Hydrolysis of serine-containing peptides at neutral pH promoted by [MoO4]2- oxyanion

Article abstract of DOI:10.1021/ic2015034

Hydrolysis of the dipeptides glycylserine (GlySer), leucylserine (LeuSer), histidylserine (HisSer), glycylalanine (GlyAla), and serylglycine (SerGly) was examined in oxomolybdate solutions by means of ¹H, ¹³C, and ⁹⁵Mo NMR spectroscopy. In the presence of a mixture of oxomolybdates, the hydrolysis of the peptide bond in GlySer proceeded under neutral pD conditions (pD = 7.0, 60 °C) with a rate constant of k _obs = 5.9 × 10^-6 s^-1. NMR spectra did not show evidence of the formation of paramagnetic species, excluding the possibility of Mo(VI) reduction to Mo(V), indicating that the cleavage of the peptide bond is purely hydrolytic. The pD dependence of k_obs exhibits a bell-shaped profile, with the fastest cleavage observed at pD 7.0. Comparison of the rate profile with the concentration profile of oxomolybdate species implicated monomolybdate MoO₄^2- as the kinetically active complex. Kinetics experiments at pD 7.0 using a fixed amount of GlySer and increasing amounts of MoO₄^2- allowed for calculation of the catalytic rate constant (k₂ = 9.25 × 10^-6 s ^-1) and the formation constant for the GlySer-MoO₄ ^2- complex (K_f = 15.25 M^-1). The origin of the hydrolytic activity of molybdate is most likely a combination of the polarization of amide oxygen in GlySer due to the binding to molybdate, followed by the intramolecular attack of the Ser hydroxyl group.

Full text of DOI:10.1021/ic2015034

Article
pubs.acs.org/IC
Hydrolysis of Serine-Containing Peptides at Neutral pH Promoted by
[MoO4]2− Oxyanion
Phuong Hien Ho, Karen Stroobants, and Tatjana N. Parac-Vogt*
Katholieke Universiteit Leuven, Department of Chemistry, Celestijnenlaan 200F, B-3001, Leuven, Belgium
S
* Supporting Information
ABSTRACT: Hydrolysis of the dipeptides glycylserine (GlySer), leucylserine (LeuSer), histidylserine (HisSer), glycylalanine
(GlyAla), and serylglycine (SerGly) was examined in oxomolybdate solutions by means of 1H, 13C, and 95Mo NMR
spectroscopy. In the presence of a mixture of oxomolybdates, the hydrolysis of the peptide bond in GlySer proceeded under
neutral pD conditions (pD = 7.0, 60 °C) with a rate constant of kobs = 5.9 × 10−6 s−1. NMR spectra did not show evidence of the
formation of paramagnetic species, excluding the possibility of Mo(VI) reduction to Mo(V), indicating that the cleavage of the
peptide bond is purely hydrolytic. The pD dependence of kobs exhibits a bell-shaped profile, with the fastest cleavage observed at
pD 7.0. Comparison of the rate profile with the concentration profile of oxomolybdate species implicated monomolybdate
MoO42− as the kinetically active complex. Kinetics experiments at pD 7.0 using a fixed amount of GlySer and increasing amounts
of MoO4 allowed for calculation of the catalytic rate constant (k2 = 9.25 × 10−6 s−1) and the formation constant for the
2−
GlySer−MoO4 complex (Kf = 15.25 M−1). The origin of the hydrolytic activity of molybdate is most likely a combination of
2−
the polarization of amide oxygen in GlySer due to the binding to molybdate, followed by the intramolecular attack of the Ser
hydroxyl group.
Besides the basic requirement that the complex should be
able to promote amide bond cleavage, the selectivity of the
cleavage remains a big challenge. Currently, there is great
interest in the development of reagents that target diverse
amino acids and promote cleavage under mild conditions.32
Selective hydrolysis at cysteine, methionine, and histidine
residues has been achieved with Pt and Pd complexes.33
Molybdocene dichloride has been shown to selectively
hydrolyze cysteine-containing peptides.34 Selective hydrolysis
of serine-containing peptides by zinc salts has been reported by
the group of Komiyama,26 and more recently Ni(II) ions have
also been used to selectively hydrolyze the X−Ser sequence in a
series of oligopeptides.28
The chemistry of molybdenum(VI) oxo-complexes has been
intensively investigated because of their importance in
biochemical and chemical applications. In neutral and alkaline
solutions, molybdate is present as the monomeric anion
MoO42−. However, acidification results in condensation and
the formation of larger polyoxomolybdate species. Depending
on the pH, concentration, and temperature, a range of different
complexes, among which [Mo7O24]6− and [Mo8O26]6− have
been fully structurally characterized, are formed.35 The
INTRODUCTION
■
Reagents that promote selective cleavage of peptides and
proteins are needed for a number of applications, such as the
study of protein function and solution structure,1−6 mapping of
enzyme active sites,7−15 ligand-induced conformational
changes,16−18 and elucidation of metal binding sites.8,9,11,13,14
Recently, the use of peptide-cleaving agents as catalytic drugs
has been proposed.19−22 However, the remarkable inertness of
the peptide bond, with an estimated half-life of up to 600 years
under physiological conditions, makes its hydrolysis a
challenging task.23 Cyanogen bromide, the most common
chemical reagent for fragmentation of proteins, has several
shortcomings: it is volatile and toxic, is applied in a 100-fold
excess over methionine residues, requires 70% formic acid as a
solvent, and gives several side reactions.24
The potential of transition metal complexes to promote
peptide bond hydrolysis was recognized long ago. A large
number of studies on the metal-promoted hydrolysis of amides
have been performed with activated amides or amino acid
esters as peptide models.25 In recent years, a number of metal
complexes including zinc(II),26 copper(II),27 nickel(II),28
palladium(II),29 cerium(IV),30 and zirconium(IV)31 have
been found to be effective at promoting the hydrolysis of
unactivated amide bonds in peptides and proteins.
