Hydrolytic activity of vanadate toward serine-containing peptides studied by kinetic experiments and DFT theory

Article abstract of DOI:10.1021/ic300761g

Hydrolysis of dipeptides glycylserine (Gly-Ser), leucylserine (Leu-Ser), histidylserine (His-Ser), glycylalanine (Gly-Ala), and serylglycine (Ser-Gly) was examined in vanadate solutions by means of ¹H, ¹³C, and ⁵¹V NMR spectroscopy. In the presence of a mixture of oxovanadates, the hydrolysis of the peptide bond in Gly-Ser proceeds under the physiological pH and temperature (37 °C, pD 7.4) with a rate constant of 8.9×10^-8 s^-1. NMR and EPR spectra did not show evidence for the formation of paramagnetic species, excluding the possibility of V(V) reduction to V(IV) and 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 hydrolysis observed at pD 7.4. Combined ¹H, ¹³C, and ⁵¹V NMR experiments revealed formation of three complexes between Gly-Ser and vanadate, of which only one complex, designated Complex 2, formed via coordination of amide oxygen and amino nitrogen to vanadate, is proposed to be hydrolytically active. Kinetic experiments at pD 7.4 performed by using a fixed amount of Gly-Ser and increasing amounts of Na ₃VO₄ allowed calculation of the formation constant for the Gly-Ser/VO₄^3- complex (K_f = 16.1 M ^-1). The structure of the hydrolytically active Complex 2 is suggested also on the basis of DFT calculations. The energy difference between Complex 2 and the major complex detected in the reaction mixture, Complex 1, is calculated to be 7.1 kcal/mol in favor of the latter. The analysis of the molecular properties of Gly-Ser and their change upon different modes of coordination to the vanadate pointed out that only in Complex 2 the amide carbon is suitable for attack by the hydroxyl group in the Ser side chain, which acts as an effective nucleophile. The origin of the hydrolytic activity of vanadate is most likely a combination of the polarization of amide oxygen in Gly-Ser due to the binding to vanadate, followed by the intramolecular attack of the Ser hydroxyl group.

Full text of DOI:10.1021/ic300761g

Article
pubs.acs.org/IC
Hydrolytic Activity of Vanadate toward Serine-Containing Peptides
Studied by Kinetic Experiments and DFT Theory
Phuong Hien Ho,† Tzvetan Mihaylov,† Kristine Pierloot,† and Tatjana N. Parac-Vogt*,†
†Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001, Leuven, Belgium
S
* Supporting Information
ABSTRACT: Hydrolysis of dipeptides glycylserine (Gly-Ser),
leucylserine (Leu-Ser), histidylserine (His-Ser), glycylalanine
(Gly-Ala), and serylglycine (Ser-Gly) was examined in
vanadate solutions by means of 1H, 13C, and 51V NMR
spectroscopy. In the presence of a mixture of oxovanadates,
the hydrolysis of the peptide bond in Gly-Ser proceeds under
the physiological pH and temperature (37 °C, pD 7.4) with a
rate constant of 8.9 × 10−8 s−1. NMR and EPR spectra did not
show evidence for the formation of paramagnetic species,
excluding the possibility of V(V) reduction to V(IV) and
indicating that the cleavage of the peptide bond is purely
hydrolytic. The pD dependence of kobs exhibits a bell-shaped
profile, with the fastest hydrolysis observed at pD 7.4. Combined 1H, 13C, and 51V NMR experiments revealed formation of three
complexes between Gly-Ser and vanadate, of which only one complex, designated Complex 2, formed via coordination of amide
oxygen and amino nitrogen to vanadate, is proposed to be hydrolytically active. Kinetic experiments at pD 7.4 performed by
using a fixed amount of Gly-Ser and increasing amounts of Na3VO4 allowed calculation of the formation constant for the Gly-
Ser/VO43‑ complex (Kf = 16.1 M−1). The structure of the hydrolytically active Complex 2 is suggested also on the basis of DFT
calculations. The energy difference between Complex 2 and the major complex detected in the reaction mixture, Complex 1, is
calculated to be 7.1 kcal/mol in favor of the latter. The analysis of the molecular properties of Gly-Ser and their change upon
different modes of coordination to the vanadate pointed out that only in Complex 2 the amide carbon is suitable for attack by the
hydroxyl group in the Ser side chain, which acts as an effective nucleophile. The origin of the hydrolytic activity of vanadate is
most likely a combination of the polarization of amide oxygen in Gly-Ser due to the binding to vanadate, followed by the
intramolecular attack of the Ser hydroxyl group.
extensively studied; however, a number of studies have emerged
during the last two decades revealing that oligomeric forms,
such as tetrameric [V4O12]4‑ (V4), pentameric [V5O15]5‑ (V5),
and decameric [V10O28]6‑ (V10) oxovanadates, also possess
important biological functions.5,6 The interconversion between
different oligomeric species is possible; however, different
biological activity was observed depending on which oligomer
is initially present in the solution.6−10 Despite a number of
studies which point toward the biological importance of
vanadate and its polyoxo forms, the molecular basis for this
activity remains largely unexplored. While it has been mainly
assumed that the mode of action of oxo-vanadates mostly
depends on their structural features such as the charge and the
size of the species,11 the potential reactivity of oxo-vanadates
toward biologically relevant substrates has been scarcely
explored.
INTRODUCTION
■
Vanadium is considered to be an essential element which has a
beneficial effect at low concentrations but becomes toxic when
present in higher concentrations.1 The biological role of
vanadium has been widely investigated; however, despite many
studies, its exact role is not yet fully understood. The recent
medicinal applications of vanadium are reflected in exploring
the use of structure−activity relationships of antidiabetes
vanadium complexes, development of vanadate compound as
antibacterial and antitumor agents, and investigation of
osteogenic properties of several vanadium compounds.2,3
Several excellent reviews on the biological and medicinal
importance of vanadium and its compounds have been
published.4
−
2‑
Vanadate, (H2VO4 /HVO4 ) which is the most commonly
occurring form of vanadium(V), has very complex solution
chemistry and tendency to form different polyoxoanions at
physiological pH, which complicates the thorough under-
standing of its biological role. The monomoric vanadate (V1) is
a strong inhibitor of myosin and several enzymes, including
ATPases, ribonucleases, and phosphatases. Due to its structural
analogy to phosphate, the monomeric form has been very
In our quest in understanding the biological role of
oxovanadates, we recently focused our attention on probing
their reactivity toward biologically relevant molecules and their
Received: April 13, 2012
Published: July 30, 2012
© 2012 American Chemical Society
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model systems. Our studies have shown that oxovanadates
efficiently hydrolyze a series of carboxyesters, as well as a
phosphoester bond in commonly used RNA and DNA model
substrates such as 2-hydroypropyl-4-nitrophenyl phosphate
(HPNP), 4-nitrophenyl phosphate (NPP), and bis-4-nitro-
phenyl phosphate (BNPP).12−14 The kinetic studies in which
the rate profile was compared with the concentration profile of
different oxovanadates present in solution, combined with
different spectroscopic techniques, allowed for the identifica-
tion of hydrolytically active species. Interestingly, depending on
the type of substrate, different oxovanadates were implicated as
kinetically active. While in the case of NPP and BNPP
hydrolysis V10 was identified as the reactive oxoanion, in the
transesterification of HPNP the V4 and V5 were implicated as
the reactive complexes.12,13 The origin of the hydrolytic activity
of polyoxovanadates was attributed to their high internal lability
and their known ability to react with phosphate derivatives.
