Amide bond hydrolysis in peptides and cyclic peptides catalyzed by a dimeric Zr(iv)-substituted Keggin type polyoxometalate

Article abstract of DOI:10.1039/c3dt50705j

Detailed kinetic studies on the hydrolysis of glycylserine (Gly-Ser) and glycylglycine (Gly-Gly) in the presence of the dimeric zirconium(iv)-substituted Keggin type polyoxometalate (Et₂NH₂)₈[{α -PW₁₁O₃₉Zr(μ-OH)(H₂O)}₂] ·7H₂O (1) were performed by a combination of ¹H, ¹³C and ³¹P NMR spectroscopy. The observed rate constants for the hydrolysis of Gly-Ser and Gly-Gly at pD 5.4 and 60 °C were 63.3 × 10^-7 s^-1 and 4.44 × 10^-7 s ^-1 respectively, representing a significant acceleration as compared to the uncatalyzed reactions. The pD dependence of the rate constant for both reactions exhibited a bell-shaped profile with the fastest hydrolysis observed in the pD range of 5.5-6.0. Interaction of 1 with Gly-Ser and Gly-Gly via their amine nitrogen and amide oxygen was proven by ¹³C NMR spectroscopy. The effective hydrolysis of Gly-Ser in the presence of 1 is most likely a combination of the polarization of the amide oxygen due to its binding to the Zr(iv) ion in 1 and the intramolecular attack of the Ser hydroxyl group on the amide carbonyl carbon. The effect of temperature, inhibitors, and ionic strength on the hydrolysis rate constant was also examined. The solution structure of 1 was investigated by means of ³¹P NMR spectroscopy, revealing that its stability is highly dependent on pH, concentration and temperature. A 2.0 mM solution of 1 was found to be fully stable under hydrolytic conditions (pD 5.4 and 60 °C) both in the presence and in the absence of the dipeptides. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3dt50705j

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Amide bond hydrolysis in peptides and cyclic peptides
catalyzed by a dimeric Zr(IV)-substituted Keggin type
polyoxometalate†
Cite this: Dalton Trans., 2013, 42, 10929
Hong Giang T. Ly, Gregory Absillis and Tatjana N. Parac-Vogt*
Detailed kinetic studies on the hydrolysis of glycylserine (Gly-Ser) and glycylglycine (Gly-Gly) in the pres-
ence of the dimeric zirconium(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr-
(μ-OH)(H2O)}2]·7H2O (1) were performed by a combination of 1H, 13C and 31P NMR spectroscopy. The
observed rate constants for the hydrolysis of Gly-Ser and Gly-Gly at pD 5.4 and 60 °C were 63.3 × 10−7 s−1
and 4.44 × 10−7 s−1 respectively, representing a significant acceleration as compared to the uncatalyzed
reactions. The pD dependence of the rate constant for both reactions exhibited a bell-shaped profile
with the fastest hydrolysis observed in the pD range of 5.5–6.0. Interaction of 1 with Gly-Ser and Gly-Gly
via their amine nitrogen and amide oxygen was proven by 13C NMR spectroscopy. The effective hydrolysis
of Gly-Ser in the presence of 1 is most likely a combination of the polarization of the amide oxygen due
to its binding to the Zr(IV) ion in 1 and the intramolecular attack of the Ser hydroxyl group on the amide
carbonyl carbon. The effect of temperature, inhibitors, and ionic strength on the hydrolysis rate constant
was also examined. The solution structure of 1 was investigated by means of 31P NMR spectroscopy,
revealing that its stability is highly dependent on pH, concentration and temperature. A 2.0 mM solution
of 1 was found to be fully stable under hydrolytic conditions (pD 5.4 and 60 °C) both in the presence
and in the absence of the dipeptides.
Received 14th March 2013,
Accepted 28th May 2013
DOI: 10.1039/c3dt50705j
www.rsc.org/dalton
area of catalysis for C–H hydroxylation and double-bond epoxi-
Introduction
dation,9 H2O2-based oxidation,10 and Lewis acid mediated
Polyoxometalates (POMs) are composed of early transition organic reactions.11 Examples of catalytic studies with lantha-
metal ions in their highest oxidation state bridged by oxo nide and transition metal-substituted POMs for peptide hydro-
ligands. They are characterized by a wide range of dynamic lysis are rare.12
molecular structures, and physical and chemical properties.1
Protein hydrolysis is an important process in biotechnology
As a result POMs are attractive for applications in many and in modern proteomics. However, the rate of peptide bond
different areas, for example, in catalysis,2 medicine,3 material hydrolysis under physiological conditions is extremely slow. At
science,4 magnetism,5 and nanotechnology.6 Their molecular, neutral pH and 25 °C, the half-life for the hydrolysis of the
chemical, and physical properties can be easily modified by peptide bond in Gly-Gly is estimated to be around 350 years.13
changing the synthesis conditions.7 It has been known that Several proteolytic enzymes are available for the hydrolysis of
POMs undergo hydrolysis to form lacunary structures, which proteins. However, they often cleave proteins into too many
can readily react with a variety of transition metal and lantha- fragments and are extremely sensitive to temperature and pH.
nide ions that refill the vacant sites resulting in metal-substi- To promote peptide bond cleavage, several metal-containing
tuted POMs.8 In metal-substituted POMs, the substituted catalysts that facilitate this process have been developed. The
metal ion can coordinate to H2O or other ligands due to its hydrolysis of peptide bonds has mostly been studied in the
unshared coordination sites. This feature makes them attrac- presence of lanthanide and transition metal salts.14 Particu-
tive as catalysts for a wide variety of reactions. The majority of larly, they promote the hydrolysis of peptides containing Ser or
the applications of metal-substituted POMs are found in the Thr at the C-terminus by polarizing the peptide bond via
coordination and intramolecular attack of the hydroxyl group
on the amide carbon resulting in an ester intermediate.
Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium.
However, a drawback of using metal ions in this kind of reac-
E-mail: Tatjana.Vogt@chem.kuleuven.be; Fax: +32 16 327992; Tel: +32 16 32 76 12
tion is the formation of insoluble gels and precipitates under
neutral pH conditions.15 In addition, several metal complexes
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3dt50705j
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of Pt(II),16 Pd(II),17 Cu(II),18 Co(III),19 Ni(II),20 Zn(II),14b,21 Ce(IV),22 formed.8j The single crystal X-ray structure of 1 shows that two
and Zr(IV) were studied for peptide bond hydrolysis.12a,23 Pt(II) water molecules, which can be potentially substituted with
and Pd(II) complexes were shown to promote selective hydro- peptide substrate, are coordinated in the first coordination
lysis at Met and His residues, but only under low pH con- sphere of the Zr(IV) ion.8j In this study we examine the reacti-
ditions.24 Complexes of Cu(II), Co(III) and Ni(II) were able vity of 1 towards the hydrolysis of Gly-Gly, which has a non-
to cleave unactivated peptide bonds in peptides and pro- coordinating side chain, and Gly-Ser, a dipeptide containing a
teins,18a,c,20b,25 but Cu(II) and Ni(II) form strong metal– hydroxyl group in the side chain. We reason that the use of the
amide nitrogen bonds at pH 4 and pH 8 respectively26 forming Keggin POM as a chelating agent for Zr(IV) will not only result
catalytically inactive complexes.
