-catalyzed click reaction as a
turn led us to expect that these complexes would be stable crucial step to introduce the reactive PNP phosphonate
luminescence labels for protein profiling.
group (PNP = p-nitrophenolate anion) using a propyl azido
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Site-Selective Ser-Hydrolase Labelling
Scheme 1. Explored routes (a) and (b) towards the synthesis of the luminescent pincer platinum phosphonate inhibitor 6; the different
tested click catalysts are shown under (c).
phosphonate reagent (4) similar to the ethyl azido phos- rather surprising, since other NCN–platinum-type com-
phonate reagent[22] developed earlier by us (Scheme 1). The plexes that contained acetylene moieties were successfully
CuI-catalyzed click reaction[61,62] between an azide and an applied under standard click reaction conditions in our lab-
alkyne has been widely studied and is known to be very oratory before.[66] Therefore it was decided to attempt an
selective, tolerant to functional groups and can be per- alternative synthesis route to 6.
formed under very mild conditions, even with unactivated
In the alternative route (b), the click reaction between
alkynes.[63,64] Due to the anticipated sensitive nature of the phosphonate 4 and propargylamine was performed in the
PNP-phosphonate group (PNP is a very good leaving first step, with aminoarenethiolate catalyst 7 giving the
group), it was initially decided to perform the click reaction highest activity when compared to the commonly used click
as the last step in the synthesis of 6 (Scheme 1, a).
catalysts 8 or 9.[61,63] The subsequent amide coupling reac-
Route (a) started with the hydrolysis of ester 1[25] to the tion of 5 with 2 in the presence of benzotriazole-1-yloxytris-
corresponding acid 2, which was isolated as a bright yellow (dimethylamino)phosphonium hexafluorophosphate (BOP)
powder. Subsequently, an amide coupling was performed gave rise to the formation of target molecule 6, which was
with propargylamine to obtain complex 3.
isolated in 77% overall yield starting from 4. Complex 6
Contrary to the general ease with which click reactions was characterized by 1H, 31P, 13C NMR and IR spec-
are performed,[61,62] initial attempts to realize the subse- troscopy, high-resolution mass spectrometry and by photo-
quent CuI-catalyzed click reaction under standard reaction physical methods (vide infra). Notably, both the organome-
conditions (acetylene/azide = 1:1, 10–20 mol-% 9 in MeCN tallic complex 2 and phosphonate 5 did not show any signs
or DMF/THF = 1:1) did not work. Therefore, it was de- of decomposition under the applied reaction conditions.
cided to screen for optimum reaction conditions by varying The resulting inhibitor 6 possesses only limited solubility in
the solvent, CuI catalyst (7,[65] 8 and 9[63]), catalyst loading common organic solvents; it was only found to be suffi-
(10–23 mol-%), concentration, reaction temperature (room ciently soluble in DMSO, DMF and in a DMF/buffer mix-
temp. to 40 °C) and time (12 h to 5 d). Based on 31P and ture for the inhibition studies, and therefore these solvents
1H NMR spectroscopic analysis, none of these screens gave were used in the subsequent enzyme inhibition and spectro-
rise to any formation of the desired product. This was scopic studies.
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R. J. M. Klein Gebbink, G. van Koten et al.
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Spectroscopic Characterization of the Various NCN Pincer
Platinum Complexes
Table 1. Photophysical data of the pincer platinum complexes 1–3
and inhibitor 6.
Solvent[a] Absorbance ε [–1cm–1] Luminescence
Quantum
yield[b,d]
The various NCN–platinum complexes (1–3, Scheme 1)
and inhibitor 6 were characterized by UV/Vis absorption
(Figure 2 and Figures S1–S3 in the Supporting Infor-
mation) and emission spectroscopy (Figure 3) and the data
are collected in Table 1. Due to both the limited solubility
of each of these complexes in common organic solvents and
their complete insolubility in water or buffer solutions, sol-
vents commonly used for spectroscopic analyses (e.g.,
CH2Cl2, hexane or THF) could not be used. As a result,
the solvent used by the Williams group (dichlorometh-
ane)[57] was not suitable for the spectroscopic studies of 1–
3 and 6. DMF appeared to be the only solvent in which
all complexes dissolved readily. Since DMF is a strongly
coordinating solvent, it is not the ideal solvent for per-
forming the spectroscopic studies; the use of coordinating
solvents renders the studied compounds less emissive (i.e.,
lower quantum yields are observed). However, it is the only
solvent that allows for the comparison of all complexes 1–
3, 6. Note that in cases in which a complex is soluble in
another solvent besides DMF, the spectroscopic data in this
solvent are reported as well (Table 1).
λmax [nm][b,c]
λmax [nm][b]
1
2
DMF
380 (399)
376 (401)
380 (400)
380 (401)
377 (402)
380 (400)
380 (400)
377 (396)
380 (402)
374 (398)
14940
10980
9840
9270
3120
8770
12320
3650
8220
9470
481[e]
480[e]
482[f]
481[f]
480[e]
485[f]
493[e]
487[e]
490[e]
482[e]
0.08
0.31
MeCN
CH2Cl2
DMF
0.38 (0.58)[25][g]
0.21
MeCN
0.08
0.17
0.05
0.04
0.15
0.04
3[h] DMF
6
DMF
MeCN
CH2Cl2
DMF[i]
[a] Sample dissolved in the indicated solvent; the solubility of all
complexes in H2O, 125 m Tris (pH 8.0), Tris (50 m), 0.1%
(m/m) Triton (pH 8.0) or 150 m NH4OAc buffer was too low
to allow for appropriate measurements. [b] Measured in deaerated
solutions. [c] The shoulders around 400 nm are listed in brackets.