These initial studies encouraged us to further explore the
reactivity of oxovanadates toward a range of different peptides,
another family of molecules with an important biological
relevance. The photooxidative cleavage of proteins in the
presence of a mixture of oxovanadates has been previously
reported.15,16 The photocleavage involving the free radical
addition to the carbon atom in the peptide bond has been
proposed; however, the exact molecular mechanism has not
been elucidated. Interactions between vanadate and dipeptides
have been also previously studied, mostly by the means of 51V
NMR spectroscopy.17−21 For peptides with noncoordinating
side chains a 6-fold coordination geometry around vanadium
has been suggested at physiological pH, with coordination of
the peptide occurring via the terminal amino and the
carboxylate groups as well as via the nitrogen in the peptide
bond. However, for the peptides containing a hydroxyl group in
the side chain, coordination of this group has been also
suggested, particularly upon the increase of pH. These findings
were also confirmed by density-functional study and molecular
dynamics calculations methods.22,23 While these studies offer
important additional insight into the mode of vanadium
binding sites in proteins, they do not report on the potential
reactivity of vanadate toward the peptide bond.
hydrolysis of a peptide bond, most of them share some basic
requirements such as overall positive charge and the presence
of coordinated water molecule, allowing for the metal
coordinated water molecules to act as an intramolecular
general acid catalyst or for the nucleophile activation of
coordinated water or hydroxide ligand. However, in our recent
report we demonstrated that despite its negative charge and
lack of coordinated water or hydroxide, the molybdate anion
efficiently hydrolyzes the peptide bond in serine containing
peptides.34 Considering the structural analogy between
molybdate and vanadate anions, and the important role of
vanadium in various biochemical processes such as the
regulation of enzymatic phosphorylations, the inhibition of
the Na, K ATPase, the chlorophylly synthesis4b in this study we
examine its reactivity toward a series of peptides and give a full
account on the mechanism of this novel reaction by combining
kinetic experiments and DFT-based molecular modeling.
RESULTS AND DISCUSSIONS
■
Hydrolysis of Peptides by Vanadium(V) Oxoanions.
The reaction between vanadate(V) oxyanions with a range of
peptides was initially examined in aqueous solutions at pD 4.4
and pD 7.4. Under these pD conditions a mixture containing
different amounts of V1, V2, V4, V5, and V10 oxoanions (shown
in Scheme 1) is present in solution.
Scheme 1. Structures of Mono-, Di-, Tetra-, Penta-, and
Decavanadate
In this study we explore the reactivity of oxovanadates
toward a range of peptides and, to the best of our knowledge,
report on the first example of a peptide bond hydrolysis
promoted by a vanadate anion under physiological pH. It is to
be noted that the remarkable inertness of peptide bond, with an
estimated half-life of up to 600 years under physiological pH
and temperature, makes its hydrolysis a challenging task.24
A
number of metal complexes including zinc(II),25 copper(II),26
nickel(II),27 palladium(II),28 cerium(IV),29 zirconium(IV),30
and vanadium(IV)31 have been found to be effective at
promoting hydrolysis of unactivated amide bonds in peptides
and proteins. Besides the basic requirement that the complex
promotes amide bond cleavage, the selectivity of the cleavage
remains a big challenge, and very few complexes were reported
to promote a residue selective hydrolysis of peptides. Pt(II) and
Pd(II) complexes were shown to promote selective hydrolysis
at cysteine, methionine, and histidine residues,32 while
molybdocene dichloride complex was used for the hydrolysis
of cysteine-containing peptides.33 Selective hydrolysis of serine-
containing peptides by zinc(II) salts has been reported by the
group of Komiyama,25 and more recently Ni(II) ions have been
also used to selectively hydrolyze X-Ser sequence.27 Despite the
wide structural variety of complexes that were active toward
The degree of peptide bond hydrolysis was determined after
140 h by integration of the proton NMR resonances of the
peptide and free amino acid products. The results (Table S1)
indicate that at pD 7.4 the conversion ranged from 3% for Gly-
Phe to 52% for Gly-Ser. It is important to note that all reactions
go to completion; however, for the sake of comparison the yield
of hydrolysis was determined after 140 h for all the examined
peptides. Among all the peptides, those with a X-Ser and X-Thr
sequence were most readily hydrolyzed (Table 1). These
peptides have a serine or threonine residue at the C-terminus,
indicating the importance of the hydroxyl group in the side
chain for efficient hydrolysis.
Based on the results presented in Table 1 and Table S1,
which show the fastest hydrolysis for serine-containing
peptides, the further work was focused on the series of
dipeptides containing serine residues, such as glycylserine (Gly-
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Gly-Ser resulted in a mixture containing 90% of Gly and Ser
and 10% of cGly-Ser. Based on the integration of the proton
NMR resonances at different reaction times the hydrolysis rate
constant of Gly-Ser was calculated: kobs = 1.3 × 10−6 s−1 at 60
°C and pD 7.4 (Figure 1). For comparison, while in the
Table 1. Extent of Peptide Bond Hydrolysis in Selected
Dipeptides (2.0 mM) Measured after 140 h in the Absence
and in the Presence of 25.0 mM Na3VO4 (pD 7.4 and 4.4) at
60 °C
conversion (%) with
Na3VO4
conversion (%)
without Na3VO4
no.
dipeptide
pD 7.4
pD 4.4
pD 7.4
pD 4.4
1
2
3
4
5
6
7
Gly-Ser
52
40
39
3
5
7
3
5
2
0
0
2
0
His-Ser
25
12
6.0
8
10
6
Leu-Ser
Ser-Gly
0
Gly-Ala
Gly-Thr
Gly-Ser-Phe
7
0
13
16
15
4
6
0
Ser), leucylserine (Leu-Ser), histidylserine (His-Ser), and
serylglycine (Ser-Gly). For comparison, glycylalanine (Gly-
Ala) was studied as well (Scheme 2).