in a homogeneous reaction mixture, but may also benefit
In our quest to understand the biological role of POMs on from the protein binding properties of the POM in future
a molecular level, we recently discovered the unprecedented experiments involving the hydrolysis of proteins, as we recently
reactivity of polyoxomolybdates and polyoxovanadates towards demonstrated in the first example of protein hydrolysis pro-
phosphoester bonds in DNA and RNA model systems as well moted by a Ce(IV)-substituted Keggin polyoxometalate.12b
as ester bonds in a series of carboxyesters.27 Furthermore, we
have also shown that peptides with an X-Ser sequence were
effectively hydrolyzed in oxomolybdate and oxovanadate solu-
Experimental procedures
Chemicals
tions.28 Although amide bond hydrolysis occurred in solutions
containing a mixture of oxoanions, the kinetic experiments
D2O, DCl, NaOD, H3PW12O40·23H2O, NaHCO3, ZrOCl2, HCl,
Et2NH2Cl, EtOH, Et2O, LiCl, Gly-Ser, Gly-Gly, oxalic, malonic,
succinic, adipic, citric and malic acid were purchased
and DFT calculations revealed that the actual active species are
monomeric oxoanions.28b The effective hydrolysis of phos-
phoester bonds in DNA and RNA model compounds was also
from Sigma-Aldrich. The 2 : 2 complex 1 was synthesized
achieved in the presence of the Zr(IV)-substituted Wells–
according to the procedure described in the literature8j,33 with
some modifications as described below. The 1 : 2 complex
(Et2NH2)10[Zr(α-PW11O39)2]·7H2O (2) was synthesized according
to the procedure described in the literature.33
Dawson POM29 and the incorporation of Hf(IV) into this POM
structure resulted in higher Lewis acidity for catalysis of
Mukaiyama aldol and Mannich-type reactions,11a demonstrat-
ing the use of the Lewis acid activity of metal-substituted
POMs for catalysis of organic reactions.11b Moreover, Zr(IV)-
substituted POMs have been used for H2O2 based oxidation
reactions,10f,30 sulfide oxidation,31 and the cyclization of
citronellal.11c Our recent efforts to develop new catalysts for
amide bond hydrolysis have been focused on the use of metal-
functionalized POMs.12 The incorporation of highly Lewis
acid metal ions into the POM framework prevents the for-
mation of inactive gels, which typically occurs at neutral and
basic pH values. We have recently shown that the sandwich
type 1 : 2 Zr(IV)-substituted Wells–Dawson POM, K15H[Zr(α2-P2-
W17O61)2]·25H2O, efficiently hydrolyzed unactivated amide
bond in glycylglycine in a fully homogeneous manner.12a The
stability of this POM was shown to be highly dependent on
the pH, concentration, temperature and reaction time. When
the 1 : 2 dimeric species is dissolved in water, multiple equili-
bria between the dimeric 2 : 2, monomeric 1 : 1, and sandwich
type 1 : 2 Zr(IV)–POM complexes are observed.8i,32 Among all
these species, the monomeric 1 : 1 form is expected to have the
highest catalytic activity, due to the presence of several water
molecules in the first coordination sphere of Zr(IV). Although
the highest catalytic activity of this POM towards Gly-Gly
hydrolysis was obtained at pD 5.0, multiple equilibria still
exist in solution, preventing a detailed understanding of the
catalytic mechanism.
Synthesis of 1
The synthesis of 1 was accomplished by a 1 : 1 molar ratio reac-
tion of the in situ-generated [Zr(α-PW11O39)2]10− with ZrOCl2 in
an aqueous HCl solution, followed by the addition of an excess
amount of solid Et2NH2Cl. H3PW12O40·23H2O (4.94 g) was dis-
solved in 25 mL of H2O, followed by addition of 1.0 M NaHCO3
until a pH value of 5.25 was obtained. The volume was
adjusted to 50 mL by adding H2O. ZrOCl2 (0.256 g) was added
and the solution was stirred for 5 minutes at room tempera-
ture. HCl (1 mL of 1.0 M) was added dropwise under vigorous
stirring. The solution was stirred for an additional 30 minutes
at room temperature. To this solution ZrOCl2 (0.256 g dis-
solved in 4 mL of 1 M HCl) was added dropwise. The solution
was stirred for 30 minutes and filtered with a folded filter
paper. The colourless filtrate was evaporated at 40 °C to
20 mL. After stirring for 1 minute at 95 °C, 2.5 g of Et2NH2Cl
was added and the mixture was stirred for 5 minutes. The
white suspension was cooled to room temperature and stirred
for 1 hour. White powder was collected and washed with EtOH
(30 mL) and Et2O (3 × 50 mL), and dried in vacuo for 2 hours.
A water-soluble white powder (4.18 g – 88%) was obtained. The
31P NMR spectrum of complex 1 in D2O shows one signal at
−13.49 ppm.
To further exploit POMs as catalysts for the hydrolysis
of peptide bonds, herein we report the use of dinuclear Zr(IV)-
Measurements
substituted Keggin POM as an artificial peptidase. By using 1H NMR spectra were recorded on a Bruker Advance 400
the monolacunary α-Keggin anion [α-PW11O39]7− as a ligand spectrometer and TMSP-d4 was used as an internal reference.
for Zr(IV), the dinuclear zirconium(IV) α-Keggin polyoxometa- 13C NMR spectra were recorded on a Bruker Advance 400
late (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (1) was spectrometer. As a reference TMS in an internal reference tube
10930 | Dalton Trans., 2013, 42, 10929–10938
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was used. 31P NMR spectra were recorded on a Bruker Advance It is expected that 1 will display a higher catalytic activity since
400 spectrometer and 25% H3PO4 in H2O in an internal refer- the Zr(IV) center in 1 has free coordination sites available for
ence tube was used.
the interaction with substrate molecules. In aqueous solution
the conversion between both complexes is a reversible process
that is dependent on the pH, temperature, time and concen-
tration. The stability of 1 and 2 in aqueous solution can be
monitored by 31P NMR spectroscopy since both complexes are
characterized by a specific 31P NMR resonance. The 31P NMR
spectra of 2.0 mM 1 at different pD values are shown in Fig. 2.