Received: July 14, 2011
Published: October 31, 2011
© 2011 American Chemical Society
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biological chemistry of oxomolybdates has been marked with
reports which revealed that their in vivo antitumoral activity on
several types of tumor cells is comparable to that of some
commercial drugs.36 More recent studies have shown that
[Mo7O24]6− exhibits potent antitumor activity against pancre-
atic cancer cells, which are extremely resistant toward most of
the known therapeutic agents.37 Several studies have revealed
that molybdate binds to nucleotides, resulting in slight
hydrolysis at high temperatures.38 Oxomolybdates have also
been shown to catalyze the hydrolysis of the labile “high
energy” phosphoanhydride bonds in ATP.39,40
In our recent studies, we examined the reactivity of
oxomolybdates toward 4-nitrophenyl phosphate (NPP), bis-
(p-nitrophenyl)phosphate (BNPP), and 2-hydroxypropyl-4-
nitrophenyl phosphate (HPNP), commonly used DNA and
RNA model phosphodiesters.41−44 The interaction between
these model systems and oxomolybdate complexes led to
purely hydrolytic cleavage of a phosphodiester bond,
demonstrating the first example of a phosphodiester bond
cleavage promoted by a negatively charged oxyanion. The
efficient hydrolysis of a range of carboxyesters in which
monomeric molybdate was identified as the effective
nucleophilic catalyst has been also reported.45−47
Gly−Tyr to 91% for His−Ser. Among all of the peptides
examined, peptides with the sequence X−Ser and X−Thr were
most readily hydrolyzed. These dipeptides have a serine or
threonine residue at their C terminus, indicating the
importance of the hydroxyl group in the side chain for efficient
hydrolysis.
On the basis of the results given in Table 1, we focused our
attention on the hydrolysis of a series of dipeptides (Scheme 1)
containing a serine residue, such as glycylserine (GlySer),
leucylserine (LeuSer), histidylserine (HisSer), and serylglycine
(SerGly). For a comparison, glycylalanine (GlyAla) was studied
as well.
The Hydrolysis of Dipeptide GlySer by Molybdate(VI)
Oxyanions. The model reaction between GlySer and
molybdate(VI) oxyanions has been performed in solutions
containing 2 mM GlySer and 120 mM Na2MoO4. During the
1
course of reaction, H NMR spectroscopy showed a gradual
intensity decrease of the GlySer resonances at 3.95 ppm (CH2
of glycine residue), 3.82−3.98 ppm (CH2 of serine residue),
and 4.35 ppm (CH of serine residue) and the appearance of
free glycine resonance at 3.58 ppm and free serine resonances
at 3.83−3.90 and 3.92−4.06 ppm, indicating that the cleavage
of the peptide bond in GlySer occurred (Figure 1).
In this study, we explore the reactivity of oxomolybdates
toward a range of peptides and discover the efficient hydrolysis
under neutral pH of the peptides containing the X−Ser
sequence. By combining kinetic experiments and NMR
measurements, we give a full account on the mechanism of
this novel reaction.
Integration of the proton NMR resonances at different time
increments allowed for the calculation of the hydrolysis rate
constant for GlySer (kobs = 5.9 × 10−6 s−1 at 60 °C, pD = 7.0;
Figure 2). This represents an acceleration of nearly 20 times
compared to the hydrolysis measured under the same
conditions but in the absence of molybdate. It is worth
mentioning that the reaction also occurred under physiological
pH and temperature (37 °C, pD = 7.0) with a rate constant of
2.5 × 10−7 s−1.
