Peptide bond hydrolysis catalyzed by the Wells-Dawson Zr(α 2-P2W17O61)2 polyoxometalate

Article abstract of DOI:10.1021/ic301364n

In this paper we report the first example of peptide hydrolysis catalyzed by a polyoxometalate complex. A series of metal-substituted Wells-Dawson polyoxometalates were synthesized, and their hydrolytic activity toward the peptide bond in glycylglycine (GG) was examined. Among these, the Zr(IV)- and Hf(IV)-substituted ones were the most reactive. Detailed kinetic studies were performed with the Zr(IV)-substituted Wells-Dawson type polyoxometalate K ₁₅H[Zr(α₂-P₂W₁₇O ₆₁)₂]·25H₂O which was shown to act as a catalyst for the hydrolysis of the peptide bond in GG. The speciation of K ₁₅H[Zr(α₂-P₂W₁₇O ₆₁)₂]·25H₂O which is highly dependent on the pD, concentration, and temperature of the solution, was fully determined with the help of ³¹P NMR spectroscopy and its influence on the GG hydrolysis rate was examined. The highest reaction rate (k_obs = 9.2 (±0.2) × 10^-5 min^-1) was observed at pD 5.0 and 60 °C. A 10-fold excess of GG was hydrolyzed in the presence of K ₁₅H[Zr(α₂-P₂W₁₇O ₆₁)₂]·25H₂O proving the principles of catalysis. ¹³C NMR data suggested the coordination of GG to the Zr(IV) center in K₁₅H[Zr(α₂-P₂W ₁₇O₆₁)₂]·25H₂O via its N-terminal amine group and amide carbonyl oxygen. These findings were confirmed by the inactivity of K₁₅H[Zr(α₂-P₂W ₁₇O₆₁)₂]·25H₂O toward the N-blocked analogue acetamidoglycylglycinate and the inhibitory effect of oxalic, malic, and citric acid. Triglycine, tetraglycine, and pentaglycine were also fully hydrolyzed in the presence of K₁₅H[Zr(α₂- P₂W₁₇O₆₁)₂]·25H₂O yielding glycine as the final product of hydrolysis. K₁₅H[Zr(α ₂-P₂W₁₇O₆₁)₂] ·25H₂O also exhibited hydrolytic activity toward a series of other dipeptides.

Full text of DOI:10.1021/ic301364n

Article
pubs.acs.org/IC
Peptide Bond Hydrolysis Catalyzed by the Wells−Dawson
Zr(α2‑P2W17O61)2 Polyoxometalate
Gregory Absillis and Tatjana N. Parac-Vogt*
Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium
S
* Supporting Information
ABSTRACT: In this paper we report the first example of peptide hydrolysis
catalyzed by a polyoxometalate complex. A series of metal-substituted
Wells−Dawson polyoxometalates were synthesized, and their hydrolytic
activity toward the peptide bond in glycylglycine (GG) was examined.
Among these, the Zr(IV)- and Hf(IV)-substituted ones were the most
reactive. Detailed kinetic studies were performed with the Zr(IV)-substituted
Wells−Dawson type polyoxometalate K15H[Zr(α2-P2W17O61)2]·25H2O
which was shown to act as a catalyst for the hydrolysis of the peptide
bond in GG. The speciation of K15H[Zr(α2-P2W17O61)2]·25H2O which is
highly dependent on the pD, concentration, and temperature of the solution,
was fully determined with the help of 31P NMR spectroscopy and its
influence on the GG hydrolysis rate was examined. The highest reaction rate
(kobs = 9.2 ( 0.2) × 10−5 min−1) was observed at pD 5.0 and 60 °C. A 10-
fold excess of GG was hydrolyzed in the presence of K15H[Zr(α2-P2W17O61)2]·25H2O proving the principles of catalysis. 13C
NMR data suggested the coordination of GG to the Zr(IV) center in K15H[Zr(α2-P2W17O61)2]·25H2O via its N-terminal amine
group and amide carbonyl oxygen. These findings were confirmed by the inactivity of K15H[Zr(α2-P2W17O61)2]·25H2O toward
the N-blocked analogue acetamidoglycylglycinate and the inhibitory effect of oxalic, malic, and citric acid. Triglycine, tetraglycine,
and pentaglycine were also fully hydrolyzed in the presence of K15H[Zr(α2-P2W17O61)2]·25H2O yielding glycine as the final
product of hydrolysis. K15H[Zr(α2-P2W17O61)2]·25H2O also exhibited hydrolytic activity toward a series of other dipeptides.