While at pD 7.4 the only observable signal at −13.49 ppm is
the dimeric complex 1,8j at pD 9.0 a new set of resonances at
−14.60 and −14.69 ppm, corresponding to the sandwich
complex 2,33,35 simultaneously appeared. The complete conver-
sion of 1 into 2 and the lacunary [α-PW11O39]7− anion (δ
−10.67 ppm)8b,36 occurred at pD 10.4. However, in the pD
range 2.0–7.4 only the dimeric complex 1 was present in solu-
tion. A slight shift of 0.13 ppm was seen in acidic media,
which might be due to the protonation of coordinated water
molecules.8j As the hydrolysis of peptide bonds was studied at
60 °C in the course of several days, the effect of time on the
species distribution at 60 °C was further investigated. Solu-
tions containing 2.0 mM 1 at pD 5.4 and 7.4 were kept at 60 °C
and their 31P NMR spectra at different time increments are
presented in Fig. S1†. After 24 h at pD 7.4 and 60 °C 25% of 2
was detected in solution. Equilibrium between these two com-
plexes was obtained after 2 days representing a ratio of ca. 3 : 2
between 1 and 2. Interestingly, no products derived from the
decomposition of 1 were detected after 20 days at 60 °C and
pD 5.4. Next, the effect of the initial concentration of 1 on the
conversion at pD 5.4 and 60 °C was investigated. The sandwich
type complex 2 was formed in the 6.0 mM solution of 1 after
one day (Fig. S2†), while the 2.0 mM solution of 1 remained
unchanged after 20 days.
Stability studies
To study the effect of pD on the stability of 1, 2.0 mM solutions
of 1 in D2O were adjusted to pD 2.0, 3.0, 4.9, 7.4, 9.0, and 10.4
by using 1.0 M DCl or 1.0 M NaOD. To study the effect of time
and temperature on the stability, 2.0 mM solutions of 1 were
adjusted to pD 5.4 and 7.4 and kept at 60 °C. To study the
effect of POM concentration on the stability of 1, 2.0 mM
and 6.0 mM solutions of 1 were adjusted to pD 5.4 and kept
at 60 °C. The pH-meter reading was corrected by the equation
pD = pH + 0.41.34
Hydrolysis studies
The hydrolysis of 2.0 mM peptide in the presence of a 2.0 mM
solution of 1 was followed by H NMR spectroscopy. Typically,
1
800 μL of a 2.5 mM solution of 1 in D2O, 100 μL of a 20.0 mM
solution of the dipeptide in D2O, and 100 μL of a 5.0 mM solu-
tion of TMSP-2,2,3,3-d4 (sodium-3-trimethylsilylpropionate) in
D2O were mixed. The pD of the final solution was adjusted to
5.40 with DCl or NaOD and was measured at the beginning
and at the end of each experiment. The pD difference was
less than 0.10 unit. The reaction samples were kept at 60 °C.
1H NMR spectra were taken after mixing and after different
time increments.
Binding studies
The binding of 1 to the dipeptides was studied by 1H and
13C NMR spectroscopy, by examining changes in the NMR
spectra of the peptide upon addition of 1. The solutions for
NMR contained 20.0 mM to which 5.0 mM of 1 in D2O
was added. The pH of the solution was adjusted to pD 6.4 or
pD 9.0 by using 1.0 M DCl or 1.0 M NaOD.
The pH dependence of the reversible conversion process of
complex 2 in aqueous solution was also investigated. The 31P
NMR spectra of the 2.0 mM solution of 2 were recorded after
pD adjustment (Fig. S3†). Only complex 2 was observed in the
Results and discussion
Stability of 1 in aqueous solution
Depending on the synthesis conditions, Zr(IV) can be incorpor-
ated into the lacunary Keggin POM, forming dimeric 2 : 2
([{α-PW11O39Zr(μ-OH)(H2O)}2]8− (1) or sandwich 1 : 2 ([Zr-
(α-PW11O39)2]10− (2) structures.8j,33 Single crystal X-ray struc-
tures show that the Zr(IV) ion in 1 has two coordinated water
molecules in the first coordination sphere, whereas the Zr(IV)
ion in 2 is saturated with two lacunary Keggin units (Fig. 1).
Fig. 1 Schematic
representation
of
(Et2NH2)8[{α-PW11O39Zr(μ-OH)-
Fig. 2 31P NMR spectra of 2.0 mM solutions of 1 at different pD values
(H2O)}2]·7H2O (1) and (Et2NH2)10[Zr(α-PW11O39)2]·7H2O (2).
recorded immediately after pD adjustment.
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Fig. 3 31P NMR spectra of a 2.0 mM solution of 2 after 10 days at 60 °C and in
the pD range 4.0–7.1.
pD range 4.0–7.1, but the monolacunary species simul-
taneously appeared at pD 8.1. The sandwich complex 2 was
stable after 10 days at 60 °C and in the pD range 4.0–7.1
(Fig. 3).
Complex 1 having two labile water molecules coordinated
to Zr(IV) is expected to be a better catalyst for peptide bond
hydrolysis since Zr(IV) can coordinate to the amide carbonyl
oxygen, polarizing the carbonyl bond, resulting in an amide
carbon that is more susceptible to nucleophilic attack. There-
fore, the hydrolysis of Gly-Gly and Gly-Ser dipeptides in the
presence of 1 was explored at pD 5.4 and 60 °C, as under these
experimental conditions only 1 was present in solution
making the solution species distribution more controllable
when compared to the Zr(IV)-substituted Wells–Dawson type
POM and therefore allowing a more detailed mechanistic
study. In order to compare the catalytic reactivity of complexes
1 and 2, the hydrolysis of Gly-Ser and Gly-Gly by 2 was also
investigated under the same experimental conditions.
Fig. 4 1H NMR spectra recorded at different reaction times during the hydro-
lysis of 2.0 mM Gly-Ser by 2.0 mM 1 at pD 5.4 and 60 °C.
Hydrolysis of Gly-Ser
The cleavage of 2.0 mM Gly-Ser by 2.0 mM 1 was examined at
1
pD 5.4 and 60 °C. H NMR spectra of the reaction recorded at
different time increments are presented in Fig. 4. The signal
at 4.41 ppm corresponds to the CH proton of serine residue
in Gly-Ser. The signal of the CH2 proton of the glycyl residue
(3.90 ppm) overlaps with that of the serine residue
(3.85–3.96 ppm) in Gly-Ser. The intensity of these signals
decreased during the course of the reaction, while an increase
in intensity of free glycine (3.57 ppm) and free serine
(3.85–3.87 and 3.96–4.00 ppm) was observed. These changes
indicate that the hydrolysis of the amide bond in Gly-Ser
occurred. Additionally, the cyclic Gly-Ser (cGly-Ser), identified
by comparison of the chemical shifts with those of pure cGly-
Ser, was also detected with a maximum concentration of 10%
in total. The percentage of Gly-Ser, Gly, Ser and cGly-Ser
is shown in Fig. 5. As can be seen from Fig. 5, the intensity of
the cGly-Ser resonances remained relatively constant during
the reaction, suggesting that the cyclization to cGly-Ser is
Fig. 5 Fraction of Gly-Ser, Gly, Ser and cGly-Ser as a function of time in the
reaction between 2.0 mM Gly-Ser and 2.0 mM 1 at pD 5.4 and 60 °C.
