Recognition of AMP, ADP and ATP through cooperative binding by Cu(II) and Zn(II) complexes containing urea and/or phenylboronic acid moieties

Article abstract of DOI:10.3390/molecules23020479

We report a series of Cu(II) and Zn(II) complexes with different ligands containing a dipicolyl unit functionalized with urea groups that may contain or not a phenylboronic acid function. These complexes were designed for the recognition of phosphorylated anions through coordination to the metal ion reinforced by hydrogen bonds involving the anion and NH groups of urea. The complexes were isolated and several adducts with pyrophosphate were characterized using X-ray diffraction measurements. Coordination of one of the urea nitrogen atoms to the metal ion promoted the hydrolysis of the ligands containing 1,3-diphenylurea units, while ligands bearing 1-ethyl-3-phenylurea groups did not hydrolyze significantly at room temperature. Spectrophotometric titrations, combined with ¹H and ³¹P NMR studies, were used in investigating the binding of phosphate, pyrophosphate (PPi), and nucleoside 5-polyphosphates (AMP, ADP, ATP, CMP, and UMP). The association constants determined in aqueous solution (pH 7.0, 0.1 M MOPS) point to a stronger association with PPi, ADP, and ATP as compared with the anions containing a single phosphate unit. The [CuL⁴]²⁺ complex shows important selectivity for pyrophosphate (PPi) over ADP and ATP.

Full text of DOI:10.3390/molecules23020479

molecules
Article
Recognition of AMP, ADP and ATP through
Cooperative Binding by Cu(II) and Zn(II)
Complexes Containing Urea and/or Phenylboronic
Acid Moieties
Israel Carreira-Barral 1,2, Isabel Fernández-Pérez 1 ID , Marta Mato-Iglesias 1, Andrés de Blas 1
,
ID
Carlos Platas-Iglesias 1 and David Esteban-Gómez 1,
*
ID
ID
1
Departamento de Química, Facultade de Ciencias, Universidade da Coruña, Centro de Investigacións
Científicas Avanzadas (CICA), 15071 A Coruña, Galicia, Spain; icarreira@ubu.es (I.C.-B.);
sabela@udc.es (I.F.-P.); mmato@udc.es (M.M.-I.); andres.blas@udc.es (A.B.);
carlos.platas.iglesias@udc.es (C.P.-I.)
Departamento de Química, Facultad de Ciencias, Universidad de Burgos, 09001 Burgos, Spain
Correspondence: david.esteban@udc.es; Tel.: +34-881-01-5597
2
*
Received: 25 January 2018; Accepted: 20 February 2018; Published: 22 February 2018
Abstract: We report a series of Cu(II) and Zn(II) complexes with different ligands containing a
dipicolyl unit functionalized with urea groups that may contain or not a phenylboronic acid function.
These complexes were designed for the recognition of phosphorylated anions through coordination to
the metal ion reinforced by hydrogen bonds involving the anion and NH groups of urea. The complexes
were isolated and several adducts with pyrophosphate were characterized using X-ray diffraction
measurements. Coordination of one of the urea nitrogen atoms to the metal ion promoted the hydrolysis
of the ligands containing 1,3-diphenylurea units, while ligands bearing 1-ethyl-3-phenylurea groups
did not hydrolyze significantly at room temperature. Spectrophotometric titrations, combined with 1H
and 31P NMR studies, were used in investigating the binding of phosphate, pyrophosphate (PPi), and
0
nucleoside 5 -polyphosphates (AMP, ADP, ATP, CMP, and UMP). The association constants determined
in aqueous solution (pH 7.0, 0.1 M MOPS) point to a stronger association with PPi, ADP, and ATP as
compared with the anions containing a single phosphate unit. The [CuL4]2+ complex shows important
selectivity for pyrophosphate (PPi) over ADP and ATP.
Keywords: molecular sensors; metal complexes; cooperative binding; multitopic receptor; hydrogen
bonding; urea group; phenylboronic moiety
1. Introduction
Phosphorylated metabolites are involved in biologically relevant processes [
of chemical energy [ ] (adenosine triphosphate, ATP, and pyrophosphate (PPi), signal transduction
and transmission [ ], activation of platelets [ ], and protein kinases [ ] (adenosine monophosphate,
AMP, and adenosine diphosphate (ADP), or DNA replication catalyzed by DNA polymerase, a process
in which PPi plays an important role [ 10]. Thus, the design and synthesis of artificial receptors
that bind phosphorylated anions is an important scientific goal with potential application in sensing,
optical imaging and in vitro clinical diagnosis [11 19]. For instance, pyrophosphate (PPi) is one of the
hydrolysis products of ATP and is released in DNA/RNA polymerase reactions [20 21]. Abnormal
1], such as transport
2
3–
5
6
7
8–
–
,
levels of this anion have been related to a number of diseases0, including arthritis and cancer [22].
The interaction of Cu(II) and Zn(II) ions with nucleoside 5 -polyphosphates (AMP, ADP, ATP, CMP,
and UMP) were studied in detail by Sigel et al. in the early 90’s. [23
–27] The formation of macrochelates
was clearly defined for adenine nucleotides, thanks to the coordination of both the phosphate chains
Molecules 2018, 23, 479; doi:10.3390/molecules23020479
www.mdpi.com/journal/molecules

Molecules 2018, 23, 479
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and the nitrogen N-7 of the purine residue. [28
,29] Since then, different types of molecular receptors
have been used for the recognition of phosphorylated anions in aqueous media [
macrocyclic positively charged receptors, generally polyammonium systems [30
containing guanidinium or imidazolium groups [34 35]; (2) Metal complexes, generally coordinatively
unsaturated Cu2+ or Zn2+ complexes, with acyclic (i.e., dipicolyl or terpyridyl derivatives [36
42] or
Schiff base ligands [43]) or macrocyclic ligands [44 46]; (3) Ditopic receptors that combine a metal
coordinating unit and hydrogen-bond donors to reinforce anion binding [47,48].
1]: (1) Linear or
–
33] or receptors
,
–
–
In a previous work, we investigated the ability of the Cu2+ and Zn2+ complexes derived from L2
to bind anions through cooperative binding, involving (i) coordination of the anion to the metal ions,
and (ii) hydrogen-bonding interactions involving the coordinated anion and the NH groups of the urea
moiety as hydrogen-bond donors [49]. More recently we have shown that the [CuL2]2+ complex binds
sulphate through such cooperative binding [50]. Thus, we envisaged that the [CuL2]2+ complex and
related derivatives could be useful for the recognition of phosphorylated anions. Herein, we report a
series of ligands containing a bis(pyridin-2-ylmethyl)amine unit for coordination to Cu2+ and Zn2+,
and a urea moiety that is expected to reinforce anion binding through hydrogen-bonding interactions.
The flexibility of the receptor was modulated by including a benzyl (L1 and L2) or ethyl (L3 and L4
spacer connecting the urea and bis(pyridin-2-ylmethyl)amine units. When considering the ability of
phenylboronic acids to bind cis-diol groups [51
53], we also prepared ligands L1 L3, and L5 in an
)
–
,
attempt to reinforce the binding to AMP, ADP, and ATP. We report a series of X-ray structures and 1H
and 31P NMR studies that provide insight into the nature of the interactions established between the
Zn2+ and Cu2+ complexes and different anions. Spectrophotometric titrations were used to determine
the stoichiometries and association constants characterizing anion binding.
2. Results
2.1. Synthesis and Characterization of the Ligands and Metal Complexes
The preparation of L2 and the corresponding Cu2+ and Zn2+ complexes using sulphate and
nitrate as counterions were described in a previous work [49]. The syntheses of L1 and L3
started from (3-aminophenyl)boronic acid (
compound in quantitative yield (Scheme 1). Compound
1-(chloromethyl)-2-isocyanatobenzene or 1-chloro-2-isocyanatoethane to yield compounds
respectively. Alkylation of bis(pyridin-2-ylmethyl)amine with (or ) provided compound
1
), which was protected using ethane-1,2-diol to give
3
3
was subsequently reacted with
5
6
and
(or
7,
8),
5
7
which were purified using MPLC on neutral Al2O3. This afforded directly ligands L1 and L3, thanks to
the deprotection of the phenylboronic acid group during the chromatographic separation. The alkylation
of bis(pyridin-2-ylmethyl)amine with 5 and 7 were found to proceed in rather low yields (Scheme 1).
The synthesis of L5 started from m-tolylboronic acid (
derivative
2), which was converted to the bromomethyl
9
following the literature procedure [53]. Alkylation of bis(pyridin-2-ylmethyl)amine with
compound
9 in acetonitrile using N,N-Diisopropylethylamine (DIPEA) as a base and subsequent
purification gave L5.
The synthesis of ligand L4 was initiated by protecting compound 11 with di-tert-butyl dicarbonate
and subsequent reaction of 12 with bis(pyridin-2-ylmethyl)amine in acetonitrile using DIPEA as a base
(Scheme 2). Deprotection of compound 13 with trifluoroacetic acid (TFA) was followed by reaction of
14 with 1-isocyanato-4-nitrobenzene affording ligand L4.
The reaction of 1 equivalent of ligand (Ln; (n = 1, 3, 4 or 5)) with 1 equiv of hydrated M(ClO4)2
salts (M = Cu or Zn) in THF (L1), acetonitrile (L3 and L5), or methanol (L4) at room temperature,
provided the desired complexes as the perchlorate salts (except for [ZnL5](ClO4)2 that was prepared in
situ for 1H-NMR measurements). In the case of L4, the [CuL4(SO4)] complex was also prepared by
a similar procedure in methanol. The complexes were isolated in 82–97% yields, with the exception
of those derived from L3, which were obtained in lower yields (49% and 59% for [ZnL3](ClO4)2 and
[CuL3](ClO4)2, respectively).