The Hydrolysis of Gly-Ser by Vanadate(V) Oxyanions.
The reaction between Gly-Ser and vanadate(V) oxyanions was
performed by adding 2.0 mM of Gly-Ser to a 20.0 mM solution
Figure 1. Kinetic profile for the hydrolysis of Gly-Ser in the presence
of Na3VO4 in a D2O solution at pD 7.4 and 60 °C.
1
of Na3VO4 at pD 7.4 and incubating the sample at 60 °C. H
NMR spectra measured at different time increments showed a
gradual intensity decrease of the signal corresponding to the
CH2 protons of the glycyl residue in Gly-Ser at 3.89 ppm and
the appearance of a CH2 resonance at 3.50 ppm attributed to
glycine. The identity of the latter peak was confirmed by
spiking experiments in which a solution of free glycine was
added to the reaction mixture, resulting in an intensity increase
of the resonance at 3.50 ppm. As can be seen in Figure S1, the
intensity of all other resonances, at 3.79−3.95 ppm (CH2 of
serine residue) and 4.35 ppm (CH of serine residue), that were
attributed to Gly-Ser also decreased during the course of
reaction, while the appearance of free serine (regions 3.75−3.83
and 3.86−4.00 ppm) was simultaneously observed. These data
unambiguously indicate that the cleavage of the peptide bond in
Gly-Ser occurred. Interestingly, during the course of hydrolytic
reaction, the presence of a small amount of cyclic Gly-Ser
(cGly-Ser) was also observed in the NMR spectrum,
characterized by a singlet at 3.55 ppm and multiplet peaks in
the region of 4.11−4.15 ppm, indicating that vanadate also
promotes cyclization of Gly-Ser. The cyclization to cGly-Ser by
vanadate is an irreversible process as no hydrolysis of cGly-Ser
into Gly and Ser was detected. Therefore, the full hydrolysis of
presence of vanadate complete hydrolysis of Gly-Ser was
observed, only 15% of Gly-Ser was hydrolyzed in the absence of
vanadate under the same conditions. It is worth mentioning
that the reaction also occurred under physiological pH and
temperature (37 °C, pD 7.4) with a rate constant of 8.9 × 10−8
s−1, under which spontaneous hydrolysis of Gly-Ser was
extremely slow.
Importantly, NMR and EPR spectra did not show any
evidence of paramagnetic species, which excludes the possibility
of oxidative cleavage and the reduction of V(V) to V(IV).
These results suggest that the reaction is purely hydrolytic in
nature and can be presented as shown in Scheme 3.
Due to the importance of pH in the hydrolysis of peptide
substrates, as well as in the speciation of different oxovanadate
anions, the effect of pD on the hydrolysis rate of Gly-Ser was
examined in detail. For comparison, the hydrolysis of Gly-Ser
was also followed in the absence of vanadate in the pD range
from 5.2 to 10.0 (Table S2), and the significant rate
acceleration clearly showed that the Gly-Ser is hydrolyzed
due to the presence of oxovanadates.
Scheme 2. Structure of Peptides Used in This Study
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Scheme 3. Schematic Representation of Gly-Ser Hydrolysis in the Presence of Vanadate
At pD 7.4 the monomeric V1 coexists in equilibrium with V2,
V4, and V5. Upon acidification the formation of the
decavanadate V10 and its protonated forms takes place in
solution. The data shown in Figure 2 indicated that the
hydrolysis was very slow in acidic media and that the rate of
Gly-Ser hydrolysis at pD 5.0 (kobs= 4.6 × 10−7 s−1) in the
presence of decavanadate was very similar to the spontaneous
hydrolysis of Gly-Ser (kobs = 2.8 × 10−7 s−1) indicating that V10
was not active toward Gly-Ser hydrolysis. The slow hydrolysis
of other dipeptides at pD 4.4 (Table 1 and Table S1) in the
presence of vanadate also suggest that V10 is a hydrolytically
inactive species. The pD dependence of kobs exhibits a bell-
shaped profile, which fastest cleavage observed at pD 7.4. The
data points were fitted to a Michaelis function (eq 1) describing
a bell-shaped dependence of the rate constant on pD, where
kobs is the measured kinetic constant and h = 10−pD and k, K1,
and K2 are parameters to be estimated35
kobs = k/(1 + h/K1 + K2/h)
(1)
The fastest Gly-Ser hydrolysis occurs at neutral pD where a
mixture of V1, V2, V4, and V5 is present in solution. The
similarity in the shape of the kinetic profile and the fraction
distribution of V2, V4, and V5 would indicate that these
oxoanions are hydrolytically active toward Gly-Ser hydrolysis.
The various forms of vanadium(V) oxo species were
determined by means of 51V NMR spectroscopy. However,
previous studies examining the equilibrium of solutions
containing a mixture of oxovanadate species revealed that
these oxovanadates interconvert on the millisecond time scale,
according to the equations below17
■
Figure 2. The pD dependence of kobs ( , solid line) for the cleavage of
2.0 mM of Gly-Ser in the presence of 20.0 mM of Na3VO4. The
fractions of different oxovanadium species have been added for
comparison.
Figure 3. 51V NMR spectra of solution containing 25 mM Na3VO4 recorded in the absence (a) and the presence (b) of 5 mM Gly-Ser at pD 7.4 and
60 °C.
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4V ⇌ V
Interestingly, hydrolysis of Ac-Gly-Ser in which the terminal
amino function is converted into an amide was more than 10
times slower than the hydrolysis of Gly-Ser measured under the
same reaction conditions (kobs 1.04 × 10−7 s−1 vs 1.33 × 10−6
s−1), which suggests that the coordination via amino group is
essential for the efficient hydrolysis of the peptide bond. This in
turn would suggest that in oligopeptides the cleavage would
occur at the N-terminal amide bond.