additional organic solvents and additives transition metal-
substituted POMs were efficient catalysts for cyclic carbonate
synthesis.12 The use of the iron-substituted [Fe4(H2O)10(β-
XW9O33)2]n− complex as an inorganic alternative to organic
and organometallic coordination complexes for the bioinspired
oxygenation of catechols was demonstrated in 2009.13 Water
splitting was achieved in the presence of a tetraruthenium
[(Ru4O4(OH)2(H2O)4(γ-SiW10O36)2]10− complex.14 H2O2-
based oxidation of organic substrates was observed in the
presence of Zr(IV),15−17 Hf(IV),17 and Ti(IV)16 substituted
POMs. Furthermore, Zr(IV)18,19 and Hf(IV)19 based POMs
were reported as catalysts for the oxidation of sulfides. Metal-
substituted POMs were also proven to be efficient catalysts for
Lewis-acid mediated reactions. In 2006 the Wells−Dawson
polyoxotungstate (TBA)5H2[α1-Ln(H2O)4-P2W17O61] (Ln =
La, Sm, Eu, and Yb) was shown to be an active catalyst for aldol
reactions of imines.20 An increase in Lewis acidity compared to
the Ln analogues was observed for (TBA)5K[α1-Hf(H2O)4-
P2W17O61] in Mukaiyama aldol and Mannich-type additions.21
Efficient chemo- and diasterioselective Lewis acid catalysis was
observed for the cyclization of citronellal derivatives by the
dialuminium substituted silicotungstate (TBA)3H[γ-
SiW10O36(Al(OH)2)(μ-OH)2].22 This study was extended to
INTRODUCTION
■
Polyoxometalates (POMs) are a versatile class of metal−oxygen
clusters endowed with a diverse range of chemical and physical
properties that can be easily modified.1 Their size, shape,
composition, charge density, solubility, redox potential, and
acid strength can be tuned depending on the type of
application. As a consequence they are widely studied in
different scientific domains such as catalysis, material science,
and medicine.2,3 Their strong Brønsted acidity and rich redox
chemistry enable POMs to act as homogeneous and
heterogeneous acid, redox, and bifunctional catalysts.4 Whereas
early research was traditionally focused on Brønsted acid
catalysis and selective oxidation by nonsubstituted POMs,
nowadays the use of metal-substituted POM derivatives as
catalysts has become a quickly emerging field. By replacing one
or more addenda atoms by a specific metal center, a POM with
a customized coordination chemistry and reactivity is obtained.
The most commonly studied type of reactions are H2O2-based
epoxidation and sulfoxidation catalyzed by metal-substituted
POMs.5−7 For example, the di-iron substituted Keggin type
silicotungstate [γ-SiW10(Fe(OH2))2O38]6−, which is structurally
related to the active site of methane monooxygenase, was
shown to catalyze the selective epoxidation of olefins and
alkanes with high H2O2 efficiency.8,9 Another commercially
important reaction is the synthesis of cyclic carbonates from
CO2 and epoxide.10,11 Studies indicated that without the use of
Received: June 26, 2012
© XXXX American Chemical Society
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tetranuclear Zr(IV) and Hf(IV) sandwich type silicotung-
states.23
In our recent study we have shown that incorporation of
Zr(IV) into the Wells−Dawson POM resulted in a catalyst for
the homogeneous hydrolysis of phosphoesters.45 In this study
we further exploit the reactivity of Zr(IV)-POMs and report on
a conceptually new approach toward the development of
artificial peptidases by using the Wells−Dawson POM as a
ligand for Zr(IV). We envision that the use of a POM as a
chelating agent for Zr(IV) will not only result in a
homogeneous reaction mixture, but may also benefit from the
protein binding properties of the POM in future experiments
involving the hydrolysis of proteins. To explore the potential of
this novel type of reactivity, in this paper we report on the
hydrolysis of several peptides and present a detailed kinetic
study of glycylglycine hydrolysis by the previously reported
compound K15H[Zr(α2-P2W17O61)2]·25H2O46 (1).
Within our lab the use of POMs as catalysts for the
hydrolysis of biologically relevant molecules was established
some years ago. It was shown that isopolyoxomolybdates and
isopolyvanadates were able to hydrolyze the phospho(di)ester
bonds in a series of DNA- and RNA-model compounds.24−30
The hydrolysis of carboxyl esters has also been achieved in the
presence of oxovanadates.31 In our recent studies we also show
that peptides with a X-Ser sequence were effectively hydrolyzed
in oxomolybdate and oxovanadate solutions.32 Although
hydrolysis occurred in solutions containing polyoxo forms of
molybdate and vanadate, the kinetic experiments and density
functional theory (DFT) calculations revealed that the actual
active species are the monomeric oxoanions.32
In our quest to further exploit POMs as catalysts for peptide
bond hydrolysis we examined the reactivity of metal-function-
alized Wells−Dawson POMs toward the dipeptide glycylgly-
cine. Metal-functionalized POMs have been shown to interact
specifically with the surface of proteins, for example, the
molecular interaction with albumin proteins has been studied in
detail.33 However, to the best of our knowledge the hydrolysis
of the peptide bonds in the presence of POMs has not been
reported so far.
EXPERIMENTAL SECTION
■
Materials. The metal-substituted POMs were synthesized accord-
ing to the procedure described in literature.46−48 Glycylglycine, glycine
anhydride, glycylglycyl amide, triglycine, tetraglycine, and pentaglycine
were purchased from Sigma-Aldrich or Bachem and were used without
any further purification. Acetamidoglycylglycinate49 and ethyl
glycylglycinate50 were synthesized according to the procedure
described in literature.
Sample Preparation. 2.0 mM dipeptide and 2.0 mM K15H[Zr(α2-
P2W17O61)2]·25H2O were dissolved in D2O and the pD value was
adjusted with DCl and NaOD. The pH-meter reading was corrected
by the equation: pD = pH-meter reading + 0.41.51 K15H[Zr(α2-
P2W17O61)2]·25H2O was not allowed to reach its equilibrium
distribution before the addition of the dipeptide. The reaction was
The hydrolysis of the peptide bond, which is one of the most
important and most required procedures in biotechnology, is a
challenging task. At pH 6.8 and 25 °C, the half-life for amide
bond hydrolysis in the dipeptide glycylglycine is estimated to be
around 350 years.34 Proteolytic enzymes and chemical reagents
are currently used to effect protein cleavage in proteomics
applications.35 However, they both suffer from some serious
shortcomings. Proteolytic enzymes tend to cleave the protein at
too many sites resulting in the formation of fragments which
are too short to be accurately assigned to the protein in
question. Furthermore, they contaminate protein digests and
often require denaturation of the protein which leads to a loss
of structural information. Chemical agents, among which
cyanogen bromide is the best known, usually operate under
harsh conditions, have to be used in a high molecular excess,
give low yields, and only exhibit partial selectivity. Clearly, new
chemical reagents with improved selectivity and efficiency are
needed to comply with the increasing demand in the fields of
proteomics and biotechnology. Although several metal ions and
complexes, including Pd(II),36 Pt(II),37 Ni(II),38 Cu(II),39
Zr(IV),40 Zn(II),41 Co(III),42 and Ce(IV)43 have been
reported to promote the hydrolytic cleavage of unactivated
amide bonds in peptides and proteins, the number of metal
complexes that are able to catalyze peptide bond hydrolysis
remains scarce.