Molecules 2018, 23, 479
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Scheme 1. Synthesis of ligands L1, L3 and L5: (i) Ethane-1,2-diol, ∆, 100% (3), 85% (4); (ii) Compound
, 1-(Chloromethyl)-2-isocyanatobenzene, Et2O, r.t., 16 h, 91%; (iii) Bis(pyridin-2-ylmethyl)amine,
3
◦
CH3CN, DIPEA, 70 C, 5 d; (iv) MPLC, neutral Al2O3, CH2Cl2:MeOH, 40% (two steps); (v) Compound
, 1-Chloro-2-isocyanatoethane, Et2O, r.t., 16 h, 89%; (vi) Bis(pyridin-2-ylmethyl)amine, CH3CN,
DIPEA, 70 ◦C, 5 d; (vii) MPLC, neutral Al2O3, CH2Cl2:MeOH, 24% (two steps); (viii) Compound
N-bromosuccinimide (NBS), dibenzoylperoxide, CCl4, , 16 h, 54%; (ix) Bis(pyridin-2-ylmethyl)amine,
CH3CN, DIPEA, ∆, 16 h; (x) MPLC, neutral Al2O3, CH2Cl2:MeOH, 42% (two steps).
3
4,
∆
◦
Scheme 2. Synthesis of ligand L4: (xi) di-tert-butyl dicarbonate, dioxane, 0 C, 48 h, 80% (12); (xii)
◦
Bis(pyridin-2-ylmethyl)amine, CH3CN, DIPEA, 70 C, 3 d, 39%; (xiii) trifluoroacetic acid (TFA:CH2Cl2
(1:1), r.t., 16 h, 96%; (xiv) 1-isocyanato-4-nitrobenzene, CHCl3, r.t., 2 h, 85%.
As expected, the IR spectra (KBr discs) feature bands due to
to the presence of the urea group in L1 L3, and L4 derivatives, while
also observed when the boronic moiety is present (L1 L3, and L5). In all cases, the bands corresponding
to the (Ph/Py) mode are present, and the coordination of the metal ions induces significant shifts with
respect to the corresponding free ligands (see Materials and Methods Section). It is noticeable that the
bands corresponding to the as(Cl-O) (stretching) and as(O-Cl-O) (bending) modes of the perchlorate
groups appear without splitting for all complexes at ca. 1100 and 625 cm−1, respectively, as befit
uncoordinated anions [54 a(S-O) and
55]. In the case of [CuL4(SO4)], the bands corresponding to the
a(O-S-O) modes are splitted, as expected for a coordinated sulphate anion with low symmetry. This is
ν
(N-H) and
ν
(C=O) modes associated
,
ν
(O-H) and
ν
(O-B-O) modes are
,
ν
ν
δ
,
ν
ν
in agreement with UV-vis diffuse reflectance spectroscopy, which provided different positions for the
d-d absorption band observed for [CuL4(SO4)] and [CuL4](ClO4)2 (706 and 633 nm, respectively).

Molecules 2018, 23, 479
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Conductivity measurements were carried out in 10−3 M methanol solutions at 25 ◦C. The molar
conductivity of the perchlorate salts (154.9–171.3 cm2 Ω−1 mol−1) are in the range reported for 2:1
electrolytes [56], indicating that these complexes are completely dissociated in methanol solution. In the
case of [CuL4(SO4)], the low solubility of this charge-neutral complex prevented us from determining
its molar conductivity value, confirming that the sulphate anion is directly bonded to the Cu2+ ion.
2.2. X-ray Crystal Structures
The solid state structures of different complexes of L4 were investigated by using X-ray diffraction
measurements. Table 1 presents the bond distances of the metal coordination environments, while
views of the structures are shown in Figures 1 and 2. The complexes differ in the nature of the metal
ion (Zn2+ or Cu2+) and the anion (sulphate or pyrophosphate). The Cu2+ ions in [CuL4(SO4)] and
[CuL4(H2PPi)] complexes present octahedral coordination with a strong Jahn-Teller distortion [57].
The ligand adopts an ‘open wing butterfly’ conformation that is similar to those observed for the Cu2+
complexes of L2 [49]. The equatorial plane of the coordination polyhedron in [CuL4(SO4)] is defined
by the donor atoms of the dipicolylamine unit [N(4), N(5) and N(6)] and an oxygen atom of the anion
[O(4)] (mean deviation from planarity: 0.04 Å), with the metal ion being placed 0.01 Å below this
plane in [CuL4(SO4)] and 0.12 Å above it in [CuL4(H2PPi)]. The apical positions in [CuL4(SO4)] are
occupied by one of the nitrogen atoms of the urea subunit [N(3)] and an oxygen atom of a sulphate
anion of an adjacent complex molecule [O(5b)] (Figure 1). Thus, the presence of µ2– :η
η1 1-sulphate
anions bridging two neighboring complex molecules leads to the formation of a coordination polymer
(Figure S1, Supplementary Materials). In [CuL4(H2PPi)], the apical positions are delineated by one of
the nitrogen atoms of the urea subunit [N(3)] and an oxygen atom of the H2PPi2− anion [O(8)], which
coordinates in a κ2-H2PPi2− mode forming a six-membered chelate ring.
(a)
(b)
Figure 1.
(
a
) X-ray crystal structure of [CuL4(SO4)] with labelling scheme; (
b) X-ray crystal structure
of [CuL4(H2PPi)] with labelling scheme. The Oak Ridge Thermal-Ellipsoid Plot Program (ORTEP)
plots are at the 30% probability level. Symmetry transformations used to generate equivalent atoms for
[CuL4(SO4)]: #2 −x + 1, y − 1/2,−z + 3/2.
The cis angles of the coordination polyhedron (81.0◦–98.8◦ in [CuL4(SO4)] and 82.4◦–98.4◦
in [CuL4(H2PPi)]) are relatively close to the one expected for a regular octahedral coordination
(90◦), whereas the corresponding trans angles (162.7◦–178.0◦ for [CuL4(SO4)] and 164.4–173.8 for
[CuL4(H2PPi)]) deviate up to 18◦ from the ideal one (180◦) (Figure S2, Supplementary Materials).
The sulphate anion in [CuL4(SO4)] interacts with the metal ion via a coordinate bond and with
the urea moiety through a moderate hydrogen-bonding interaction, the latter involving one of the NH
groups of the urea unit as hydrogen-bond donor and an oxygen atom of the anion as hydrogen-bond
◦
acceptor [N(2)· · · O(6) 2.768(3) Å; N(2)-H(2N)· · · O(6), 1.97(3) Å; N(2)-H(2N)· · · O(6) 161(3) ]. The value
of the S(1)-O(6)-H(2N) angle (116.8◦) is close to the ideal one (122◦
±
10◦) [58]. The interaction
of the anion with another complex molecule causes sulphate to move away from the N(3)-H(3N)

Molecules 2018, 23, 479
5 of 25
fragment, which interacts with an oxygen atom of another sulphate anion via an intermolecular
hydrogen-bonding interaction.
Table 1. Bond distances (Å) of the metal coordination environments.
[CuL4(SO4)]
[CuL4(H2PPi)]
[{CuL4}2(µ-PPi)]
[{ZnL4}2(µ-PPi)]
M(1)-N(3)
M(1)-N(4)
M(1)-N(5)
M(1)-N(6)
M(1)-O(4)
M(1)-O(5)
M(1)-O(8)
2.728(3)
2.044(3)
2.005(3)
2.000(2)
1.960(2)
2.247(2)
-
2.670(1)
2.039(1)
1.994(1)
1.991(1)
1.930(1)
-
-
-
2.100(2)
1.990(2)
1.989(2)
1.938(1)
2.340(6)
2.045(7)
2.041(6)
2.021(5)
-
2.263(1)
2.172(2)
1.949(5)
We have also obtained single crystals suitable for X-ray diffraction measurements of
[{ZnL4}2( -PPi)] and [{CuL4}2(
-PPi)] (Figure 2), in which the fully deprotonated pyrophosphate anion
acts as a bridging µ2κ4-PPi4− ligand forming two six-membered chelate rings [59
60]. This bridging
mode of the anion has been previously reported by Hong and coworkers in the solid state for dinuclear
Zn2+ complexes derived from di(2-picolyl)amine [61 62]. The ligand in [{CuL4}2(
-PPi)] adopts an
µ
µ
,
,
µ
’open wing butterfly’ conformation (Figure 2), which results in a square pyramidal geometry around
the Cu2+ ion. The basal plane is defined by the three donor atoms of the dipicolylamine unit [N(4),
N(5), and N(6) for Cu(1) and N(10), N(11) and N(12) for Cu(2)] and an oxygen atom of pyrophosphate
[O(4) or O(9)] [mean deviation from planarity: 0.03 Å for Cu(1) and 0.05 Å for Cu(2)], while the apical
position is occupied by another oxygen atom of the anion [O(8) or O(5)]. The metal ion is placed
0.29 Å [Cu(1)] or 0.28 Å [Cu(2)] above the basal plane. The cis angles of this plane (82.8◦–95.7◦) and
those involving the oxygen atom occupying the apical position, the metal centre and the donor atoms
of the equatorial plane (93.3◦–102.5◦), as well as the trans angles (157.4◦–166.8◦), are relatively close
to the ideal ones (90 and 180◦, respectively). The index of trigonality
Cu(1) and 0.16 for Cu(2) ( = 0 for an ideal square pyramidal geometry and
bipyramidal environment) [63] is in agreement with the square pyramidal polyhedron.
τ, which amounts to 0.12 for
τ
τ
= 1 for a regular trigonal
(a)
(b)
Figure 2.
(
a
) X-ray crystal structure of [{ZnL4}2(
µ-PPi)] with labelling scheme; (b) X-ray crystal structure
of [{CuL4}2(µ-PPi)] with labelling scheme. The ORTEP plots are at the 30% probability level.