[d] Uncertainty Ϯ20%. [e] Value was obtained from measurements
using [Ru(bpy)3]Cl2 in H2O (φ = 0.028) as internal standard.
[f] Value was obtained from measurements using [Ru(bpy)3](PF6)2
in MeCN (φ = 0.062) as internal standard. [g] The quantum yield
based on spectroscopic analysis with our equipment was lower than
the one reported previously.[25] The observed difference, which is
within the uncertainty region, could be due to the different degas-
sing methods or internal standards used. [h] The solubility of com-
plex 3 in MeCN and CH2Cl2 was too low to allow for measure-
ments. [i] A 150 m NH4OAc buffer/DMF mix (1:1 v/v) was used.
The UV/Vis absorption spectra of 1–3 and 6 in DMF
display very similar features with a strong absorbance re-
gion between 350 and 440 nm (Figure 2). All complexes
have an absorbance maximum between 374 and 380 nm,
which seems to be almost independent of the nature of the
solvent used (Table 1, vide infra). The strong features in the
1
absorbance region from 350 to 440 nm are assigned to π–
π* transitions of the ligands and to charge-transfer transi-
tions.[57]
All complexes are luminescent in solution and the emis-
sion spectra display very similar features with maxima be-
tween 480 and 493 nm (Figure 3). The quantum yields in
different solvents vary from 0.04 to 0.38, with the highest
quantum yield for the parent ester complex 1 in dichloro-
methane and lower quantum yields for inhibitor 6 (Table 1).
The emission maxima are slightly redshifted with a small
Stokes shift increasing in the order 1 = 2 Ͻ 3 Ͻ 6. The
emission energy is also redshifted when changing the para
substituent of the NCN–platinum complex from –C(O)-
OMe (complex 1) to –C(O)N(H)–R (3 and inhibitor 6),
which is due to the electronic nature of the different substit-
uents.[25,27,67,68] The quantum yield for 6 in DMF is slightly
decreased in comparison to 1–3.
Figure 2. Normalized absorption spectra of NCN–platinum com-
plexes 1–3 and inhibitor 6 in DMF.
Lipase Labelling Studies with Inhibitor 6
The pincer platinum phosphonate inhibitor 6 was applied
as a site-selective inhibitor in labelling studies of different
serine hydrolases. Due to the poor solubility of 6, previous
protocols to study artificial enzymes[19–23] were adapted for
Figure 3. Normalized emission spectra of NCN–platinum com-
plexes 1–3 and inhibitor 6 in DMF.
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Site-Selective Ser-Hydrolase Labelling
protein-labelling studies with luminescent probe 6. As a
model protein for the labelling studies, cutinase from
Fusarium solani pisi (21 kDa) was chosen. Cutinase is a li-
pase with high esterase activity that is also known to be
very reactive towards phosphonate inhibitors.[19,20,22] There-
fore, a solution of phosphonate 6 (6 equiv., 10 m) in DMF
was added to a solution of cutinase in buffer (200 µ, pH
8.0). After incubation overnight, the excess amount of in-
hibitor was removed by dialysis with buffer (see the Experi-
mental Section for details) and an activity test of the re-
sulting solution was performed. This test showed no resid-
ual ester-hydrolysis activity for cutinase, thereby indicating
complete inhibition of cutinase by 6 as a result of the for-
mation of cut-6.[19–21] Illumination of the inhibited cutinase
batches at 257 nm showed that the enzyme solutions were
luminescent after inhibition and dialysis (Figure 4, negative
photograph), thereby indicating successful protein labelling
(cut-6) and a high stability of the organometallic label 6
under the described conditions. After exposure of the cut-
6 solution to UV light over 5 min, the solution was still
luminescent. Storage of the cut-6 protein solution in the
refrigerator over 6 weeks also did not affect its emission
properties.
Figure 5. (A) Luminescence (negative photograph) and (B) Coom-
assie stained gel of cutinase (Cut), Candida antarctica lipase B
(CALB) and Bacillus subtilis lipase A (BSLA) without (–) and after
(+) exposure to 6 overnight. As the samples were not dialyzed prior
to gel electrophoresis, the excess amount of unreacted 6 at the lower
end of the gels (below the 11 kDa line) is visible.
The luminescent gel photograph shows that luminescent
labelling of cutinase, CALB and BSLA did indeed occur
(Figure 5, A). The Coomassie stained photograph of the
same gel shows that protein was also present in the samples
without 6 (Figure 5, B). Since SDS-PAGE is run under de-
natured protein conditions, these experiments clearly prove
the covalent labelling of the different proteins by 6. The
different protein-6 molecules were still luminescent after gel-
electrophoresis conditions, thereby showing again the sta-
bility of inhibitor 6.
Spectroscopy with cut-6 at Different pH Values and under
Denatured Conditions
Different, commonly used organic dyes like, for example,
fluorescein, are not very photostable and their luminescence
emission properties often depend on the polarity and pH
value of their environment.[13,14] Furthermore, the active
site His188 residue in cutinase [Figure 1, pKa(His) ≈ 6.5]
could be (de)protonated, thereby potentially exerting an in-
fluence on the spectroscopic properties of the luminescent
Figure 4. Negative photograph of cutinase solutions (200 µ) un-
der UV light (excitation wavelength 257 nm). Left: after incubation
with inhibitor 6 and subsequent dialysis (cut-6). Right: cutinase
solution without inhibitor.