It is important to note that the NMR spectra did not show
evidence of any paramagnetic species, which excludes the
possibility of oxidative cleavage and the reduction of Mo(VI) to
Mo(V). The appearance of “blue species” was not observed
during any point of the reaction, indicating that the hydroxyl
group in the Ser side-chain did not cause oxidation, as has been
previously observed in reactions of oxomolybdates with some
alcohols.48 The “molybdenum blue” species are obtained by the
reduction of Mo(VI) in the presence of reducing agents,
including organic acids and sugars, and are characterized by a
typical electronic absorption spectrum with λmax at 748 nm.49,50
Although the general term “molybdenum blue” is often used,
not always completely identical solutions are formed; however,
the common feature for all of the “molybdenum blues” is their
characteristic blue color that originates from the electronic
transition of the intervalence (MoV →MoIV) charge transfer
type.51 The absence of such blue species suggests that the
reaction is purely hydrolytic in nature and can be presented as
shown in Scheme S1 (Supporting Information).
Effect of pH on the Hydrolysis of GlySer. The pH plays
an important role in the speciation of oxomolybdate anions,
and molybdate solutions often result in a complex mixture of
different species. Acidification of [MoO4]2− to a pH below 7.0
results in the formation of the heptamolybdate [Mo7O24]6−
cluster and its protonated forms. 95Mo NMR and Raman
spectroscopy studies have shown that [MoO4]2− and
[Mo7O24]6− anions coexist in the pH range 4.5−6.5. Further
acidification produces [Mo8O26]4− and different protonated
forms of this polyoxoanion. The effect of pD on the cleavage
reaction was examined in order to correlate the rate-pH profile
with the species distribution diagram. The fact that the
RESULTS AND DISCUSSION
■
The reactivity of molybdate (VI) oxyanions toward a range of
peptides was initially examined in aqueous solutions at pD 7.0
and 60 °C. The extent of hydrolysis after 60 h was determined
from the 1H NMR spectra by integration of the NMR
resonances of the peptide and free amino acid products at
different time increments. As the results in Table 1 show, the
conversion was mainly dependent on the sequence of the
peptide. The conversion ranged from 2% for Gly−Ala and
Table 1. The Hydrolysis of a Series of Dipeptides (2 mM)
Measured in the Absence and in the Presence of 120 mM
Na2MoO4 (pD = 7.0, 60°C) after 60 h
conversion (%) with
Na2MoO4
conversion (%) without
Na2MoO4
entry
dipeptide
1
2
GlySer
68
91
43
60
3
7
10
6
HisSer
3
LeuSer
GlyThr
SerGly
4
6
5
0
6
CysGly
GlySerPhe
AspAla
GlyAla
17
25
3
0
7
0
8
0
9
2
0
10
11
12
13
14
15
16
GluCysGly
GlyTyr
GlyGly
AlaHis
4
0
2
0
4
0
5
0
GlyAsn
GlyMet
LysGly
3
0
2
0
2
0
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2−
Scheme 1. Structures of the Dipeptides and MoO4 Used in the Study
1
Figure 1. H NMR spectra of the reaction between 2 mM GlySer and 120 mM Na2MoO4 at pD 7.0 and 60 °C measured at different time
increments.
solutions, the rates were plotted as a function of pD, which was
obtained by adding 0.41 pH units to the recorded pH value.52
The hydrolysis of GlySer was measured at 60 °C in the pD
range 4.0−8.8, both in the presence and in the absence of
molybdate. The data (Table S1, Supporting Information)
clearly indicated that the acceleration of the hydrolysis was due
to the presence of oxomolybdate and not due to the acid or
base catalysis. This is illustrated in Figure 3, where the values of
k
obs and the fraction of polyoxometalate species are plotted as a
function of pD. The concentration of molybdate under a given
pD, ionic strength, and temperature was calculated by using a
thermodynamic model previously described by our group.43
The distribution of Mo species calculated by this model agrees
very well with the distribution that was determined by several
experimental methods.
As can be seen in Figure 3, the pD dependence of kobs
exhibits a bell-shaped profile, with the fastest cleavage observed
at pD 7.0. The data points were fitted to the Michaelis function
(eq 1) describing a bell-shaped dependence of the rate constant
on pH.53
Figure 2. Kinetic profile for the hydrolysis of 2 mM GlySer in the
presence of 120 mM Na2MoO4 in a D2O solution at pD 7.0 and 60
°C.
(1)
reactivity follows the pD profile of a certain species suggests
that kobs is proportional to the concentration of that species.
Since the kinetic measurements were performed in D2O
In eq 1, kobs is the measured kinetic constant; h = 10−pH; and k,
K1, and K2 are parameters to be estimated. Comparison of the
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Figure 4. Influence of Na2MoO4 concentration on kobs for the cleavage
of 2 mM GlySer at T = 60 °C and pD 7.0.
Figure 3. The pD dependence of kobs (solid line) for the cleavage of
GlySer (2 mM) in the presence of Na2MoO4 (120 mM) at 60 °C. The
fractions of various oxo-molybdenum species as a function of pD have
been added for comparison (dashed lines).
