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Metallocarbonyl complexes of bromo- and dibromomaleimide synthesis and biochemical application.

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Full Paper
Received: 13 October 2011
Revised: 23 November 2011
Accepted: 10 December 2011
Published online in Wiley Online Library
( DOI 10.1002/aoc.1872
Metallocarbonyl complexes of bromo- and
dibromomaleimide: synthesis and
biochemical application
Bogna Rudolf,a* Michele Salmain,b,c Emilia Fornald,e
and Agnieszka Rybarczyk-Pirekf
Bromomaleimides react with cysteine residues to form thiomaleimides that can be further cleaved with TCEP (tris(2-carboxyethyl)
phosphine) to regenerate the cysteine derivatives. Herein we report the preparation of new organometallic Fe complexes
containing monobromo and dibromo maleimide ligands. Both of these complexes were characterised by X-ray diffraction.
Organometallic bromomaleimide derivatives were reacted with the thiol-containing biomolecules: cysteine ethyl ester
hydrochloride, glutathione and papain. These cysteine-containing molecules underwent a substitution reaction with
metallocarbonyl bromo- or dibromo maleimide complexes, followed by an addition reaction to the thio-maleimide double
bond if thiol was added in excess. The metallocarbonyl mono-bromomaleimide complex was shown to inhibit the peptidase
activity of the enzyme papain. The resulting papain–maleimide product could be cleaved by addition of TCEP to regenerate
the catalytically active enzyme. Copyright © 2012 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: metallocarbonyl complexes; papain inhibitors; biometalloorganic chemistry; bromomaleimides
ligand substitution reactions with N-, S- or Se-containing residues
of the enzyme target, as is the case for cysteine proteases
inhibitors.[9] Alternatively, some substitutionally inert metal
complexes were shown to act as enzyme inhibitors thanks to
their particular scaffold, the metal centre creating a unique
structural organization of the surrounding ligands in the 3D
space. In this line, highly potent and specific ruthenium-based
inhibitors have been identified for various protein kinases.[10]
The maleimide motif is widely used for the selective alkylation
of thiols in the pH 6.5–7.5 range and there are numerous
N-functionalized maleimide reagents applied for cysteine modification.[1] Among these, (Z5-C5H5)Fe(CO)2(Z1-N-maleimidato) 1
was applied as protein marker.[2,3] This metallocarbonyl complex
displays characteristic strong absorption bands nC=O, appearing
in the 1950–2060 cm 1 mid-IR spectral region, which is usually
free of any absorption of biomolecules or biological matrices. This
feature made it useful as an IR-detectable marker to follow
several biochemical processes as hormone–receptor or antigen–
antibody interactions. Metallocarbonyl compounds including
the maleimidato entity can be easily conjugated to bioactive
compounds since they react with thiol groups in the presence
of other functional groups in the biomolecules.[3–5]
More recently, we also reported that complex 1 and its
molybdenum and tungsten analogues acted as irreversible
inhibitors of the cysteine endoproteinase papain by irreversible
alkylation of the sulfhydryl group present at its active site.[6] More
generally, an increasing number of metal-based compounds
have been shown to display enzyme inhibition abilities, with
potential applications in medicine.[7,8] Some metal complexes
with labile ligands were shown to act as enzyme inhibitors by
Appl. Organometal. Chem. 2012, 26, 80–85
* Correspondence to: Bogna Rudolf, Department of Organic Chemistry,
University of Lodz, 91-403 Lodz, Poland. E-mail:
a Department of Organic Chemistry, University of Lodz, 91-403 Lodz, Poland
b Chimie ParisTech, Laboratoire Charles Friedel, 75005 Paris, France
c CNRS UMR 7223, 75005 Paris, France
d Chemistry Department, Faculty of Mathematics and Life Sciences, John Paul II
Catholic University of Lublin, 20-718 Lublin, Poland
e Laboratory of Separation and Spectroscopic Method Applications, Center for
Interdisciplinary Research, John Paul II Catholic University of Lublin, 20-718
Lublin, Poland
f Department of Crystallography and Crystal Chemistry University of Lodz,
91-403 Lodz, Poland
Copyright © 2012 John Wiley & Sons, Ltd.
