Zn(II) enhances nucleotide binding and dephosphorylation in the presence of a poly(ethylene imine) dendrimer

Article abstract of DOI:10.1016/j.ica.2013.11.008

The interaction of a second-generation poly(ethylene imine) dendrimer (L) with the nucleotides AMP, ADP and ATP was analysed by means of potentiometric titrations (0.1 M Me₄NCl, 298.1 K), in the absence and in the presence of Zn²⁺, to perform speciation of the systems and determination of complex stability constants. Protonated forms of L interact with anionic forms of the nucleotides giving rise to stable anion complexes. In the presence of Zn²⁺, ternary complexes (ion-pair complexes) become the main species in solution, the presence of each partner enhancing the binding of the other one (positive cooperativity effect). The stability constants determined by the same method and under the same experimental conditions for the formation of PO₄^3-, P₂O₇ ^4- and P₃O₁₀^5- complexes with L showed a strict similarity between the binding of AMP, ADP and ATP and their inorganic counterparts, indicating that the main interaction of nucleotides with protonated ligand forms takes places through their inorganic tails. Dephosphorylation of ATP to form ADP and phosphate was monitored by means of ³¹P NMR spectra at pH 3 and 9 in the presence of L and of its Zn ²⁺ complexes. The rate of ATP dephosphorylation is enhanced by about 2 times at pH 3 and 5 times at pH 9 in the presence of L and by about 4 times at pH 3 and 6 times at pH 9 in the presence of its metal complex, relative to the uncatalysed process, the dephosphorylation reactions being much faster in the acidic media where successive cleavage of ADP to give AMP and phosphate was also observed. Depending on the pH, the observed dephosphorylation enhancements have been interpreted in terms of formation of phosphoramidate intermediates, formation of ATP-Zn²⁺ coordinative bonds and positive allosteric effects due to metal ion coordination.

Full text of DOI:10.1016/j.ica.2013.11.008

Inorganica Chimica Acta xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Zn(II) enhances nucleotide binding and dephosphorylation
in the presence of a poly(ethylene imine) dendrimer
⇑
Carla Bazzicalupi, Antonio Bianchi , Claudia Giorgi, Barbara Valtancoli
Department of Chemistry ‘‘Ugo Schiff’’, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
The interaction of a second-generation poly(ethylene imine) dendrimer (L) with the nucleotides AMP,
4
ADP and ATP was analysed by means of potentiometric titrations (0.1 M Me NCl, 298.1 K), in the absence
and in the presence of Zn2 , to perform speciation of the systems and determination of complex stability
+
Keywords:
constants. Protonated forms of L interact with anionic forms of the nucleotides giving rise to stable anion
complexes. In the presence of Zn , ternary complexes (ion-pair complexes) become the main species in
Anion complexes
Ion-pair complexes
Nucleotides
ATP dephosphorylation
Poly(ethylene imine)
Dendrimers
2+
solution, the presence of each partner enhancing the binding of the other one (positive cooperativity
effect). The stability constants determined by the same method and under the same experimental condi-
3ꢀ
4ꢀ
5ꢀ
tions for the formation of PO
4
2
, P O
7
3
and P O10 complexes with L showed a strict similarity between
the binding of AMP, ADP and ATP and their inorganic counterparts, indicating that the main interaction of
nucleotides with protonated ligand forms takes places through their inorganic tails.
Dephosphorylation of ATP to form ADP and phosphate was monitored by means of 31P NMR spectra at
2
+
pH 3 and 9 in the presence of L and of its Zn complexes. The rate of ATP dephosphorylation is enhanced
by about 2 times at pH 3 and 5 times at pH 9 in the presence of L and by about 4 times at pH 3 and 6 times
at pH 9 in the presence of its metal complex, relative to the uncatalysed process, the dephosphorylation
reactions being much faster in the acidic media where successive cleavage of ADP to give AMP and
phosphate was also observed. Depending on the pH, the observed dephosphorylation enhancements have
been interpreted in terms of formation of phosphoramidate intermediates, formation of ATP–Zn2
coordinative bonds and positive allosteric effects due to metal ion coordination.
+
Ó 2013 Published by Elsevier B.V.
1
. Introduction
The appreciation that anions play important roles in both
Since the seminal works of Kimura and Lehn on nucleotide
binding by polyammonium macrocycles in the earlier ’80s [8,9],
the use of synthetic receptors has become a common strategy to
mimic biological binding and recognition of nucleotides by pro-
teins [1,13,14]. Proteins employ a variety of forces to bind anions,
their binding sites consisting of both polar and apolar groups such
as cationic, hydrogen bonding residues, aliphatic chains and
aromatic functionalities giving rise to stacking interactions. The
polyfunctional character of proteins can be replicated in synthetic
receptors by the introduction of appropriate functional groups and
the topological control of their molecular arrangement. A snapshot
of the nucleotide–synthetic receptor interaction, showing the
variety of binding forces, was recently obtained by X-ray determi-
abiotic and biological systems has attracted a great deal of interest
toward the use of synthetic receptors for the binding and recogni-
tion of anionic substrates [1–7]. Among anions of biological inter-
est, the purine mononucleotides ATP, ADP, AMP, and the related
inorganic phosphates, were among the first targets of synthetic
receptors [8,9]. It is known that ATP, along with ADP and inorganic
phosphate, from which they are formed, take part in more chemi-
cal reactions than any other compound except water [10]. ATP is
mainly associated with the transport of chemical energy within
cells for metabolism, but it is also involved in signal transduction
and transmission pathways [11], and is incorporated into nucleic
acids by polymerases in the process of DNA replication and tran-
scription [12]. ATP is produced by ATP synthase from inorganic
phosphate and ADP or AMP and is converted into his precursors
by metabolic processes that use it as an energy source.
nation of the first crystal structure showing a nucleotide (thymi-
0
dine 5 -triphosphate) adduct with
a polyammonium ligand
containing aromatic groups [15]. Cationic groups of anion binding
sites of proteins include metal ions. A 2007 review, dealing with
the binding motifs found in proteins for recognition of phosphate
containing substrates, revealed that 547 of the 3003 protein struc-
tures identified in the RCSB protein database as phosphate binders
contain a metal coordinating to the anionic group in the substrate
⇑
Corresponding author.