The influence of the ionic strength on the rate constant was
studied by following the hydrolytic reaction in the presence of
different amounts of NaClO4 salt. An increase in the NaClO4
concentration resulted in the increase of the GlySer hydrolysis
rate constant, resulting in a positive salt effect. Nonlinearity of
the plot in which log(k/ko) was plotted as a function of the
ionic strength implied that the salt concentration not only
influenced the hydrolysis rate but that NaClO4 may also have
exhibited a secondary effect on the composition and stability of
species in solution (Figure S1, Supporting Information).
The effect of temperature on the hydrolytic reaction was
rate constant profile with the concentration profile of
oxomolybdate species shows that, up to pD 7.0, the best
overlap of the kobs profile is observed with the concentration of
MoO42−. Although in the acidic pD range, other molybdenum-
(VI) species such as [Mo7O24]6− and [Mo8O26]4− are present
in solution; they seem to be catalytically much less active. Slow
hydrolysis at lower pD values, where [Mo7O24]6− and
[Mo8O26]4− predominate, implies low catalytic activity of
determined by measuring the temperature dependence of kobs
.
2−
these species. The hypothesis that monomeric MoO4 is the
As expected, at higher temperatures, increased rates have been
observed. Activation Gibbs function (ΔG‡ = 114.20 kJ mol−1 at
37 °C), enthalpy (ΔH‡ = 90.6 kJ mol−1), and entropy (ΔS‡ =
−76.34 J mol−1 K−1) were obtained from the Arrhenius and
Eyring plots (Figure S2, Supporting Information). The negative
value of the activation entropy is in agreement with the
formation of the complex [GlySer/MoO42−]. However, the
activation parameters need to be interpreted with caution, as
they are usually derived from the composite rate constants that
also include contribution from the binding of the substrate to
the catalyst.
hydrolytically active species was further strengthened by the
fact that full hydrolysis of GlySer was observed at pD 7.5 (kobs
=
4.9 × 10−6 s−1, 60 °C) where MoO4 was the only
molybdenum(VI) species present in solution.
2−
Binding of GlySer to MoO42−. The rate increase of
2−
GlySer hydrolysis in the presence of MoO4 suggests that
interaction between molybdate and the peptide must take place
in solution. The binding constant between molybdate and
GlySer was determined by kinetic experiments using a fixed
amount of GlySer and increasing amounts of molybdate at pD
7.0 and 60 °C. Assuming that the monomeric molybdate is the
hydrolytically active species, the binding constant between
GlySer and molybdate could be determined by fitting the data
shown in Figure 4 to a general catalytic scheme presented in
eq 1:
Coordination of GlySer to Molybdate. The binding
between GlySer and molybdate was further examined by 95Mo,
1
13C, and H NMR spectroscopy. The addition of GlySer to
MoO42− solution did not cause detectable changes in the 95Mo
NMR spectrum, which is probably due to very low NMR
sensitivity of this nucleus. However, the 1H spectrum was more
(1a)
1
informative, as a number of H NMR GlySer resonances were
2−
shifted upon mixing with MoO4 (Figure S3, Supporting
Information). The binding was also examined by 13C NMR at
pD 7.0 and pD 8.4, and the results (Table S2, Supporting
Information) indicated that, at pD 7.0, the largest shifts were
observed for the CH2 (C1) and carbonyl CO group (C2) in
the glycyl residue and the carboxylate COO− group (C2′) in
the serine residue, while the CH group (C′1) of the serine
The values for the catalytic (k2 = 9.3 × 10−6 s−1) and
formation constants for the [GlySer/MoO42−] complex (Kf =
k−1/k1 = 15.25 M−1) were obtained by a computer generated
least-squares fit of kobs to eq 2.
2−
residue was not much affected by addition of MoO4 (see
Scheme 1 for atom designation). The larger shifts of carbon C1,
(2)
C2, and C2′ resonances in 13C NMR spectra suggest that
2−
Interestingly, the value of Kf is similar to the formation constant
previously determined for the binding between GlySer and
mononuclear complexes formed from a cis,cis-1,3,5-triamino-
cyclohexane ligand L, [CuL]2+, which has a binding constant of
15.97 (M−1).27a
MoO4 binds to GlySer via the amino nitrogen, the amide
oxygen, and the carboxylate oxygen. The NMR chemical shifts
observed at pD 8.4 also implicate coordination of the amino
nitrogen and the carboxylate oxygen atoms; however,
significant shifting of the α-carbon of Ser residue suggests
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that at higher pD values not the amide oxygen but the amide
nitrogen coordinates to MoO42−. Furthermore, while at pD 7.0
a weak interaction between the C3′ hydroxyl group of serine
GlySer. However, the binding of malic acid to MoO42− appears
to be rather weak since a 25 molar excess of the inhibitor
resulted only in a 26% decrease of the rate constant. Due to the
presence of an additional carboxylic group, the binding of citric
2−
and MoO4 could be plausible; at pD 8.4, this interaction is
2−
very unlikely. While the carbon atoms adjacent to the
coordinating groups experience shifts from 0.2 to 0.75 ppm,
the shift of the C3 carbon adjacent to the hydroxyl group is
only 0.05 ppm.
acid to MoO4 is stronger compared to malic acid, and
consequently, its presence resulted in a 52% decrease of the
reaction rate constant. The inhibitory effect of aliphatic
dicarboxylic acids was rather small. Their bidentate mode of
binding to molybdate is less effective compared to the
tridentate binding of GlySer that occurs via amino nitrogen,
the amide oxygen and the carboxylate oxygen.