Molecules 2018, 23, 479
6 of 25
The ligand L4 in [{ZnL4}2(
µ
-PPi)] adopts a 0bent propeller0 conformation and wraps around the
metal ions providing trigonal bipyramidal coordination environments. The equatorial plane of the
bipyramid is defined by the nitrogen atoms of the pyridine units [N(5) and N(6) for Zn(1) and N(11)
and N(12) for Zn(2)] and an oxygen atom of the pyrophosphate anion [O(8) for Zn(1) and O(5) for
Zn(2)]. The apical positions are occupied by the amine nitrogen atom and one of the oxygen atoms
of the anion [N(4) and O(4) for Zn(1) and N(10) and O(9) for Zn(2)]. The Zn2+ ion is placed 0.24 Å
[Zn(1)] or 0.25 Å [Zn(2)] above the equatorial plane. The angles involving the donor atoms of this
plane (113.9◦–124.7◦), as well as the trans angle (170.4◦–170.6◦) are close to the ones expected for a
◦
◦
regular trigonal bipyramid (120 and 180 , respectively). The index of trigonality
both metals [63], which is in agreement with the indicated geometry.
τ amounts to 0.76 for
The PPi4− anions in both [{ZnL4}2( -PPi)] and [{CuL4}2(
µ µ-PPi)] show a cooperative binding in
which the anion interacts simultaneously with the metal ion via coordinate bonds, and with the urea
moiety through Y-shape hydrogen-bonding interactions. The N-H(N)-O angles and distances (Table 2)
show that these interactions are rather symmetrical and can be regarded as moderate, although the
N
· · · O distances point to a slightly weaker interaction in the Zn2+ complex. In all cases, the P-O-H(N)
angles (109◦–122◦) are close to the ideal value for a tetrahedral anion (122◦ ± 12◦) [58].
Table 2. Intramolecular hydrogen-bonding interactions observed in the solid state structures of
[CuL4(SO4)], [{CuL4}2(µ-PPi)] and [{ZnL4}2(µ-PPi)].
◦
D-H· · · A/
d(D-H)/Å
d(H· · · A)/Å
d(D· · · A)/Å
[CuL4(SO4)]
N(2)-H(2N)· · · O(6)
N(2)-H(2N)· · · O(10)
N(3)-H(3N)· · · O(8)
N(8)-H(8N)· · · O(6)
N(9)-H(9N)· · · O(5)
N(2)-H(2N)· · · O(10)
N(3)-H(3N)· · · O(8)
N(8)-H(8N)· · · O(6)
N(9)-H(9N)· · · O(5)
0.82(3)
0.79(2)
0.74(3)
0.79(2)
0.82(3)
0.73(9)
0.76(9)
0.82(9)
0.75(9)
1.97(3)
2.04(3)
2.08(3)
1.98(3)
1.94(3)
2.12(9)
2.13(9)
2.05(9)
2.15(9)
2.768(3)
2.830(3)
2.797(2)
2.765(2)
2.751(2)
2.850(9)
2.880(9)
2.870(8)
2.876(9)
161(3)
175(2)
161(3)
173(2)
169(2)
172(10)
168(10)
178(9)
163(10)
[{CuL4}2(µ-PPi)]
[{ZnL4}2(µ-PPi)]
2.3. Stability of the Complexes Toward Hydrolysis
1
The H-NMR spectra of the Zn2+ complexes of both L1 and L2 recorded in wet dmso-d6
were observed to change over time, which caused an increase of the number and complexity
of the signals (Figures S3 and S4, Supplementary Materials). This suggests that both [ZnL1]2+
and [ZnL2]2+ complexes hydrolyse in aqueous solution to form the [ZnL6]2+ complex (Scheme 3),
2-aminophenylboronic acid or 3-nitroaniline, and carbon dioxide. The presence of a 3-nitroaniline
molecule in the molecular structure of [CuL2(
µ-SO4)]2 reported recently also points in this direction [50].
In the case of [ZnL2](ClO4)2, new resonances appear in the 1H-NMR spectrum from 7.35 to 6.60 ppm,
none of which correspond to the complex or the free ligand. The presence of a signal at 6.94 ppm
due to 3-nitroaniline confirmed the hydrolysis of the complex. The relative intensity of the signals
attributable to the [ZnL2]2+ species and 3-nitroaniline indicate that 6% of the complex has hydrolysed
after four hours at room temperature, while after 48, 96, and 168 h this percentage increases to 28%,
43%, and 49%, respectively. A similar situation was observed for [ZnL1]2+, which hydrolysed to
a slightly lower extent after 4 h (~5%). 1H DOSY experiments performed for [ZnL2]2+ 168 h after
dissolution of the complex allowed discriminating the three species that are present in solution on the
basis of their different diffusion coefficients (Figure 3). Taking the diffusion coefficient of dmso-d5 as
a reference (D = 6.81
of 1.34
10−10 m2 −1, 1.85
3-nitroaniline, respectively.
×
10−10 m2
·
s
−1) [64], these studies provided diffusion coefficients D at 25 ◦C
−1, and 3.64 10−10 m2 s−1 for [ZnL2]2+, [ZnL6]2+, and
×
·
s
×
10−10 m2
·
s
×
·

Molecules 2018, 23, 479
7 of 25
Scheme 3. Ligands and phosphorylated anions discussed in the present work.
The hydrolysis of the urea unit in [CuL2]2+ was monitored in aqueous solution by following the
absorption band of 3-nitroaniline ( max = 357 nm,
= 1486 M−1 cm−1) over time and employing the
λ
ε
·
method of initial rates (Figure S5, Supplementary Materials). The observed pseudo-first-order rate
constants (kobs) and half-life times (t1/2) were determined by fitting the data obtained at 390 nm using
◦
two temperatures (25 and 50 C, I = 0.1 M NaClO4) and two pH values (5.5 and 7.0). In line with
previous studies [65], our results did not reveal significant pH dependence of the kobs values in this
pH range, which were determined to be 4.5
±
0.1
×
10−5
·
s
−1 at 25 ◦C and 7.30
±
0.04
×
10−4 s−1 at
50 ◦C. The corresponding half-life times are 258 min at 25 ◦C and 19 min at 50 ◦C. Thus, the [CuL2]2+
complex experiences significant hydrolysis in aqueous media at room temperature. Analogous studies
performed for the [CuL4]2+ complex provided rate constants of 3.12
±
0.01
×
10−9
·
s−1 and 1.265
±
0.002
×
10−7
·
s−1 at 25 and 50 ◦C, respectively, which correspond to half-life times of ~89 (25 ◦C)
and ~2 (50 ◦C) months. Thus, the replacement of the 1-(3-nitrophenyl)-3-phenylurea unit of L2 by a
1-ethyl-3-(3-nitrophenyl)urea group increases the stability toward hydrolysis of the corresponding
Cu2+ complex by 3–4 orders of magnitude.
Most of the studies regarding the decomposition of ureas focused on its hydrolysis to ammonium
and carbamate ions [66,67] (the latter is unstable and its decomposition gives rise to the formation of
ammonium ions, carbon dioxide, and water [68]) by urease [69]. Kinetic experiments performed in
◦
water at pH 7 and 38 C showed that the value of kobs for this reaction decreases as the substitution on
the urea moiety increases (from 5913 s−1 for urea to 0.075 s−1 for N-methylurea [70
,
71]). The value
reported for the uncatalyzed hydrolysis of tetramethylurea (6.0
×
10−11 −1) points to a very slow
s
reaction in the absence of urease [72]. Thus, we conclude that the relatively high rate constants
determined for the hydrolysis of the urea groups in [ZnL1]2+, [ZnL2]2+, and [CuL2]2+ are related to the
coordination of a nitrogen atom of the urea unit to the metal ion.

Molecules 2018, 23, 479
8 of 25
Figure 3. DOSY 1H-NMR spectrum (500 MHz, 298 K) recorded for [ZnL2]2+ 168 h after dissolution of
the complex in dmso-d6.
2.4. Binding to Phosphorylated Anions
The binding of phosphorylated anions phosphate, AMP, CMP, UMP, PPi, ADP, and ATP to
[CuL3](ClO4)2, [CuL4](ClO4)2, and [CuL5](ClO4)2 was investigated by using spectrophotometric
titrations in aqueous solutions (pH = 7.0, MOPS buffer). The absorption spectra of the complexes
show a d-d absorption band with a maximum at 615 ([CuL3]2+), 654 ([CuL4]2+), and 643 nm ([CuL5]2+),
typical of dxz, dyz
→ dx2−y2 transitions in a tetragonal ligand field [73]. This is consistent with the
Jahn-Teller distorted octahedral environment, where two coordination positions are likely occupied
by water molecules, and supported by the molar conductivity values of these complexes in methanol
solution. Addition of phosphate, AMP, CMP, or UMP causes slight changes in the d-d absorption
band of the complexes, in particular, a slight bathochromic shift of the absorption maximum and
small changes in the molar extinction coefficients (Figure 4, see also Figures S6–S8, Supplementary
Materials). This indicates that the complexes retain the distorted octahedral coordination upon anion
binding. A completely different situation is however observed upon addition of PPi, ADP, and ATP,
as these anions provoke both an important bathochromic shift of the d-d absorption band and the
appearance of a shoulder in the low energy side. This is characteristic of Cu2+ complexes having
trigonal bipyramidal coordination geometries, which in the case of [CuL2]2+ complexes, was related
to the de-coordination of the weakly bonded urea nitrogen atom [49]. This is in line with the X-ray
structure of [{ZnL4}2(
bidentate fashion and establishes hydrogen-bonding interactions with the urea group.
µ-PPi)] shown in Figure 2, in which the anion coordinates to the metal ion in a