E-mail address: antonio.bianchi@unifi.it (A. Bianchi).
[
16].
0
020-1693/$ - see front matter Ó 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.ica.2013.11.008
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2
C. Bazzicalupi et al. / Inorganica Chimica Acta xxx (2013) xxx–xxx
One of the most interesting points of nucleotide receptors
and anion–metal ion interactions can participate in stabilising
these ion-pair complexes.
chemistry is the capacity that some synthetic receptors have to in-
duce catalytic processes mimicking the behaviour of ATP-ases or
kinases [1–7,14]. The macrocyclic polyamines O-BISDIEN [8,17],
Considering the peculiar binding properties of L, we found it
interesting to analyse the effect of the dendritic structure of this
ligand on the binding of AMP, ADP and ATP, as well as on the pos-
sible catalytic activity that it might have in ATP dephosphorylation
in the absence and in the presence of the biologically significant
[
7
21]aneN [18] and the meta-cyclophane [19] shown in Fig. 1, for
example, rank as the abiotic receptors producing highest enhance-
ments of ATP cleavage. The insertion of metal ions into the receptor
structure may affect the receptor ability to enhance nucleotide
dephosphorylation. For instance, investigation of the effect of the
2
+
metal ion Zn . For the sake of comparison, the analysis was
3ꢀ
4ꢀ
5ꢀ
10
extended to the binding of PO
4
2
, P O
7
and P
3
O
which are
biologically significant metal ions Ca2 , Mg , and Zn , added to
O-BISDIEN, revealed striking influences on ATP cleavage and on
the formation of the phosphoramidate intermediate and pyrophos-
+
2+
2+
the inorganic counterparts of these nucleotides. We describe here
the results of this study.
phate [20]. While addition of both Ca2 and Mg increased the
observed percentage of phosphoramidate, only Ca provided a sig-
nificant acceleration of ATP dephosphorylation, almost doubling
the first-order rate constant found for free O-BISDIEN in the same
experimental conditions. Conversely, the presence of Zn , as well
as of Cd , to O-BISDIEN/ATP solutions decreased the rate of nucle-
otide cleavage.
+
2+
2
. Experimental
2+
2.1. Materials
2+
Ligand L was synthesised as previously described [23].
2
+
2.2. Potentiometric measurements
Most of polyammonium receptors employed for nucleotide
binding and activation studies were macrocycles [1–9,13–15,17–
0] although also several linear [21] and few branched [22] mole-
+
All pH-metric measurements (pH = ꢀlog[H ]) employed for the
2
determination of equilibrium constants were carried out in 0.1 M
cules were also considered.
4
NMe Cl solutions at 298.1 ± 0.1 K, by using the equipment and
In recent papers [23,24], we described the coordination proper-
ties of the second-generation poly(ethylene imine) dendrimer (L),
showing that this iperbranched ligand is able to form stable com-
the methodology that has been already described [23]. The com-
bined Hamilton glass electrode (LIQ-GLASS 238000/08) was cali-
brated as a hydrogen concentration probe by titrating known
amounts of HCl with CO -free NMe OH solutions and determining
2
+
2+
2+
2+
2+
plexes with both metal ions (Ni , Cu , Zn , Cd and Pb ) [23]
ꢀ
2ꢀ
2ꢀ
2ꢀ
ꢀ
4
2
4
and anions (NO
3
, SO
4
, SeO
4
, HPO
4
2
and H PO ) [24]. Fur-
the equivalent point by Gran’s method [25] which allows one to
thermore, due to the many amine groups present in the molecules,
L forms highly protonated metal complexes that, in turn, bind an-
ions giving rise to ion-pair complexes of considerable stability.
Molecular modelling calculations showed that both anion–ligand
o
determine the standard potential E and the ionic product of water
(
pK
measurements were performed for each system in the pH range
.5–10.5. The computer program HYPERQUAD [26] was used to calcu-
late the equilibrium constants from e.m.f. data. The concentration
w 4
= 13.83(1) at 298.1 ± 0.1 K in 0.1 M NMe Cl). At least three
2
ꢀ3
of the ligand was 1 ꢁ 10 M in all measurements. The concentra-
tion of anions [A] in anion coordination experiments was in the
range = [L] 6 [A] 6 5[L]. Three measurements were performed for
each anion and the titration curves were treated both separately
or as a unique set without significant variations in the calculated
stability constants. The final stability constants were obtained by
processing the whole set of curves. The measurement for the
simultaneous binding of metal ions and anions were performed
with a metal ion concentration [M] = 0.8[L], to ensure that only
mononuclear complexes were formed, while the concentration of
the anion was again [L] 6 [A] 6 5[L]. Three measurements were
performed for each system by using the same procedure. Different
equilibrium models for the complex systems were generated by
eliminating and introducing different species, including species
with 1:2 and 1:3 receptor:anion stoichiometries. Only those mod-
NH
2
H
H
NH
2
N
O
O
N
N
H
2
N
H
N
NH
N
N
NH
2
N
N
H
H
N
L
O-BISDIEN
H
2
N
NH
2
H
N
H
H
N
N
NH
2
HN
NH
N
N
N
els for which the HYPERQUAD program furnished a variance of the
2
N
N
H
2
residuals
r
6 9 were considered acceptable. Such condition was
O
O
P
O
8
N
CH
2
7
O
[
21]aneN
7
unambiguously met by a single model for each system. The equi-
O
P
O
O
P
O
-
librium constants for ligand protonation, for the formation of
-
-
-
O
O
O
1
’
2+
H
H
Zn complexes with L, for anion protonation and for the formation
2
+
H
H
of Zn complexes with anions used in calculation were deter-
mined by us in previous works or taken from the literature
OH OH
[
23,27,28].