Encouraged by these results, it was decided to investigate metal dye in cut-6. To investigate the chemical robustness
the labelling properties of 6 towards other proteins[22,23] and and the influence of the environment polarity and of the
to assay the formation and stability of the covalent protein- pH value on the spectroscopic properties of protein label 6,
6 adducts by performing gel electrophoresis. For this study, it was decided to analyze a cut-6 solution at different pH
the serine hydrolases Bacillus subtilis lipase A (BSLA)[69] values in Tris/Triton buffer and under denatured conditions.
(19 kDa), commercially available Candida antarctica lipase The emission spectrum of cut-6 at pH 5, 7 and 9 in Tris/
B (CALB) (33 kDa) and cutinase as reference protein were Triton buffer is shown in Figure 6. For all measurements
chosen.
the quantum yield remained constant within the experimen-
The solutions of the respective enzymes in buffer (pH tal error (0.20–0.12, uncertainty Ϯ20%).
8.0) were incubated with a solution of luminescent inhibitor
The spectroscopic properties of cut-6 at pH 5, 7 and 9
6 (in DMF) overnight. Also, a control experiment without are very similar with nearly identical absorbance (376–
6 was performed simultaneously. These solutions were used 378 nm) and emission (470–471 nm) maxima (Figure 6,
(i.e., without any further treatment) in gel electrophoresis Table 2). The absorbance and emission maxima of cut-6 in
using a sodium dodecyl sulfate polyacrylamide gel electro- Tris/Triton buffer are blueshifted in comparison to inhibitor
phoresis (SDS-PAGE) gel. The luminescence and Coomas- 6 in DMF or in a DMF/buffer mixture (Table 1, ∆λAbs
=
sie stained negative photograph of the gel are shown in Fig- 2–4 nm, ∆λEm = 11–12 nm), thereby indicating the differ-
ure 5.
ence of the hydrophobic and sterically constraint environ-
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R. J. M. Klein Gebbink, G. van Koten et al.
FULL PAPER
are demonstrated. It turns out that inhibitor 6 is a very
stable, luminescent organometallic complex, which can be
selectively used for the labelling of various lipases. Com-
plexes 1–3 and 6 display the expected photophysical proper-
ties, with the emission energy and quantum yield decreasing
after introduction of the amide functionality (for 3 and 6),
but maintaining a clearly luminescent complex. The lumi-
nescent properties of 6 can potentially be used in novel
spectroscopic applications [e.g., luminescence resonance en-
ergy transfer (LRET)][52] with an organic dye and subse-
quent phosphorescence emission by this dye, thereby ex-
panding the spectroscopic toolbox for studying biomolec-
ules.
Figure 6. Normalized luminescence spectra of cut-6 at pH 5, 7 and
9 in Tris/Triton buffer; for comparison, the spectrum of inhibitor 6
in DMF/buffer (1:1 v/v, buffer: Tris/Triton, pH 8.0) has been in-
cluded.
Previous studies by our group[22] have shown that phos-
phonate inhibitors are selective toward serine hydrolases
and do not react with other proteins that lack the reactive
active-site residue. Furthermore, we illustrated that such in-
hibitors can even be used to selectively label proteins in liv-
ing cells and as such have been used for protein isolation
and identification. This opens up the possibility to use 6 as
a luminescent lipase-specific label in cell lysates and for fu-
ture proteomic applications. Currently, we are investigating
how the properties of organometallic pincer complexes can
be used in this area (e.g., by exploiting the unique coordina-
tion properties of the platinum metal centre).[33]
In conclusion, this study shows that organometallic
pincer–platinum complexes[73] can be used for diagnostic
targeting of proteins, which in combination with a serine
hydrolase reactive phosphonate inhibitor opens up new ac-
tivity-based protein profiling possibilities. The proof-of-
principle methodology demonstrated here is by no means
limited to serine hydrolases; the chemical versatility of
pincer complexes also allows attachment to other biochemi-
cal probes like, for example, carbohydrates or oligopep-
tides.[27,28,58,74,75]
ment of the protein-active site[21,70–72] compared to the
highly polar solvents. The higher quantum yield of cut-6 at
pH 5, 7 and 9 than for 6 in DMF or DMF/buffer (Table 1)
was also attributed to the hydrophobic environment of the
cutinase active site[21,70–72] into which the metal moiety is
embedded in cut-6. The emission spectra showed an ad-
ditional broad maximum around 630 nm for cut-6 at pH 5,
7 and 9, but not for 6 in DMF/buffer or 1 in DMF (Fig-
ure S5 in the Supporting Information), which was attrib-
uted to excimer formation of cut-6 in aqueous solution.
This excimer formation can be facilitated by the hydro-
phobic packing of the cutinase active sites in aqueous me-
dia, as it is a common feature observed for lipases.[21,70–72]
The aggregate formation of cut-6 could also be the reason
for the observed broadness of the spectra in Figure 6.