Molecules 2018, 23, 479
9 of 25
The titration profiles obtained with AMP, CMP, and UMP were satisfactory analyzed using a
1:1 binding model, providing the association constants listed in Table 3. In the case of PPi, ADP and
ATP satisfactory fittings required an equilibrium model, including both 2:1 and 1:1 species ([CuLn]2+
Anion, n = 3, 4 or 5). The high resolution mass spectra recorded by electrospray ionization (ESI+) for
solutions of [CuL4]2+ in the presence of one equivalent of PPi, ADP, or ATP show peaks due to the 2:1
and 1:1 entities (Table S1 and Figure S9, Supplementary Materials), which supports the model used for
the determination of association constants. A similar situation was observed for phosphate, which
also forms 2:1 and 1:1 complexes. The association constants obtained from the analysis of the titration
curves are reported in Table 3. The anions containing a single phosphate unit (phosphate, AMP, CMP,
and UMP) provide similar Log K11 values, which show that the nature of the nucleobase does not
affect significantly the association constants. Thus, most likely the nucleobase is not participating
significantly in the recognition process as observed for CMP and UMP in previous studies [28]. In the
case of AMP, similar values of the stability constants were obtained for the corresponding adducts,
so the formation of a macrochelate ring involving both the phosphate and the nitrogen N-7 of the
adenosine moiety does not seem to take place. This could be related to the arrangement of the ligands
around the Cu2+ ion in the complexes, which may block the effective approach of the nitrogen N-7 of
adenosine to the inner sphere of the Cu2+ ion, thus inhibiting its coordination [29].
:
Figure 4. Family of UV/vis spectra taken during the course of the titration of [CuL4]2+ (5
in H2O, pH 7.0, MOPS 0.1 M, 25 ◦C) with standard solutions (0.5 M) of: (
) NaH2PO4; ( ) Na2AMP;
) Na2ADP; and, ( ) Na2ATP. Insets: titration profiles at selected wavelengths vs equivalents of anion
×
10−3
M
a
b
(
c
d
and species distribution diagram.
The comparison of association constants obtained with different equilibrium models may be
misleading. Thus, we used pA values defined as pA =
−
log[Anion]free using an anion concentration
of 0.001 M and a concentration of [CuLn]2+ complex of 0.01 M (pH 7.0). The definition of pA is similar
to that of pM often used to assess the relative stabilities of metal complexes in aqueous media [74].
The pA values obtained for all of the monophosphate anions are rather similar (Figure 5, see also Table 3),

Molecules 2018, 23, 479
10 of 25
with no particular selectivity for any of them being observed. When considering the spectral changes
that were displayed during the titrations, this suggests monodentate coordination of the phosphate
anion to the metal center in the 1:1 complexes. The values obtained for the anions containing two or
three phosphate units are clearly higher, particularly in the case of [CuL4]2+ and [CuL5]2+. This can be
attributed, at least in part, to a bidentate coordination of the anion to the metal center, which for [CuL3]2+
and [CuL4]2+ triggers the de-coordination of the urea nitrogen atom. However, the nature of the receptor
results in rather different stabilities. The pA values obtained for [CuL3]2+ and [CuL5]2+ show a similar
trend, with the highest stability being observed for PPi. However, [CuL5]2+ shows higher pA values
than [CuL3]2+, perhaps because of the more open structure of [CuL5]2+ related to the absence of the urea
group. The high selectivity of [CuL5]2+ for PPi over ADP and ATP suggests that the cis-diol groups of
the nucleotides are not forming esters with the phenylboronic acid moiety of the ligand.
Table 3. Association constants (log K values) and pA values obtained from spectrophotometric titrations
1
in aqueous solutions (pH 7, MOPS buffer).
Phosphate
AMP
CMP
UMP
PPi
ADP
ATP
[CuL3]2+
[CuL4]2+
[CuL5]2+
Log K11
Log K21
pA
4.20(1)
-
5.16
4.35(1)
-
5.31
3.59(1)
-
4.56
4.02(1)
-
4.98
4.82(4)
4.14(5)
7.77
4.02(1)
2.42(4)
5.44
4.28(1)
3.00(4)
6.15
2
2
Log K11
Log K21
pA
3.44(1)
1.72(7)
4.56
3.80(1)
-
4.76
3.31(1)
-
4.29
6.83(9)
4.8(1)
10.44
6.26(5)
4.32(6)
9.39
Log K11
Log K21
pA
3.45(3)
3.67(7)
5.94
3.31(3)
-
4.29
3.51(1)
-
4.48
3.00(1)
-
4.00
6.1(3)
-
11.01
4.89(2)
3.81(2)
7.52
4.61(1)
3.41(3)
6.85
1 pA values defined as pA=
during the titration prevented the determination of association constants.
−
log[Anion]free, with [Anion] = 0.001 M and [(CuLn)2+] = 0.01 M (pH 7.0). 2 Precipitation
Figure 5. pA values (pA=
−
log[Anion]free, with [Anion] = 0.001 M and [(CuLn)2+] = 0.01 M and pH
7.0) calculated for the association of [CuLn]2+ complexes (n = 3, 4 or 5) with phosphorylated anions
with the equilibrium constants listed in Table 3.
Unfortunately, the addition of PPi to aqueous solutions of [CuL4]2+ caused the precipitation of the
2:1 aduct, which is subsequently redissolved upon addition of an excess of the anion. This prevented us
from determining the corresponding association constants. Nevertheless, the pA values characterizing
the association of [CuL4]2+ with ADP and ATP are clearly higher than those that were obtained for
[CuL5]2+ and [CuL3]2+. We attribute this to the electron withdrawing effect of the –NO2 group of
L4, which results in: (1) A weaker coordination of the urea group to the metal ions, so that it can be
more easily displaced by anion coordination; and (2) A cooperative binding through coordination of

Molecules 2018, 23, 479
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the anion to the metal center and hydrogen-bonding interactions involving the urea NH donors and
oxygen atoms of the anion (as evidenced in the X-ray structures shown in Figure 2). These interactions
are likely weaker in the case of [CuL3]2+, and might be additionally hindered by the presence of the
boronic group in position 2 with respect to the urea unit.
In the case of [CuL3]2+ and [ZnL3]2+ complexes, the high resolution mass spectra recorded
in dmso in the presence of one equivalent of AMP, ADP, and ATP show peaks due to 1:1 entities
(Figure S10, Supplementary Materials). The position and isotopic profiles of the peaks indicate that
the phenylboronic moiety has reacted with the cis-diol units present in the sugar ring of nucleotides
in all cases. Similar measurements performed in aqueous solution did not evidence the formation
of phenylboronic esters in the recognition of the nucleotides in this medium, suggesting that this
cooperative interaction is not present in aqueous solution.
2.5. NMR Studies
The interaction of the [ZnL4]2+ complex with phosphorylated anions was investigated by using
1H-NMR spectroscopy in dmso-d6 solution, which was selected as solvent in view of the low solubility
1
of the complex in aqueous media. The H-NMR signals of the urea protons were particularly
informative with respect to the interaction of the anion with the complex (Figure 6). These signals are
observed at 9.40 and 6.52 ppm in the spectrum of the [ZnL4]2+ complex, dissolved as the perchlorate
salt. The addition of phosphate or AMP does not provoke significant changes in the chemical
shifts of the urea protons. However, the signal at 9.40 ppm experiences important shifts to lower
fields upon addition of one equivalent of PPi or ADP, shifting to 10.40 and 10.33 ppm, respectively.
In parallel, the signal at 6.52 ppm undergoes smaller downfield shifts to 6.89 and 6.81 ppm, respectively.
These spectral changes reflect the establishment of hydrogen-bonding interactions involving the urea
fragment and the anion [75]. Interestingly, the addition of ATP provokes smaller shifts of the signals
of urea protons to 9.53 and 6.88 ppm, which is in line with the lower pA value determined for the
association of ATP and [CuL4]2+. A similar behavior is also observed for the [ZnL3]2+ complex, which
contains a phenylboronic function (Figure S11, Supplementary Materials).
Figure 6. 1H-NMR spectra (300 MHz, 298 K) recorded for the [ZnL4]2+ complex in the presence of
different anions as their sodium salts in dmso-d6.

Molecules 2018, 23, 479
12 of 25
The corresponding 31P NMR spectrum of the ATP adduct (Figure 7) shows three signals at
20.92 and 9.05 ppm due to the Pα, Pβ, and Pγ nuclei, respectively. The corresponding chemical
shifts measured for the uncoordinated ATP anion are 10.90, 22.42, and 9.26 ppm. The small
−
9.55,
−
−
−
−
−
chemical shift induced by coordination in the signal of Pγ and the relatively large chemical shifts
experienced by Pα and Pβ indicates that only the two inner phosphate groups of ATP play a significant
role in the coordination to the metal ion. In the case of ADP coordination to the metal ion induces
important upfield shifts of the signals of both Pα and Pβ, from
5.56 to 7.46 ppm (Pβ). Taken together, these results indicate that both ADP and ATP coordinate to
the metal ion through the phosphate units containing Pα and Pβ, likely in a similar way as observed
in the solid state for [{ZnL4}2( -PPi)] and [{CuL4}2(
-PPi)] (Figure 2). It is also worth noting that the
−
7.29 to
−
9.13 ppm (Pα) and from
−
−
µ
µ
spectrum recorded in the presence of ATP of PPi shows a small signal at 0 ppm due to the presence
of phosphoric acid, indicating that the complex catalyzes the hydrolysis of the PPi anion and the
triphosphate unit of ATP. In the case of [ZnL3]2+, similar 31P chemical shifts are observed, but the
higher relative intensity of the signal at 0 ppm for polyphosphorylated anions indicates a higher extent
of the hydrolysis process (Figure S12, Supplementary Materials).
The 31P NMR spectra recorded in the presence of phosphate shows two very broad signals, which
evidences the presence of different species in solution involved in dynamic processes. In the case of
AMP, the 31P NMR spectra show at least three signals for both [ZnL3]2+ and [ZnL4]2+ complexes, none
of which coincides with that of free AMP, again suggesting the presence of several species in solution.
Figure 7. 31P NMR spectra (500 MHz, 298 K) recorded for [ZnL4]2+ complex in the presence of AMP,
ADP, ATP and phosphate as their sodium salts in dmso-d6. The 31P NMR spectra of free metabolites
are shown as colored traces.