The high Lewis acidity of Zr(IV) imparted by the +IV
oxidation state, combined with its oxophilicity, kinetic lability,
fast ligand-exchange kinetics, and formation of complexes with
flexible geometries and high coordination numbers, makes
Zr(IV) especially suitable for the hydrolysis of the amide bond
in peptides and proteins. However, above pH 5.0, aqueous
solutions of Zr(IV) tend to form insoluble gels and precipitates,
often resulting in low hydrolysis yields.43 To overcome this
problem complexation of Zr(IV) with the azacrown ether 4,13-
diaza-18-crown-6 was used in studies reported by Grant and co-
workers.40,44 This resulted in faster peptide bond hydrolysis,
however, despite the reactivity increase, the formation of
insoluble gels was still observed.
1
kept at 60 °C and followed at different time increments by H NMR.
Between consecutive measurements, the NMR tube was kept at 60 °C.
After complete hydrolysis of the dipeptide, the pD was measured
again. Typically, a value pDinitial 0.2 was obtained. The decrease of
the percentage of the dipeptide was fitted to a monoexponential
function, and the error was calculated to be in the range of 2−7%.
NMR Spectroscopy. 1H NMR spectra were recorded on a Bruker
Avance 400 (SW = 12.11 ppm, TD = 32768) and Bruker Avance 600
(SW = 20.55 ppm, TD = 65536) spectrometer. As a reference 0.5 mM
3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid was present in the reaction
mixture. 13C NMR spectra were recorded on a Bruker Avance 400
(100.61 MHz, SW = 238.90 ppm, TD = 32768) and a Bruker Avance
600 (150.90 MHz, SW = 238.92 ppm, TD = 32768) spectrometer. As
a reference 1% TMS in CDCl3 in an internal reference tube was used.
31P NMR spectra were recorded on a Bruker Avance 400 (161.98
MHz, SW = 399.62, TD = 65536) and a Bruker Avance 600 (242.94
MHz, SW = 395.82 ppm, TD = 65536) spectrometer. As a reference
25% H3PO4 in D2O in an internal reference tube was used. The
spectra were recorded at 298 K on a 5 mm PABBI 1H-BB Z-GRD and
BBO 600 MHz S3 5 mm with Z-gradient; BTO probe for respectively
the 400 and 600 MHz instrument.
RESULTS AND DISCUSSION
■
Metal-Substituted Wells−Dawson POMs. Several metal-
substituted Wells−Dawson type POMs containing Mn(III),
Fe(III), Co(II), Ni(II), Cu(III), Y(III), La(III), Eu(III),
Yb(III), Zr(IV), and Hf(IV) were synthesized, and their
reactivity toward peptide bond hydrolysis of glycylglycine (GG)
was examined. After prolonged reaction times and in the
presence of an excess of the metal-substituted POM only
POMs containing Zr(IV) and Hf(IV) showed significant
activity (1:1 POM:GG, 2.0 mM, 50 °C, pD 7.2 for Y(III),
La(III), Eu(III), Yb(III), Zr(IV), Hf(IV) − 10:1 POM:GG 2.0
mM, 50 °C, pD 5.50 for Mn(III), Fe(III), Co(II), Ni(II),
Cu(III)). The lack of reactivity observed for the POMs
containing first row transition metal ions (Mn(III), Fe(III),
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Co(II), Ni(II), and Cu(III)) can be explained by the
coordination environment of the metal ion.47,52 In these
POMs the metal ion has a 6-fold octahedral coordination
environment in which the polyoxometalate ligand acts as a
pentadentate ligand. The sixth coordination site is occupied by
water which can be substituted by other ligands.47,52 However,
previous studies have shown that effective hydrolysis of
peptides usually requires the presence of at least two free
coordination sites on the metal center: one for the attachment
of the peptide and the other for the internal delivery of the
water molecule which acts as an effective nucleophile.53 In
contrast to first row transition metals, rare earth ions are
characterized by higher coordination numbers and after
incorporation into the Wells−Dawson POM more free
coordination sites around the metal ion are present. However,
despite this fact, the Y(III), La(III), Eu(III), and Yb(III)
substituted POMs showed low activity toward GG hydrolysis.
This might be due to a decreased Lewis acidity resulting from
incorporation into the POM structure20 or due to the
formation of dimeric POM species in solution.48 On the
other hand, Zr(IV) and Hf(IV) were active toward hydrolysis
of the peptide bond in GG. At pD 7.2 and 50 °C 2.0 mM of
glycylglycine was fully hydrolyzed in the presence of 2.0 mM of
the Zr(IV)- and Hf(IV)-substituted Wells−Dawson type POM.
Therefore the Zr(IV)-substituted Wells−Dawson POM K15H-
[Zr(α2-P2W17O61)2]·25H2O (1) was selected to explore in
detail this novel type of reactivity.