Adenosine 5’-monophosphate (AMP)
H
H
N
N
3
1
2
.3. P NMR measurements
Adenosine 5’-diphosphate (ADP)
Adenosine 5’-triphosphate (ATP)
HN
NH
31P (161 MHz) NMR measurements were performed in D
solutions at 298 K on a Bruker-Advance III 400 MHz spectrometer.
Chemical shifts were recorded relative to 85% H PO as an external
reference. Small amounts of 0.01 M NaOD and DCl were used to ad-
just the solutions pD. The pH was calculated from the measured pD
value by using the relationship pH = pD ꢀ 0.40 [29]. The kinetics of
2
O
N
N
H
3
4
meta-cyclophane
Fig. 1. Ligand (L) and nucleotide drawings. Abiotic receptors producing highest
enhancements of ATP dephosphorylation.
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C. Bazzicalupi et al. / Inorganica Chimica Acta xxx (2013) xxx–xxx
Table 2
3
ATP dephosphorylation were determined by keeping 0.01 M solu-
3
ꢀ
4ꢀ
5ꢀ
10
tions of ATP and 0.001 M in the catalyst (L or ZnL2 ) at pH 3 and
+
Stability constants of the complexes formed by L with PO4 , P O
and P O
.
2
7
3
a
0.1 M Me
4
NCl, 298.1 ± 0.1 K.
9
at 70 °C for increasing times. The reaction mixtures were rapidly
3
1
nꢀ
43ꢀ
74ꢀ
3
O
105ꢀ
quenched to room temperature before recording the P NMR
spectra.
Anion (A
HL+ + HA
)
PO
P
2
O
P
(nꢀ1)ꢀ
(2ꢀn)+
3.13(8)b
3.22(7)
= [(HL)HA]
2
3
+
+
(nꢀ1)ꢀ
(3ꢀn)+
H
2
H
3
H
4
H
3
H
5
H
4
H
6
H
5
H
6
H
6
H
7
H
7
H
8
H
7
L
L
L
L
L
L
L
L
L
L
L
L
L
L
+ HA
2
= [(H L)HA]
nꢀ
(3ꢀn)+
(4ꢀn)+
+ A = [(H
3
L)A]
L)A]
3.84(5)
3.47(4)
4.20(5)
5.01(5)
2.4. Modelling calculations
4+
nꢀ
+ A = [(H
4
3
5
4
6
+
+
+
+
(nꢀ1)ꢀ
(4ꢀn)+
(5ꢀn)+
+ HA
= [(H
3
L)HA]
3.38(8)
4.18(8)
nꢀ
(5ꢀn)+
Simulated annealing procedures (10 ps equilibrating time, 10 ps
+ A = [(H
5
L)A]
5.92(4)
7.35(4)
6.87(4)
(nꢀ1)ꢀ
+ HA
= [(H
4
L)HA]
5.13(4)
running time and 10 ps cooling time, running temperature 600 K,
starting and ending temperature 0 K) were carried out to analyse
nꢀ
(6ꢀn)+
+ A = [(H
6
L)A]
5+
6+
6+
(nꢀ1)ꢀ
(6ꢀn)+
(7ꢀn)+
+ HA
= [(H
= [(H
5
L)HA]
L)HA]
L)H
L)HA]
L)H
3.56(6)
4.04(4)
4.08(5)
5.10(5)
4.64(6)
4.39(4)
2
+
3ꢀ
2ꢀ
ꢀ
the adducts formed by H
3
L
with ADP , HADP and H
2
ADP ,
(nꢀ1)ꢀ
+ HA
6
4
ꢀ
3ꢀ
2ꢀ
(nꢀ2)ꢀ
(8ꢀn)+
or ATP , HATP and H
2
ATP . The AMBER forcefield, as imple-
mented in the version 7.51 of HYPERCHEM [30], was used for all calcu-
lations. Atomic charges were estimated at the semiempirical level
PM3 method) [31] and an implicit simulation of aqueous environ-
ment (variable dielectric constant = 4R) was applied.
0 conformers per adduct were obtained and the conformers
+ H
+ HA
2
A
= [(H
6
2
A]
7
7
8
+
+
+
(nꢀ1)ꢀ
(8ꢀn)+
= [(H
7
7.37(5)
5.95(4)
5.82(4)
6.34(4)
(nꢀ2)ꢀ
(nꢀ2)ꢀ
(nꢀ3)ꢀ
(9ꢀn)+
+ H
+ H
+ H
2
A
2
A
3
A
= [(H
7
2
A]
A]
A]
4.10(4)
3.40(4)
(10ꢀn)+
(10ꢀn)+
=[(H
8
L)H
L)H
2
(
7+
= [(H
7
3
e
a
43ꢀ
Values for PO
are taken from Ref [24].
8
b
Values in parentheses are standard deviations on the last significant figures.
featuring similar conformations were grouped in families. Only
the families within 2 kcal/mol from the minimum were then
selected.
Table 3
Stability constants of the complexes formed by ZnL2 with AMP, ADP and ATP. 0.1 M
+
3
. Results and discussion
Me
4
NCl, 298.1 ± 0.1 K.