The proposed coordination environment of molybdenum-
(VI) is analogous to that previously reported for molybdate
complexes with malate and citrate ligands in which a MoO3 unit
is bound to three heteroatoms originating from the ligand.54
While this requires cleavage of one bond between
Hydrolysis of Ac−GlySer and GlySer−OEt. The amino
group in Ac−GlySer is acetylated and mimics an internal amide
bond. Interestingly, the hydrolysis of Ac−GlySer was about 20
times slower (kobs = 1.5 × 10−7 s−1, pD = 7.0, 60 °C) than the
hydrolysis of GlySer measured under the same conditions. This
implies that the presence of a free amino group is essential for
2−
molybdenum(VI) and oxygen in MoO4 and the increase of
the coordination number of molybdenum(VI) from four to six,
this is not unusual, as previous studies between vanadate,
VO43−, and dipeptides have shown that coordination of
peptides also leads to the cleavage of a V−O bond and
expansion of the coordination number of vanadium(V) from
four to six.55
1
efficient hydrolysis. According to 13C and H NMR data, the
amino group in GlySer coordinates to MoO42−, resulting in
effective tridentate coordination for peptide hydrolysis to occur.
Due to the much higher pKa value of the proton on the amide
Since molybdate is known to interact with amino acids and
organic acids,35 the interaction of molybdate with glycine and
serine, which are the products of GlySer hydrolysis, was also
examined. 13C NMR spectra of glycine and serine recorded in
the absence and presence of molybdate were considerably
different, indicating that interaction took place in solution
(Table S3, Supporting Information). The binding of glycine
and serine to MoO42− most likely occurs via the amine nitrogen
and carboxylate oxygen, which was confirmed by large chemical
shifts (0.45 and 1.25 ppm) of the carbonyl carbon atoms of
glycine and serine, respectively. Although present in solution as
products of amide bond cleavage, glycine and serine do not
seem to significantly inhibit the GlySer hydrolysis. The addition
of a 25 M excess of Gly or Ser to the reaction mixture had no
significant effect on the hydrolysis rate, most likely due to the
preferential binding of molybdate to the GlySer.
2−
nitrogen, the coordination of this nitrogen atom to MoO4 is
unlikely to occur at pD = 7.0. Further indication that the
2−
attachment of the amino group to MoO4 is essential for
effective hydrolysis comes from the pD profile of the hydrolysis
rate constant of GlySer. The decrease of the hydrolysis rate
under acidic conditions (Figure 3) may also be explained by the
protonation of the amino group, which hinders coordination to
2−
MoO4
.
In the case of GlySer−OEt, in which the carboxylic group is
1
converted into an ester, H NMR spectra showed the presence
of ethanol resonances and indicated that full hydrolysis of the
ester group occurred within 24 h. The hydrolysis of the peptide
bond occurred only after GlySer−OEt was converted into
2−
GlySer. These results suggest that MoO4 also effectively
promotes the hydrolysis of esters, which may not be surprising
considering the much higher kinetic lability of the ester bond
compared to the peptide bond.
Effect of Inhibitors on the Hydrolysis Rate Constant
of GlySer. The inhibitory effect of several nonreactive
substrate analogues was examined. In the screening experi-
ments, the rate constant for the hydrolysis of GlySer (2 mM)
Hydrolysis of HisSer. Among all examined peptides,
HisSer was the most readily hydrolyzed (Table 1). At pD 7.0
and 60 °C, the rate constant for hydrolysis was 1.1 × 10−5 s−1,
meaning that complete hydrolysis was achieved in less than four
days. Similarly to GlySer hydrolysis, the pD dependence of kobs
exhibits a bell-shaped profile, with the fastest cleavage observed
at pD 7.0 (Figure 5).
2−
by MoO4 (60 mM) was determined in the presence of an
excess of several organic acids that are expected to bind to
molybdate (Table 2). According to the results in Table 2,
among all of the examined substrates only malic and citric acid
2−
reduced the effectiveness of MoO4 toward the hydrolysis of
The 1H NMR spectrum of HisSer in the presence of MoO4
2−
indicated that the H6 proton in the imidazole ring experienced
the largest shift (Table S4, Supporting Information), suggesting
that HisSer coordinates to Mo(VI) via its imidazole nitrogen.