New organometallic Fe complexes containing bromomaleimide ligands
Nevertheless, utilization of maleimides for protein labelling
have some limitations. The double bond of the maleimide entity
undergoes an irreversible reaction with sulfhydryl groups to
form stable thioethers, which prevents the possibility for the
controlled disassembly of the conjugate to release the unmodified protein. This feature may be important for in vitro and
in vivo applications.[11]
Recently, it was reported that bromomaleimides react rapidly
and selectively with cysteine residues to form thiomaleimides
that can be further cleaved with tris(2-carboxyethyl)phosphine
(TCEP) to regenerate the cysteine derivative (Scheme 1). J. R.
Baker and colleagues also showed that bromomaleimides offer
opportunities for up to three points of attachment (instead of
two offered by maleimide), increasing the number of chemical
and biological entities that can be attached to the system.[11,12]
Searching for a new method to incorporate metallocarbonyl
labels into biomolecules in a reversible manner, we synthesized
the mono- and dibromomaleimide metallocarbonyl complexes
2, 3. We then examined their reaction with the low-molecularweight thiols cysteine ethyl ester and glutathione. We found
that the bromomaleimide compounds 2 and 3 underwent a
substitution reaction of bromine atom(s) followed by the addition
of another thiolate anion to the maleimide double bond when the
thiol was added in excess. We also observed that modification
of thiols by bromomaleimide derivatives was reversible under
the specific conditions. The reaction of thiol with an equimolar
amount of bromomaleimide led to the thiomaleimide derivative,
which could be cleaved by TCEP, resulting in the starting thiol.
In this work, we also studied the ability of complex 2 to alkylate
site-specifically the single free cysteine residue of papain. Papain
is a vegetal cysteine endoproteinase that is closely related in
structure and mechanism of action to mammalian enzymes (cathepsins,
calpains, caspases) involved in important pathologies (degenerative diseases, cancers). As these enzymes are interesting drug
targets, this stimulated the design of specific inhibitors. Most of
known papain inhibitors are molecules carrying an electrophilic
group that react with the sulfhydryl group of Cys25 (which is a
part of the enzyme’s active site) to form covalent adducts.[13–15]
Papain was reacted with complex 2 and the protein adduct was
characterized by electrospray ionization–mass spectrometry (ESI-MS)
and IR spectroscopy. The formation of papain–metallocarbonyl
maleimide conjugate was observed. The obtained conjugate was
subsequently cleaved by TCEP and the peptidase activity of the
enzyme was restored.
Results and Discussion
Synthesis of Metallocarbonyl Bromomaleimide Complexes 2
and 3
The iron half-sandwich complex (Z5-cyclopentadienyl)Fe(CO)2
(Z1-N-maleimidato) 1 bearing the maleimide moiety was previously
obtained by photochemical reaction of (Z5-cyclopentadienyl)Fe
(CO)2I with maleimide.[2] Applying the same method, we synthesized the metallocarbonyl bromomaleimide derivatives 2 and 3
by reaction of (Z5-cyclopentadienyl)Fe(CO)2I with bromomaleimide and dibromomaleimide (Scheme 2) in the presence of
diisopropylamine. Both compounds 2 and 3 featured water
solubility useful for biochemical application. However, we observed a decreased water solubility when comparing complex 1
(12.0 mg ml 1) to its analogues with one bromine atom 2 (0.75
mg ml 1) and two bromine atoms 3 (0.14 mg ml 1).
The resulting metallocarbonyl derivatives 2 and 3 were
characterized by classical spectroscopic means. The 1H NMR
spectrum of 2 displayed a singlet at 6.81 ppm assigned to
the olefin proton, which was absent in the 1H NMR spectrum of
3. The metallocarbonyl complexes 2 and 3 displayed two
characteristic and intense absorption bands at 2043 and 1992
cm 1 on their IR spectra.
The molecular structure of 2 and 3 was determined by X-ray
crystallography. X-ray-grade crystals were obtained from layered
dichloromethane–heptane. Molecular diagrams are shown in
Figs 1 and 2. Crystal data, selected bond lengths and angles
are given in supporting information Tables S1 and S2. Both
compounds 2 and 3 crystallized in the centrosymmetric
monoclinic space groups: P21/n and P21/c, respectively. Molecules 2 and 3 differ from each other only in the substituent at
position 3 of the heteroatomic five-membered ring. In general,
their three-dimensional shape is almost the same. In both
compounds, the iron Fe1 atom is bonded to the cyclopentadienyl
ring (C6–C7–C8–C9–C10), two carbonyl ligands (C11–O11 and
C12–O12) and the nitrogen atom N1 of the maleimide moiety.