nꢀ
The equilibrium constants for the interaction of anions with L
Anion (A
)
AMP
ADP
ATP
and its mononuclear Zn2 complexes were determined by
computer analysis of potentiometric titration data performed by
means of the program HYPERQUAD [26]. This computer program
furnished the complex stability constants according to the
+
(HZnL)3 + A = [(HZnL)A]
+
nꢀ
(3ꢀn)+
2.46(6)a 3.19(6)
ZnL)4 + A = [(H
+
nꢀ
ZnL)A]
ZnL)A]
(4ꢀn)+
2.99(6)
3.52(5)
4.81(5)
4.47(4)
5.28(4)
4.41(5)
5.48(5)
4.66(5)
5.63(5)
4.3(5)
3.73(4) 3.70(4)
4.14(5) 4.52(4)
6.05(5) 6.76(4)
5.76(6) 7.00(6)
6.66(4) 8.61(4)
5.74(4) 7.16(5)
5.23(4) 6.04(5)
5.63(5) 5.65(5)
3.92(5) 3.95(4)
4.55(6) 4.11(4)
2.35(7)
(H
2
3
3
4
5
4
2
ZnL)5 + A = [(H
+
nꢀ
(5ꢀn)+
(
(
(
(
H
H
H
H
3
ZnL)5 + HA
+
(nꢀ1)ꢀ
= [(H
ZnL)HA]
(6ꢀn)+
(7ꢀn)+
3
6+
nꢀ
(6ꢀn)+
ZnL) + A = [(H
4
ZnL)A]
nꢀ
m+2ꢀn)+
+
(mꢀn)+
nꢀ
2+
+
equilibria A + L + mH = (ALH
m
)
and A + Zn + L + mH =
7+
nꢀ
(7ꢀn)+
ZnL) + A = [(H
5
ZnL)A]
(
6+
(nꢀ1)ꢀ
(
m
AZnLH )
. Such equilibria are indicative of the stoichiome-
(H
ZnL) + HA
4
= [(H ZnL)HA]
(
(
(
7ꢀn)+
+
(8ꢀn)+
[
[
[
[
(H
(H
(H
(H
4
5
6
7
ZnL)HA]
ZnL)HA]
ZnL)HA]
ZnL)HA]
+ H = [(H
+ H = [(H
+ H = [(H
5
ZnL)HA]
6
ZnL)HA]
7
ZnL)HA]
try of the complexation equilibria but do not provide any
information about the location of the m protons in the complex
species. In previous studies, it was shown that the formation of
anion complexes does not modify the protonation pattern of the
8ꢀn)+
9ꢀn)+
+
(9ꢀn)+
+
(10ꢀn)+
(10ꢀn)+
+
(11ꢀn)+
+ H = [(H
7
ZnL)H
2
A]
(
11ꢀn)+
+
(12ꢀn)+
[(H ZnL)H A]
7
2
+ H = [(H ZnL)H A]
8
2
ligand, although
a modest general shift of NMR signals,
a
Values in parentheses are standard deviations on the last significant figures.
corresponding to the increase of ligand basicity brought about by
anion complexation, was observed [18a,21a,32]. Accordingly, the
location of protons in the complexes was assumed to be regulated
by the basicity of the interacting species and the relevant stability
constants, listed in Tables 1–3, were calculated according to this
role.
mainly controlled by electrostatic forces. A first exception to this
role is observed when the stability of the complexes formed by
6+
7+
3ꢀ
H
6
L
and H
7
L
is considered, the complexes of HATP
and
2
ꢀ
HADP with the less charged ligand species being more stable
ꢀ
3.1. Interaction of L with nucleotides and inorganic phosphates
that those with the more charged ones and the stability of HAMP
complexes being identical for the two differently charged ligand
forms (Table 1). It was previously shown that in H6L6+ protonation
involves the six external primary nitrogen atoms of the ligand
while the successive protonation stage giving rise to H7L7+ takes
place on the central tertiary nitrogen. We can expect that some
modification of the receptor structure occurs with the seventh pro-
tonation stage which deteriorates the nucleotide-receptor match-
ing. Indeed, modelling calculations performed on the complexes
The equilibrium constants for the interaction of AMP, ADP and
ATP with protonated forms of L are listed in Table 1. As can be seen
from this table, the ligand needs to be at least in its tetraprotonated
4
+
H
4
L
form to interact with these nucleotides. With a few excep-
tions, the stability of the complexes increases with the positive
charge of the ligand (increasing ligand protonation) and the nega-
tive charge of the nucleotide, evidencing that the interaction is
3ꢀ
6+
7+
of HATP with H
6
L
and H
7
L
revealed that protonation of the
central ligand nitrogen atom gives rise to a significant modification
of the ligand arrangement affecting the strength of the nucleotide-
receptor interaction. Fig. 2 shows the minimum energy structures
Table 1
Stability constants of the complexes formed by L with AMP, ADP and ATP. 0.1 M
Me
4
NCl, 298.1 ± 0.1 K.
3
+
4+
nꢀ
calculated for the [(H
with an implicit simulation of the aqueous environment (see
Section 2). According to these structures, in [(H
L)HATP]3+, the
6 7
L)HATP] and [(H L)HATP] complexes
Anion (A
)
AMP
ADP
ATP
4
5
6
6
7
7
8
+
+
+
+
+
+
+
nꢀ
(4ꢀn)+
(5ꢀn)+
(6ꢀn)+
2.75(4)a
2.38(4)
3.29(4)
4.12(3)
4.10(5)
4.81(4)
4.95(3)
H
H
H
H
H
H
H
4
L
5
L
6
L
6
L
7
L
7
L
8
L
+ A = [(H
4
5
6
L)A]
L)A]
L)A]
= [(H
= [(H
2.90(5)
3.25(5)
3.78(5)
4.79(6)
4.17(5)
4.32(4)
4.13(5)
3.72(2)
4.23(2)
5.10(2)
5.80(2)
5.03(3)
4.94(2)
4.79(2)
nꢀ
6
+ A = [(H
nꢀ
+ A = [(H
six external ammonium groups of the ligand assumes a convergent
arrangement, thus forming 10 salt-bridges with the phosphate
(nꢀ1)ꢀ
(7ꢀn)+
(8ꢀn)+
+ HA
+ HA
6
L)HA]
L)HA]
(nꢀ1)ꢀ
3ꢀ
7
chain of HATP . The resulting complex is further stabilised by
(nꢀ2)ꢀ
(nꢀ2)ꢀ
(9ꢀn)+
+ H
+ H
2
A
A
= [(H
7
L)H
2
A]
(10ꢀn)+
an intramolecular Hꢂ ꢂ ꢂ
p
interaction between the terminal OH
and the aromatic ring of the nucleotide
Fig. 2a). On the other hand, upon protonation of the central
2
=[(H
8
L)H A]
2
3ꢀ
groups of HATP
a
Values in parentheses are standard deviations on the last significant figures.