Table 2. The absorbance λmax, luminescence λmax and quantum
yield values for cut-6 at pH 5, 7 and 9 in Tris/Triton buffer.
pH[a]
Absorbance Luminescence Quantum
λmax [nm]
λmax [nm]
yield[b]
cut-6
cut-6
cut-6
6
5
7
9
376
378
377
374
471
470
471
482
0.20
0.13
0.12
0.04
Experimental Section
8 (DMF/buffer)
General Comments: All reagents were used as supplied from Acros
or Sigma–Aldrich, unless stated otherwise. MeCN and CH2Cl2
were distilled from CaH2. DIEA = N,N-diisopropylethylamine
(Hünig’s base). All synthetic procedures were performed under an
inert atmosphere using Schlenk techniques and distilled solvents,
unless stated otherwise. The enzyme inhibition studies and dialysis
experiments were performed in air. Water for the preparation of
the buffer solutions was filtered with the Milli-Q filtration system
(Millipore, Quantum Ultrapure) prior to use. The dialysis cassettes
(Slide-A-Lyzer, 10,000 MWCO, 0.1–0.5 mL or 0.5–3 mL) for the
purification of enzyme NCN–platinum hybrids were purchased
from Pierce. Cutinase mutant N172K was provided by Unilever.
BSLA was provided by Prof. Wim J. Quax and co-workers.[69]
CALB (batch PPW 4534) was purchased from Novo Nordisk A/S,
Copenhagen, Denmark. Electrospray ionization mass spectra and
high-resolution electrospray ionization mass spectra were recorded
with a Micromass LCT spectrometer. All samples were introduced
with a nanoflow electrospray source (Protana, Odense, Denmark)
and all spectra were calibrated with a CsI solution (50 mgmL–1 in
MQ/H2O). Matrix-assisted laser desorption ionization mass spec-
[a] Measurements were performed in 0.1% (m/m) Triton, 50 m
Tris buffer at the indicated pH value. [b] Uncertainty Ϯ20%.
The study of cut-6 at different pH values showed that the
dye is remarkably stable in the pH range 5–9, thus making
it widely applicable.[21,70–72] The protonated (pH Ͻ 6.5) or
deprotonated (pH Ͼ 6.5) state of the 188His residue in cu-
tinase did not significantly affect the spectroscopic proper-
ties of the dye. This spectroscopic behaviour differs from
commonly used luminescent organic dyes like fluorescein
and rhodamine,[11,42] being pH dependent, and from some
other biological transition metal labels like redox-active
probes (e.g., ferrocene derivatives),[37] which have been re-
ported to be very sensitive to changes in pH.
Conclusion
The design, syntheses and spectroscopic analyses of lu-
minescent inhibitor 6 and its use in protein labelling studies trometry experiments were performed with a Voyager-DE Pro (Ap-
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Site-Selective Ser-Hydrolase Labelling
plied Biosystems) instrument in the reflector mode with trihydroxy
anthracene in MeCN as matrix. Photographs of SDS-PAGE gels
were taken with a BioRad GelDoc 2000 workstation (exciation
wavelength 257 nm). Elemental microanalyses were performed by
Dornis and Kolbe, Mikroanalytisches Laboratorium (Mülheim a.d.
Ruhr, Germany). NMR spectroscopic measurements were per-
formed with a Varian Inova 300 MHz or Varian Oxford 400 MHz
spectrometer at 298 K. The syntheses of 1[25] and 7[65] were carried
out as reported.
CH CH ) ppm. IR (liquid): ν = 2985.6, 2941.4, 2096.2, 1445.1,
˜
2 3
1394.9, 1368.9, 1349.3, 1256.2, 1018.4, 967.2, 848.5, 749.2,
711.2 cm–1. Due to the hydrolysis sensitivity of (3-azidopropyl)-
phosphonochloridate, the crude product was used directly in the
next synthesis step.
Ethyl 4-Nitrophenyl (3-Azidopropyl)phosphonate (4): A solution of
p-nitrophenol (0.7152 g, 5.1416 mmol, 1 equiv.) and distilled trieth-
ylamine (2.6014 g, 3.57 mL, 0.0257 mol, 5 equiv.) in dry benzene
(50 mL) was added dropwise to a solution of (3-azidopropyl)phos-
phonochloridate (1.0879 g, 5.1416 mmol, 1 equiv.) in dry benzene
(20 mL), after which the mixture was stirred at room temp. for 2 h.
All volatiles were removed in vacuo, after which the residue was
dissolved in Et2O (150 mL) and washed with 1  K2CO3
(2ϫ100 mL), brine (2ϫ100 mL) and H2O (3ϫ100 mL). The or-
ganic layer was dried with Na2SO4 and after filtration and removal
of all volatiles in vacuo a slightly orange oil was obtained (1.5998 g,
5.0911 mmol, 99%). The 1H NMR spectroscopic signals were as-
signed with the aid of COSY spectra. 1H NMR (CDCl3, 298 K,
Electronic Spectroscopic Measurements: UV/Vis spectra were re-
corded with a Varian Cary 50 Scan UV/Vis spectrometer. Lumines-
cence spectra were obtained on a SPEX fluorolog spectrometer
with [Ru(bpy)3](PF6)2 in degassed MeCN as standard (φr = 0.062).
Emission quantum yields were measured by the method of Crosby
and Demas[76] in degassed solvents and calculated by φS
=
φr(Br/Bs)(ns/nr)2(Ds/Dr), in which n is the refractive index of the sol-
vents and D is the integrated intensity. The quantity B is calculated
by B = 1–10–AL, in which A is the absorbance and L is the optical
path length.