Molecules 2018, 23, 479
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To check whether the structures in solution of [ZnL4(H2PPi)] and [{ZnL4}2(
µ-PPi)] complexes
correspond to those observed in solid state, the geometries of both monomeric and dimeric species were
modeled by means of DFT calculations (TPSSh/SVP) in dmso solution (Figure S13, Supplementary
Materials). In both cases, the possibility that the pyrophosphate anion was totally deprotonated or
doubly protonated was considered, and the corresponding 31P NMR spectra of the four minimum
energy geometries were calculated at the TPSSh/TZVP level (Table 4). The minimum energy geometry
obtained for the [{ZnL4}2(
µ-PPi)] species is in agreement with the one previously described in the
solid state, where identical conformations of the ligands backbone (‘bent propeller’), coordination
polyhedron around the Zn(II) metal ion (trigonal bipyramid), and intramolecular hydrogen-bonding
interactions are present. In the case of the [ZnL4(H2PPi)] species, the obtained geometry differs
considerably from that observed for the [CuL4(H2PPi)] complex in the solid state. The coordination
polyhedron around the Zn2+ metal ion can be now best described as a square pyramid, with the ligand
in an ‘open wing butterfly‘ conformation. Despite this, it is noticeable to remark that in both geometries
the pyrophosphate anion is coordinated to the Zn(II) metal ions forming six-membered chelate rings
in κ µ µ-PPi)], respectively. Hydrogen
2-H2PPi2− or µ2κ4-PPi4− modes for [ZnL4( -PPi)] and [{ZnL4}2(
bond interactions between the oxoanion and the urea moieties are observed in both cases.
The comparison of the calculated chemical shifts with the experimental values recorded in the
presence of 0.5 or 1 equivalents of pyrophosphate anion are in agreement with those calculated
for a fully deprotonated form in the case of the [{ZnL4}2(
µ-PPi)] dimeric complex, and with a
doubly protonated form for the [ZnL4(H2PPi)] monomeric complex. The good agreement between
experimental and calculated data suggests that both complexes adopt the calculated minimum energy
geometries in solution.
Table 4. 31P measured chemical shifts (dmso-d6) and calculated chemical shifts using the GIAO method
at the TPSSh/TZVP level (see Computational Details) for [ZnL4(H2PPi)] and [{ZnL4}2(
µ-PPi)] species
(fully deprotonated and doubly protonated forms of the pyrophosphate anion were considered).
δ (Calculated)
Deprotonated Form
δ (Calculated)
Doubly Protonated Form
Species
δ (Experimental)
[{ZnL4}2(µ-PPi)]
−5.00
−7.75
−5.90
−10.37
−6.74
[ZnL4(H2PPi)]
−72.15
3. Materials and Methods
3.1. General Methods
Elemental analyses were carried out on a ThermoQuest Flash EA 1112 elemental analyzer.
ESI-TOF low resolution mass spectra (MS) were recorded from MeOH/MeCN/CHCl3, MeOH/MeCN,
MeCN/CHCl3, or MeCN solutions, while the high resolution MS were recorded from MeCN/H2O or
MeCN/dmso solutions, in both cases using a LC-Q-q-TOF Applied Biosystems QSTAR Elite spectrometer
in the positive mode. IR spectra were recorded using a Bruker Vector 22 spectrophotometer equipped
1
with a Golden Gate at◦tenuated total reflectance (ATR) accessory (Specac). H, 13C and 31P NMR spectra
were recorded at 25 C on Bruker Avance 300 and Bruker Avance 500 spectrometers, and spectral
assignments were based in part on 2D COSY, HSQC, and HMBC experiments. UV/vis spectra were
recorded with a Perkin-Elmer Lambda 900 spectrophotometer. The spectra obtained from solutions
were recorded with quartz cells (path length: 1 cm) and the cell holder was thermostatted at 25.0 ◦C,
through circulating water. Anion binding studies were performed by monitoring the spectral changes
of 10−3 M solutions of [CuL3](ClO4)2 or [CuL5](ClO4)2 complexes in H2O at pH 7.0 upon addition of a
0.1 M solution of the corresponding sodium salt containing a 10−3 M concentration of the corresponding
complex at pH 7.0. For the [CuL4](ClO4)2 complex, the concentrations of the titration solutions and the
solutions of the sodium salts were 5
in all cases. Binding constants were obtained by using a simultaneous fit of the UV/vis absorption
×
10−3 M and 0.5 M, respectively. The pH was buffered with MOPS

Molecules 2018, 23, 479
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spectral changes at 7–12 selected wavelengths in the range 500–1200 nm. A minimum of 26 absorbance
data points at each of these wavelengths was used, and all spectrophotometric titration curves were
fitted with the HYPERQUAD 2008 (HypSpec) program [76].
Reagents
(3-Aminophenyl)boronic acid (1), 3-tolylboronic acid (2), 2-bromoethanamine hydrobromide (11),
ethane-1,2-diol, di-tert-butyl dicarbonate, bis(pyridin-2-ylmethyl)amine, 1-isocyanato-4-nitrobenzene,
and 1-chloro-2-isocyanatoethane were obtained from commercial sources and used as received.
Solvents were of reagent grade and used without further purification.
Caution: Although we have experienced no difficulties with the perchlorate salts, these should be
regarded as potentially explosive and handled with care [77].
3.2. Synthesis
3-(1,3,2-Dioxaborolan-2-yl)aniline (3). A solution of (3-aminophenyl)boronic acid (1) (2.536 g, 16.04 mmol)
and ethane-1,2-diol (1.169 mL, 20.85 mmol) in acetonitrile (50 mL) was heated to reflux with stirring
for 6 h. The solvent was removed by rotary evaporation and the resulting oil was dried under vacuum,
after which it became a pale brown solid (3) that was used without further purification (2.605 g, 100%).
δH (solvent CDCl3, 500 MHz, 298 K): 7.23–7.17 (m, 2H), 7.14 (m, 1H), 6.80 (ddd, 1H, 3J = 7.7 Hz, 4J =
2.6 Hz, 4J = 1.4 Hz), 4.35 (s, 4H), 3.49 (s, 2H). δC (solvent CDCl3, 125.8 MHz, 298 K): 145.8, 128.9, 125.2,
121.3, 118.4, 66.1. MS-ESI+, m/z (%BPI): [
59.0; H, 6.2; N, 8.6%. Found: C, 58.6; H, 6.3; N, 8.5%. IR: 3444, 3361, 3215
1625, 1579, 1484, 1443 ν(C=C), 1338 ν(C-BO2), 1210 ν(O-B-O) cm−1
3
+ H]+, 164.1 (47%). Elem. Anal. Calcd for C8H10BNO2: C,
(N-H), 2981, 2910 (C-H),
ν
ν
.
2-(3-Tolyl)-1,3,2-dioxaborolane ( ). This compound was prepared by adapting the experimental
4
procedure found in the literature [53]. A solution of 3-tolylboronic acid ( ) (1.829 g, 13.05 mmol)
2
and ethane-1,2-diol (0.951 mL, 16.96 mmol) in toluene (50 mL) was heated to reflux with stirring for
16 h using a Dean-Stark apparatus for water removal. The resulting solution was filtered while hot
and the filtrate concentrated to dryness. The residue was purified by column chromatography on SiO2
with CH2Cl2 as the eluent to give pure product
500 MHz, 298 K): 7.67 (s, 1H), 7.65 (m, 1H), 7.32–7.31 (m, 2H), 4.37 (s, 4H), 2.39 (s, 3H). δC (solvent
CDCl3, 125.8 MHz, 298 K): 137.3, 135.6, 132.4, 131.9, 127.9, 66.1, 21.4. MS-ESI+, m/z (%BPI): [ + H]+,
163.1 (38%). Elem. Anal. Calcd for C9H11BO2: C, 66.7; H, 6.8%. Found: C, 66.6; H, 6.8%. IR: 3023, 2974,
2908 ν(C-H), 1607, 1583, 1481 ν(C=C), 1335 ν(C-BO2), 1206 ν(O-B-O) cm−1
4 as a pale yellow oil (1.691 g, 80%). δH (solvent CDCl3,
4
.
1-(3-(1,3,2-Dioxaborolan-2-yl)phenyl)-3-(2-(chloromethyl)phenyl)urea ( ). A solution of compound 3
5
(1.400 g, 8.590 mmol) and 1-(chloromethyl)-2-isocyanatobenzene (1.184 mL, 8.590 mmol) in diethyl
ether (40 mL) was stirred at room temperature for 16 h. The precipitate formed was isolated by
filtration and washed with diethyl ether (3
×
5 mL) to give 2.594 g of the desired compound (91%)
as a white solid. δH (solvent dmso-d6, 500 MHz, 298 K): 9.38 (s), 9.20 (s), 8.25 (s), 8.21 (s), 7.88 (m),
7.69–7.64 (m), 7.61 (m), 7.43 (m), 7.34–7.31 (m), 7.27–7.19 (m), 7.06 (m), 4.85 (s), 4.35 (s), 4.32 (s), 3.39
(s). δC (solvent dmso-d6, 125.8 MHz, 298 K): 152.7, 139.4, 137.5, 130.6, 129.2, 128.5, 127.9, 127.6, 124.0,
123.1, 122.4, 121.1, 65.9, 65.7, 62.8, 43.5. MS-ESI+, m/z (%BPI): [
5
+ H]+, 331.1 (7%); [5 − PG + H]+, 305.1
(23%); [5 − Cl]+, 295.1 (9%); [5 − PG
−
Cl]+, 269.1 (85%). Elem. Anal. Calcd for C16H16BClN2O3: C,
58.1; H, 4.9; N, 8.5%. Found: C, 58.4; H, 4.9; N, 8.3%. IR: 3285 ν(N-H), 2979, 2912 ν(C-H), 1637 ν(C=O),
1606, 1586, 1489, 1477 ν(C=C), 1560 δ(N-H), 1335 ν(C-BO2), 1214 ν(O-B-O), 672 ν(C-Cl) cm−1
.
1-(3-(1,3,2-Dioxaborolan-2-yl)phenyl)-3-(2-chloroethyl)urea ( ). A solution of compound (1.400 g,
7
3
8.590 mmol) and 1-chloro-2-isocyanatoethane (0.755 mL, 8.590 mmol) in diethyl ether (40 mL) was
stirred at room temperature for 16 h. The precipitate formed was isolated by filtration and washed
with diethyl ether (3
(solvent dmso-d6, 500 MHz, 298 K): 8.70 (s, 1H), 7.76 (m, 1H), 7.55
(t, 1H, 3J = 5.7 Hz), 4.31 (s, 4H), 3.66 (t, 2H, 3J = 6.2 Hz), 3.42 (c, 2H, 3J = 6.0 Hz). δC (solvent dmso-d6,
×
5 mL) to give 2.052 g of the desired compound (89%) as a white solid. δH
−
7.52 (m, 1H), 7.27–7.25 (m, 2H), 6.39