Previous studies have shown that while at neutral pH the
Gly-Ser binds to vanadate as shown in Complex 1, the increase
of pH results in the formation of another complex, designated
Complex 3, in which coordination of Gly-Ser occurs via its
amine nitrogen, amide nitrogen, and hydroxyl oxygen. 51V
NMR spectra of Gly-Ser measured in the presence of vanadate
at pD 9.3 indeed indicated the appearance of a new peak at
−493 ppm, as reported in the literature.17,19 1H and 13C NMR
studies (Table S5) supported the structure of Complex 3
shown in Scheme 5. The C3′ carbon experienced a large shift of
13.5 ppm, suggesting the coordination of the hydroxyl oxygen
in the Ser side chain to vanadium.
1
4
5V ⇌ V
1
5
2V ⇌ V
1
2
In order get more insight into the nature of the hydrolytically
active species, the interaction between Gly-Ser and different
oxovanadates was followed by 1H, 13C, and 51V NMR
spectroscopy. 51V NMR spectra of vanadate solutions were
recorded in the absence and the presence of 5.0 mM of Gly-Ser,
and the data shown in Figure 3 indicate the appearance of a
broad 51V resonance at −506 pm, which is at the same position
as previously reported for a complex formed between V1 and
Gly-Ser.19 In this complex, designated Complex 1, the
coordination of the peptide occurs via its amine nitrogen,
amide nitrogen, and carboxylate oxygen. A detailed analysis of
the 51V NMR spectra indicated that while the V2, V4, and V5
resonances remained unaffected upon addition of Gly-Ser, the
V1 resonance at −557.39 ppm is shifted by 0.57 ppm and
broadened by 25 Hz at its half-width, suggesting its interaction
with the peptide. The broadening and shifting of the V1
resonance implies that another complex (designated Complex
2), that is in a fast exchange with the free Gly-Ser on the NMR
time scale, is simultaneously formed. Further information about
the structure of Complex 1 and Complex 2 was obtained by 1H
and 13C NMR spectroscopy. Similarly to the 51V NMR results,
an additional set of Gly-Ser peaks was observed in both 1H and
13C NMR spectra, indicating formation of Complex 1. The
NMR data shown in Table S3 are consistent with the
coordination of Gly-Ser to vanadate via its amine nitrogen,
amide nitrogen, and carboxylate oxygen as reported in previous
studies, thus resulting in the structure of Complex 1 as shown
in Scheme 5. The formation of Complex 1 was impeded with
the protection of either the N-terminal or C-terminal of Gly-
Ser, strongly suggesting that both the amine and carboxylate
group are involved in complex formation. Interestingly, besides
Scheme 5. Proposed Structures of Complex 1 and Complex
3
Moreover, substitution of the amide nitrogen as in
glycylsarcosine did not result in formation of Complex 2 and
Complex 3, strongly suggesting the involvement of amide
nitrogen in complex formation.20 As previous studies suggested,
these complexes have both a deprotonated amine (NH2)
nitrogen and a deprotonated amide (OCN−) nitrogen.21 Solid-
state and solution studies confirm the loss of the amide proton
in various other metal complexes.36,37 With all the data above,
the structures of two Gly-Ser complexes that are formed at
different pH regimes were postulated as shown in Scheme 5.
Binding Vanadate to Gly-Ser. The increase of the
chemical shift difference between the free and bound CH2
group of the glycyl residue in Gly-Ser upon addition of Na3VO4
suggests the formation of a complex in which the dipeptide is in
fast exchange between the free and bound form. The binding
constant between vanadate and Gly-Ser in Complex 2 was
determined by NMR titration experiments in which a fixed
amount of Gly-Ser and increasing amounts of vanadate at pD
7.4 were used. The binding constant between Gly-Ser and
vanadate was determined by fitting the data shown in Figure 4
to the scheme presented in eq 2.
the appearance of a new set of peaks in H and 13C NMR,
1
shifting of the Gly-Ser resonances was observed in both NMR
methods. According to Table S4 the largest shifts were
observed for the CH2 (C1) and carbonyl CO group (C2)
of the glycyl residue, while the CH (C1′), CH2 (C3′), and
carboxylate COO− group (C2′) of the serine residue were not
affected by the addition of Na3VO4. The shift of the carbon C1
and C2 resonance suggests that Gly-Ser binds to vanadate via
the amine nitrogen and the amide oxygen forming a complex
designated Complex 2, which is in fast exchange with the free
peptide on the NMR time scale (Scheme 4).
Scheme 4. Proposed Structure of Complex 2
The values for the Δm1ax = 32 Hz, where Δmax represents the
maximum difference in H NMR chemical shift between the
free and bound CH2 group of the glycyl residue in Gly-Ser
occurring upon addition of Na3VO4 (Δmax= ΔboundGly‑Ser
−
Δ
freeGly‑Ser) and the formation constant for Complex 2 [Gly-
3‑
Ser/VO4 ] (Kf = 16.1 M−1), were obtained by a computer
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Figure 5. Influence of salt concentration on kobs for the reaction
between 2.0 mM Gly-Ser and Na3VO4 at pD 7.4 and 60 °C.
Figure 4. Effect of Na3VO4 concentration on the chemical shift
difference between the free and bound CH2 group of the glycyl residue
in Gly-Ser.
expected to bind to vanadate (Table 2). Among all the
examined substrates only malonic, malic, and citric acid
generated least-squares fit of kobs to eq 3 which is applied for
the binding in fast exchange38
Table 2. Rate Constants of Gly-Ser (2.0 mM) Peptide Bond
Hydrolysis Measured in the Presence of Na3VO4 (20.0 mM)
and Different Inhibitors (50.0 mM) at pD 7.4 and 60 °C
+[VO 3−
]
4
Gly‐Ser HooooooooI [Gly‐Ser...VO 3−
]
(2)
(3)
4
entry
inhibitor
formula
kobs
1
1
1
1
2
3
4
5
6
7
8
no hibitor
alanine
1.33 × 10−6
9.03 × 10−7
7.22 × 10−7
8.36 × 10−7
8.17 × 10−7
9.22 × 10−7
7.53 × 10−7
5.47 × 10−7
=
+
Δ
Δ
maxKf[Na3VO ]
Δmax
4
H2NCH2COOH
malonic acid HOOCCH2COOH
succinic acid HOOC (CH2)2COOH
glutaric acid HOOC (CH2)3COOH
It is interesting to note that the value of the Kf is very similar
to the formation constant previously determined for the
binding between Gly-Gly and vanadate (17 M−1).17
Since vanadate is known to interact with amino acids,39 the
interaction of vanadate with glycine and serine, which are the
adipic acid
malic acid
citric acid
HOOC (CH2)4COOH
HOOCCH2CH(OH)COOH
HOOCCH2C(COOH)
OHCH2COOH
1
products of Gly-Ser hydrolysis, was also examined. H NMR
spectra of glycine and serine exhibited slight shifts upon
addition of vanadate, indicating that weak interactions took
place in solution. The binding of glycine and serine most likely
occurs via their amine nitrogen and carboxylate oxygen which
was confirmed by 0.01 ppm shifts of protons Hα of glycine and
serine in the presence of vanadate (Table S6).
efficiently reduced vanadate promoted Gly-Ser hydrolysis.