3
400 MHz): δ = 1.31 (t, JH,H = 7.0 Hz, 3 H, P-O-CH2CH3), 1.90–
2.10 (m,
4
H, PCH2CH2CH2N3, PCH2CH2CH2N3 or
3
Diethyl (3-Azidopropyl)phosphonate: Azide exchange resin (44.88 g,
surface loading 3.8 mmolg–1, 0.17 mol, 10 equiv.) was added to a
solution of diethyl (3-bromopropyl)phosphonate (4.42 g,
0.017 mol, 1 equiv.) in DMF (250 mL), after which the suspension
was stirred at 40 °C for 3 d. The beads were filtered off and washed
with Et2O (2ϫ200 mL), and the solution was filtered through Ce-
lite (2ϫ), after which all volatiles were removed in vacuo. The crude
product was distilled with the second fraction containing the prod-
uct (5.7 mbar, 105 °C, 2.79 g, 0.013 mol, 74%). The 1H NMR spec-
PCH2CH2CH2N3), 3.42 (t, JH,H = 6.4 Hz, 2 H, PCH2CH2CH2N3
or PCH2CH2CH2N3), 4.10–4.31 (m, 2 H, P-O-CH2CH3), 7.38 (d,
3JH,H = 9.6 Hz, 2 H), 8.22 (d, 3JH,H = 9.6 Hz, 2 H) ppm. 31P NMR
(CDCl3, 298 K, 162 MHz): δ = 29.78 ppm. 13C NMR (CDCl3,
298 K, 101 MHz): δ = 16.53 (d, JC,P = 5.8 Hz, P-O-CH2CH3),
22.42 (d, JC,P = 5.0 Hz, PCH2CH2CH2N3), 23.40 (d, JC,P
3
3
1
=
2
143.4 Hz, PCH2), 51.38 (d, JC,P = 17.4 Hz, PCH2CH2), 63.42 (d,
2JC,P = 7.1 Hz, P-O-CH2CH3), 121.2 (d, JC,P = 4.6 Hz, ArC2,
3
ArC6), 125.9 (ArC3, ArC5), 144.8 (ArC4), 155.7 (d, 2JC,P = 8.2 Hz,
P-O-ArC1) ppm. MS (ES+, MeCN) for C11H15N4O5P (314.0780)
[M]+: m/z calcd. for [M + H]+ 315.0853; found 315.0858; calcd. for
[M + Na]+ 337.0672; found 337.0678. C11H15N4O5P: calcd. C
42.04, H 4.81, N 17.83, P 9.86; found C 41.96, H 4.76, N 17.78, P
1
troscopic signals were assigned with the aid of COSY spectra. H
NMR (CDCl3, 298 K, 400 MHz): δ = 1.30 (t, 3JH,H = 4.4 Hz, 6 H,
3
2ϫP-O-CH2CH3), 1.73–1.89 (m, 4 H, 2ϫCH2), 3.35 (t, JH,H
=
6.6 Hz, 2 H, CH2), 4.02–4.12 (m, 4 H, 2ϫCH2CH3) ppm. 31P
NMR (CDCl3, 298 K, 162 MHz): δ = 31.84 ppm. 13C NMR
9.94. IR (liquid): ν = 3479.4, 3114.5, 3081.4, 2985.0, 2939.3, 2873.2,
˜
3
(CDCl3, 298 K, 101 MHz): δ = 16.60 (d, JC,P = 5.8 Hz, 2ϫP-O-
2455.0, 2101.9, 1613.8, 1591.7, 1520.1, 1488.9, 1446.0, 1347.2,
1226.2, 1162.6, 1111.2, 1031.4, 914.8, 861.6, 752.8, 689.1, 636.9,
608.3 cm–1.
3
CH2CH3), 22.58 (d, JC,P = 4.5 Hz, PCH2CH2CH2N3) 23.02 (d,
1JC,P = 143.0 Hz, PCH2), 51.61 (d, JC,P = 16.6 Hz, PCH2CH2),
2
2
61.80 (d, JC,P = 6.2 Hz, 2ϫP-O-CH2CH3) ppm. MS (ES+,
Ethyl 4-Nitrophenyl {3-[4-(Aminomethyl)-1H-1,2,3-triazol-1-yl]-
propyl}phosphonate (5): Propargylamine (127.7 µL, 0.0020 mol,
0.1098 g, 1.4 equiv.), the copper amino arenethiolate catalyst
(0.0082 g, 0.0356 mmol, 0.025 equiv.) and more dichloromethane
(1 mL) were added to a solution of phosphonate 4 (0.4475 g,
0.0014 mol, 1.0 equiv.) in degassed dichloromethane (1 mL). The
solution was stirred overnight, after which all volatiles were evapo-
rated in vacuo and the residue was dissolved in CH2Cl2 (50 mL).
The organic layer was washed with 1  K2CO3 (2ϫ50 mL), brine
(2ϫ50 mL) and water (2ϫ50 mL). After drying with Na2SO4 and
filtration through Celite, all volatiles were evaporated in vacuo. The
oil was dissolved in a minimum of dichloromethane and precipi-
tated with hexane. After drying in vacuo, a yellow oil was obtained
MeCN) for C7H16N3O3P (221.0929) [M]+: m/z calcd. for [M +
H]+ 222.1002; found 222.1007; calcd. for [M + Na]+ 244.0821;
found 244.0827. C7H16N3O3P: calcd. C 38.01, H 7.29, N 19.00, P
14.00; found C 37.91, H 7.21, N 18.85, P 13.91. IR (liquid): ν =
˜
2094.7, 1445.2, 1392.3, 1368.5, 1349.2, 1237.7, 1019.0, 952.3, 781.5,
704.0 cm–1.