Aqueous Solution Behavior of 1. Incorporation of
Zr(IV) into the monolacunary α2-Wells−Dawson POM results
in its coordination to four oxygen atoms in the vacancy of the
POM. In contrast to other transition metal ions which are
usually tetra- or hexa-coordinated, Zr(IV) shows higher
coordination numbers. As a result, incorporation of Zr(IV)
into the Wells−Dawson type POM results in the formation of a
1:2 species in which Zr(IV) is 8-coordinated. The singe crystal
X-ray structure of 1 shows that one Zr(IV) ion coordinates two
Wells−Dawson units.46 This species is expected to have low
catalytic activity in solution because of the absence of water
molecules that can be replaced by the substrate in the
coordination sphere of Zr(IV). However, when the 1:2 species
is dissolved in water multiple equilibria between the dimeric
2:2, monomeric 1:1, and sandwich type 1:2 Zr(IV):POM
complexes are observed, depending on the pH, temperature,
and concentration (Scheme 1).54−56 Among all the species
present in solution, the 1:1 monomer is anticipated to have the
most favorable catalytic properties because of several free
coordination sites that are available for interaction with the
substrate.57
Since all POMs shown in Scheme 1 are characterized by a
specific 31P NMR resonance, the species distribution at
different solution conditions can be monitored by 31P NMR
spectroscopy. As can be seen from Figure 1 and Supporting
Figure 1. pD dependence of the distribution of 2.0 mM of 1 (1:2, solid
squares, and 1:1, solid circles) obtained by 31P NMR recorded
immediately after mixing and pH adjustment.
Information, Figure S1 a decrease in the amount of 1:2
(characterized by 31P NMR signals at −9.34 ppm and −13.95
ppm)46 is observed when the pD is lowered. While at pD 7.0
the 1:2 sandwich type is the only species present in solution, at
pD 3.0 the 1:1 monomer (characterized by 31P NMR signals at
−10.04 ppm and −13.76 ppm)56 and the 1:2 species are
present in a 70:30 ratio. At pD 1.5 only the 1:1 monomer is
present. These data are consistent with a study performed by
Nomiya et al.54 In that study it was concluded that the
acidification of an aqueous solution containing the 1:2
compound resulted in the formation of the 2:2 or 1:1 species,
as well as the saturated Wells−Dawson POM [α-P2W18O62]6−.
The latter species was also observed in our study, and it was
characterized by a single 31P NMR resonance at −12.85 ppm. It
can be concluded that lower pD values are expected to be
beneficial for the catalytic activity of 1 since they favor the 1:1
species over the 1:2 complex. In the following step the
influence of the starting concentration of 1 on the species
distribution was investigated. Supporting Information, Figure
S2 and Table S1 show the species distribution as a function of
the initial concentration of 1 at pD 5.0. For a 10.0 mM solution
of 1 only the 31P NMR resonances of the 1:2 compound were
observed. However, the presence of [α-P2W18O62]6− implies
that a small fraction of the 1:2 compound is converted into the
1:1 species. While [α-P2W18O62]6− is characterized by two
chemically equivalent P atoms, the 1:1 species consists of 2
nonequivalent P atoms resulting in a lower signal intensity and
will therefore not be detected at low concentrations. While for
an initial concentration of 2.0 mM of 1, the 1:2 and 1:1 species
were present in a 80:20 ratio, the inverse (20:80) is observed
for a 0.5 mM solution. Therefore it can be concluded that low
concentrations of 1 result in higher fractions of the desired 1:1
monomer. Samples containing 2.0 mM and 10.0 mM of 1 were
kept at pD 5.0 and 60 °C for a prolonged period to investigate
the species distribution as a function of time. Surprisingly, for
the 2.0 mM sample, no 1:1 monomer was detected after 1 day.
Instead, two resonances corresponding to the 2:2 species were
Scheme 1. Equilibria between the 2:2, 1:1, and 1:2
Zr(IV):POM Species
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observed. Over the course of several days the intensity of these
signals increased, while the intensity of the 1:2 resonances
decreased. Supporting Information, Table S2 summarizes the
species distribution both for 2.0 mM and 10.0 mM of 1 as a
function of reaction time at 60 °C. The data suggest that for
both a 2.0 mM and 10.0 mM solution the equilibrium
distribution between the 2:2 dimer and 1:2 species is obtained
after 2 days at 60 °C. Interestingly, for the 2.0 mM solutions
both species are present in a 50:50 ratio, while for 10.0 mM the
2:2 and 1:2 species are distributed in a 20:80 ratio. As the
hydrolysis of peptide was monitored at elevated temperatures,
the effect of temperature on the POM species distribution of a
2.0 mM solution of 1 at pD 5.0 was also examined (Supporting
Information, Figure S3). At temperatures below 50 °C the 1:1
monomer and 1:2 species were detected. Above 70 °C only the
2:2 dimer and the 1:2 complex were present. For the 60 °C
sample an intermediate distribution was observed. A chemical
shift difference in between that of the 1:1 monomer and 2:2
dimer was obtained. The broadening of the resonance signal at
−13.81 ppm confirmed the interchange between the 1:1 and
2:2 species.
occurs.43,58 Indeed, upon adding glycine anhydride (cGG) to
the reaction mixture, an intensity increase of the resonance at
4.04 ppm was observed. This undoubtedly demonstrates that
cyclization of GG also occurred in the presence of 1. Figure 3
Figure 3. Hydrolysis of 2.0 mM of GG in the presence of 2.0 mM of 1
at pD 5.0 and 60 °C. Percentage of GG (solid squares), G (solid
circles), and cGG (solid triangles) as a function of reaction time.