Respective bond lengths and angles are the same within
experimental error. The Cg1–Fe1 distance (Cg1 corresponds to
the centre of gravity of the cyclopentadienyl ring) was equal to
1.723(3) Å in 2 and 1.719(4) Å in 3, while the C6–Cg1–Fe1–N1
torsion angles are 174.9(4) and 168.0(6) for 2 and 3, respectively. In the case of an ideal Cs molecular symmetry, these angles
should be equal to 180 .
cysteine derivative
cysteine derivative
Scheme 1. Formation of thiobromomaleimide and conversion to cysteine derivative.
h / dipa
2 R=H
3 R = Br
Scheme 2. Synthesis of bromomaleimide complexes 2 and 3.
Appl. Organometal. Chem. 2012, 26, 80–85
Copyright © 2012 John Wiley & Sons, Ltd.
B. Rudolf et al.
bond (Scheme 3). Depending on the relative amount of the two
substrates, a different proportion of the two products was
obtained as determined by LC-MS (Table 1). With one equiv. of
CysOEt, compound 4 was the major product of the reaction,
whereas with 2 equiv. of CysOEt the amount of compound 5
was twice that of compound 4.
When the mixture of 4 and 5 was treated with TCEP, we found
that bioconjugate 4 almost disappeared (in 98%), whereas
compound 5 remained unchanged, as observed by LC-MS
(Scheme 4). We also detected cysteine ethyl ester in the reaction
mixture (Scheme 4).
Reaction of 3 with Glutathione
Figure 1. Molecular drawing of 2 with atom labelling scheme. Probability
level 30%.
We examined whether the dibromomaleimide complex 3 reacted
with the tripeptide glutathione. Complex 3 was allowed to
react with two equivalents of glutathione in phosphate buffer
(pH 6.2) and the reaction mixture was analysed by LC-MS after
1 and 24 h. Three products 6–8 with one, two and three molecules of glutathione attached to the metallocarbonyl complex
(Scheme 5) were detected in the reaction mixture. The major
product of reaction was shown to be compound 7, resulting from
the substitution of the two bromine substituents (Table 1).
Papain Inhibition Study
Figure 2. Molecular drawing of 3 with atom labelling scheme. Probability
level 30%.
There are small differences in C=O bond lengths (but still
within 3s criteria) as only the C2–O2 carbonyl group formed
intermolecular contacts. In both crystal structures, molecules
are linked by C&bond;Br . . . O=C halogen bonds, forming a
one-dimensional chain of molecules. Details of intermolecular
interactions are given in supporting information Table S3.
Reaction of 2 with Cysteine Ethyl Ester Hydrochloride
We presumed that the bromomaleimide metallocarbonyl complexes 2 and 3 would react with cysteine derivatives, giving thiomaleimide products. To test this hypothesis, we carried out two
reactions between the bromomaleimide complex 2 and one
and two molar equivalents of cysteine ethyl ester hydrochloride
in DMF (Scheme 3). After 2 h, the reaction mixtures were analysed
by liquid chromatography–mass spectrometry (LC-MS). For both
experiments, we observed the formation of complex 4, where
the Br atom was substituted by S of cysteine ethyl ester, together
with complex 5, where the substitution reaction was followed by
the addition of cysteine ethyl ester to the C&dbond;C double
We have already reported the reaction of papain with the
metallocarbonyl maleimide complex 1 and its molybdenum and
tungsten analogues.[6] We showed that these three complexes
were able to alkylate the thiolate group of the single free cysteine
residue of this enzyme, namely Cys25. As a consequence, all
three compounds behave as irreversible inhibitors of papain. It
was thus interesting to examine whether complexes 2 and 3
containing the bromomaleimide moiety were also able to inhibit
this enzyme. We performed the inhibition experiments with
compounds 2 and 3, but unfortunately the dibromo derivative
3 was too poorly soluble in water and precipitated in the
reactional conditions. Freshly purified papain was allowed to
react overnight with a eightfold molar excess of complex 2. The
protein adduct was purified by gel filtration to remove excess
reagent and the peptidase activity, measured on a chromogenic
substrate, was equal to 20% of the starting enzyme preparation.
After concentration of the solution, the sample was analysed by
Table 1. Relative amount of cysteine ethyl ester and glutathione
adducts determined by LC-MS
2 + 1 equiv. cysteine ethyl ester
2 + 2 equiv. cysteine ethyl ester
3 + 2 equiv. glutathione, 1 h
3 + 2 equiv. glutathione, 24 h
Scheme 3. Reaction of 2 with cysteine ethyl ester.