irreversible or the cleavage of cGly-Ser is too slow compared to
Gly-Ser hydrolysis. To clarify this issue, the hydrolysis of
2.0 mM cGly-Ser in the presence of 2.0 mM 1 at pD 5.4 and
60 °C was explored. Ring-opening of cGly-Ser occurred to form
Gly-Ser and Ser-Gly (identified by spiking the authentic
sample), which further hydrolyzed forming Gly and Ser
(Fig. S4† and Fig. 6). This finding proves that the cyclization
of Gly-Ser is a reversible process. Remarkably, no gel or
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gradual decrease of the two Gly-Gly resonances (3.86 and
3.89 ppm) and a steady increase of the free glycine resonance
at 3.56 ppm. In addition, the cyclic Gly-Gly (cGly-Gly) at
4.04 ppm was observed during the course of the hydrolytic
reaction. The percentage of Gly-Gly, Gly and cGly-Gly is shown
in Fig. 7. The data show that the fraction of cGly-Gly gradually
decreased to zero after reaching a maximum of 11%. The
degradation of cGly-Gly can directly form Gly by breaking two
chemical bonds at the same time or indirectly form Gly via
Gly-Gly. To gain more insights into the disappearance of cGly-
Gly, we examined the hydrolysis of 2.0 mM cGly-Gly in the
presence of 2.0 mM 1 at pD 5.4 and 60 °C. Fig. S6† shows the
1H NMR spectra recorded at various reaction times of the cGly-
Gly cleavage. The percentage of cGly-Gly, Gly-Gly and Gly is
presented in Fig. 8. As can be seen, 27% of cGly-Gly is con-
verted to Gly-Gly after one day. Notably, no free glycine was
detected after one day and it could only be seen after sufficient
amounts of Gly-Gly were formed, which were subsequently
hydrolyzed to free glycine. This finding suggests that simul-
taneously breaking two amide bonds in cGly-Gly is unfavour-
able and that the cyclization of Gly-Gly to cGly-Gly by 1 is a
reversible process. The hydrolysis of cGly-Gly by 1 is presented
in Scheme 1.
Fig. 6 Fraction of cGly-Ser, Gly-Ser, Ser-Gly and Gly as a function of time in the
reaction between 2.0 mM cGly-Ser and 2.0 mM 1 at pD 5.4 and 60 °C.
precipitation was observed in any of the reactions in the pres-
ence of 1, indicating that hydrolysis of the peptide bond
occurs under fully homogenous conditions. Notably, the pres-
ence of 1 does not only accelerate the peptide bond cleavage,
but also promotes the cyclization of Gly-Ser which did not
occur in the control reactions.
Complete cleavage of Gly-Ser was obtained, resulting in 5%
of cGly-Ser and 95% of Gly and Ser in the mixture (Fig. 5).
By fitting the decrease in the concentration of Gly-Ser to a
monoexponential function, the rate constant kobs = 63.3 × 10−7 s−1
1
(t = 30 h) for the hydrolysis was calculated. It is important
2
to note that despite the fact that the reaction rate appears
slow, it represents a significant acceleration of nearly 50 times
compared to the uncatalyzed reaction. In Gly-Ser samples
without the catalyst no hydrolysis of the peptide bond was
observed in the time period during which full peptide bond
hydrolysis occurred in the presence of 1 (Table S2†). Similarly,
the observed rate constant of 3.33 × 10−7 s−1 (t = 577 h) for the
1
2
hydrolysis of cGly-Ser was obtained, which is about 20 times
slower compared to Gly-Ser cleavage. Under the same experi-
mental conditions the hydrolysis of 2.0 mM Gly-Ser in the Fig. 7 Fraction of Gly-Gly, Gly, and cGly-Gly as a function of time in the reaction
between 2.0 mM Gly-Gly and 2.0 mM 1 at pD 5.4 and 60 °C.
presence of 2.0 mM 2 occurred with a rate constant of 3.05 ×
10−7 s−1, which is only about two times larger than the uncata-
lyzed reaction (Table S2†), but about 20 times smaller than the
observed rate constant in the presence of 1 under the same
reaction conditions. This indicates that the dimeric complex 1
with free coordinated water molecules and unshared coordi-
nation sites available for interaction with the peptide is far
more active than the sandwich type complex 2. Despite the fact
that no water molecules are coordinated to the Zr(IV) ion in 2,
the limited reactivity can be explained by the fact that binding
of the substrate can displace the equilibrium towards the cata-
lytically active 1 : 1 species.37
Hydrolysis of Gly-Gly
A mixture containing 2.0 mM Gly-Gly and 2.0 mM solution of
1 was reacted at 60 °C and pD 5.4. The 1H NMR spectra
Fig. 8 Fraction of cGly-Gly, Gly-Gly and Gly as a function of time in the reaction
(Fig. S5†) measured at different time increments showed a between 2.0 mM cGly-Gly and 2.0 mM 1 at pD 5.4 and 60 °C.
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Scheme 1 Hydrolysis of cGly-Gly by 1.
Similarly to Gly-Ser hydrolysis, no gel formation or precipi-
tation was observed in any of the reactions in the presence of
1. The observed rate constant of 4.44 × 10−7 s−1 for the clea-
vage of Gly-Gly was calculated by fitting the decrease in Gly-Gly
concentration to a monoexponential function. Although the
hydrolysis of Gly-Gly is relatively slow and has a half-life of
approximately 18 days, this represents a significant accelera-
tion compared to the uncatalyzed reaction in which only 25%
of Gly-Gly was hydrolyzed after seven months under identical
conditions. Additionally, under the same experimental con-
ditions the hydrolysis of 2.0 mM Gly-Gly in the presence of
2.0 mM 2 was also studied. After 15 days at pD 5.4 and 60 °C
only 3% of Gly-Gly was hydrolyzed. The very low activity of 2
compared to 1 is most likely due to the lack of free coordi-
nation sites at Zr(IV) as discussed earlier in the text. Signifi-
cantly, the 31P NMR spectra of 2.0 mM 1 in the presence of
2.0 mM Gly-Ser or in the presence of 2.0 mM Gly-Gly at pD 5.4
recorded at the end of the hydrolytic reactions showed only
one peak corresponding to 1, indicating its stability in the
presence of the dipeptides (Fig. S7†).
Fig. 9 13C NMR spectrum of Gly-Ser in the presence (*) and absence (°) of 1 at
pD 6.4 (the signal at 42.4 ppm belongs to the CH2 carbon of the ethyl group of
the diethyl ammonium counter ions of 1).
Table 1 13C NMR chemical shift values (ppm) of 20.0 mM Gly-Ser in the pres-
ence and in the absence of 5.0 mM 1
pD 6.4
pD 9.0
Δδ
Δδ
13C NMR Gly-Ser Gly-Ser + 1 (ppm) Gly-Ser Gly-Ser + 1 (ppm)
δ1
δ2
δ3
δ4
δ5
40.72
167.11 166.84
57.31 57.29
176.00 175.98
61.97 61.98
40.64
0.08
0.27
0.02
0.01
0.01
42.06
170.49 167.34
57.14 57.33
176.11 176.05
62.06 62.05
40.95
1.11
3.15
0.19
0.06
0.01
Rate constants for the hydrolysis of Gly-Ser and Gly-Gly by
other metal-based catalysts are presented in Table S3.† A direct
comparison is difficult due to the different reaction conditions
(pH, temperature, and concentration) used in these studies.