However, the binding of malonic and malic acid to vanadate
appears to be rather weak, since a 25 molar excess of the
inhibitor resulted only in respectively a 46 and 44% decrease of
the rate constant. Due to the presence of an additional
carboxylic group, the binding of citric acid to vanadate is
stronger compared to malonic and malic acid, and, con-
sequently, its presence resulted in a decrease of Gly-Ser
hydrolysis by 58%. The inhibitory effect of other aliphatic
dicarboxylic acids was rather small. Their bidentate mode of
binding to vanadate is less effective as the tridentate binding of
Gly-Ser that occurs via its amine nitrogen, amide oxygen, and
carboxylate oxygen. These results are in accordance with the
previous studies which suggest that binding of peptides to
vanadate is 1−2 orders of magnitude stronger compared to the
binding of oxalate and lactate.19,40
Effect of Reaction Conditions and Inhibitors on the
Gly-Ser Hydrolysis. The influence of ionic strength on the
rate constant was studied by following the hydrolytic reaction
in the presence of different amounts of NaClO4. A strong
negative effect on the rate of Gly-Ser hydrolysis was observed
upon adding increasing amounts of NaClO4 (Figure 5). In the
presence of 2.0 mM NaClO4, the rate constant of the hydrolysis
is ca. three times lower compared to the rate constant
determined in the absence of NaClO4. This may be due to
the fact that the presence of high concentrations of salt impedes
electrostatic interactions between vanadate and the peptide.
However, addition of salt also influences the equilibrium
between different oxovanadate species as it is known that in the
presence of high concentrations of NaClO4, polyoxovanadates
V4, V5 forms become more prominent. Since 51V NMR spectra
indicated that vanadate is the species which effectively interacts
with Gly-Ser, the decrease of its concentration in favor of
polyoxo species may also influence the reaction rate.
The effect of temperature on the hydrolytic reaction was
determined by measuring the temperature dependence of kobs
.
As expected, at higher temperatures increased rates were
observed. The activation Gibbs function (ΔG‡ = 117 kJ mol−1
at 37 °C), enthalpy (ΔH‡ = 91 kJ mol−1), and entropy (ΔS‡ =
−84 J mol−1 K−1) were obtained from the Arrhenius and Eyring
plots (Figure S3). The negative value of the activation entropy
is in agreement with the formation of the complex [Gly-Ser/
The inhibitory effect of several nonreactive substrate
analogues was examined by determining the rate constant for
the hydrolysis of Gly-Ser (2.0 mM) by vanadate (20.0 mM) in
the presence of an excess of several organic acids that are
3‑
VO4 ]. However, the activation parameters need to be
interpreted with caution, as they are usually derived from the
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composite rate constants that also include contribution from
the binding of the substrate to the catalyst.
Scheme 6. Intramolecular Attack of the Ser Hydroxyl Group
on the Amide Carbonyl Carbon in Gly-Ser and Ser-Gly
Mechanism of the Hydrolysis. 51V NMR studies
indicated that in a mixture containing different oxovanadate
species, monomeric vanadate is the only anion that interacts
1
with dipeptides. H and 13C NMR experiments indicated that
monomeric vanadate can form three type of complexes with
Gly-Ser, depending on the pH of the solution. While two
complexes (Complex 1 and Complex 3) have been previously
described, the existence of Complex 2 has not been reported so
far. In Complexes 1 and 3 the amide nitrogen is coordinated to
vanadate which leads to stabilization of the peptide bond.17
This makes Complexes 1 and 3 very unlikely candidates to act
as the hydrolytically active species. In contrary, in Complex 2,
this coordination is not observed, and binding via the carbonyl
oxygen is proposed. The interaction between the carbonyl
oxygen of the peptide bond and metal ions is known to polarize
this bond making the amide carbon more susceptible to
nucleophilic attack of water which leads to peptide bond
hydrolysis. The kinetic studies performed at different pD values
suggest that although high pD should be beneficial for the
presence of monomeric vanadate as it decreases the amount of
inactive oligomeric forms, the reaction rate decreases
presumably due to the formation of the inactive Complex 3.
From the screening experiments performed with 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 hydrolysis. As shown in Table S1 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 nucleophile.
The hypothesis that the hydrolysis of X-Ser dipeptides is due to
a N→O acyl rearrangement is strongly supported by the fact
that hydrolysis of Gly-Ser was about 2 orders of magnitude
faster than the hydrolysis of Ser-Gly (Table 3). While the
sterically impedes the intramolecular attack of the Ser hydroxyl
group on the amide carbonyl carbon.
The acceleration of Gly-Ser hydrolysis in the presence of
vanadate suggested that this oxyanion, in addition to the
hydroxyl group within the peptide, plays an important role in
the hydrolysis of the dipeptide. The role of vanadate is most
likely to polarize the carbonyl group by coordinating to it and
to facilitate the attack of the nucleophile. All the data above
allow to postulate a possible mechanism for the hydrolysis of
Gly-Ser in the presence of vanadate as shown in Scheme 7.
A similar mechanism to that shown in Scheme 7 has been
previously proposed for Zn(II)25 and molybdate34 assisted
hydrolysis of GlySer. While the exact comparison is difficult to
make due to the different conditions (pH, temperature,
concentration, type of serine peptides) the rate of peptide
hydrolysis in serine-containing peptides promoted by vanadate
is in the same order of magnitude as previously reported for
Zn(II) and molybdate salts.
Vanadate and Zn(II) have rather distinct coordination
chemistry, however, they are both oxophilic41 and have the
tendency to coordinate to the carbonyl group and cause its
polarization. Comparison of the Gly-Ser hydrolysis observed in
the presence of molybdate and vanadate reveals that the rate
constant measured in the presence of vanadate is about four
times slower than that measured in the presence of molybdate
under similar conditions.34 While in the hydrolytically active
Complex 2 formed by vanadate, only the bidentate
coordination of Gly-Ser has been proposed, the tridentate
coordination of the peptide has been observed in the
hydrolytically active complex formed in the presence of
molybdate. The different mode of coordination might be the
cause for slightly higher hydrolytic activity of molybdate.