Copyright © 2012 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2012, 26, 80–85
New organometallic Fe complexes containing bromomaleimide ligands
Scheme 4. Cleavage of thiomaleimide 4 with TCEP.
Scheme 5. Reaction of 3 with glutathione.
IR spectroscopy (Fig. 3). The presence of two nCO bands at 2053
and 1993 cm 1 indicated that the metallocarbonyl probe was
covalently attached to papain. The protein conjugate was also
analysed by mass spectroscopy in ESI mode. Two species were
identified from the multiply charged ions present in the mass
spectrum (supporting information Fig. S1 and Table 2). The higher
mass species corresponded to the expected 1:1 protein conjugate
with the metallocarbonyl label. The lower mass species was
readily assigned to the papain–maleimide adduct that resulted
from the cleavage of the Fe&bond;N bond. Such a cleavage had
been previously observed for the papain–1 conjugate.[6]
Treatment of the Protein Conjugate by TCEP
An aqueous solution of papain–2 conjugate was treated with 1
mM TCEP and the peptidase activity of the solution was measured
over a period of 24 h on a chromogenic substrate (Fig. 4). The
enzymatic activity, which was initially null, gradually increased
until it reached 1.4 U mg 1 protein after 24 h, ~35% activity of
fully active papain. The mixture was dialysed in water and the
protein solution was again submitted to mass spectroscopy in
ESI mode (Fig. S2, Table 2). A new species corresponding to
native papain was detected in the solution together with
papain–2 conjugate and papain–maleimide (resulting again from
cleavage of the Fe&bond;N bond during mass analysis). The
presence of a fraction of papain–2 conjugate treated with TCEP
Table 2. ESI-MS analysis of papain–2 adduct before and after
reaction with TCEP
After treatment with
TCEP for 24 h
Appl. Organometal. Chem. 2012, 26, 80–85
Copyright © 2012 John Wiley & Sons, Ltd.
23 518
23 693
23 422
23 518
23 693
23 544 4
23 708 4
23 433 3
23 544 4
23 708 4
Figure 3. IR spectrum of papain–2 adduct in the nCO region.
Mass (Da)
B. Rudolf et al.
specific activity (u/mg protein)
prepared using a published procedure.[16] Papain was purchased
from Fluka (ref. 76220) and purified by affinity chromatography
according to a previously described procedure.[6] L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNa) was purchased from Bachem.
LC-MS Analysis
Figure 4. Enzymatic activity of papain after treatment with TCEP.
explains why full enzymatic activity was not totally recovered
after 24 h of reaction.
General Remarks
H and 13C NMR spectra were recorded in CDCl3 on Gemini 200BB
(Varian) and Avance 600 MHz (Bruker) spectrometers and
referenced to internal tetramethylsilane. The IR spectra were
recorded in CHCl3 on an FT-IR NEXUS spectrometer (Thermo
Nicolet). Elemental analysis was performed at the Analytical
Services of the Centre of Molecular and Macromolecular Studies
of the Polish Academy of the Sciences (Lodz). All solvents were
purified according to standard procedures. Chromatographic
separations were performed on silica gel Merck 60 (230–400
mesh ASTM). All reactions were carried out under argon. The
FT-IR spectrum of papain bioconjugate was recorded on a Tensor
27 FT-IR spectrometer (Bruker) equipped with a 6 mm diameter
membrane holder perpendicularly positioned with respect to
the IR beam. The aqueous solution of protein conjugate (6 10
ml, 63 mM) was deposited on a 6 mm diameter nitrocellulose
membrane and allowed to dry in air. The ESI mass spectra of proteins were acquired by on a triple-quadrupole mass spectrometer
API 3000 LC-MS/MS system (Applied Biosystems, PE Sciex) in
positive ion mode with the following instrumental parameters:
declustering potential 20 V, capillary voltage 5000 V, source
temperature 400 C. Samples for mass spectrometric analysis
were prepared in water (630 or 850 mM) and acidified with 10%
formic acid (ACS, Merck). Samples (25 ml) were introduced by flow
injection analysis (FIA) using a liquid chromatography system
(Agilent 1100 series) without column in the turbo ionspray source
by using H2O/CH3OH (1:1) as eluent (200 ml min 1). Molecular
masses were calculated from m/z peaks in the charge distribution
profile of the multiply charged ions using Hypermass 11 script of
Analyst 1.1 software (Applied Biosystems, PE Sciex).