Hydrolysis of Gly-Gly (GG). GG was fully hydrolyzed to
glycine (G) in the presence of equimolar amounts of 1 at pD
1
5.0 and 60 °C. The reaction was followed by recording H
NMR spectra (Figure 2) at different reaction times. A gradual
shows the percentage of GG, G, and cGG as a function of
reaction time, calculated on the basis of the intensity of their 1H
NMR resonances. As can be seen from Figure 3, the amount of
cGG reaches a maximum of 7.50%, before gradually decreasing
to zero. However, since the kinetic experiments were
performed immediately upon mixing 1 with GG it is possible
that part of the cGG formation is correlated to the equilibration
process of 1.
No precipitation or turbidity was observed in any of the
reactions with 1, indicating that the Wells−Dawson POM
ligand is able to stabilize Zr(IV) in aqueous solutions. The
observed rate constant (kobs) for the hydrolysis of GG was
calculated by fitting the increase in the concentration of G to a
monoexponential function. Its value, kobs = 9.2 ( 0.2) × 10−5
min−1 (t1/2 = 5 days) represents a significant acceleration
compared to the blank reaction. In the absence of 1 no
hydrolysis of the amide bond in GG was observed after two
months at 60 °C. Therefore it is difficult to precisely calculate
the rate acceleration compared to the uncatalyzed cleavage.
However, based on literature data, a half-life of about 5 years
was estimated for the uncatalyzed reaction under similar
conditions.34 No hydrolysis was observed in the presence of the
lacunary [α2-P2W17O61]10−, implying that the presence of
Zr(IV) is essential for the hydrolytic activity. The hydrolysis of
2.0 mM of GG in the presence of 2.0 mM of ZrCl4 was also
studied at pD 5.0 and 60 °C and while after about 4 days free
1
glycine was observed in H NMR, the data were difficult to
quantify as the reaction mixture was not homogeneous because
of gel formation. This is consistent with the previous studies
investigating the aqueous solution behavior of Zr(IV) in which
the formation of polymeric [Zr8(OH)20(H2O)24]12+ species
upon dissolving Zr(IV) in aqueous solutions was observed.59
The disappearance of the cGG peak suggests that 1 promotes
its formation but also its hydrolysis. The reversibility of
diketopiperazine formation was examined by following the
hydrolysis of 2.0 mM of cGG (Supporting Information, Figure
S4) in the presence of 2.0 mM of 1 at pD 5.0 and 60 °C. As can
be seen from Supporting Information, Figure S5 the addition of
1 to cGG resulted in complete conversion of cGG into G.
1
Figure 2. H NMR spectra recorded at various reaction times during
the hydrolysis of 2.0 mM of GG in the presence of 2.0 mM of 1 at pD
5.0 and 60 °C. (A) After mixing, (B) after 14 days, (C) after 24 days
and (D) 23 days.
decrease of the two GG resonances at 3.82 ppm and 3.89 ppm
and an increase of the G resonance at 3.56 ppm were observed
over time. In addition, a fourth resonance at 4.04 ppm was
detected in the course of the hydrolytic reaction. Several studies
reporting the hydrolysis of dipeptides demonstrated that in
addition to amide bond hydrolysis, diketopiperazine formation
D
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However, an induction period for the formation of G was
observed (inset Supporting Information, Figure S5) as
hydrolysis to G could only be observed after sufficient amounts
of GG were formed, suggesting that GG acts as an intermediate
in the conversion of cGG to G. This is not surprising since
hydrolysis of cGG to G would require the breaking of two
chemical bonds at the same time. As it was the case for GG, no
hydrolysis in the presence of [α2-P2W17O61]10− was detected.
Additionally, no conversion to GG, followed by hydrolysis to G,
was observed in the absence of 1. On the basis of these
observations, we propose the reaction scheme presented in
Scheme 2.
(Supporting Information, Figure S8) allows for the calculation
of the enthalpy of activation, ΔH⧧ = 96.14 kJ mol−1, and
entropy of activation, ΔS⧧ = −33.53 J mol−1 K−1. At 60 °C the
Gibbs free energy of activation is 107.31 kJ mol−1. The negative
value of ΔS⧧ is the result of the coordination of GG to the
metal center.
Hydrolysis of Gly-Gly Derivatives. The influence of 1 on
the hydrolysis of several GG derivatives was investigated to get
more insight in the binding mode of GG to 1. For this purpose
GG derivatives that were modified at the N- and C-terminal
end were prepared. Figure 4 gives an overview of the substrates
that were investigated.
Scheme 2. Hydrolysis of GG in the Presence of 1
Figure 4. (A) acetamidoglycylglycinate (AcGG), (B) ethyl glycylgly-
cinate (GGOEt), and (C) glycylglycyl amide (GGNH2).
1H NMR spectra of a solution containing 2.0 mM of
acetamidoglycylglycinate (AcGG) in the presence of 2.0 mM of
1 did not show any evidence of amide bond hydrolysis after 7
days at pD 5.0 and 60 °C. This suggests that the free amino-
terminus is of importance for the interaction of the substrate
with the POM. The interaction may occur via coordination to
the metal center or via secondary interactions of the protonated
amino group with the negatively charged surface of 1. When
ethyl glycylglycinate (GGOEt) was used, full hydrolysis of the
ethyl ester bond was observed within a few hours prior to
1
The effect of salt ions on kobs was investigated by adding LiCl
to a reaction mixture containing 2.0 mM of GG and 2.0 mM of
1. Supporting Information, Figure S6 shows that upon adding
increasing amounts of LiCl the hydrolysis rate constant
decreased. In the presence of 1.0 M of LiCl, a 3-fold decrease
in kobs was observed. This results in an increase in half-life from
5 to 15 days. The presence of LiCl could influence the POM
speciation or affect the electrostatic interaction between 1 and
GG. To differentiate between these two possibilities, the
speciation of 1 in the presence of LiCl was further examined.