13C NMR measurements (Table S5, Supporting Information)
confirmed that besides the imidazole nitrogen atom, the amine
nitrogen, amide oxygen, and carboxylate oxygen atoms are also
involved in the binding to molybdate (Scheme 2). However, at
pD values below 5.3, the coordination of the imidazole nitrogen
becomes unfavorable, and the coordination mode becomes the
same as observed for GlySer. The slow hydrolysis of HisSer at
low pD values may be the combination of this change in
coordination and the decrease of MoO42− concentration in the
acidic media.
Table 2. Rate Constants of GlySer (2 mM) Peptide Bond
Hydrolysis Measured in the Presence of Na2MoO4 (120
mM) and Different Inhibitors (50 mM) at pD = 7.0 and 60
°C
kobs (10−6
entry
inhibitor
formula
s−1
)
1
2
3
4
5
6
7
8
no inhibitor
alanine
3.86
3.64
3.72
3.87
3.80
3.84
2.85
1.67
H2NCH2COOH
malonic acid HOOCCH2COOH
succinic acid HOOC (CH2)2COOH
glutaric acid HOOC (CH2)3COOH
adipic acid
malic acid
citric acid
HOOC (CH2)4COOH
HOOCCH2CH(OH)COOH
The Mechanism of X−Ser Peptide Hydrolysis. From
the screening experiments of a range of different dipeptides, it
was evident that the presence of the hydroxide group in the side
chain of X−Ser-type dipeptides was essential for efficient
HOOCCH2C(COOH)
OHCH2COOH
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Scheme 3. Intramolecular Attack of Ser Hydroxyl Group on
Amide Carbonyl Carbon in GlySer and SerGly
Scheme 4. Possible Mechanism of GlySer Hydrolysis
2
Promoted by MoO4
Figure 5. The pD dependence of kobs (solid line) for the cleavage of
HisSer (2 mM) in the presence of Na2MoO4 (120 mM) at 60 °C.
Fractions of oxo-molybdenum species as a function of pD have been
added for comparison (dashed lines).
Scheme 2. Possible Mechanism of HisSer Hydrolysis
2−
Promoted by MoO4
2−
peptide and MoO4 is not sufficient to effect the hydrolysis;
this further supports the hypothesis that the intramolecular
attack of the Ser hydroxyl group is essential for the cleavage of
the amide bond.
hydrolysis. For example, in comparison to GlySer, the
hydrolysis of GlyAla, which has a similar bulkiness of the side
chain but lacks a hydroxyl group, was approximately two orders
of magnitude slower. As shown in Table 1, all dipeptides that
were readily hydrolyzed have a serine residue at the C-terminus,
suggesting the important role of the hydroxyl group in the Ser
side chain as an intramolecular nucelophile. The hypothesis
that the hydrolysis of X−Ser dipeptides is due to a N→O acyl
rearrangement is strongly supported by the fact that the
hydrolysis of GlySer was about 2 orders of magnitude faster
than the hydrolysis of SerGly. While the intramolecular attack
of the Ser hydroxyl group on the amide carbonyl carbon is
possible in GlySer, it is impossible in SerGly. In the case of
SerGly, the intramolecular attack of the Ser hydroxyl group
results in a five-membered ring transition state, while in the
case of SerGly the intramolecular attack should form an
unfavorable four-membered ring transition state (Scheme 3).26b
The acceleration of GlySer hydrolysis in the presence of
Slower hydrolysis of GlySerPhe compared to GlySer is most
likely due to the less effective coordination of this tripeptide to
molybdate. While the carboxylate group in GlySer is available
for coordination, resulting in the formation of a chelating
complex shown in Scheme 3, this carboxylate group is
transformed into an amide group in the tripeptide, which is
less effective as a ligand for molybdate at neutral pH. However,
it is also possible that the presence of the bulky phenyl group in
the side chain of the GlySerPhe tripeptide sterically impedes
the intramolecular attack of the Ser hydroxyl group on the
amide carbonyl carbon.