Ethyl (3-Azidopropyl)phosphonochloridate: Freshly distilled oxalyl
chloride (9.9 mL, 14.90 g, 0.12 mol, 10 equiv.) was added to a solu-
tion of diethyl (3-azidopropyl)phosphonate (2.60 g, 0.012 mol,
1 equiv.) in Et2O (100 mL), which had been cooled to –10 °C, after
which the solution was stirred at room temp. The progress of the
reaction was monitored by 31P NMR spectroscopy. After 3 d, fresh
oxalyl chloride (9.9 mL) was added. After 11 additional days all
volatiles were evaporated in vacuo, thus yielding a slightly yellowish
oil (2.25 g, 0.011 mol, 91%). The 1H NMR spectroscopic signals
were assigned with the aid of COSY spectra. 1H NMR (CDCl3,
298 K, 400 MHz): δ = 1.38 (t, 3JH,H = 4.6 Hz, 3 H, P-O-CH2CH3),
1
(0.4598 g, 1.2450 mmol, 87%). The H NMR spectroscopic signals
were assigned with the aid of COSY spectra. 1H NMR (CDCl3,
298 K, 400 MHz): δ = 1.28 (t, 3JH,H = 7.0 Hz, 3 H, P-O-CH2CH3),
1.90–1.98 (m,
2 H, PCH2CH2CH2N3), 2.24–2.35 (m, 2 H,
2
PCH2CH2CH2N3), 3.35 (d, JH,H = 511.1 Hz, 2 H, NH2), 4.02–
3
1.90–2.02 (m,
2
H, PCH2CH2CH2N3), 2.17–2.25 (m,
2
H, 4.27 (m, 2 H, P-O-CH2CH3), 4.46 (t, JH,H = 6.8 Hz, 3 H,
3
3
PCH2CH2CH2N3 or PCH2CH2CH2N3), 3.42 (t, JH,H = 6.4 Hz, PCH2CH2CH2N3), 7.34 (d, JH,H = 9.2 Hz, 2 H, ArH), 7.54 (s, 1
2 H, PCH2CH2CH2N3 or PCH2CH2CH2N3), 4.15–4.35 (m, 2 H, H, triazoleH), 8.20 (d, JH,H = 9.2 Hz, 2 H, ArH) ppm. 31P NMR
3
CH2CH3) ppm. 31P NMR (CDCl3, 298 K, 162 MHz):
δ
=
(CDCl3, 298 K, 162 MHz): δ = 29.00 ppm. 13C NMR (CDCl3,
43.82 ppm. 13C NMR (CDCl3, 298 K, 101 MHz): δ = 16.12 (d,
298 K, 101 MHz): δ = 16.56 (d, JC,P = 6.2 Hz, P-O-CH2CH3),
3
3JC,P
=
7.0 Hz, P-O-CH2CH3), 22.59 (d, JC,P
=
5.0 Hz,
23.26 (d, JC,P = 143.8 Hz, PCH2), 23.66 (d, JC,P = 4.6 Hz,
3
1
3
PCH2CH2CH2N3), 31.00 (d, JC,P = 126.4 Hz, PCH2), 51.08 (d, PCH2CH2CH2N3), 49.98 (d, 2JC,P = 17.0 Hz, PCH2CH2), 63.64 (d,
1
2JC,P = 19.1 Hz, PCH2CH2), 63.62 (d, JC,P = 8.3 Hz, P-O-
2JC,P = 7.0 Hz, P-O-CH2CH3), 115.9 (triazole C=CH), 121.7 (CH2-
2
Eur. J. Inorg. Chem. 2010, 1929–1938
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
1935

R. J. M. Klein Gebbink, G. van Koten et al.
FULL PAPER
3
NH2), 126.4 (triazole C=CH), 121.2 (d, JC,P = 4.5 Hz, ArC2, washed with 1  K2CO3 (2ϫ50 mL), brine (2ϫ50 mL) and water
ArC6), 126.0 (ArC3, ArC5), 144.9 (ArC4), 155.5 (d, 2JC,P = 8.6 Hz,
(2ϫ50 mL). After drying with Na2SO4 and filtration through Ce-
P-O-ArC1) ppm. MS (ES+, MeCN) for C14H20N5O5P (369.3129) lite, all volatiles were evaporated in vacuo to obtain a yellow solid
[M]+: m/z calcd. for [M + H]+ 370.1275; found 370.1280; calcd. for
[M + Na]+ 392.1094; found 392.1100. HRMS (ES+, MeCN) for
C14H20N5O5P (369.3129) [M]+: m/z calcd. for [M + H]+ 370.1275;
(0.1239 g, 0.1446 mmol, 88%). The 1H NMR spectroscopic signals
were assigned with the aid of COSY and long-range COSY spectra.