Molecules 2018, 23, 479
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125.8 MHz, 298 K): 155.1, 155.0, 139.9, 139.4, 128.4, 127.7, 127.4, 127.2, 123.8, 123.7, 120.9, 119.8, 65.6,
62.8, 44.5, 44.4, 41.2. MS-ESI+, m/z (%BPI): [
Anal. Calcd for C11H14BClN2O3: C, 49.2; H, 5.3; N, 10.4%. Found: C, 49.3; H, 5.2; N, 10.4%. IR: 3329
7
+ H]+, 269.1 (48%); [7 − PG + H]+, 243.1 (100%). Elem.
ν
(N-H), 2978, 2914
ν(C-H), 1629 ν(C=O), 1605, 1486, 1476 ν(C=C), 1564 δ(N-H), 1333 ν(C-BO2), 1214
ν(O-B-O), 614 ν(C-Cl) cm-1.
2-(3-(Bromomethyl)phenyl)-1,3,2-dioxaborolane (
9). This compound was prepared by adapting the
experimental procedure found in the literature [53]. A mixture of compound
4
(1.570 g, 9.693 mmol),
NBS (1.830 g, 10.18 mmol) and PDB (0.056 g, 0.173 mmol) in CCl4 (40 mL) was heated to reflux with
stirring for 16 h. The reaction mixture was allowed to cool to room temperature, the precipitated
succinimide was removed by filtration and the filtrate was concentrated under reduced pressure.
The residue was purified by column chromatography on SiO2 with CH2Cl2 as the eluent to give pure
product 9 as a pale yellow oil (1.039 g, 45%). δH (solvent CDCl3, 500 MHz, 298 K): 7.84 (m, 1H), 7.74
(m, 1H), 7.51 (m, 1H), 7.37 (t, 1H, 3J = 7.5 Hz), 4.51 (s, 2H), 4.39 (m, 4H). δC (solvent CDCl3, 125.8 MHz,
298 K): 136.3, 135.5, 134.9, 132.3, 128.5, 66.2, 33.6. MS-ESI+, m/z (%BPI): [9 − Br]+, 161.1 (100%). Elem.
Anal. Calcd for C9H10BBrO2: C, 44.9; H, 4.2%. Found: C, 45.0; H, 4.3%. IR: 2976, 2908 ν(C-H), 1605,
1583 ν(C=C), 1338 ν(C-BO2), 1216 ν(O-B-O), 602 ν(C-Br) cm−1
.
tert-Butyl-(2-bromoethyl)carbamate (12). This compound was prepared according to the literature
procedure [78]. A suspension of compound 11 (2.500 g, 12.08 mmol) in 40 mL of dioxane was stirred at
0 ◦C for 30 min, and subsequently a solution of di-tert-butyl dicarbonate (2.663 g, 12.08 mmol) and
triethylamine (1.701 mL, 12.08 mmol) in 15 mL of dioxane was added slowly. The mixture was stirred
at 0 ◦C for two hours, and at room temperature for two days. The solid formed was filtered and the
filtrate concentrated to dryness. The resulting oil was dissolved in chloroform (50 mL) and washed
with water (3
×
15 mL). The organic layer was dried over anhydrous sodium sulfate and the solvent
removed by rotary evaporation. The residue was dried under vacuum for several hours to give 2.152 g
(80%) of 12 as a pale yellow oil. δH (solvent CDCl3, 500 MHz, 298 K): 5.02 (s, 1H), 3.50 (m, 2H), 3.42
(m, 2H), 1.42 (s, 9H). δC (solvent CDCl3, 125.8 MHz, 298 K): 155.7, 79.9, 42.5, 32.8, 28.4. MS-ESI+, m/z
(%BPI): [12 + Na]+, 246.0 (1%); [12 − Boc + H]+, 124.0 (68%). Elem. Anal. Calcd for C7H14BrNO2: C,
37.5; H, 6.3; N, 6.3%. Found: C, 37.1; H, 5.7; N, 6.8%. IR: 3336
1507 ν(N-H), 1162 ν (N-C) cm−1
ν(N-H), 2977, 2932 ν(C-H), 1689 ν(C=O),
.
a
tert-Butyl-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)carbamate (13). A solution of compound 12 (2.030 g, 9.060
mmol), bis(pyridin-2-ylmethyl)amine (1.528 mL, 8.237 mmol), N,N-diisopropylethylamine (2.898 mL,
16.47 mmol) and a catalytic amount of KI in acetonitrile (100 mL) was heated to reflux with stirring
for three days. The resulting solution was filtered while hot and the filtrate concentrated to dryness.
The residue was extracted with chloroform (5
×
50 mL) and water (25 mL), the organic layers combined
and dried over anhydrous sodium sulfate, and the solvent evaporated under reduced pressure.
The resulting oil was purified on a CombiFlash RF-200, using a 48 g-neutral alumina column and
CH2Cl2 as eluent to obtain pure product 13 as an orange oil which was dried under vacuum for several
hours (1.092 g, 39%). δH (solvent CDCl3, 500 MHz, 298 K): 8.51 (m, 2H), 7.60 (m, 2H), 7.38 (d, 2H, 3J
= 7.8 Hz), 7.12 (m, 2H), 5.80 (s, 1H), 3.83 (s, 4H), 3.20 (m, 2H), 2.67 (m, 2H), 1.41 (s, 9H). δC (solvent
CDCl3, 125.8 MHz, 298 K): 159.3, 156.3, 149.2, 136.6, 123.2, 122.2, 78.8, 60.3, 53.7, 38.6, 28.6. MS-ESI+,
m/z (%BPI): [13 + Na]+, 365.2 (100%); [13 + H]+, 343.2 (19%). Elem. Anal. Calcd for C19H26N4O2:
C, 66.6; H, 7.7; N, 16.4%. Found: C, 66.3; H, 7.7; N: 15.6%. IR: 3315
ν(N-H), 3051–2821 ν(C-H), 1699
ν(C=O), 1590, 1569 ν(Py), 1520 ν(N-H), 1167 ν (N-C), 757 ν(C-H) cm−1
.
a
N0,N0-Bis(pyridin-2-ylmethyl)ethane-1,2-diamine (14). A solution of compound 13 (1.075 g, 3.139 mmol)
and trifluoroacetic acid (10 mL, 129.3 mmol) in chloroform (10 mL) was stirred at room temperature for
16 h. The solvent was evaporated under reduced pressure and the residue was dissolved in chloroform
(30 mL). The acid excess was neutralized with an aqueous solution of NaOH (1 M) and the organic
layer washed with distilled water (2 × 15 mL). The organic phase was dried over anhydrous sodium