In general, the best metal ions for the hydrolysis of the amide
bond are strong Lewis acids characterized by high positive
charge and small size.31,32 A similar mechanism to that shown
in Scheme 4 has been previously proposed for Zn(II)-assisted
hydrolysis of GlySer.26b While oxomolybdate and Zn(II) have
rather distinct coordination chemistry, they both have the
tendency to coordinate to the carbonyl group and cause its
polarization. Oxomolybdate has been shown to hydrolyze
carbon and phosphorus esters,45−47 and it was suggested that
the origin of this hydrolytic activity lies in the ability of Mo(VI)
in oxomolybdate to serve as an electrophilic catalyst that
interacts with the oxygen atom of the carbonyl and the
phosphoester group. It is therefore plausible that a similar
interaction can occur with the carbonyl group of the amide
bond. The rate of peptide hydrolysis in serine-containing
peptides promoted by oxomolybdate compares roughly to
those previously reported for Zn(II) and Ni(II) salts. While the
exact comparison is difficult to make due to the different
2−
MoO4 suggested that the oxyanion, in addition to the
hydroxyl group within the peptide, plays an important role in
2−
the hydrolysis of the dipeptide. The role of MoO4 is most
likely to polarize the carbonyl group by coordinating to it and
to facilitate the attack of nucleophile. The coordination of
molybdate to the NH2 group seems to play an important role
since the rate constant for the cleavage of GlySer is much faster
than that of Ac−GlySer. This coordination seems to assist the
effective interaction of the carbonyl group with MoO42−. All of
the data above allow one to postulate a possible mechanism for
the hydrolysis of GlySer, as shown in Scheme 4. Slow
hydrolysis of LysGly, which is positively charged at pD = 7.0,
suggests that a favorable electrostatic interaction between the
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Inorganic Chemistry
Article
conditions (pH, temperature, concentration, type of serine
peptides) used in our study and the previous study, Table S6
(Supporting Information) gives the summery of available
kinetic data.
makes it more susceptible toward hydrolysis. This implies that
water from the solvent acts as a nucleophile and that the
catalyst acts through destabilization of the phosphodiester bond
through its incorporation into the polyoxometalate skeleton.
While the structural analogy between phosphate and molybdate
forms the basis of this interaction, it is easy to understand that
such interaction between structurally distinct molybdate and
peptides is less plausible. Indeed, to the best of our knowledge,
there are no studies reporting on complexation between
[Mo7O24]6−, which is a dominant species under mildly acidic
solutions, and peptides. On the other hand, monomeric
molybdate MoO42−, which predominates at neutral and basic
pH, has a tendency to coordinate to polydentate ligands.
Therefore, the different coordination affinities of monomeric
and polyoxo forms of molybdate toward phosphoesters and
peptides are most likely the reason for their different
reactivaties exhibited toward these classes of compounds.
The gradual decrease in hydrolysis rate constant at pD > 7
can be explained by the formation of a hydrolytically inactive
complex between an oxyanion and X−Ser peptides observed in
basic solutions.56 Many studies showed that the formation of
high stability peptide complexes is linked to the involvement of
the deprotonated amide function. A large number of other
metal ions were also reported to form M−N amide bonds in
peptides, including cobalt(II) and cobalt(III), zinc(II),
platinum(II) and platinum(IV), vanadium(V), and chromium-
(III).57 In this case, the pKa of the peptide amide nitrogen is
lowered by the positive charge of metal ions. Metals bind
strongly to deprotonated amide nitrogens and are much less
polarizing than protons. As a result, substitution of the amide
hydrogen atom for a metal greatly reduces the susceptibility of
the amide carbonyl carbon atom toward nucleophilic attack.58
13C NMR measurements of a GlySer solution at pD 8.4 (Table
S2, Supporting Information) have shown that in the presence
of MoO42−, the carbon atoms in the CH2 and carbonyl CO
group of the glycyl residue show large shifts. This is consistent
with the deprotonation of the amide group and its coordination
2−
Hydrolysis of GlySer by Other MO4 (M = Cr, W)
2−
Oxyanions. Besides MoO42−, two other iso-structural MO4
oxyanions, CrO4 and WO42−, were examined for their
2−
efficiency toward the hydrolysis of GlySer. The rate constants
are compared in Table 3.
Table 3. Rate Constants for the Hydrolysis of 2 mM GlySer
in the Presence of 120 mM Concentration of Different
Oxyanions (pD = 7.0 and 60 °C)
2−
to MoO4 (Scheme 5).
Scheme 5. Active and Inactive Complexes [Mo−GlySer]
oxyanion
kobs (s−1
)
5.9 × 10−6
4.4 × 10−7
6.5 × 10−7
2−
MoO4
2−
CrO4
2−
WO4
As can be seen from Table 3, compared to MoO42−, both
CrO42− and WO42− were less effective toward the hydrolysis of
the peptide bond in GlySer. The size of MO42− increases in the
order CrO42− < MoO42− ≈ WO42−. Therefore, solvation of the
MO42− species in H2O is the most significant for CrO42−, which
hinders effective coordination to GlySer and results in low
reactivity. Previous studies have shown that tungsten chelates
are significantly more labile with respect to individual metal−
ligand bonds than molybdenum chelates.59 13C NMR studies of
The faster hydrolysis of HisSer compared to other X−Ser
dipeptides containing noncoordinating side chains is most
likely due to the effective coordination of the imidazole
2−
2−
GlySer performed in the presence and the absence of WO4
nitrogen to MoO4 and the formation of a more stable
showed smaller shifts of all 13C NMR resonances as compared
to MoO42− (Table S7, Supporting Information). The lability of
the [WO42−/GlySer] complex most likely results in lower
reactivity toward GlySer peptide bond hydrolysis.
complex. However, it is also plausible that imadozole nitrogen
acts as a base, accepting the hydroxyl proton from Ser residue
and facilitating the intramolecular attack. The slower hydrolysis
rate constant for LeuSer can be explained by the steric
hindrance caused by the Leu side chain, which inhibits
nucleophilic attack and results in slower peptide hydrolysis.