3
1H NMR ([D6]DMSO, 298 K, 400 MHz): δ = 1.20 (t, JH,H
=
found 370.1233. IR (solid): ν = 2937.8, 1612.4, 1590.2, 1519.6,
7.0 Hz, 3 H, P-O-CH2CH3), 1.98–2.10 (m, 4 H, PCH2CH2CH2N3),
˜
3
1490.0, 1345.4, 1218.6, 1161.5, 1105.8, 1029.4, 911.1, 853.2, 751.0, 4.09–4.15 (m, 2 H, P-O-CH2CH3), 4.44 (t, JH,H = 6.8 Hz, 3 H,
688.3 cm–1.
PCH2CH2CH2N3), 4.57 (d, 4JH,H = 5.6 Hz, 2 H, CH2NH), 7.42 (d,
3
3JH,H = 9.2 Hz, 2 H, PNPArH), 7.59 (t, JH,H = 5.9 Hz, 2 H, H5-
[4-(Methoxycarbonyl)-2,6-bis(pyridin-2-yl)phenyl]platinum(II) Chlo-
ride (1): This complex was prepared according to the literature pro-
3
py), 8.07 (s, 1 H, triazoleH), 8.12 (d, JH,H = 7.2 Hz, 2 H, H3-py),
3
8.24 (d, JH,H = 9.2 Hz, 2 H, PNPArH), 8.24–8.27 (m, 4 H, t, H4-
3
cedure.[25] 1H NMR (400 MHz, CDCl3): δ = 9.38 (dd, JH,H
=
3
3
py, H3, H6), 8.97 (t, JH,H = 7.0 Hz, 1 H, NH), 9.12 (d, JH,H
=
5.6 Hz, 4JH,H = 1.6 Hz, JPt,H = 35.19, 46.79 Hz, 2 H, H6-py), 8.14
2
5.9 Hz,
162 MHz):
2
H, H6-py) ppm. 31P NMR ([D6]DMSO, 298 K,
(s, JPt,H = 4.0 Hz, 2 H, H4 and H6), 8.00 (dt, JH,H = 7.80 Hz,
4
3
δ
=
30.19 ppm. 13C NMR ([D6]DMSO, 298 K,
4JH,H = 1.6 Hz, 2 H, H4-py) 7.81 (d, JH,H = 5.6, 7.8 Hz, 2 H, H3-
4
3
101 MHz): δ = 16.81 (d, JC,P = 5.0 Hz, P-O-CH2CH3), 22.89 (d,
py), 7.36 (ddd, JH,H = 5.6, 7.8 Hz, JH,H = 1.6 Hz, 2 H, H5-py),
4
4
1JC,P = 140.8 Hz, PCH2), 23.82 (d, 3JC,P = 4.6 Hz, PCH2CH2CH2),
3.94 (s, 3 H, CH3) ppm.
2
2
49.79 (d, JC,P = 19.0 Hz, PCH2CH2), 63.45 (d, JC,P = 7.2 Hz, P-
O-CH2CH3), 116.5 (triazole C=CH), 121.3 (CH2-NH2), 122.1
(ArC2, ArC6), 123.9, 124.4, 124.6, 125.3, 126.5 (triazole C=CH),
126.9 (ArC3, ArC5), 130.1, 141.0, 141.4, 144.8, 145.7, 152.1, 156.1
[4-Carboxy-2,6-bis(pyridin-2-yl)phenyl]platinum(II) Chloride (2):
KOH (1.08 g, 19.24 mmol, 100 equiv.) was dissolved in MeOH
(5 mL).
[4-(Methoxycarbonyl)-2,6-bis(pyridin-2-yl)phenyl]plati-
2
num(II) chloride (100 mg, 0.192 mmol, 1 equiv.) was suspended in
this solution. The suspension was stirred at 40 °C for 24 h. A 4 
HCl (9.62 mL, 38.48 mmol, 200 equiv.) solution was added to the
suspension. The yellow precipitate was washed with water
(3ϫ20 mL) and MeOH (2ϫ20 mL) and the solvents evaporated
(d, JC,P = 8.6 Hz, P-O-ArC1), 166.2, 166.5 ppm. MS (ES+,
MeCN) for C31H29N7O6PPt (856.1253) [M]+: m/z calcd. for [M –
Cl]+ 821.1559; found 821.1586; calcd. for [M + Na]+ 880.1105;
found 880.1238. HRMS (ES+, MeCN) for C31H29N7O6PPt
(856.1253) [M]+: m/z calcd. for [M – Cl]+ 821.1565; found 821.1575.
to dryness.
A yellow solid was obtained (78.3 mg, 80%,
IR (solid): ν = 3071.5, 1644.3, 1609.0, 1591.1, 1519.4, 1475.7,
˜
0.155 mmol) 1H NMR (400 MHz, CDCl3): δ = 13.00 (s, 1 H), 9.12
(d, 3JH,H = 5.8 Hz, 3JH,Pt = 34.0 Hz, 2 H, py-H6), 8.28 (m, 4 H, H2
and H4 and py-H3), 8.23 (dt, 3JH,H = 7.4 Hz, 4JH,H = 1.6 Hz, 2 H),
1344.3, 1219.0, 1159.1, 1110.7, 1027.3, 910.2, 860.0, 751.1,
687.7 cm–1.
Inhibition of Cutinase with Complex 6: A solution of complex 6
(30 µL, 10 m, 0.3 µmol, 3.0 equiv.) in DMF or MeCN was added
to a solution of cutinase (0.5 mL, 200 µ, 0.1 µmol, 1.0 equiv.) in
Tris (50 m) and 0.1% (m/m) Triton buffer (pH 8.0). After 2 h,
more inhibitor 6 (30 µL, 10 m, 0.3 µmol, 3.0 equiv.) was added.