While the hydrolysis of Gly-Ser catalyzed by 1 was approxi-
mately two times faster than the one in the presence of the
Zr(IV)–Wells–Dawson POM, hydrolysis of Gly-Gly was three
times faster when the Zr(IV)–Wells–Dawson POM was used
instead of 1. The Zr(IV)/4,13-Diaza-18-crown-6 system displayed
a higher reactivity both for Gly-Ser and Gly-Gly hydrolysis
when compared to 1. However, the use of 1 resulted in homo-
geneous reaction conditions, while the Zr(IV)/4,13-Diaza-
18-crown-6 system led to heterogeneous reaction conditions
due to the formation of Zr(IV) gels.
0.08 ppm at the C-1 carbon was observed. Additionally, the 13
C
NMR chemical shift of the C-terminal carboxylate group was
unaffected by the addition of 1, confirming the non-involve-
ment of the carboxylate group in coordination. Such a coordi-
nation is stronger at pD 9.0 since large shifts of 3.15 and
1.11 ppm were observed at the amide carbonyl carbon and the
Cα of Gly residue respectively (Table 1 and Fig. S8†). In
addition, the C-3 atom experienced a shift of 0.19 ppm. This
could be a result of binding to the amide nitrogen forming a
hydrolytically inactive complex in which Zr(IV) binds to Gly-Ser
via both amine and amide nitrogens. The tendency for
forming such an inactive peptide complex in basic media was
also observed for Cu2+ and Ni2+.39
Similarly, the binding of Gly-Gly to 1 via the amine nitrogen
and amide oxygen at pD 6.4 causes changes in 13C NMR
chemical shifts of 0.10 and 0.34 ppm at the C-1 and amide
carbons respectively (Table S1 and Fig. S9†). Stronger inter-
action was also obtained at pD 9.0 and the formation of a
hydrolytically inactive complex caused a shift of 0.19 ppm at
the C-3 carbon (Fig. S10†). Thus hydrolytically active and inac-
tive complexes of Gly-Ser and Gly-Gly to 1 were proposed in
Scheme 2.
Binding modes of 1 with dipeptides
The binding mode of 1 was constructed on the basis of the
shifts in 13C NMR spectra of dipeptides upon addition of 1. At
pD 6.4 the highest shift was obtained for the amide carbonyl
carbon in Gly-Ser dipeptide (Fig. 9 and Table 1), indicating
that the interaction occurs at the amide oxygen. This inter-
action is important for the hydrolysis of peptide bonds since it
increases the partial positive charge on the amide carbon and
renders it more susceptible to nucleophilic attack by water.38 It
has been known that the amine group plays a key role in the
Effect of pD on the hydrolysis of Gly-Ser and Gly-Gly
coordination since the hydrolysis was not obtained for N-term- The influence of pD on the rate constant for the reaction
inal blocked peptides.12a,22,28a As a consequence, a shift of between 2.0 mM Gly-Ser and 2.0 mM 1 at 60 °C was further
10934 | Dalton Trans., 2013, 42, 10929–10938
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nitrogen, while at higher pH values the formation of inactive
complexes is also observed.
Effect of concentration of 1 on the hydrolysis of Gly-Gly and
Gly-Ser
The influence of the POM concentration on the observed rate
constant for the hydrolysis of 2.0 mM Gly-Gly by 1 at pD 5.4
and 60 °C was further studied (Fig. 11). This study reveals that
2.0 mM is the optimal concentration of 1 for the cleavage of
the peptide bond in Gly-Gly. A 30% decrease of the rate con-
stant was observed when a 6.0 mM solution of 1 was used. The
conversion of 1 into the much less active 2 is considered to be
the main reason leading to a decrease in reaction rate at high
concentrations of 1. Similar results were obtained for the
hydrolysis of 2.0 mM Gly-Ser by 1 at pD 5.4 and 60 °C
(Fig. S11†). Noticeably, 1 is not only able to promote Gly-Gly
and Gly-Ser hydrolysis, but also capable of hydrolyzing an
excess amount of the substrate. In both reactions, a 10-fold
excess of dipeptides was completely hydrolyzed by 0.2 mM 1,
indicating that a catalytic turnover is possible.
Scheme 2 Hydrolytically active (a) and inactive (b) complexes.
Effect of experimental conditions and inhibitors on the
hydrolysis of Gly-Gly and Gly-Ser
Fig. 10 pD dependence of the rate constant for the hydrolysis of 2.0 mM Gly-
Ser (a) and 2.0 mM Gly-Gly (b) in the presence of 2.0 mM 1 at 60 °C.
The effect of temperature on the hydrolysis of the amide bond
in the two dipeptides at pD 5.4 was determined by measuring
the observed reaction rate constant at different temperatures
ranging from 37 °C to 70 °C. The experimental activation
energy parameter (Ea) was obtained from the Arrhenius plot
(Fig. S12a and S13a†). The activation values of these two reac-
tions are similar and in good agreement with previously pub-
lished results.12a For comparison, the activation energy
(109.93 kJ mol−1) of Gly-Ser in the absence of 1 at pD 5.4 was
calculated (Fig. S14†). Apparently, the presence of the catalyst
lowers the activation energy for the hydrolysis of Gly-Ser by
20 kJ mol−1. Linear fitting of ln(kobs/T) as a function of 1/T
(Fig. S12b and S13b†) allows for the calculation of the enthalpy
and the entropy of activation (Table 2). The Gibbs energies of
activation (ΔG#) for the hydrolysis of Gly-Gly and Gly-Ser at
investigated. Fig. 10a shows a bell-shape profile with a
maximum at pD 5.5. The highest rate of the reaction at this pD
value is related to the species distribution in aqueous solution.
Only the active dimeric complex 1 containing coordination
sites that can anchor Gly-Ser exists in aqueous solution under
identical conditions (pD 5.4 and 60 °C). The rate constant
decreases at higher pD values due to the formation of an inac-
tive complex at high pD values, as discussed above. Further-
more, higher pH values also favour the conversion of 1 into 2,
which is proven to have a lower catalytic activity. In acidic
media, although only the dimeric complex 1 is detectable, a
decrease in reaction rate is observed. This is most likely due
to the fact that coordination of Zr(IV) to the amine nitrogen
is hindered due to protonation of this group in acidic
solutions.
The hydrolysis of 2.0 mM Gly-Ser in the presence of 2.0 mM
ZrOCl2 salt at pD 5.4 or pD 8.4 and 60 °C was also investigated,
and gel formation in both reaction mixtures was observed.
Rate constants of 10.83 × 10−7 s−1 and 8.05 × 10−7 s−1 at pD 5.4
and pD 8.4, respectively, were determined, which are approxi-
mately 6 times slower compared to the hydrolysis in the
presence of 1.
The effect of pD on the observed rate constant of Gly-Gly
shows
a similar trend as obtained in Gly-Ser cleavage
(Fig. 10b). The fasted hydrolytic rate occurs at pD 6.0 where
complex 1 is dominant in solution. Similarly to Gly-Ser, the
coordination of 1 to Gly-Gly was studied by 13C NMR spec-
troscopy and the results (Table S1 and Fig. S9†) are analogous
to those obtained for Gly-Ser. At pD 6.4, the coordination of
Gly-Gly to 1 occurs via the amide carbonyl oxygen and amine
Fig. 11 Influence of the concentration of 1 on the observed rate constant for
the hydrolysis of 2.0 mM Gly-Gly at pD 5.4 and 60 °C.