Molecules 2018, 23, 479
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sulfate and the solvent removed by rotary evaporation to give the pure product 14 as an orange oil
that was dried under vacuum for several hours (0.727 g, 96%). δH (solvent CDCl3, 500 MHz, 298 K):
8.50 (m, 2H), 7.62 (m, 2H), 7.45 (d, 2H, 3J = 7.8 Hz), 7.12 (m, 2H), 3.82 (s, 4H), 2.77 (t, 2H, 3J = 6.1 Hz),
2.65 (t, 2H, 3J = 6.0 Hz), 2.06 (s, 2H). δC (solvent CDCl3, 125.8 MHz, 298 K): 159.7, 149.2, 136.5, 123.1,
122.1, 60.8, 57.4, 39.7. MS-ESI+, m/z (%BPI): [14 + H]+, 243.2 (100%). Elem. Anal. Calcd for C14H18N4:
C, 69.4; H, 7.5; N, 23.1%. Found: C, 69.1; H, 6.9; N, 22.8%. IR: 3360
ν(N-H), 3054–2823 ν(C-H), 1589,
1568, 1473, 1432 ν(Py), 756 ν(C-H) cm−1
.
(3-(3-(2-((Bis(pyridin-2-ylmethyl)amino)methyl)phenyl)ureido)phenyl)boronic acid (L1). A solution of
compound (1.150 g, 3.479 mmol), bis(pyridin-2-ylmethyl)amine (0.587 mL, 3.163 mmol),
5
N,N-diisopropylethylamine (1.113 mL, 6.325 mmol) and a catalytic amount of KI in acetonitrile (50 mL)
was heated to reflux with stirring for three days. The resulting solution was filtered while hot and the
filtrate concentrated to dryness. The residue was extracted with chloroform (5
×
50 mL) and water
(25 mL), the organic extracts combined and dried over anhydrous sodium sulfate, and the solvent
evaporated under reduced pressure. The resulting oil was purified on a CombiFlash RF-200, using a
48 g-neutral alumina column and CHCl3/MeOH as eluent (from 0 to 3%; the desired product eluted
from 2 to 3%) to obtain pure product L1 as a pale brown solid (0.607 g, 40%). δH (solvent dmso-d6,
3
4
500 MHz, 298 K): 10.30 (s, 1H, H14), 8.96 (s, 1H, H16), 8.50 (ddd, 2H, H1, J = 4.8 Hz, J = 1.7 Hz,
4J = 0.8 Hz), 8.03 (m, H12), 8.01 (s, H22), 7.77 (m, 1H, H23), 7.76–7.73 (m, H20), 7.71 (td, H3, 3J = 7.7 Hz,
4J = 1.8 Hz), 7.46 (m, 1H, H18), 7.40 (m, 2H, H4), 7.30 (t, 1H, H19, 3J = 7.7 Hz), 7.27–7.20 (m, 5H, H2, H9
and H11), 6.93 (td, 1H, H10, 3J = 7.4 Hz, 4J = 1.2 Hz), 3.80 (s, 4H, H6), 3.72 (s, 2H, H7). (See Figure S14
in Supplementary Materials for labelling scheme of the ligand). δC (solvent dmso-d6, 125.8 MHz, 298
K): 157.7 C5, 152.6 C15, 149.0 C1, 139.4 C13, 139.2 C17, 136.9 C3, 129.9 C9, 127.9 C19, 127.9 C18, 127.8
C11, 125.6 C8, 124.2 C23, 123.6 C4, 122.5 C2, 121.5 C10, 120.2 C20, 119.4 C12, 58.5 C6, 56.4 C7. MS-ESI+,
m/z (%BPI): [L1 + H]+, 468.2 (100%). Elem. Anal. Calcd for C26H26BN5O3: C, 64.3; H, 5.8; N, 14.4%.
Found: C, 64.5; H, 5.1; N: 14.6%. IR: 3290
ν(N-H/O-H), 3050–2830 ν(C-H), 1695 ν(C=O), 1593, 1571,
1477 ν(Ph/Py), 1538 ν(N-H), 1207 ν(O-B-O), 749 ν(C-H) cm−1
.
(3-(3-(2-(Bis(pyridin-2-ylmethyl)amino)ethyl)ureido)phenyl)boronic acid
(
L3).
A
solution of
compound (0.350 g, 1.303 mmol), bis(pyridin-2-ylmethyl)amine (0.220 mL, 1.185 mmol) and
7
N,N-diisopropylethylamine (0.417 mL, 2.370 mmol) in acetonitrile (30 mL) was heated to reflux with
stirring for four days. The resulting solution was filtered while hot and the filtrate concentrated to
dryness. The residue was extracted with chloroform (5
×
20 mL) and water (10 mL). During this
process a brown solid insoluble in both phases was formed. This solid was filtered off, the organic
extracts were combined, dried over anhydrous sodium sulfate and the solvent evaporated under
reduced pressure. The resulting oil was purified on a CombiFlash RF-200, using an 8 g-neutral alumina
column and CH2Cl2/MeOH as eluent (from 0 to 5%; the desired product eluted from 3 to 5%) to
obtain pure product L3 as a pale yellow solid (0.113 g, 24%). δH (solvent dmso-d6, 500 MHz, 298 K):
8.55 (s, 1H, H11), 8.48 (m, 2H, H1), 7.96 (s, 2H, H17), 7.72 (td, 2H, H3, 3J = 7.7 Hz, 4J = 1.7 Hz), 7.60–7.56
(m, 4H, H4, H15 and H18), 7.33 (m, 1H, H13), 7.23 (m, 2H, H2), 7.18 (t, 1H, H14, 3J = 7.6 Hz), 6.15 (m,
1H, H9), 3.81 (s, 4H, H6), 3.25 (c, 2H, H8, 3J = 5.9 Hz), 2.59 (t, 2H, H7, 3J = 6.0 Hz). (See Figure S14 in
Supplementary Materials for labelling scheme of the ligand). δC (solvent dmso-d6, 125.8 MHz, 298 K):
159.0 C5, 155.3 C10, 148.8 C1, 139.7 C12, 136.5 C3, 134.7 C16, 127.7 C14, 127.0 C13, 123.8 C15, 122.8 C4,
122.2 C2, 119.8 C18, 59.6 C6, 53.8 C7, 37.0 C8. MS-ESI+, m/z (%BPI): [L3 + H]+, 406.2 (100%). Elem.
Anal. Calcd for C21H24BN5O3: C, 62.2; H, 6.0; N, 17.3%. Found: C, 61.9; H, 6.1; N: 17.7%. IR: 3303
ν
(N-H/O-H), 3054–2849
ν(C-H), 1659 ν(C=O), 1593, 1477 ν(Ph/Py), 1551 ν(N-H), 1338 ν(C-BO2), 757
ν(C-H) cm−1
.
1-(2-(Bis(pyridin-2-ylmethyl)amino)ethyl)-3-(4-nitrophenyl)urea (L4). A solution of compound 14 (0.713
g, 2.942 mmol) and 1-isocyanato-4-nitrobenzene (0.498 g, 2.942 mmol) in chloroform (40 mL) was
stirred at room temperature for two hours. The solvent was evaporated under reduced pressure and
the residue was purified on a CombiFlash RF-200, using a 12 g-silica gel column and CHCl3/MeOH

Molecules 2018, 23, 479
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as eluent (from 0 to 4% of methanol; the desired compound eluted at 4%) to give L4 as a yellow oil
that became a solid after remaining under vacuum for several days (1.014 g, 85%). δH (solvent CDCl3,
500 MHz, 298 K): 8.95 (s, 1H, H11), 8.46 (m, 2H, H1), 8.00 (m, 2H, H14), 7.56–7.51 (m, 4H, H3 and H13),
7.26 (d, H4, 3J = 7.8 Hz), 7.09 (m, 2H, H2), 6.99 (s, 1H, H9), 3.73 (s, 4H, H6), 3.26 (c, 2H, H8, 3J = 5.1 Hz),
2.61 (t, 2H, H7, 3J = 5.9 Hz). (See Figure S14 in Supplementary Materials for labelling scheme of the
ligand). δC (solvent CDCl3, 125.8 MHz, 298 K): 158.1 C5, 155.3 C10, 149.1 C1, 146.7 C12, 141.3 C15,
136.9 C3, 125.1 C14, 123.9 C4, 122.5 C2, 117.2 C13, 60.0 C6, 53.0 C7, 37.3 C8. MS-ESI+, m/z (%BPI): [L4
+ H]+, 407.2 (100%). Elem. Anal. Calcd for C21H22N6O3: C, 62.1; H, 5.5; N, 20.7. Found: C, 62.4; H,
5.3; N, 20.0%. IR: 3317
ν(N-H), 3078–2804 ν(C-H), 1643 ν(C=O), 1614, 1591, 1475, 1433 ν(Ph/Py), 1553
ν (NO ), 1328 ν (NO ), 749 ν(C-H) cm−1
.
a
s
2
2
(3-((Bis(pyridin-2-ylmethyl)amino)methyl)phenyl)boronic acid (L5). A solution of compound
9 (0.310
g, 1.287 mmol), bis(pyridin-2-ylmethyl)amine (0.217 mL, 1.170 mmol), N,N-diisopropylethylamine
(0.412 mL, 2.340 mmol) and a catalytic amount of KI in acetonitrile (50 mL) was heated to reflux
with stirring for 16 h. The resulting solution was filtered while hot and the filtrate concentrated to
dryness. The residue was extracted with chloroform (5
×
25 mL) and water (15 mL), the organic
layers combined and dried over anhidrous sodium sulfate, and the solvent evaporated under reduced
pressure. The resulting oil was purified on a CombiFlash RF-200, using a 48 g-neutral alumina column
and CH2Cl2/MeOH as eluent (from 0 to 5%; the desired product eluted from 4% to 5%) to obtain pure
product L5 as a pale yellow solid (0.165 g, 42%). δH (solvent dmso-d6, 500 MHz, 298 K): 8.48 (ddd,
2H, H1, 3J = 4.8 Hz, 4J = 1.8 Hz, 4J = 0.8 Hz), 8.04 (s, 2H, H13), 7.80 (m, H14), 7.77 (td, H3, 3J = 7.7 Hz,
4J = 1.8 Hz), 7.67 (m, 1H, H11), 7.59 (m, 2H, H4), 7.47 (m, 1H, H9), 7.31 (t, 1H, H10, 3J = 7.5 Hz), 7.24
(m, 2H, H2), 3.70 (s, 4H, H6), 3.62 (s, 2H, H7). (See Figure S14 in Supplementary Materials for labelling
scheme of the ligand). δC (solvent dmso-d6, 125.8 MHz, 298 K): 159.2 C5, 148.8 C1, 137.5 C8, 136.6 C3,
134.6 C14, 134.1 C12, 132.9 C11, 130.4 C9, 127.4 C10, 122.5 C4, 122.2 C2, 59.1 C6, 57.9 C7. MS-ESI+, m/z
(%BPI): [L5 + Na]+, 356.2 (67%); [L5 + H]+, 334.2 (100%). Elem. Anal. Calcd for C19H20BN3O2: C, 68.5;
H, 6.1; N, 12.6%. Found: C, 68.2; H, 6.0; N: 11.0%. IR: 3049–2850
1362 ν(C-BO2), 758 ν(C-H) cm−1
ν(C-H), 1593, 1571, 1475 ν(Ph/Py),
.
General procedure for the preparation of [ML1](ClO4)2 (M = Cu or Zn). A solution of hydrated
Cu(ClO4)2 or Zn(ClO4)2 (0.103 mmol) in THF (2 mL) was added to a solution of L1 (0.050 g, 0.103 mmol)
in the same solvent (5 mL). The mixture was stirred at room temperature for an hour and the solvent
was removed under reduced pressure. The solid was treated with diethyl ether (5 mL), filtrated and
dried under vacuum.
[CuL1](ClO4)2·H2O ([CuL1](ClO4)2). Dark green solid. Yield: 0.077 g, 96%. MS-ESI+, m/z (%BPI):
[CuL1(ClO4)]+, 629.1 (3%); [Cu(L1-H)]+, 529.1 (41%). Elem. Anal. Calcd for C26H26BCl2CuN5O11
H2O:
·
C, 41.8; H, 3.8; N, 9.4%. Found: C, 42.1; H, 3.8; N, 9.1%. ΛM (MeOH, 10−3 M, 25 C): 154.9
◦
cm2·Ω−1
·
mol−1 (2:1 electrolyte). IR: 3358
ν(N-H/O-H), 1651 ν(C=O), 1614 ν(Ph/Py), 1549 ν(N-H),
1345
ν
(C-BO2), 1053
ν
a(Cl-O), 619
ν
a(O-Cl-O) cm−1. UV/vis diffuse reflectance spectroscopy: 633 nm.
[ZnL1](ClO4)2 ([ZnL1](ClO4)2). Light brown solid. Yield: 0.073 g, 97%. MS-ESI+, m/z (%BPI):
[ZnL1(ClO4)]+, 630.1 (4%); [Zn(L1-H)]+, 530.1 (100%); [ZnL1]2+, 265.6 (71%). Elem. Anal. Calcd
−3
for C26H26BCl2N5O11Zn: C, 42.7; H, 3.6; N, 9.6%. Found: C, 42.6; H, 3.7; N: 9.3%. ΛM (MeOH, 10 M,
25 ◦C): 164.0 cm2·Ω−1
·
mol−1 (2:1 electrolyte). IR: 3354
ν
(N-H/O-H), 1612
ν(Ph/Py), 1559 ν(N-H),
1343 ν(C-BO2), 1055 νa(Cl-O), 620 ν . δH (solvent dmso-d6, 500 MHz, 298 K): 8.60 (m,
a(O-Cl-O) cm−1
2H), 8.42 (s, 1H), 8.15 (s, 1H), 8.01 (s), 7.97 (m), 7.60–7.55 (m, 5H), 7.49–7.35 (m, 6H), 7.22 (m, 1H), 4.22
(d, 2H, H6A, 2J = 16.0 Hz), 3.82 (s, 2H), 3.67 (d, 2H, H6B, 2J = 16.0 Hz). δC (solvent dmso-d6, 125.8 MHz,
298 K): 154.4, 153.6, 147.6, 140.5, 138.6, 138.5, 134.8, 133.3, 129.4, 128.3, 127.9, 127.8, 127.5, 125.7, 124.9,
124.7, 124.4, 120.4, 55.4, 51.7.