Bromomalemide was synthesized as previously described.[12]
Dibromomalemide, glutathione, cysteine ethyl ester and TCEP
were purchased from Sigma-Aldrich. (Z5-C5H5)Fe(CO)2I was
Liquid chromatography–high-resolution accurate mass spectrometry (HPLC-HRMS) analyses were carried out using an Agilent
Technologies liquid chromatograph 1200 series composed of a
nano and capillary pump, a thermostat and a microautosampler
coupled to an Agilent Technologies 6538 UHD accurate mass
quadrupole time-of-flight liquid chromatograph–mass spectrometer equipped with an HPLC-chip cube. Ions formed in the
chip cube nano-electrospray were acquired in positive ion scan
mode. Instrument control, data acquisition and analysis were
performed using Agilent Mass Hunter software, version B.03.
Chromatographic separations were carried out on a largecapacity (160 nl) 150 mm C18 chip using a linear gradient of
aqueous 0.1% formic acid and 0.1% formic acid in acetonitrile.
Synthesis of (5-C5H5)Fe(CO)2 1-N-bromomaleimidato 2 and (Z5-C5H5)Fe
(CO)2 Z1-N-dibromomaleimidato 3
A stirred, water–ice-cooled and argon-saturated solution of
(Z5-C5H5)Fe(CO)2I (116 mg, 0.38 mmol), bromomaleimide
(66 mg, 0.34 mmol) or dibromomaleimide (96 mg, 0.38 mmol)
in toluene (35 ml) containing diisopropylamine (0.045 ml) was
illuminated with visible light (4 100 W tungsten lamps) for 3
h. The initial black colour of the solution turned yellow upon
illumination. The greyish solid formed was filtered off and the
filtrate was evaporated to dryness under reduced pressure.
The residue was dissolved in dichloromethane and purified
by column chromatography. A black band containing a small
amount of the starting (Z5-C5H5)Fe(CO)2I was eluted with
chloroform, followed by a yellow band of 2 or 3 eluted with
dichloromethane–methanol 9:1. The products were crystallized
from dichloromethane–heptane.
2: (43 mg, 36%). 1H NMR (200 MHz, d in ppm): 5.09 (s, 5H, Cp)
6,81 (s, 1H, olefin). 13C NMR (150 MHz, d in ppm) 211.91
(Fe&bond;C&tbond;O), 181.04 (C&dbond;O), 177.3, (C&dbond;O),
136.25 (&bond;(Br)&bond;C&dbond;C), 133.62 (&bond;C&dbond;
CH, olefin), 84.83 (Cp). IR (cm 1): 2052, 1998 (C&tbond;O), 1659
(CO imide). Anal. Found C, 37.61; H, 1.83; N, 3.98. Calcd for
C11H6BrFeNO4 C, 37.54; H, 1.72; N, 3.98%.
3: (38 mg, 40%). 1H NMR (200 MHz, d in ppm): 5.07 (s, 5H, Cp). 13C
NMR (150 MHz, d in ppm) 211.60 (Fe&bond;C&tbond;O), 175.19
(C&dbond;O), 131.96 (&bond;(Br)&bond;C&dbond;C), 84.85 (Cp), IR
(cm 1): 2046, 1996 (C&tbond;O), 1669 (CO imide). Anal. Found C,
30.67; H, 1.18; Br, 37.21; N, 3.28. Calcd for C11H5Br2FeNO4 C, 30.67;
H, 1.17; Br, 37.09; N, 3.25%.
X-Ray Structural Determination of 2–3
The crystal structures of 2 and 3 were solved by direct methods
using SHELXS-86[17] and refined by full-matrix least-square
method on F2 using SHELXL97.[18] After refinement with isotropic
displacement parameters, refinement was continued with
anisotropic displacement parameters for all non-hydrogen
atoms. The positions of hydrogen atoms were calculated with
idealized geometry. All the hydrogen atoms were refined with
the use of a rigid body model with isotropic displacement
Copyright © 2012 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2012, 26, 80–85
New organometallic Fe complexes containing bromomaleimide ligands
parameters equal to 1.2 of the equivalent displacement parameters of the parent atoms they are attached to. A summary
of crystallographic relevant data is given in supporting information Table S1. The molecular geometry was calculated using
PLATON.[19] Selected bond distances, angles, and geometry of
hydrogen bonds are summarized in supporting information
Table S2. Detailed crystallographic data can be obtained from
the Cambridge Crystallographic Data Centre, Deposition Numbers
CCDC 842697 and 842698.