After incubation overnight, the solution was dialyzed over 24 h
with NH4OAc buffer (150 m, 2ϫ400 mL) and an activity test was
performed.
3
7.60 (ddd, JH,H = 5.8, 7.4 Hz, 2 H, py-H5) ppm. 13C NMR
(75 MHz, DMSO): δ = 168.4 (C=O), 167.8 (C6-py), 166.3 (C4-py),
156.1 (C2), 152.0 (quat), 141.3 (quat), 141.3 (C4 and C6), 126.7 (C4-
py), 125.5 (quat), 121.52 (quat) ppm. MALDI-TOF MS: calcd. for
C20H14N3OPt: 470.0468; found 469.8722 [M – Cl]+. HRMS (ES+,
MeCN) for C17H11ClN2O2Pt (505.0157) [M]+: m/z calcd. for [M –
Cl + H2O]+ 488.0574; found 488.0559.
[4-(Prop-2-ynylcarbamoyl)-2,6-bis(pyridin-2-yl)phenyl]platinum(II)
Chloride (3): Compound 1 (73.3 mg, 0.145 mmol, 1 equiv.), propar-
gylamine (15.97 mg, 0.290 mmol, 2 equiv.) and DIEA (23.41 mg,
0.181 mmol, 1.25 equiv.) were dissolved in DMF (20 mL) and co-
oled for 10 min with ice to 0 °C. Subsequently, BOP (67.25 mg,
0.152 mmol, 1.05 equiv.) was added. After 24 h, all volatiles were
removed under reduced pressure. The resulting yellow powder was
washed with CHCl3 (3ϫ20 mL) and Et2O (2ϫ20 mL) and dried
under reduced pressure (60.0 mg, 76%, 0.12 mmol). 1H NMR
Activity Tests: A solution of p-nitrophenyl butyrate (7.0 µL, 50 m)
in MeCN was added to different batches of Tris (50 m) and 0.1%
(m/m) Triton buffer (1.493 mL, pH 8.0). Portions of inhibited and
uninhibited cutinase (0.5 µL, 200 µ, 0.1 nmol) were added to these
substrate solutions and the release of p-nitrophenolate was mea-
sured at 400 nm over 10 min.
Inhibitions for the Gel-Electrophoresis Experiments: A solution of 6
(20.0 m, 50.0 µL, 1.0 mmol) in DMF was added to solutions of
cutinase (1.0 mg/mL), CALA (0.8 mg/mL) and CALB (2.0 mg/mL)
in Tris (50 m) and 0.1% (m/m) Triton buffer (200 µL each). The
samples were incubated in the refridgerator overnight, after which
the samples were analyzed with SDS-PAGE. For this, the protein
samples (20 µL) were loaded onto 12.5% SDS-PA gels and sepa-
rated by gel electrophoresis.[77] Subsequently, the gel was analyzed
under a UV lamp (257 nm, GelDoc), after which the gel was
stained with Coomassie.
3
3
(400 MHz, CDCl3): δ = 9.13 (d, JH,H = 5.9 Hz, JH–Pt = 35.2 Hz,
3
3
2 H, H6-py), 8.88 (t, JH,H = 5.4 Hz, 1 H, NH), 8.26 (t, JH,H
=
=
8.0 Hz, 2 H, H4-py), 8.25 (s, 2 H, H3 and H6), 8.15, (d, JH,H
3
8.0 Hz, 2 H, H3-py), 7.60 (t, 3JH,H = 5.9 Hz, 2 H, H5-py), 4.12 (dd,
3JH,H = 5.4 Hz, JH,H = 2.4 Hz, 2 H, NH–CH2), 3.17 (t, JH,H
=
4
4
2.4 Hz, 1 H, CϵC–H) ppm. 13C NMR (75 MHz, DMSO): δ =
166.6, 152.2, 141.1, 129.8, 124.8, 121.0 ppm. MALDI-TOF MS:
calcd. for C20H14N3OPt: 507.0785; found 507.1562 [M – Cl]+. Ele-
mental analysis calcd. (%) for C18H13ClN2O2Pt: C 44.25, H 2.60,
N 7.74, Pt 35.93; found: C 44.12, H 2.48, N 7.79, Pt 36.10.
Supporting Information (see also the footnote on the first page of
this article): Additional absorption and emission spectra of com-
pounds 1, 2, 6 and cut-6 in different solvents.
Complex 6: Complex 2 (0.0831 g, 0.1643 mmol, 1.0 equiv.) and
more DMF (5 mL) was added to phosphonate 5 (0.0607 g,
0.1643 mmol, 1.0 equiv.) dissolved in dry DMF (1 mL). The solu-
tion was cooled to 0 °C, after which DIEA (33.9 µL, 0.2054 mmol,
1.25 equiv.) and BOP (0.0763 g, 0.1725 mmol, 1.05 equiv.) were
added. After being stirred overnight, all volatiles were evaporated
in vacuo. The solid residue was dissolved in CH2Cl2 (50 mL) and
Acknowledgments
Utrecht University (support to B. W.) and NRSC Catalysis (sup-
port to H. P. D.) are kindly acknowledged for financial support. Dr.
1936
www.eurjic.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2010, 1929–1938

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gift of CuI aminoarenethiolate catalyst. Dr. Sipke Wadman is
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