(
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4
C. Bazzicalupi et al. / Inorganica Chimica Acta xxx (2013) xxx–xxx
74ꢀ 105ꢀ
) corresponding to the phosphate chains of
3ꢀ
(
PO
4
, P
2
O
3
, P O
3ꢀ
AMP, ADP and ATP. The equilibrium data for PO
mined in a previous work [24].
4
were deter-
As shown by this table, the ligand requires a lower positive
charge (lower protonation) to achieve an observable interaction
3
ꢀ
4ꢀ
5ꢀ
with PO
4
2
, P O
7
3
and P O10 , relative to AMP, ADP and ATP. In
2.19
4+
1
.84 1.92
fact, while the ligand must be at least tetraprotonated (H
bind the nucleotides, HL and H
form complexes with PO
4
L
) to
+
3+
3
L
offer enough stabilisation to
1.85
2.23
3ꢀ
4ꢀ
5ꢀ
1.92
1.83
4
and with P
2
O
7
3
and P O10 , respec-
1.90
2.27
tively (Table 2). An inspection to Table 2, demonstrates that also
for these inorganic phosphates the complex stability experience a
general increase with the charge of the interaction partners. Never-
theless, as already observed for nucleotides, there are some cases
in which complex stability is not completely controlled by
charge–charge attraction and hydrogen bonding may account for
the apparent deviations from the electrostatic role. For instance,
1.99
2.27
4
+
2ꢀ
4
4
H L
forms with HPO
a complex of relatively higher stability
a
5
+
5
than H L
in agreement with a decreasing propensity of the
ligand, with increasing protonation, to accept the formation of par-
ticularly favourable Nꢂ ꢂ ꢂHO–P hydrogen bonds.
The equilibrium data reported in Tables 1 and 2 evidence close
similarities in the binding of AMP, ADP, ATP and their inorganic
counterparts by L. When the anions are in the same protonation
state, the stability orders ATP > ADP > AMP and triphosphate >
diphosphate > phosphate are strictly followed, and only for the
diprotonated forms of the smaller AMP and phosphate substrates
an extra stabilisation, that can be ascribed to a complete inclusion
of the anionic groups into the ligand void [24], is observed. Accord-
ingly, we can infer that the association of the nucleotides with the
protonated forms of L is fundamentally controlled by the interac-
tion of their phosphate tails that interact with the ligand through
electrostatic and hydrogen bond forces.
1
.83
1.83
2
.06
1
.96
1
.84
.89
1
.87
1
1.86
2
+
2.10
3.2. Interaction of ZnL with AMP, ADP and ATP
In a previous paper, we reported that L is able to form Zn2+ com-
plexes with a variety of stoichiometries, including species with 1:1,
1:2 and 2:3 metal-to-ligand molar ratios [24]. For the sake of sim-
plicity, in the present study we only considered the interaction of
nucleotide with mononuclear complexes. Accordingly, a 1:0.8 me-
tal-to-ligand molar ratio was adopted in our measurements to en-
sure that polynuclear species were not formed in significant
amount. The equilibrium data for the complexation equilibria de-
rived by computer analysis of these measurements are listed in
Table 3.
b
L)HATP]3+ (a) and [(H
6 7
Fig. 2. Minimum energy structures calculated for [(H L)
HATP]4+ (b). Distances in Å.
nitrogen atom, H
and forms only 8 salt-bridges with HATP in [(H
7
L7+ loses the convergent arrangement of H
6
L6+
3ꢀ
4+
7
L)HATP] along
with an additional hydrogen bond with a nitrogen atom of the
adenosine residue (Fig. 2b). Therefore, a weaker interaction is ob-
L)HATP]4 , relative
+
The first characteristic of this complex system, coming into evi-
dence at a first inspection to Table 3, is the ability of L to assemble
served between the interacting partners in [(H
7
3
+
7+
6 7
to [(H L)HATP] , despite the higher positive charge of the H L
ligand form, in agreement with the stability data obtained by
means of potentiometric measurements.
+
ternary complexes in a protonation range that, spanning from HL
to H
7
+
7
L , exceeds the protonation range found for the formation of
It was previously evidenced that electrostatic attraction in the
absence of hydrogen bonding can furnish insufficient stabilisation
to the formation of nucleotide complexes with polyammonium li-
gands in aqueous solution [18a,33]. Furthermore, it was also
shown that the typology of hydrogen bonds can provide different
contributions to the stability of such complexes. In particular, pro-
tonation of phosphate anions in complexes with polyamine/pol-
yammonium receptors can increase the complex stability thanks
to the favourable contribution, mostly of enthalpic nature, brought
about by hydrogen bonding to unprotonated ligand nitrogen atoms
via the P-OH donors of the phosphate groups [22a,22b,32,27]. As a
matter of fact, also for the complex systems here studied, proton-
ation of the nucleotides, while reducing their negative charge, does
not produce a weakening of the complexes but more commonly
gives rise to an increase of complex stability.
metal complexes which extend, at most, up to the pentaprotonated
5
+
5+
H
5
L
species. This means that, while H
5
L
is the most charged
2
+
ligand form able to bind Zn [23], the participation in the com-
plexation process of nucleotides enhances the ability of L to form
metal complexes in acidic media. For instance, the distribution dia-
grams for the systems L/Zn and L/Zn /ATP complexes show that
complexation in the presence of ATP is still effective in high pro-
portion at very acidic pH’s (Fig. 3b), while in the absence of ATP
no complexes are practically formed below pH 5 (Fig. 3a). There-
fore, at variance to what occurs for Zn complexes, only three
nitrogen atoms not involved in protonation are sufficient to fix
the metal in the ternary complexes with nucleotides.