31P NMR results show that increasing the LiCl concentration
up to 1.0 M did not change the solution speciation of 1 and
point toward the importance of electrostatic interactions for the
effective hydrolysis of GG.
The reaction between 2.0 mM of GG and 2.0 mM of 1 was
also studied at various temperatures at pD 5.0 (Supporting
Information, Figure S7). Increasing the reaction temperature
from 37 to 80 °C resulted in a 100-fold increase of the reaction
rate constant and a decrease of the half-life to 0.5 days. When
the data in Supporting Information, Figure S7 were fitted to the
Arrhenius equation both the pre-exponential factor, 1.6
( 0.4)·1013 min−1, and the experimentally determined
activation energy, 91.0 ( 0.7) kJ mol−1, were obtained.
Comparison of the obtained activation energy to the one in
the absence of any catalyst is difficult since very few reports on
the activation energy for hydrolysis of dipeptides are available.
However, high temperature studies on the natural hydrolysis of
GG at near neutral pH resulted in an estimation of 96 kJ mol−1
for the conversion of GG to G+G.34 This implies that in the
presence of 1 the activation energy for the hydrolysis of GG is
lowered by 3−5 kJ mol−1. Linear fitting of ln(kobs/T) as a
function of the reciprocal temperature to the Eyring equation
amide bond hydrolysis. H NMR spectra clearly indicate the
formation of ethanol in the beginning of the reaction. Once all
GGOEt was converted to GG a similar hydrolysis profile to that
of GG was observed (Supporting Information, Figure S9). In
the absence of 1, no hydrolysis of both the ester and the amide
bond was observed after 5 days at 60 °C and pD 5.0. For
glycylglycyl amide (GGNH2), hydrolysis of the C-terminal
amide functionality resulted in the formation of GG. This
process was slow compared to ester bond hydrolysis in GGOEt,
and as can be seen from Supporting Information, Figure S10
hydrolysis of GG occurred before all GGNH2 was converted
into GG. No free glycine amide (GNH2) was detected during
the course of the reaction indicating that the hydrolysis of the
terminal G-NH2 amide bond occurs preferentially to internal
G-G amide bond hydrolysis in GGNH2.
Peptide Binding to 1. 13C NMR studies (Figure 5 and
Table 1) performed on the mixture of GG with 1 suggest that
both carbonyl functionalities of GG interact with 1, with the
largest shift being observed for the amide carbonyl. This is
Figure 5. 13C NMR spectrum of GG in the presence (*) and absence
(°) of 1.
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Table 1. 13C NMR Chemical Shift Values (ppm) of GG in
the Presence and Absence of 1
30.0 mM GG
10.0 mM GG + 10.0 mM 1
Δ(ppm)
C1(CO)
C3(CO)
C2(CH2)
C4(CH2)
176.30
166.93
43.23
176.42
167.14
43.36
0.12
0.16
0.13
0.28
40.58
40.86
important since coordination of the amide carbonyl to Zr(IV)
facilitates amide bond hydrolysis by activating the amide carbon
toward nucleophilic attack by water molecules. As a result of
this coordination the 13C NMR resonance of the methylene
group adjacent to the amide carbonyl experienced the largest
shift. This shift is also consistent with the observation that the
N-terminal amine functionality plays on important role in the
coordination, and is consistent with the lack of hydrolysis
observed in AcGG, a N-terminal blocked derivative of GG. The
C-terminal carbonyl carbon also experienced a shift. However
this shift appears not to be the result of interaction of GG with
the Zr(IV) center in 1. Coordination of GG via its amine
nitrogen and carbonyl amide oxygen renders coordination of
the C-terminal carbonyl group sterically impossible because of
the geometrical constraints imposed by the energetically
favored trans configuration of the amide bond. The observed
shift is most likely the result of a concentration effect observed
for the 13C NMR shift values of a pure GG solution. When the
13C NMR shifts of a 10.0 mM solution of GG were compared
to those of a 30.0 mM solution of GG all carbon atoms had
identical chemical shift values except for the C-terminal
carbonyl carbon. A difference of 0.12 ppm, similar to the one
in the presence of 1, was observed and is most likely caused by
the intermolecular hydrogen bonding that occurs in peptide
solutions.60 These observations led us to propose that the
coordination of Zr(IV) occurs via the amine nitrogen atom and
amide oxygen. In a next step the binding of AcGG to 1 was
examined. As can be seen from Supporting Information, Figure
S11 and Table S3, only the resonances corresponding to the C-
terminal carboxyl group and the adjacent methylene group are
significantly shifted. In contrast to GG, no coordination of the
amide carbonyl is observed. This mode of coordination is not
favorable for the activation of the amide bond toward
hydrolysis and may explain the lack of reactivity of 1 toward
AcGG.
Figure 6. pD dependence of the rate constant for the hydrolysis of 2.0
mM of GG in the presence of 2.0 mM of 1 at 60 °C (solid squares).
lowered from 7.0 to 0.5. In the 1:1 species Zr(IV) is
coordinated to 3 water molecules that can be replaced by
GG. This makes the 1:1 species structurally most suitable for
catalytic activity, and therefore an increase of its concentration
results in a rate constant increase. However, at pD values lower
than 5.0, where the 1:1 species is becoming the most abundant
species, a reaction rate decrease is observed. This can be
explained by the fact that coordination of the N-terminal amine
group to the Zr(IV) center in GG requires the Zr(IV)-assisted
deprotonation of the amine group. Since this process is
impeded when the pD is decreased, a weaker interaction
between GG and 1 is expected at lower pD-values resulting in a
decrease of kobs. Since the maximum rate was observed at pD
5.0, all further experiments were performed at this pD.