Since our recent studies on the reactivity of oxomolybdates
toward phosphodiesters have shown that the fastest hydrolysis
occurred under mildly acidic conditions and implicated
[Mo7O24]6− as the hydrolytically active complex,41−44 it is
interesting to address the reasons that might account for the
different pD and speciation trends exhibited by peptides versus
activated phosphodiesters. The interaction between a phos-
phodiester and [Mo7O24]6− leads to an intramolecular exchange
process which results in partial detachment of one MoO4
tetrahedron and allows for the attachment of the structurally
related phosphodiester tetrahedron into the polyoxometalate
structure. The incorporation of the phosphodiester group into
the polyoxomolybdate skeleton results in the sharing of its
oxygen atoms with the Mo(VI) center that can cause
polarization and strain of the P−O ester bond, which in turn
CONCLUSIONS
■
In conclusion, we report the first example of hydrolytic peptide
bond cleavage promoted by a negatively charged oxyanion. To
the best of our knowledge, the hydrolytic cleavage of the
peptide bond has been so far achieved only by metal cations or
positively charged metal complexes. The hydrolytic effect of
MoO42− can be attributed to its ability to efficiently coordinate
X−Ser peptides and polarize their carbonyl group toward the
internal nucleophilic attack by the hydroxyl group of the Ser
residue. Although the hydrolysis occurred in solutions
containing polyoxo forms of molybdate, the kinetic studies
indentified the monomeric molybdate as the hydrolytically
active complex. We are currently investigating the reactivity of
2−
MoO4 toward the hydrolysis of different oligopeptides and
proteins. This and future studies focusing on the reactivity of
oxomolybdates toward relevant biomolecules are important, as
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Article
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EXPERIMENTAL SECTION
Materials. Glycylserine (GlySer), leucylserine (LeuSer), histidyl-
serine (HisSer), glycylalanine (GlyAla), serylglycine (SerGly), and
sodium molydate were purchased from Acros and used without further
■
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1
purification. The pD of the solutions for the H NMR studies was
adjusted with D2SO4 and NaOD, both from Acros. Ac−GlySer and
GlySer−OEt were synthesized using a standard procedure.60
NMR Spectroscopy. 1H and 13C spectra were recorded on Bruker
Advance 400 and Bruker Advance 600 spectrometers. D2O with 0.05
wt % 3-(trimethylsilyl) propionic acid as an internal standard was used
as a solvent. A solution of TMS in CDCl3 was employed as an external
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Kinetics. In a typical kinetic experiment, the hydrolysis of a 2 mM
peptide in the presence of 120 mM molybdate was followed by H
1
NMR spectroscopy. For example, the typical reaction mixture was
prepared by dissolving 0.32 mg of GlySer in 1 mL of D2O to which 92
mg of Na2MoO4 × 2 H2O was added. The pH of the final mixture was
adjusted by adding small amounts (typically 1−5 μL) of 2 M D2SO4 or
1.5 M NaOD. The pH of the solution was measured in the beginning
and at the end of the hydrolytic reaction, and the difference was
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1
in the H NMR spectra at different time intervals. The observed first
order rate constants (kobs) were calculated by the integral method from
at least 90% conversion. This included integrating proton NMR
resonances of free glycine and plotting them as a function of time. The
linear fitting method (ln[A] = kobs × t + C), where A is the
concentration of the substrate and t is the time at which the
concentration measured was used. The R values were generally higher
than 0.98. The Michaelis−Menten kinetic measurements were
performed on samples which contain a fixed concentration of GlySer
(2 mM), while the concentration of molybdate was increased from 0
to 625 mM.
The influence of the ionic strength on the reaction rate was studied
by following the reaction between 2 mM GlySer and 120 mM
Na2MoO4 (pD = 7.0, 60 °C) in the presence of different amounts of
NaClO4. As before, the rate constants were determined by the
integration of the 1H NMR signal intensities of the CH2 group of free
glycine.
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ASSOCIATED CONTENT
* Supporting Information
Additional tables, figures, and schemes. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
(29) (a) Milovic, N. M.; Kostic, N. M. J. Am. Chem. Soc. 2003, 125,
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AUTHOR INFORMATION
■
6, 3906−3913.
Corresponding Author
*E-mail: Tatjana.Vogt@chem.kuleuven.be.
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2004, 43, 6130.
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ACKNOWLEDGMENTS
■
T.N.PV. thanks K. U. Leuven for the financial support
(START1/09/028). P.H.H. thanks the Vietnamese Govern-
ment and K. U. Leuven for a doctoral fellowship. K.S. thanks
F.W.O. Flanders for the doctoral fellowship.
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