2
+
2+
2
+
If on the one hand the presence of these anions promotes the
formation of metal complexes with the ligand in higher proton-
ation state, extending metal complexation to more acidic solutions,
on the other hand, the presence of the metal ions favours the bind-
ing of nucleotides by the ligand in lower protonation state (Tables
Table 2 collects the equilibrium constants obtained for the
interaction of protonated form of L with the inorganic phosphates
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C. Bazzicalupi et al. / Inorganica Chimica Acta xxx (2013) xxx–xxx
5
1
00
100
Zn2+
ZnH
3
L5+
ZnH
ZnL2+
80
60
40
8
0
0
0
0
L4+
Σ[ZnH LAMP]
m
%
2
6
ZnH
4
L6+
ZnHL3+
%
a
a
4
2
L7+
5
Σ
[H LAMP]
ZnH
4
m
20
0
0
2
6
8
10
L)ATP]2+
12
2
4
6
8
10
12
pH
pH
1
00
ATP]8+
[
(ZnH
8
L)H
(ZnH
2
[(ZnH
4
100
80
5
+
[
6
L)HATP]
8
0
0
0
0
ZnL2+
7
+
[
(ZnH
7
2
L)H ATP]
%
[(ZnH
5
L)ATP]3+
Σ
[ZnH LADP]
m
6
6
4
2
0
0
0
[
(ZnH
5
L)HATP]4+
[(ZnH L)ATP]
2
b
b
4
2
Σ
[H LADP]
m
[(ZnH L)ATP]+
3
0
0
2
4
6
8
10
12
pH
2
4
6
8
10
12
pH
Fig. 3. Distribution diagrams of the complexes formed in the systems Zn2+/L (a) and
2+
Zn /L/ATP (b) as a function of pH. All reagents, 0.01 M.
1
8
6
4
2
0
00
0
Σ
[ZnH LATP]
m
1
and 3), extending anion complexation to more basic solutions. As
2+
0
a matter of fact, while in the absence of Zn the ligand requires to
be at least in the tetraprotonated H
4+
4
L
form to bind nucleotides, in
c
0
the presence of this metal ion nucleotide complexes start being
formed with the mono- and diprotonated ligand forms.
The extension of the pH region in which complexation takes
place is one evidence of the positive cooperativity of Zn and
nucleotides in the formation of these ternary complexes. A further
Σ
[H LATP]
m
0
2
+
2
4
6
8
10
12
clear evidence is the general trend of complex stability showing
pH
m+
that nucleotides are better coordinated by protonated H
m
L
L
2+
m+
Fig. 4. Competitive distribution diagrams calculated for the systems (a) L/Zn2+/
ligand forms in the presence of Zn , as well as the ability of H
m
AMP, (b) L/Zn2 /ADP, (c) L/Zn /ATP. [L] = 0.02 M, [Zn ] = [nucleotide] = 0.01 M.
+
2+
2+
2
+
m+.
m
to bind Zn is enhanced when the nucleotides are bound to H L
Percentage of free nucleotides is not shown.
Just to make a specific example of this trend, we can note that the
4
ꢀ
5+
equilibrium constant for the interaction of ATP
with H
5
L
5
+
4ꢀ
+
(
logK = 4.23 for H
5
L
+ ATP = [(H L)ATP] , Table 1) is enhanced
5
2+
5+
preorganisation generated by separated coordination to Zn2+ and
to nucleotides. Quite similar cooperativity features were already
found for the formation of ion-pair complexes of L with Cu
by about 4.4 logarithmic units when Zn is coordinated to H
5
L
(
logK = 8.61 for (H
5
ZnL)7 + ATP = [(H
+
4ꢀ
5
ZnL)ATP] , Table 3), as
3+
2
+
well as an analogous increase of stability is observed when the
,
,
equilibrium constant for the interaction of Zn2 with [(H
+
L)ATP]
+
Zn , Cd and Pb and inorganic anions such as NO
2+
2+
2+
ꢀ
, SO
2ꢀ
4
5
3
2ꢀ
3ꢀ
4
is compared with the equilibrium constant for the binding of
SeO
4
and PO
. In those cases, modelling calculations suggested
2
+
5+
Zn to H
5
L
.
that both direct coordination of the anionic species to the metal
ions and positive allosteric effects are involved in the stabilisation
of tertiary (ion-pair) complexes [24]. By analogy, it seems likely
that similar contributions are also operative in determining the
Different effects can contribute to the generation of such
positive binding cooperativity. The most obvious is the increased
nꢀ
electrostatic attraction exerted on the nucleotide anions (A
=
and the de-
relative
2ꢀ
3ꢀ
4ꢀ
(m+2)+
m+
2+
AMP , ADP , ATP ) by ZnH
m
L
relative to H
m
L
LA
cooperativity observed in the formation of nucleotide-Zn com-
(
mꢀn)+
creased repulsion exerted on the metal ion by H
m
plexes with L.
m+
m
to H L
. A further contribution is expected to originate from the
A clear picture of the enhancement of nucleotide binding in the
+
m+
(m+2)+
+
2+
different localisation of H ions in H
m
L
and ZnH
m
L
. H ions
presence of Zn at the different pH’s can be obtained by calculat-
m+
m
in H L
are located as far apart as possible to minimise the charge
ing competitive distribution diagrams for systems containing L,
Zn and nucleotide in 2:1:1 M ratio and representing the percent-
(
m+2)+
2+
density, while in MH
m
L
they are necessarily located in a re-
stricted region of the ligand, giving rise to the localisation of a
greater positive charge, thus enhancing anion attraction. Addi-
age of nucleotide bound to the ligand (all forms) and to the Zn2+
complex (all forms) as a function of pH [34]. Under similar condi-
tions, the nucleotide can distribute between the ligand and the
metal complex according to its coordination preference. Such dis-
tribution diagrams calculated for AMP, ADP and ATP ( 0.01M) are
displayed in Fig. 4. As can be seen from the figure, there is a general
preference for the coordination of nucleotides to the metal
2+
tional contributions may come from direct Zn -nucleotide coordi-
nation within the complex, an event that becomes more and more
likely as the protonation state of the ligand in the ternary complex
increases, namely when the ligand becomes less involved in metal
coordination, and from positive allosteric effects due to ligand
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6
C. Bazzicalupi et al. / Inorganica Chimica Acta xxx (2013) xxx–xxx
complexes. Only in the case of the system L/Zn2+/ATP there is a
minimal inversion of this tendency around pH 9 (Fig. 4c) where
Table 4
Rate constants (k) determined at 343.1 K for ATP dephosphoryl-
ation in the absence and in the presence of L and its mononuclear
+
5+
4ꢀ
the formation of [Zn(H
3
L)ATP] (logK = 4.52 for Zn(H
3
L) + ATP
)
2+
Zn complexes.