The gradual decrease in hydrolysis rate constant at pD > 7.4
can be explained by the increased formation of the hydrolyti-
cally inactive Complex 3, which starts to form around this pD.
This complex has two unfavorable features for the hydrolysis of
the peptide bond, as it involves coordination of the
deprotonated amide nitrogen and hydroxyl oxygen to the
metal ion. Substitution of the amide hydrogen atom for a metal
greatly reduces the susceptibility of the amide carbonyl carbon
atom toward nucleophilic attack, and, at the same time,
coordination of the hydroxyl oxygen to the metal makes the
nucleophilic attack to the amide bond impossible.
Table 3. Rate Constants of the Hydrolysis of Dipeptides in
the Presence of Na3VO4 at pD 7.4 and 60 °C
peptide
Gly-Ser
kobs (s−1
)
1.33 × 10−6
9.72 × 10−7
1.15 × 10−6
6.46 × 10−8
5.22 × 10−8
2.60 × 10−7
Leu-Ser
His-Ser
Gly-Ala
Ser-Gly
Gly-Ser-Phe
intramolecular attack of the Ser hydroxyl group on the amide
carbonyl carbon is possible in Gly-Ser, it is impossible in Ser-
Gly. In the case of Gly-Ser the intramolecular attack of the Ser
hydroxyl group results in a five-membered ring transition state,
while in the case of Ser-Gly the intramolecular attack forms an
unfavorable four-membered ring transition state (Scheme 6). In
comparison to Gly-Ser, the hydrolysis of Gly-Ala in the
presence of vanadate was approximately two order magnitude
slower. The similarity in the backbone of Gly-Ser and Gly-Ala
except for the hydroxyl group in the Ser side chain strongly
suggests that this group is essential for effective hydrolysis.
The slower hydrolysis for Leu-Ser can be explained by the
steric hindrance caused by the Leu side chain which inhibits
nucleophilic attack and results in slower peptide hydrolysis.
Similarly, in the case of Gly-Ser-Phe the presence of the bulky
phenyl group in the side chain of the GlySerPhe tripeptide
Our recent studies on the reactivity of oxovanadates toward
carboxylic esters also identified monomeric vanadate as the
hydrolytically active species.14 The origin of the hydrolytic
activity of vanadate was attributed to a combination of its
nucleophilic nature and the chelating effect of the vanadium
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Scheme 7. Proposed Mechanism of Gly-Ser Hydrolysis Promoted by Vanadate
which can lead to the stabilization of the transition state. On
the contrary, polyoxo forms of vanadate (V4, V5, and V10) were
implicated as active species in the hydrolysis of phosphoest-
ers.12,13 The high lability of the polyoxo forms results in partial
detachment of one VO4 tetrahedron and allows for the
attachment of the structurally related phosphodiester tetrahe-
dron into the polyoxometalate structure. The incorporation of
the phosphodiester group into the polyoxovanadate skeleton
results in the sharing of its oxygen atoms with V(V) center that
can cause polarization and strain of the P−O ester bond, that in
turn makes it more susceptible toward hydrolysis. The
structural analogy between phosphate and vanadate forms the
basis of this interaction; however, such interaction is less
plausible between structurally distinct vanadate and peptides.
Indeed, to the best of our knowledge there are no studies
reporting on inclusion of peptides into polyoxovanadate
structures. A complex formed between V10 and Gly-Gly has
been structurally characterized, and it was found that the
peptide interact noncovalently with the polyoxometalate
structure via hydrogen bonding.42 On the other hand,
monomeric vanadate has a tendency to coordinate to
polydentate ligands and to peptides.17,39 Therefore, the
different coordination affinities of monomeric and polyoxo
forms of vanadate toward phosphoesters and peptides are most
likely the reason for the different reactivity exhibited toward
these classes of compounds.
formations optimized in aqueous media are depicted in Figure
6.
The theoretically predicted structures of Complexes 1 and 3
in the gas phase were previously described.22 DFT-based Car−
Parrinello molecular dynamics simulations and NMR calcu-
lations of the vanadate-glycylglycine complex that is analogous
to Complex 1 reveal that in solution this compound should be
formulated as five-coordinate anionic [VO2(Gly-Gly′)]−
Molecular Modeling of Gly-Ser and Its Vanadate
Complexes. To support the experimental data and the
suggestion that bidentate coordination of Gly-Ser to the
vanadate ion through both the amine and carbonyl group might
take place in the course of the hydrolysis reaction, we
undertook DFT-based molecular modeling of Complex 2. For
the sake of comparison the molecular structures of Complexes
1 and 3 as well as of that of the Gly-Ser anion and Gly-Ser
zwitterion are also considered. Their lowest energy con-
Figure 6. IEFPCM-B3LYP/6-311++G(2d,2p) optimized geometries
of Gly-Ser and its vanadate complexes.
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1
Table 4. Experimental and Theoretical H and 13C NMR Chemical Shifts (δ, ppm) Calculated at the IEFPCM-B3LYP/6-311+
+G(2d,2p) Level of Theory
Gly-Ser anion
theor.
Gly-Ser zwitterion
exp. theor.
45.16
complex 2
theor.
complex 1
theor.
complex 3
theor.
atom
exp.
42.97
exp.
46.84
exp.
47.90
C1
49.81
179.37
58.20
184.54
68.18
40.76
167.10
57.29
175.97
62.0
49.57
52.37
186.53
78.03
189.69
71.58
53.83
186.90
74.63
187.71
84.05
C2
172.61
57.02
175.91
62.11
3.49
169.76
59.58
183.87
59.53
179.80
67.94
182.99
62.26
3.59
172.56
69.83
179.59
75.60
3.60
C1′
C2′
C3′
182.84
67.75
182.27
67.35
Ha (average)
3.39/3.72
(3.55)
3.85
3.92/3.94
(3.93)
3.68/3.84
(3.76)
3.51/3.53
(3.52)
3.43/3.55
(3.49)
Ha′
4.29
4.06
4.33
4.09
4.14
4.69
5.04
4.58
5.14
Hb′ (average) 3.80/3.84 3.35/3.98
3.82/3.88 3.49/4.08
(3.79)
3.52/4.12
(3.82)
3.95/4.11 4.20/4.41
(4.31)
4.39/4.55 4.48/5.32
(4.90)
(3.67)
1
Table 5. Relative Experimental and Theoretical H and 13C NMR Chemical Shifts (Δδ, ppm) Calculated at the IEFPCM-
B3LYP/6-311++G(2d,2p) Level of Theory
complex 2
complex 1
complex 3
a
a
b
atom
exp.
theor.