Reaction of 2 with Cysteine Ethyl Ester Hydrochloride
A solution of cysteine ethyl ester hydrochloride (16 mg, 0.085
mmol or 85 mg, 0.046 mmol) with complex 2 (30 mg, 0.085
mmol) in DMF was stirred for 2 h. After this time the solvent
was evaporated. Formation of 4 and 5 was confirmed by LC-MS.
Theoretical monoisotopic [M + H]+ mass of 4: 421.0156, measured
mass 421.0153 (0.36 ppm). Theoretical monoisotopic [M + 2H]+2
mass of 5: 285.5367, measured mass: 285.5369 ( 0.43 ppm)
Cleavage with TCEP
The reaction mixture of 2 and cysteine ethyl ester (where cysteine
ethyl ester was not detected) was treated with tenfold molar
excess of TCEP in phosphate buffer pH 8. After 10 min of stirring
the solvent was evaporated and the mixture, analysed by LC-MS,
showed the peak of cysteine ethyl ester. Theoretical monoisotopic
[M + H]+ mass of cysteine ethyl ester: 150.0583, measured mass
150.0583 (0.15 ppm). The peak of 4 almost disappeared.
following a previously described procedure.[6] After 24 h, the
solution was dialysed in water and analysed by ESI-MS.
The monobromo and dibromo metallocarbonyl complexes 2 and
3 can be used for reversible cysteine derivative modification.
Reaction with an equimolar quantity of thiol gave the thiomaleimide complex, which could be cleaved by TCEP to regenerate
the thiol substrate. In the case an excess of thiol was used, the
substitution reaction was followed by the addition to the thiomaleimide double bond. The substitution process could be an entry
to new reversible labelling of proteins by metallocarbonyl complexes. Furthermore, this method enabled us to attach up to
three glutathione derivatives to one metallocarbonyl entity
(dibromomaleimide complex 3). Bromomaleimide complex 2
reacted with papain, giving the metallocarbonyl thiomaleimide–
papain bioconjugate. Treatment of the protein conjugate with
TCEP induced cleavage of the C&bond;S bond, resulting in partial
recovery of papain’s peptidase activity.
The Polish Ministry of Science and Education is gratefully acknowledged for financial support (Grant PBZ-KBN 118/T09/12).
B. Rudolf also acknowledges the Centre National de la Recherche
Scientifique for a 3-month researcher position in Paris. Céline
Fosse (Laboratoire de spectroscopie de masse, Chimie Paristech)
is gratefully acknowledged for the ESI-MS analysis of proteins.
Reaction of 3 with Glutathione
A solution of glutathione (2.9 mg, 0.0095 mmol) in phosphate
buffer (pH 6.2) was mixed with a solution of complex 3 (2.15
mg, 0.0049 mmol) in acetonitrile and stirred for 1 h or 24 h. After
this time the solvent was evaporated. Formation of the products
6, 7 and 8 was confirmed by LC-MS. Theoretical monoisotopic
[M + H]+ mass for 6: 656.9586; measured [M + H]+ mass: 656.9581
(0.76 ppm). Theoretical monoisotopic [M + H]+ and [M + 2H]+2
masses for 7: 884.1161 and 442.5617, respectively; measured
[M + H]+ and [M + 2H]+2 masses: 884.1160 ( 0.09 ppm) and
442.5618 (0.15ppm), respectively. Theoretical monoisotopic
[M + 3H]+3 mass for 8: 397.7380; measured [M + 3H]+3 mass:
397.7382 ( 0.34 ppm).
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Papain Inhibition Study
Affinity purified papain (3.5 mM, 75 ml) was allowed to react with
8 molar equiv. of 2 (2 mmol) in phosphate buffer pH 7.0 overnight
at room temperature. The solution was concentrated to 8 ml
by ultrafiltration with a stirred cell (Millipore) equipped with a
YM-10 10 kDa NMWL membrane (Millipore) and was further
passed on a gel filtration column Hiprep 10/26 desalting, GE
Healthcare) using 0.15 M NaCl as eluent. The fractions containing
the protein were pooled, and the solution was concentrated
again by ultrafiltration and dialyzed in water.
Cleavage by TCEP
An aqueous solution of protein conjugate (63 mM, 400 ml) was
treated with 1 mM TCEP and the peptidase activity was measured
during the reaction with the chromogenic substrate PFLNa
Appl. Organometal. Chem. 2012, 26, 80–85
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brom, synthesis, application, dibromomaleimide, metallocarbonyl, complexes, biochemical
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