+
weakly competes with the formation of [(H
6
L)ATP]2 (logK = 5.10
ꢀ
3
6
+
4ꢀ
pH
k (min 1 ꢁ 10 )
6
for H L
+ ATP ). At lower pH values, the system shows the larg-
2
+
0.68(2)a
0.041(4)
1.3(1)
0.19(1)
2.8(2)
est enhancement of nucleotide binding in the presence of Zn rel-
ative to the metal free system, ATP being almost completely bound
to the metal complex below pH 5. Also for ADP the largest binding
enhancement due to metal ion coordination is observed in acidic
media where the percentage of coordinated nucleotide increases
by about four times in the presence of Zn2 . In the case of AMP,
the enhancement is more modest. For all systems, the binding dif-
ferences vanish at high pH since under such conditions both the
metal free ligand and the Zn2 complexes no longer interact with
nucleotides.
ATP
ATP
3
9
3
9
3
9
L + ATP
L + ATP
L + ATP + Zn
2+
+
L + ATP + Zn2
0.25(1)
+
a
Values in parentheses are standard deviations on the last
significant figures.
+
much faster in the acidic medium, the presence of L and its Zn2+
complexes produces larger rate enhancements in the alkaline med-
2
+
2
+
ium. Despite L and its Zn complexes are significantly less efficient
than the most effective synthetic catalysts so far studied, which are
able to enhance the ATP dephosphorilation rate by about 100 times
[8,19], their catalytic effect falls within the range shown by most of
synthetic compounds, both macrocyclic and acyclic, that prove
efficient in the activation of ATP dephosphorylation [13,14,21].
When the catalyst contains unprotonated amine groups, nucle-
otide dephosphorylation was shown to proceed through the
formation of a labile phosphoramidate intermediate [8,17–19].
Since at pH 9 L and its mononuclear Zn complexes contain a
number of amine groups not involved in protonation, neither in
metal coordination, a similar dephosphorylation pathway is very
likely to occur and, as a matter of fact, the cleavage reaction shows
a significant acceleration.
At pH 3, L still contains three unprotonated nitrogen atoms [24],
and phosphoramidate formation is still possible, even if it is less
probable. Conversely, at this pH, the Zn complex does not contain
free amine groups [23], so that the formation of the phosphoram-
idate intermediate is inhibited. As discussed above, under these pH
conditions, ATP is expected to be directly coordinated to the metal
3.3. ATP dephosphorylation in the presence of L and ZnL
The process of ATP dephosphorylation to produce ADP and inor-
ganic phosphate was monitored by means of 3 P NMR measure-
1
ments in aqueous solutions at pH 3 and 9 at 343 K in the
absence and in the presence of L and its mononuclear Zn2+ com-
2
+
plexes. The relevant spectra are shown in Fig. 5 for ATP/L/Zn at
pH 3 and in Figs. S1–S3 (Supplementary material) for the other sys-
tems. As can be seen in these figures, at both pH values, the pro-
gression of the dephosphorylation reaction is manifested by the
decrease of ATP signals and the concomitant evolution of ADP
and inorganic phosphate signals. Under acidic condition (Figs. 5
and S2) also the successive dephosphorylation of ADP to produce
AMP and inorganic phosphate is observed.
2
+
b
The signal of the P atom of ATP was used to quantify the
amount of ATP that undergoes cleavage with time, showing that
the dephosphorylation reaction proceeds through a first order ki-
2
+
netic according to the equation ln([ATP]/[ATP]
0
) = ꢀkt, where
[
ATP] and [ATP] are the concentration of ATP at t and t = 0 times,
0
respectively. The values of the rate constants (k) determined in the
3
1
presence of L and its mononuclear Zn2+ complexes are reported in
ion. Indeed, the P NMR spectra reported in Fig. 5 and S1 show
that the P and P signals of ATP are affected by the addition of
Zn , while the P signal does not experience significant modifica-
c
b
Table 4, along with the rate constants for the spontaneous ATP
dephosphorylation measured under the same experimental condi-
tions in the absence of catalysts. These data evidence that the rate
of spontaneous ATP dephosphorylation is enhanced by about 2
times at pH 3 and 5 times at pH 9 in the presence of L and by about
2
+
a
tions, suggesting that only the terminal phosphate groups are in-
volved in metal coordination. The interaction of ATP phosphate
2
+
groups with Zn may offer an alternative route to the cleavage
process. The electron-withdrawing effect of the coordinated Zn2+
ion produces an increase of positive charge on the terminal P atom
of ATP, thus favouring the nucleophilic attack by water molecules
to form ADP and inorganic phosphate.