Catalytic Turnover. Figure 7 shows the influence of the
concentration of 1 on the rate constant for the hydrolysis of 2.0
For GGOEt the biggest shifts are observed for the amide
carbonyl and the adjacent methylene group (Supporting
Information, Figure S12 and Table S4). Coordination of the
ester carbonyl oxygen atom explains the ester bond hydrolysis
observed during the first hours of reaction. Despite the smaller
shift after the addition of 1, ester bond hydrolysis proceeded at
a significantly higher reaction rate compared to amide bond
hydrolysis. This is because ester bonds are more kinetically
labile and more susceptible toward hydrolysis compared to
peptide bonds.
pD Dependence of kobs. The pD dependence of the GG
hydrolysis rate constant (Figure 6) shows a bell-shape profile
with a maximum at pD 5.0. When the pD was decreased from
8.0 to 5.0, an increase in kobs was observed, while below pD 5.0
a decrease of kobs was evidenced. This bell-shape like behavior is
a typical sign of two opposing effects influencing kobs. The
reaction rate increase observed can be related to the aqueous
solution species distribution diagram shown in Figure 1. It was
shown that the 1:1 monomer content increased when the pD is
Figure 7. Influence of the concentration of 1 on the rate constant for
the hydrolysis of 2.0 mM of GG at pD 5.0 and 60 °C.
mM of GG at pD 5.0 and 60 °C. These concentration studies
revealed that 1 is not only able to significantly accelerate GG
hydrolysis, but is also capable of hydrolyzing a larger molar
excess of GG. In the presence of 0.2 mM of 1, 2.0 mM of GG
was completely hydrolyzed to G. Although modest, this
turnover of 10 proves the principles of catalysis. The highest
rate constant was obtained when GG and 1 were present in a
1:1 (2.0 mM) ratio. When more than 2.0 mM of 1 was used, a
decrease in kobs was observed. This finding can be explained
keeping in mind both the concentration and the time
F
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dependence of the equilibria. For a 10.0 mM sample of 1, only
the 1:2 species was present after dissolving 1 and pD
adjustment to pD 5.0. However, when the sample was kept
at 60 °C for several days, the 2:2 dimer was formed in a 20:80
ratio compared to the 1:2 species. For the 2.0 mM sample on
the other hand, 20% of the 1:1 monomer and 80% of the 1:2
species were present after dissolving and pD adjustment to pD
5.0, and a 50:50 ratio of the 2:2 dimer compared to the 1:2
species was observed after a few days at 60 °C. Therefore, the
decrease in concentration of the 2:2 dimer, in which Zr(IV) has
free coordination sites available for the binding of GG, explains
slower reaction rates observed upon increasing the concen-
tration of 1 from 2.0 mM to 10.0 mM.
Inhibition Studies. To gain more insight into the
coordination of GG to 1, the hydrolysis of GG in the presence
of several nonreactive substrate analogues was examined. To
achieve efficient coordination of the inhibitor with the metal
center, dicarboxylic acids with varying aliphatic chain lengths
were chosen as candidates for inhibition experiments. In
addition, malic and citric acid were also examined. These
inhibitors are known to act as multidendate ligands for many
metal ions (Figure 8).61
largest effect was observed in the presence of oxalic acid, where
no hydrolysis was observed. While malonic and glutaric acid
strongly inhibited hydrolysis, the inhibition effect was much
smaller in the presence of succinic and adipic acid.
Coordination of the three carboxylate groups of citric acid to
Zr(IV) most likely prevents the binding and Lewis acid
activation of GG. For the dicarboxylic acids, it was assumed that
when the aliphatic chain length was increased, the binding
affinity of the inhibitor decreased most likely because of the
formation of a larger, more flexible chelate ring. However, the
hydrolysis rate constant measured in the presence of glutaric
acid did not follow the proposed trend. When the structure of
succinic acid is compared to that of malic acid, it can be seen
that the only difference is the presence of an additional
hydroxyl group in malic acid. The presence of this hydroxyl
group is apparently responsible for the significant difference in
inhibitory effect observed between these two acids. The
tridendate coordination between malic acid and the 1:1
monomer was earlier observed by Fedin et al.56 This study
shows that carbonyl groups efficiently bind to the Zr(IV) center
in 1. This is important since the coordination of GG to 1 and
its activation toward nucleophilic attack is mainly governed by
carbonyl group interaction.
Hydrolysis Mechanism. 13C NMR interaction studies and
inhibition experiments show that the amide carbonyl plays a
vital role in the coordination of GG to the Zr(IV) ion in 1.
Coordination of the amide carbonyl leads to Lewis acid
activation of the amide bond and makes it more susceptible
toward nucleophilic attack by water resulting in the hydrolysis
of GG (Scheme 3). Both the attack of coordinated water, which
Scheme 3. Mechanism for the Hydrolysis of GG in the
Presence of 1: Nucleophilic Attack of Solvent Water (Left)
and Coordinated Water (Right)
Figure 8. Chemical structure of the inhibitors used in this study.
Table 2 gives an overview of the rate constants for the
hydrolysis of GG in the presence of the inhibitor. Malic and
citric acid fully inhibited the hydrolysis of GG, while for the
dicarboxylic acids a decrease of the reaction rate constant was
observed depending on the length of the aliphatic chain. The
is an internal nucleophile, and that of solvent water are possible
and these two mechanisms are kinetically indistinguishable.35
The N-terminal amine group is considered as the second
coordinating entity, which seems to be essential for the effective
attachment of the peptide as the N-blocked analogue AcGG
was not hydrolyzed in the presence of 1.