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Table 2 Experimental activation parameters of the hydrolysis of Gly-Gly and
Gly-Ser by 1 at pD 5.4
Ea
ΔH#
ΔS#
ΔG# at 310 K
Dipeptide
(kJ mol−1
)
(kJ mol−1
)
(J mol−1 K−1
)
(kJ mol−1
)
Gly-Gly
Gly-Ser
90.03
89.97
87.33
87.22
−104.18
−83.71
119.63
113.17
Scheme 3 Proposed mechanism for Gly-Gly hydrolysis in the presence of 1.
310 K are 119.63 and 113.17 kJ mol−1 respectively. The negative
activation entropy obtained is most likely the result of the
coordination of the dipeptides to the Zr(IV) center.
The influence of ionic strength on the rate constant of Gly-
Gly and Gly-Ser hydrolysis was investigated by adding LiCl to a
reaction mixture containing 2.0 mM Gly-Gly or 2.0 mM Gly-Ser
and 2.0 mM 1 at pD 5.4 and 60 °C. LiCl has a negative effect
on the hydrolysis of both dipeptides (Fig. S15†) as high LiCl
concentrations cause a decrease in the reaction rate. The pres-
ence of 0.6 M LiCl causes a 3-fold decrease in the rate con-
stant. Complex 1 is stable in the presence of the LiCl salt since
the 31P NMR spectroscopy of 1 in the presence of 1.0 M LiCl at
pD 5.4 and 60 °C after 5 days does not show any traces of its
conversion into 2 (Fig. S16†). Therefore the decrease in kobs
caused by the salt is most likely due to hindering of the elec-
trostatic interaction between the dipeptide and 1.
Several non-reactive substrate analogues (Fig. S17†) were
chosen to study the inhibitive effect on the binding between
dipeptides and 1. Due to the oxophilicity of Zr(IV), dicarboxylic
acids with multiple donor groups are expected to coordinate to
the Zr(IV) center. As can be seen from Table 3, the presence of
these dicarboxylic acids causes a decrease in the overall rate
constant for the hydrolysis of the examined dipeptides. Par-
ticularly, the strongest inhibition effect was obtained in the
presence of oxalic acid as well as the carboxylic acids contain-
ing an alcohol donor group adjacent to one of the carboxylate
groups (malic and citric acid), whereas dicarboxylic acids with
a longer aliphatic chain length caused less inhibition. The
competitive inhibition effect of the former on the hydrolysis of
Gly-Gly and Gly-Ser can be explained by the formation of their
strong complexes with five- and six-membered chelate rings
with Zr(IV). For dicarboxylic acids with a long aliphatic chain
Scheme 4 Proposed mechanism for Gly-Ser hydrolysis in the presence of 1.
length, their bidentate mode of binding to Zr(IV) is less
effective, causing a weaker inhibitor effect as observed in both
hydrolytic reactions.
Hydrolysis mechanism
The binding studies of 1 to the dipeptides and inhibition and
kinetic experiments showed the important role of the amide
carbonyl oxygen in metal coordination. This coordination
polarizes the amide carbon and makes it more susceptible to
nucleophilic attack by water, resulting in the hydrolysis of Gly-
Ser and Gly-Gly. In addition, the coordination to the N-termi-
nus is indispensable for effective hydrolysis.12a,28a The mech-
anism for the hydrolysis of Gly-Gly by 1 is proposed in
Scheme 3, assuming that the peptide coordinates to one Zr(IV).
It is, however, also plausible that the peptide bridges two Zr(IV)
ions in 1, but the available experimental evidence is not
sufficient to distinguish between these two possibilities. The
more effective hydrolysis of Gly-Ser can be a result of the polar-
ized amide carbon due to the binding to the Zr(IV) center, fol-
lowed by the intramolecular attack of the Ser hydroxyl group
(Scheme 4) on the amide carbonyl carbon producing a five-
membered cyclic transition state which is rearranged to an
ester intermediate.
Table 3 The observed rate constants of the hydrolysis of 2.0 mM Gly-Gly or
2.0 mM Gly-Ser by 2.0 mM 1 in the presence of 5.0 mM inhibitor at pD 5.4 and
60 °C
107 × kobs (s−1
)
Inhibitor
(5.0 mM)
2.0 mM Gly-Gly +
2.0 mM 1
2.0 mM Gly-Ser +
2.0 mM 1
Conclusion
No inhibitor
Oxalic acid
Citric acid
4.44
0.28
0.56
0.83
1.94
2.78
3.61
63.33
3.33
6.39
In conclusion, we report on the detailed mechanism of amide
bond hydrolysis catalyzed by the dimeric Zr(IV)-substituted
Keggin type polyoxometalate. Homogeneous hydrolysis of the
peptide bond in Gly-Ser, a hydroxyl-containing side chain
dipeptide, and Gly-Gly with a non-coordinating side chain was
Malic acid
4.17
Malonic acid
Succinic acid
Adipic acid
23.89
42.50
55.28
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achieved in the presence of the POM. The highest catalytic
activity of this POM towards peptide bond hydrolysis was
obtained at pD 5.5–6.0, at which no traces of dissociation
of complex 1 were observed resulting in a more controllable
species distribution and thereby allowing a more profound
mechanistic study when compared to the Zr(IV)-substituted
Wells–Dawson type polyoxometalate typically characterized by
multiple equilibria at pD 5.5–6.0. Coordination of 1 to dipep-
tides via the amine nitrogen and amide carbonyl oxygen is pro-
posed, resulting in polarization of the peptide bond and
making it more susceptible to hydrolysis. This and our pre-
vious findings reporting on the peptidase activity of POMs
provide new and growing opportunities for metal-substituted
POM as a novel class of catalyst for peptide bond hydrolysis.
The hydrolysis of different proteins by 1 is currently being
investigated.
H. J. Thomas, Appl. Catal., A, 2003, 256, 37;
(d) L. C. W. Baker and D. C. Glick, Chem. Rev., 1998, 98, 3;
(e) Q.-H. Luo, R. C. Howell, M. Dankova, J. Bartis,
C. W. Williams, W. D. Horrocks, V. G. Young,
A. L. Rheingold, L. C. Francesconi and M. R. Antonio,
Inorg. Chem., 2001, 40, 1894; (f) J. Wang and Q. Hao,
J. Alloys Compd., 2009, 482, 235; (g) G. Süss-Fink, Angew.
Chem., Int. Ed., 2008, 47, 5888; (h) Y. V. Geletii, B. Botar,
P. Kögerler, D. A. Hillesheim, D. G. Musaev and C. L. Hill,
Angew. Chem., Int. Ed., 2008, 47, 3896; (i) M. Sokolov,
N. Izarova, E. Peresypkina, A. Virovets and V. Fedin, Russ.
Chem. Bull., 2009, 58, 507; ( j) K. Nomiya, Y. Saku,
S. Yamada, W. Takahashi, H. Sekiya, A. Shinohara,
M. Ishimaru and Y. Sakai, Dalton Trans., 2009, 5504;
(k) Y. Saku, Y. Sakai and K. Nomiya, Inorg. Chem. Commun.,
2009, 12, 650; (l) Y. Saku, Y. Sakai, A. Shinohara,
K. Hayashi, S. Yoshida, C. N. Kato, K. Yoza and K. Nomiya,
Dalton Trans., 2009, 805; (m) A. J. Gaunt, I. May,
D. Collison, K. Travis Holman and M. T. Pope, J. Mol.
Struct., 2003, 656, 101.