At pH 9, Zn is coordinated by five nitrogen atoms of L [24] and,
accordingly, ATP coordination to the metal in the ternary com-
plexes would be possible, although it does not seem to be very
4
times at pH 3 and 6 times at pH 9 in the presence of its metal
complex. That is, although the dephosphorylation reactions are
2
+
8
3
10 min
60 min
2
+
likely because ATP is known to bind Zn in a chelate mode by
using two or three phosphate groups [35]. Indeed, a comparison
3
1
of the P NMR spectra in Figs. S2 and S3 evidences that the intro-
duction of the metal ion at pH 9 does not produce significant ef-
fects on the ATP signals, suggesting that the phosphate groups of
the nucleotide are not involved in metal coordination. Accordingly,
we should conclude that, at pH 9, phosphoramidate formation
plays the main role in ATP dephosphorylation. Nevertheless, it
was demonstrated that also the size of the nucleotide receptors
2
70 min
PAMP
1
80 min
ADP
ATP
ADP
P
β
P
α
ADP
P
β
P
i
[
18b] and the topology of the binding sites [18b] may have major
9
0 min
effects in determining the ability of this type of ligands in enhanc-
ATP
ATP
2+
P
β
ing ATP dephosphorylation. Since the coordination of Zn modifies
P
γ
P
α
0
min
-5
significantly the ligand conformation and the localisation of
ammonium groups in protonated species [23,24], we expect that
a favourable contribution to the acceleration of ATP dephosphoryl-
ation observed at pH 9 might come from allotropic effects pro-
0
-10
-15
-20
ppm
Fig. 5. Evolution with time of the 31P NMR spectrum in the system L/Zn2+/ATP in
O at pH 3, 343.1 K. [L] = [Zn ] = [ATP] = 0.01 M.
2
+
2+
D
2
duced by Zn coordination.
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C. Bazzicalupi et al. / Inorganica Chimica Acta xxx (2013) xxx–xxx
7
Similar reasoning can be used to account for the formation of
AMP upon dephosphorylation of ADP at pH 3.
[7] H. Dugas, Bioorganic Chemistry: A Chemical Approach to Enzyme Action,
Springer, New York, 1996.
[
[
8] E. Kimura, M. Kodama, T. Yatsunami, J. Am. Chem. Soc. 104 (1982) 3182.
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[
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4
. Conclusion
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(
(
b) D.G. Hardie, S.A. Hawley, Bioessays 23 (2001) 1112;
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In summary, we have confirmed the ability of the G-2 poly(eth-
[
12] C. Castro, E.D. Smidansky, J.J. Arnold, K.R. Maksimchuk, I. Moustafa, A. Uchida,
M. Götte, W. Konigsberg, C.E. Cameron, Nat. Struct. Mol. Biol. 16 (2009) 212.
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ylene imine) dendrimer L to form both anion and ion-pair com-
3
ꢀ
4ꢀ
5ꢀ
plexes by studying its interaction with PO
4
2
, P O
7
3
, P O
10
,
[
AMP, ADP and ATP in the absence (all anions) and in the presence
2
+
(
nucleotides) of Zn ions. When the anions are in the same proton-
ation state, the anion complexes formed by the nucleotides follow
the same order of stability shown by their inorganic counterparts
[15] C. Bazzicalupi, A. Bencini, A. Bianchi, E. Faggi, C. Giorgi, S. Santarelli, B.
Valtancoli, J. Am. Chem. Soc. 130 (2008) 2440.
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(
ATP > ADP > AMP and triphosphate > diphosphate > phosphate),
3
indicating that the association of these nucleotides with the pro-
tonated forms of L is fundamentally controlled by the interaction
of their phosphate tails that interact with the ligand through
1
[
(
2
+
electrostatic and hydrogen bond forces. In the presence of Zn
,
Deffo, F. Takusagawa, M.P. Mertes, K. Bowman Mertes, P. Paoletti, Bioorg.
Chem. 20 (1992) 8;
the ligand becomes a better nucleotide receptor and a positive
cooperativity is observed in the formation of the ternary com-
plexes (ion-pair complexes), the presence of each partner enhanc-
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(
c) A. Andrés, C. Bazzicalupi, A. Bencini, A. Bianchi, V. Fusi, E. García-España, C.
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The metal free ligand promotes the dephosphorylation of ATP
under both acidic (pH 3) and alkaline (pH 9) conditions with two-
fold and fivefold rate enhancements at pH 3 and 5, respectively,
relative to the uncatalysed dephosphorylation processes. Rather
surprisingly, the presence of Zn2 ions gives rise to further rate
enhancements, in contrast to the commonly observed inhibiting
properties of this metal ion, the dephosphorylation rate increasing
by 4 times at pH 3 and 6 times at pH 9, relative to the uncatalysed
dephosphorylation processes. That is, although the dephosphoryl-
ation reactions are much faster in the acidic medium, the presence
(
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(
2000) 1187.
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[
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(
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2
+
of L and its Zn complexes produces larger rate enhancements
relative to the uncatalysed processes) in the alkaline medium. It
deserves to be stressed here that, a pH 9, both the metal-free ligand
(
(
(
2
+
and it Zn complex show similar binding ability toward ATP
Fig. 4c), showing that the ATP dephosphorylation properties of
(
(
García-España, F. Pina, C. Soriano, Helv. Chim. Acta 86 (2003) 3118;
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(
(
these compounds are not controlled by the strength of the
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Depending on pH, formation of phosphoramidate intermedi-
ates, direct coordination of ATP to Zn2 and positive allosteric
effects seem to be responsible of these dephosphorylation
enhancements. The observed catalytic effects fall within the range
shown by most of synthetic compounds, both acyclic and macrocy-
clic, that prove efficient in the activation of ATP dephosphorylation.
+
(i) Y. Guo, Q. Ge, H. Lin, H.K. Lin, S. Zhu, C. Zhou, Biophys. Chem. 105 (2003)
19;
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1
(
´
Inorg. Chim. Acta 339 (2002) 307;
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Marinelli, B. Valtancoli, Dalton (2003) 2564.
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Luis, M.A. Niño, J.A. Ramírez, J. Chem. Soc., Perkin Trans. 2 (1991) 1445;
(
b) M.T. Albelda, E. García-España, H.R. Jiménez, J.M. Llinares, C. Soriano, A.
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Appendix A. Supplementary material
(
5
(
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2013.11.008.
(
(
2011) 2814;
e) P. Arranz Mascaros, C. Bazzicalupi, A. Bianchi, C. Giorgi, M.D. Gutíerrez
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