Hydrolysis of Other Dipeptides. The reactivity of 1
toward other dipeptides with a Gly-X sequence was examined,
where X = Ala, Val, Leu, Ile, Phe, Ser, Thr, Tyr, Glu, Gln, Asn,
Asp, His, Lys, Arg, Met, and Cys. All dipeptides were fully
hydrolyzed, and we are currently investigating the dependence
of the rate constant on the nature of the X side chain. For
example Gly-Ser was hydrolyzed at a significantly higher rate
(kobs = 2.08 × 10−4 min−1 at pD 5.0 and 60 °C) as compared to
Gly-Gly, because of an intramolecular N→O acyl rearrange-
ment which leads to peptide bond hydrolysis.41 Dipeptides
containing bulky residues such as Gly-Leu were hydrolyzed
much slower (kobs = 8.56 × 10−6 min−1) presumably because of
Table 2. Hydrolysis of 2.0 mM of GG in the Presence of 2.0
mM of 1 and 20.0 mM of the Inhibitor at pD 5.0 and 60 °C
inhibitor
no. of CH2 groups
kobs (min−1
)
no inhibitor
oxalic acid
malonic acid
succinic acid
glutaric acid
adipic acid
malic acid
9.2 ( 0.2)·× 10−5
no hydrolysis
1.76 ( 0.05)·× 10−5
7.3 ( 0.2)·× 10−5
4.0 ( 0.2)·× 10−5
9.7 ( 0.4)·× 10−5
no hydrolysis
0
1
2
3
4
citric Acid
no hydrolysis
G
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the steric hindrance of the side chain of the Leu residue, which
prevents effective delivery of the nucleophile. A more detailed
study on the hydrolysis of dipeptides by 1 is currently
underway.
Supporting Information, Figure S15 and Table S5 summarize
the 13C chemical shifts of 3G both in the presence and in the
absence of 1. Both amide carbonyls and corresponding
methylene groups displayed the biggest shift. On the basis of
the 13C chemical shift values for 4G, reported in Supporting
Information, Figure S16 and Table S6, the assumption that 1
displays exo- rather than endopeptidase activity cannot be
validated. In the case of exopeptidase activity a clear preference
for coordination to amide carbon 3 and/or 7 is expected. Since
all three amide carbons display a significant shift in the presence
of 1 no distinction between exo- and endopeptidase activity can
be made. Instead, 4G might act as a bridging ligand between
two POMs or display multiple coordination modes to Zr(IV).
The insolubility of 5G at concentrations (10.0 mM) needed for
13C NMR made a 13C NMR study of the binding of 5G to 1
impossible.
Hydrolysis of Triglycine, Tetraglycine, and Pentagly-
cine. The hydrolytic activity of 1 was further tested on longer
peptides consisting of glycine residues. Supporting Information,
1
Figure S13 shows the assignment of the H NMR spectra
recorded during the hydrolysis of triglycine (3G) in the
presence of 1. Quantitative analysis of these spectra was made
possible since each reactant (except for cGG) was characterized
by at least one isolated 1H NMR resonance. The kinetic profile
of the species observed during the course of hydrolytic reaction
is shown in Supporting Information, Figure S14. GG and G
were formed in equal amounts in the early stages of the
reaction. Once GG is formed, parallel pathways in which 3G
and GG are hydrolyzed can occur. Interestingly, the hydrolysis
of GG into G occurred before all 3G was converted to GG.
Furthermore, cyclization of GG to cGG was observed.
Eventually, all 3G was fully converted to G. Figure 9 shows
CONCLUSIONS
■
Homogeneous hydrolysis of the highly inert amide bond in the
dipeptide glycylglycine was observed in the presence of the
Zr(IV)-substituted Wells−Dawson type POM. The hydrolysis
rate constant represents a significant acceleration compared to
the blank reaction in which no hydrolysis was observed after
several months under the same reaction conditions. Several
dipeptides and oligopeptides were also successfully hydrolyzed
by 1, demonstrating that the catalyst can be applied to a range
of different peptide bond containing substrates. The hydrolytic
activity of the POM toward proteins is currently under
investigation, with the ultimate goal of developing metal-
substituted POMs as a novel class of artificial proteases.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C, and 31P NMR data. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 9. Hydrolysis of 2.0 mM of 4G in the presence of 2.0 mM of 1
at pD 5.0 and 60 °C. Percentage of 4G (left pointing solid triangles),
3G (solid diamonds), GG (solid squares), and G (solid circles) as a
function of reaction time.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Tatjana.Vogt@chem.kuleuven.be.
■
Notes
The authors declare no competing financial interest.
the time dependence of the distribution of all species that were
present during the hydrolysis of 2.0 mM of tetraglycine (4G) in
the presence of 2.0 mM of 1 at 60 °C and pD 5.0. For the
hydrolysis of 4G an induction period for the formation of GG
was observed. Initially only conversion of 4G to 3G+G was
detected. An alternative pathway would be the conversion of
4G to GG+GG. Since no GG was observed during the first 24 h
of reaction, it can be concluded that 1 shows exopeptidase
rather than endopeptidase reactivity. This is further confirmed
by the relative position of the percentage maxima of 3G and
GG. Whereas the maximum amount of 3G is present after 3
days, the maximum in GG concentration was only observed
after 8 days. At the end of reaction a full conversion of 4G into
G was observed.
ACKNOWLEDGMENTS
■
G.A. thanks the FWO-Flanders (Belgium) for the postdoctoral
fellowship. T.N.P.-V. acknowledges KU Leuven for the financial
support (Research Fund START1/09/028).
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