Acknowledgements
T.N.P.V. thanks KU Leuven for financial support (START1/09/
028). H.G.T.L. thanks the Vietnamese Government and KU
9 D. Kumar, E. Derat, A. M. Khenkin, R. Neumann and
S. Shaik, J. Am. Chem. Soc., 2005, 127, 17712.
Leuven for a doctoral fellowship. G.A. thanks the FWO for a 10 (a) O. V. Zalomaeva, O. A. Kholdeeva and A. B. Sorokin,
postdoctoral fellowship.
Green Chem., 2006, 8, 883; (b) F. Hussain, B. S. Bassil,
U. Kortz, O. A. Kholdeeva, M. N. Timofeeva, P. de Oliveira,
B. Keita and L. Nadjo, Chem.–Eur. J., 2007, 13, 4733;
(c) O. A. Kholdeeva and R. I. Maksimovskaya, J. Mol. Catal.
A: Chem., 2007, 262, 7; (d) N. V. Maksimchuk,
M. N. Timofeeva, M. S. Melgunov, A. N. Shmakov,
Y. A. Chesalov, D. N. Dybtsev, V. P. Fedin and
O. A. Kholdeeva, J. Catal., 2008, 257, 315; (e) T. Trubitsyna
and O. Kholdeeva, Kinet. Catal., 2008, 49, 371;
(f) O. A. Kholdeeva, G. M. Maksimov, R. I. Maksimovskaya,
M. P. Vanina, T. A. Trubitsina, D. Y. Naumov, B. A. Kolesov,
N. S. Antonova, J. J. Carbo and J. M. Poblet, Inorg. Chem.,
2006, 45, 7224.
Notes and references
1 (a) N. Mizuno and K. Kamata, Coord. Chem. Rev., 2011, 255,
2358; (b) J. T. Rhule, C. L. Hill and D. A. Judd, Chem. Rev.,
1998, 98, 327; (c) D.-L. Long, E. Burkholder and L. Cronin,
Chem. Soc. Rev., 2007, 36, 105.
2 (a) A. Proust, R. Thouvenot and P. Gouzerh, Chem.
Commun., 2008, 1837; (b) C. L. Hill, J. Mol. Catal. A: Chem.,
2007, 262, 2; (c) C. L. Hill and C. M. Prosser-McCartha,
Coord. Chem. Rev., 1995, 143, 407; (d) I. Kozhevnikov, Cata-
lysis by polyoxometalates, John Wiley & Sons, Ltd, Chiche- 11 (a) C. Boglio, K. Micoine, P. Rémy, B. Hasenknopf,
ster, 2002.
S. Thorimbert, E. Lacôte, M. Malacria, C. Afonso and
J.-C. Tabet, Chem.–Eur. J., 2007, 13, 5426; (b) C. Boglio,
G. Lemiere, B. Hasenknopf, S. Thorimbert, E. Lacote and
M. Malacria, Angew. Chem., Int. Ed., 2006, 45, 3324;
(c) Y. Kikukawa, S. Yamaguchi, K. Tsuchida, Y. Nakagawa,
K. Uehara, K. Yamaguchi and N. Mizuno, J. Am. Chem. Soc.,
2008, 130, 5472.
3 J. T. Rhule, C. L. Hill, D. A. Judd and R. F. Schinazi, Chem.
Rev., 1998, 98, 327.
4 (a) T. Yamase, Chem. Rev., 1998, 98, 307; (b) E. Coronado
and P. Day, Chem. Rev., 2004, 104, 5419; (c) E. Coronado
and J. R. Galan-Mascaros, J. Mater. Chem., 2005, 15, 66;
(d) E. Coronado, C. Giménez-Saiz and C. J. Gómez-García,
Coord. Chem. Rev., 2005, 249, 1776.
5 U. Kortz, A. Müller, J. van Slageren, J. Schnack, N. S. Dalal
and M. Dressel, Coord. Chem. Rev., 2009, 253, 2315.
6 D.-L. Long, R. Tsunashima and L. Cronin, Angew. Chem.,
Int. Ed., 2010, 49, 1736.
12 (a) G. Absillis and T. N. Parac-Vogt, Inorg. Chem., 2012, 51,
9902; (b) K. Stroobants, E. Moelants, H. G. T. Ly, P. Proost,
K. Bartik and T. N. Parac-Vogt, Chem.–Eur. J., 2013, 19,
2848; (c) H. G. T. Ly, G. Absillis, S. R. Bajpe, J. A. Martens
and T. N. Parac-Vogt, Eur. J. Inorg. Chem., 2013, DOI:
10.1002/ejic.201300270.
7 J. J. Borras-Almenar, E. Coronado, A. Muller and
M. T. Pope, Polyoxometalate molecular science, Kluwer 13 A. Radzicka and R. Wolfenden, J. Am. Chem. Soc., 1996,
Academic publishers, Dordrecht, 2003. 118, 6105.
8 (a) K. Nomiya, Y. Sakai and S. Matsunaga, Eur. J. Inorg. 14 (a) M. Komiyama and T. Takarada, Met. Ions. Biol. Syst.,
Chem., 2011, 2011, 179; (b) M. Sokolov, E. Chubarova,
E. Peresypkina, A. Virovets and V. Fedin, Russ. Chem. Bull.,
2007, 56, 220; (c) L. E. Briand, G. T. Baronetti and
2003, 40, 355; (b) M. Yashiro, Y. Sonobe, A. Yamamura,
T. Takarada, M. Komiyama and Y. Fujii, Org. Biomol.
Chem., 2003, 1, 629.
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 10929–10938 | 10937

View Article Online
Paper
Dalton Transactions
15 M. Kassai, R. G. Ravi, S. J. Shealy and K. B. Grant, Inorg.
Chem., 2004, 43, 6130.
16 N. V. Kaminskaia and N. M. Kostić, Inorg. Chem., 2001, 40,
2368.
17 (a) L. Zhu and N. M. Kostić, Inorg. Chim. Acta, 2002, 339,
104; (b) D. P. Ašanin, S. Rajković, D. Molnar-Gabor and
M. I. Djuran, Monatsh. Chem./Chemical Monthly, 2004, 135,
1445; (c) N. M. Milović and N. M. Kostić, J. Am. Chem. Soc.,
2002, 124, 4759.