4.41
exp.
6.08
theor.
7.21
exp.
4.93
theor.
4.03
C1
0.16
0.37
−0.01
0.03
0.02
0.02
0
C2
14.11
−0.06
−0.57
−0.40
12.70
10.65
7.02
16.77
18.45
6.85
−0.05
12.81
3.68
7.53
C1′
C2′
C3′
Ha
16.42
3.17
0.26
3.83
13.49
0.11
15.87
−0.24/−0.10
−0.26
0.36
−0.40/−0.41
0.95
0.05/−0.17
1.07
Ha′
Hb′
0.05
0.29
0
0.03/0.04
0.13/0.23
0.71/0.32
0.59/0.71
1.13/1.35
a
b
Calculated with respect to Gly-Ser zwitterion. Calculated with respect to Gly-Ser anion.
species.23 Based on these findings in this study Complexes 1−3
are modeled as anionic species with a five-coordinated
vanadium. Among the three complexes considered Complex
1 has the lowest free energy in the aqueous phase. As it was
previously found39 the tridentate coordination of Gly-Ser
through N1, N2, and O3 in complex 3 is less stable than the
corresponding N1, N2, and O2 coordination in Complex 1.
The free energy in solution of Complex 3 is calculated to be
higher with respect to complex 1 by 8.4 kcal/mol. Noteworthy,
the free energy of Complex 2 is computed to be higher with
respect to the major complex (Complex 1) by only 7.1 kcal/
mol. Thus, on the basis of the calculations the formation of
Complex 2 should be thermodynamically feasible under the
reaction conditions.
Further, in support of the experimental evidence for
bidentate coordination of Gly-Ser the 1H and 13C NMR
chemical shifts for the three complexes and the free ligand were
calculated and compared with the experimental spectra. The
theoretical chemical shifts along with the experimental data are
summarized in Table 4. A comparison of the results for the Gly-
Ser anion (pD = 9.3) and Gly-Ser zwitterion (pD = 7.4) as well
as for the Complexes 1 and 3 reveal very good agreement
between the calculated and the experimental 1H and 13C NMR
shifts. The 13C NMR chemical shifts for these species are
predicted with a standard error of 2−3 ppm and a R2 = 0.99.
However, a better understanding of the NMR changes in Gly-
Ser which occur upon coordination could be obtained
considering the relative chemical shifts, Δδ (Table 5). With
exception of Δδ values for C1 in complex 2 and for C2 in
complex 3 all other theoretical values are in qualitative
agreement with the experiment. It should be noted that for
Complex 2 the calculations predict the largest Δδ value for C2
followed by C1 and almost no shifting for the other atoms.
Qualitatively this is exactly what is experimentally observed,
however in smaller magnitude. A possible explanation for the
smaller experimental shifting of C2 and C1 could be found on
the basis of lower kinetic stability of Complex 2 and
comparatively short lifetime of this complex.
For a detailed understanding of the role of the vanadate or
how Gly-Ser becomes hydrolytically active/inactive upon
coordination a comparative analysis of various molecular
properties of Gly-Ser and its vanadate complexes were
performed. Selected geometrical parameters, atomic charges,
bond orders, and the bonding orbitals polarization (from NBO
analysis) are collected in Table 6. A comparison of the bonding
orbitals polarizations (BOP) of Gly-Ser with those of
Complexes 1−3 reveals that the σC2−N2 and σC2−O2
bonding orbitals remain slightly affected upon coordination,
whereas the πC2−O2 is more sensitive and hence more
informative. The coordination of the Gly-Ser carbonyl oxygen
to the vanadium atom produce additional polarization of the
CO bond. Thus, the πCO bonding orbital, with
percentage of occupancy 26.18(C)/73.82(O) in the Gly-Ser
zwitterion, becomes more polarized in Complex 2, 22.29(C)/
77.71(O), the CO bond order decreases from 1.55 to 1.40,
and the bond length increases with 0.021 Å. Conversely, going
again from the Gly-Ser zwitterion to Complex 2, the C2−N2
bond order increase from 1.31 to 1.37 and the C2−N2 bond
length is calculated to be shorter by 0.011 Å. As a result of
electron density reorganization upon coordination the atomic
charge of C2 in Complex 2 becomes more positive, as
compared to the free ligand, and hence more susceptible for
nucleophilic attack (in particular, intramolecular attack of the
alcoholic function). In Complexes 1 and 3 the atomic charges
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Table 6. Relative Free Energy in Aqueous Solution (ΔGsol, kcal/mol), Selected Geometrical Parameters (Bond Lengths in Å,
Angles in deg), Wiberg Bond Order, Bonding Orbitals Polarization (BOP, %), and Atomic Charges Calculated at the IEFPCM-
B3LYP/6-311++(2d,2p) Level of Theory
quantity
ΔGsol
Geom. param.
R(C2−N2)
R(C2−O2)
R(N3−C1′)
R(O2. O2′. O3′)−V
R(N1−V)
Gly-Ser anion
Gly-Ser zwitterion
complex 2
7.1
complex 1
0.0
complex 3
8.4
1.345
1.328
1.317
1.256
1.458
2.153
2.212
-
1.341
1.238
1.454
1.961
2.155
2.047
−2.2
1.337
1.241
1.445
1.886
2.182
2.072
0.3
1.234
1.235
1.452
1.456
-
-
-
-
R(N2−V)
-
-
D(O2−C2−N2−C1′)
Wiberg BO
C2−N2
C2−O2
(O2. O2′. O3′)−V
N1−V
4.3
6.3
6.5
1.25
1.31
1.37
1.40
0.38
0.43
-
1.29
1.54
0.63
0.51
0.62
1.30
1.53
0.83
0.48
0.59
1.57
1.55
-
-
-
-
-
-
N2−V
NBO BOP
C2−N2
C2−O2
σ 37.64/62.36
σ 34.34/65.66
π 27.43/72.57
σ 38.26/61.74
σ 35.31/64.69
π 26.18/73.82
σ 38.32/61.68
σ 34.46/65.54
π 22.29/77.71
σ 38.34/61.66
σ 34.92/65.08
π 26.31/73.69
σ 38.38/61.62
σ 34.92/65.08
π 26.03/73.97
Charge
NBO
C2
0.691
−0.627
−0.721
-
0.683
−0.604
−0.699
-
0.715
0.684
0.684
N2
−0.572
−0.638
0.895
−0.641
−0.730
0.849
−0.638
−0.740
0.837
O2
V
Hirshfeld
C2
0.166
−0.079
−0.359
-
0.185
−0.062
−0.307
-
0.214
0.156
0.148
N2
−0.047
−0.231
0.465
−0.180
−0.384
0.441
−0.176
−0.403
0.401
O2
V
of C2 retain almost the same values (according to NBO
analysis) or become even less positive (according to Hirshfeld
analysis) as compared to the free ligand.