18 (a) M. C. B. de Oliveira, M. Scarpellini, A. Neves, H. Terenzi,
A. J. Bortoluzzi, B. Szpoganics, A. Greatti, A. S. Mangrich,
E. M. de Souza, P. M. Fernandez and M. R. Soares, Inorg.
Chem. Soc., 2008, 130, 17400; (e) L. V. Lokeren,
E. Cartuyvels, G. Absillis, R. Willem and T. N. Parac-Vogt,
Chem. Commun., 2008, 2774; (f) N. Steens, A. M. Ramadan
and T. N. Parac-Vogt, Chem. Commun., 2009, 965;
(g) E. Cartuyvels, K. Van Hecke, L. Van Meervelt, C. Görller-
Walrand and T. N. Parac-Vogt, J. Inorg. Biochem., 2008, 102,
1589; (h) T. Mihaylov, T. Parac-Vogt and K. Pierloot, Inorg.
Chem., 2012, 51, 9619.
28 (a) P. H. Ho, K. Stroobants and T. N. Parac-Vogt, Inorg.
Chem., 2011, 50, 12025; (b) P. H. Ho, T. Mihaylov,
K. Pierloot and T. N. Parac-Vogt, Inorg. Chem., 2012, 51,
8848.
Chem., 2005, 44, 921; (b) E. L. Hegg and J. N. Burstyn, 29 S. Vanhaecht, G. Absillis and T. N. Parac-Vogt, Dalton
J. Am. Chem. Soc., 1995, 117, 7015; (c) Y. Fujii, T. Kiss, Trans., 2012, 41, 10028.
T. Gajda, X. S. Tan, T. Sato, Y. Nakano, Y. Hayashi and 30 (a) G. Al-Kadamany, S. S. Mal, B. Milev, B. G. Donoeva,
M. Yashiro, J. Biol. Inorg. Chem., 2002, 7, 843.
19 M.-J. Rhee and C. B. Storm, J. Inorg. Biochem., 1979, 11, 17.
20 (a) A. M. Protas, A. Bonna, E. Kopera and W. Bal, J. Inorg.
R. I. Maksimovskaya, O. A. Kholdeeva and U. Kortz,
Chem.–Eur. J., 2010, 16, 11797; (b) O. A. Kholdeeva and
R. I. Maksimovskaya, J. Mol. Catal. A: Chem., 2007, 262, 7.
Biochem., 2011, 105, 10; (b) E. Kopera, A. Krezel, 31 (a) C. Jahier, S. S. Mal, U. Kortz and S. Nlate, Eur. J. Inorg.
A. M. Protas, A. Belczyk, A. Bonna, A. Wyslouch-Cieszynska,
J. Poznanski and W. Bal, Inorg. Chem., 2010, 49, 6636.
21 M. T. Bordignon Luiz, B. Szpoganicz, M. Rizzoto,
M. G. Basallote and A. E. Martell, Inorg. Chim. Acta, 1999,
287, 134.
22 T. Takarada, M. Yashiro and M. Komiyama, Chem.–Eur. J.,
2000, 6, 3906.
23 M. Kassai and K. B. Grant, Inorg. Chem. Commun., 2008, 11,
521.
Chem., 2010, 1559; (b) M. Carraro, N. Nsouli, H. Oelrich,
A. Sartorel, A. Soraru, S. S. Mal, G. Scorrano, L. Walder,
U. Kortz and M. Bonchio, Chem.–Eur. J., 2011, 17, 8371;
(c) S. S. Mal, N. H. Nsouli, M. Carraro, A. Sartorel,
G. Scorrano, H. Oelrich, L. Walder, M. Bonchio and
U. Kortz, Inorg. Chem., 2010, 49, 7.
32 (a) M. N. Sokolov, N. V. Izarova, E. V. Peresypkina,
D. A. Mainichev and V. P. Fedin, Inorg. Chim. Acta, 2009,
362, 3756; (b) Y. Saku, Y. Sakai and K. Nomiya, Inorg. Chim.
Acta, 2010, 363, 967.
24 (a) M. I. Djuran and S. U. Milinkovic, Polyhedron, 1999, 18,
3611; (b) T. N. Parac, G. M. Ullmann and N. M. Kostic, 33 C. N. Kato, A. Shinohara, K. Hayashi and K. Nomiya, Inorg.
J. Am. Chem. Soc., 1999, 121, 3127; (c) T. N. Parac and Chem., 2006, 45, 8108.
N. M. Kostic, J. Am. Chem. Soc., 1996, 118, 5946; 34 P. K. Glasoe and F. A. Long, J. Phys. Chem., 1960, 64, 188.
(d) T. N. Parac and N. M. Kostić, J. Am. Chem. Soc., 1996, 35 L. Cai, Y. Li, C. Yu, H. Ji, Y. Liu and S. Liu, Inorg. Chim.
118, 51; (e) T. N. Parac and N. M. Kostić, Inorg. Chem.,
1998, 37, 2141.
25 (a) J. W. Jeon, S. J. Son, C. E. Yoo, I. S. Hong and J. Suh,
Acta, 2009, 362, 2895.
36 G. M. Maksimov and R. I. Maksimovskaya, Polyhedron,
1996, 15, 4275.
Bioorg. Med. Chem., 2003, 11, 2901; (b) A. Krezel, E. Kopera, 37 (a) N. Dupre, P. Remy, K. Micoine, C. Boglio,
A. M. Protas, J. Poznanski, A. Wyslouch-Cieszynska and
W. Bal, J. Am. Chem. Soc., 2010, 132, 3355; (c) A. Krezel,
M. Mylonas, E. Kopera and W. Bal, Acta Biochim. Pol., 2006,
53, 721.
S. Thorimbert, E. Lacote, B. Hasenknopf and M. Malacria,
Chem.–Eur. J., 2010, 16, 7256; (b) C. Boglio, B. Hasenknopf,
G. Lenoble, P. Remy, P. Gouzerh, S. Thorimbert, E. Lacote,
M. Malacria and R. Thouvenot, Chem.–Eur. J., 2008, 14,
1532.
26 R. B. Martin, Met. Ions. Biol. Syst., 2001, 38, 1.
27 (a) N. Steens, A. M. Ramadan, G. Absillis and T. N. Parac- 38 S. Scheiner and W. N. Lipscomb, J. Am. Chem. Soc., 1977,
Vogt, Dalton Trans., 2010, 39, 585; (b) G. Absillis, R. Van 99, 3466.
Deun and T. N. Parac-Vogt, Inorg. Chem., 2011, 50, 11552; 39 (a) H. Sigel, Angew. Chem., Int. Ed. Engl., 1968, 7, 137;
(c) P. H. Ho, E. Breynaert, C. E. A. Kirschhock and T. N.
Parac-Vogt, Dalton Trans., 2011, 40, 295; (d) G. Absillis,
E. Cartuyvels, R. Van Deun and T. N. Parac-Vogt, J. Am.
(b) I. J. Grant and R. W. Hay, Aust. J. Chem., 1965, 18,
1189; (c) H. Sigel and R. B. Martin, Chem. Rev., 1982, 82,
385.
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