possible. While the aim of our studies is not necessarily to
propose the use of vanadate for practical purposes in the
hydrolysis of peptides and proteins, but it is a rather a
remarkable fundamental finding that despite the lack of some
basic requirements commonly assumed for an artificial
metallopeptidase such as positive charge and the presence of
water or hydroxide in the first coordination spehre, the
vanadate anion is able to selectively hydrolyze a series of X-Ser
peptides under physiological pH and temperature. This and
future studies focusing on the reactivity of oxovanadates toward
relevant biomolecules may shed more light on the molecular
origin of the biological activity of vanadium.
CONCLUSION
■
In conclusion, we report on the first example of peptide bond
hydrolysis promoted by the vanadate(V) oxyanion. The
hydrolytic effect of vanadate can be attributed to its ability to
efficiently coordinate X-Ser peptides and polarize carbonyl
groups toward the internal nucleophilic attack by a hydroxyl
group of Ser residue. Although the hydrolysis occurred in
solutions containing polyoxo-forms of vanadate, the NMR and
kinetic studies indentified the monomeric vanadate as the
source for the hydrolytically active species. A previously
unreported complex in which coordination of the peptide to
vanadate occurs via amine nitrogen and peptide carbonyl
oxygen atoms has been discovered. This complex is proposed
to be the effective hydrolytic species, and its existence has been
supported by theoretical calculations which suggest that the
formation of Complex 2 is thermodynamically feasible under
the reaction conditions. We are currently investigating the
reactivity of vanadate toward the hydrolysis of a range of
different oligopeptides and proteins. It is interesting to mention
that although the studies with short peptides presented in this
work suggest that the cleavage should preferentially occur at the
N-terminal amide bond, the preliminary studies with proteins
indicate that the hydrolysis of the internal amide bonds is also
EXPERIMENTAL SECTION
■
Materials. Glycylserine (Gly-Ser), leucylserine (Leu-Ser),
histidylserine (His-Ser), glycylalanine (Gly-Ala), serylglycine (Ser-
Gly), and sodium vanadate were purchased from Acros and used
without further purification. The pH of the solutions for the 1H NMR
studies was adjusted with D2SO4 and NaOD, both from Acros.
NMR Spectroscopy. 1H, 13C, and 51V NMR spectra were recorded
on a Bruker Advance 400 and Bruker Advance 600 spectrometers.
D2O with 0.05 wt % 3-(trimethylsilyl)propionic acid as an internal
standard for 1H NMR was used as a solvent. A solution of VOCl3 and
a solution of TMS in CDCl3 were employed as an external 51V and 13
NMR references.
C
Kinetics. In a typical kinetic experiment, the hydrolysis of 2 mM a
1
peptide in the presence of 25 mM of vanadate was followed by H
NMR spectroscopy. For example, the typical reaction mixture was
prepared by dissolving 0.32 mg of Gly-Ser in 1 mL of D2O to which
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4.6 mg of Na3VO4 × 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 typically less than 0.1 unit. The pD value of the solution was
obtained by adding 0.41 to the pH reading, according to formula pD =
pH + 0.41. The reaction samples were kept at constant temperature
(typically 60 °C), and the rate constants for the hydrolysis were
determined by following the appearance of the free glycine resonance
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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
resonance 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 concentration
was measured, was used. The R-values were generally higher than 0.98.
The influence of the ionic strength on the reaction rate was studied by
following the reaction between 2 mM Gly-Ser and 25 mM Na3VO4
(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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Computational Procedure. All geometrical optimizations and
molecular properties were calculated with the Gaussian0943 program
package. Conformational analysis of the Gly-Ser anion, Gly-Ser
zwitterion, and Complexes 1, 2, and 3 were performed with the hybrid
density functional B3LYP44−46 and basis 6-31+G(d,p) both in the gas
phase and in water solution, utilizing the IEFPCM47,48 method as
implemented in Gaussian09 (using the default options). The
vanadium electrons in Complexes 1−3 were described with relativistic
Stuttgart pseudopotential49 (SDD, describing ten core electrons) and
the appropriate contracted basis set (8s7p6d1f)/[6s5p3d1f]. The
lowest energy conformations were refined with the same density
functional in solution and basis set 6-311++G(2d,2p) for all atoms.
The molecular properties were calculated at the final geometries using
the same level of theory, IEFPCM-B3LYP/6-311++G(2d,2p). To
estimate the atomic charges both Hirshfeld50−52 and natural bond
orbital (NBO) analysis53,54 were performed. The polarization of the
bonding orbitals and the Wiberg bond orders55 are computed applying
(13) Steens, N.; Ramadan, A. M.; Absillis, G.; Parac-Vogt, T. N.
Dalton Trans. 2010, 39 (2), 585−592.
(14) Ho, P. H.; Breynaert, E.; Kirschhock, C. E. A.; Parac-Vogt, T. N.
Dalton Trans. 2011, 40 (1), 295−300.
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1
the NBO scheme. The H and 13C isotropic shielding constants (σi)
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ASSOCIATED CONTENT
* Supporting Information
Tables S1−S7 and Figures S1−S3. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
*E-mail: Tatjana.Vogt@chem.kuleuven.be.
Notes
■
The authors declare no competing financial interest.
(27) (a) Krezel, A.; Kopera, E.; Protas, A. M.; Poznanski, J.;
Wyslouch-Cieszynska, A.; Bal, W. J. Am. Chem. Soc. 2010, 132 (10),
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ACKNOWLEDGMENTS
■
T.N.P.V. thanks KU Leuven for the financial support
(START1/09/028). P. H. Ho thanks the Vietnamese Govern-
ment and KU Leuven for a doctoral Fellowship. T.N.P.V. and
K.P. thank FWO Flanders for a research grant (G.0260.12).
T.M. thanks KU Leuven for financial support under a BOF-F+
contract connected to the GOA ″Multicentre Quantum
Chemistry″ project.
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