Molecules 2018, 23, 479
18 of 25
General procedure for the preparation of [ML3](ClO4)2 (M = Cu or Zn). A solution of hydrated
Cu(ClO4)2 or Zn(ClO4)2 (0.123 mmol) in acetonitrile (2 mL) was added to a solution of L3 (0.050 g,
0.123 mmol) in the same solvent (5 mL). The mixture was stirred at room temperature for 16 h and the
solvent was removed under reduced pressure. The solid was treated with diethyl ether (5 mL), filtered,
and dried under vacuum.
[CuL3](ClO4)2·2H2O ([CuL3](ClO4)2). Dark green solid. Yield: 0.052 g, 59%. MS-ESI+, m/z (%BPI):
[CuL3(ClO4)]+, 567.1 (16%); [Cu(L3-H)]+, 467.1 (8%); [CuL3]2+, 234.1 (100%). Elem. Anal. Calcd for
C21H24BCl2CuN5O11 2H2O: C, 35.8; H, 4.0; N, 10.0%. Found: C, 35.7; H, 3.6; N, 10.1%. ΛM (MeOH,
·
10−3 M, 25 ◦C): 170.2 cm2·Ω−1
·
mol−1 (2:1 electrolyte). IR: 3364
ν(N-H/O-H), 1613 ν(Ph/Py), 1556
ν
(N-H), 1341
ν
(C-BO2), 1052
ν
a(Cl-O), 619
ν
a(O-Cl-O) cm−1. UV/vis diffuse reflectance spectroscopy:
585 nm.
[ZnL3](ClO4)2·H2O ([ZnL3](ClO4)2). Light brown solid. Yield: 0.041 g, 49%. MS-ESI+, m/z
(%BPI): [Zn(L3-H)(OMe)]+, 482.1 (2%); [ZnL3(OMe)]2+, 241.6 (100%). Elem. Anal. Calcd for
C21H24BCl2N5O11Zn H2O: C, 36.7; H, 3.8; N, 10.2%. Found: C, 36.5; H, 4.0; N: 9.8%. ΛM (MeOH,
·
10−3 M, 25 ◦C): 167.2 cm2·Ω−1
·
mol−1 (2:1 electrolyte). IR: 3363
ν(N-H/O-H), 1612 ν(Ph/Py), 1565
ν
(N-H), 1341
ν
(C-BO2), 1054
ν
a(Cl-O), 619 δH (solvent dmso-d6, 300 MHz, 298 K):
ν
a(O-Cl-O) cm−1
.
8.60 (m), 8.52 (s), 8.08 (m), 7.95 (s), 7.62–7.49 (m, 6H), 7.37 (m, 1H), 7.20 (m, 1H), 6.24 (s, 1H), 4.37 (d,
H6A, 2J = 16.0 Hz), 4.05 (d, H6B, 2J = 16.0 Hz), 3.60 (m), 2.83 (m). δC (solvent dmso-d6, 75.5 MHz,
298 K): 155.7, 154.6, 147.6, 140.6, 139.2, 134.8, 127.8, 127.5, 124.7, 124.5, 124.3, 120.3, 57.2, 54.9, 34.7.
General procedure for the preparation of [ML4](ClO4)2 (M = Cu or Zn) and [CuL4(SO4)]. A solution
of hydrated Cu(ClO4)2, Zn(ClO4)2 or Cu(SO4) (0.123 mmol) in methanol (2 mL) was added to a solution
of L4 (0.050 g, 0.123 mmol) in the same solvent (5 mL). The mixture was stirred at room temperature for
16 h, and the precipitate formed was isolated by filtration, washed with methanol (2 mL) and diethyl
ether (2 mL), and dried under vacuum. For the Zn2+ complex no precipitate was obtained, so the
solvent was evaporated under reduced pressure and chloroform (5 mL) was added. The precipitate
formed was stirred at room temperature for 16 h, filtered, washed with 5 mL of chloroform and 5 mL
of diethyl ether, and dried under vacuum.
[CuL4](ClO4)2·2H2O ([CuL4](ClO4)2). Light blue solid. Yield: 0.075 g, 86%. MS-ESI+, m/z (%BPI):
[CuL4(ClO4)]+, 568.0 (83%); [Cu(L4-H)]+, 468.1 (22%); [CuL4]2+, 234.6 (100%). Elem. Anal. Calcd for
C21H22Cl2CuN6O11 2H2O: C, 35.8; H, 3.7; N, 11.9%. Found: C, 35.9; H, 3.4; N, 11.5%. ΛM (MeOH,
·
10−3 M, 25 ◦C): 144.5 cm2·Ω−1
·
mol−1. IR: 3584, 3410, 3342
a(NO2), 1334 s(NO2), 1093, 1061
ν
(N-H), 3089–2780
ν
(C-H), 1697
ν(C=O),
1613, 1597
ν
(Ph/Py), 1550
cm−1. UV/vis diffuse reflectance spectroscopy: 633 nm.
ν
(N-H), 1504
ν
ν
ν
a(Cl-O), 620 ν
a(O-Cl-O)
[CuL4](SO4)·1.5H2O ([CuL4(SO4)]). Light blue solid. Yield: 0.047 g, 64%. MS-ESI+, m/z (%BPI):
[Cu(L4+H)(SO4)]+, 566.1 (20%); [Cu(L4-H)]+, 468.1 (3%); [CuL4]2+, 234.6 (52%). Elem. Anal. Calcd
for C21H22CuN6O7S
10−3 M, 25 ◦C): the low solubility of this complex in methanol has prevented us from determining
the conductivity value for this compound. IR: 3254 (N-H), 3117–2943 (C-H), 1692 (C=O), 1612,
1595 (Ph/Py), 1562 (N-H), 1502 a(NO2), 1324 s(NO2), 1106, 1049, 1029 a(S-O), 600, 584 a(O-S-O)
·
1.5H2O: C, 42.5; H, 4.3; N, 14.2%. Found: C, 42.7; H, 3.9; N, 13.9%. ΛM (MeOH,
ν
ν
ν
ν
ν
ν
ν
ν
ν
cm−1. UV/vis diffuse reflectance spectroscopy: 706 nm. Slow evaporation of a solution of the
complex in a dimethylformamide/water mixture provided dark blue single crystals suitable for X-ray
diffraction analysis.
[ZnL4](ClO4)2·CHCl3 ([ZnL4](ClO4)2). Light yellow solid. Yield 0.092 g, 95%. MS-ESI+, m/z (%BPI):
[ZnL4(ClO4)]+, 569.1 (74%); [Zn(L4-H)]+, 469.1 (100%); [ZnL4]2+, 235.1 (83%). Elem. Anal. Calcd f−o3r
C21H22Cl2N6O11Zn CHCl3: C, 33.4; H, 2.9; N, 10.6%. Found: C, 33.8; H, 3.1; N: 10.7%. ΛM (MeOH, 10
·
M, 25 ◦C): 161.9 cm2·Ω−1
·
mol−1 (2:1 electrolyte). IR: 3372
ν
(N-H), 1647
ν
(C=O), 1614
ν
(Ph/Py), 1556
ν
(N-H), 1506
ν
a(NO2), 1329
ν
s(NO2), 1094, 1051 a(Cl-O), 619
ν
ν
a(O-Cl-O) cm−1
.
δH (solvent dmso-d6,
300 MHz, 298 K): 9.39 (s, 1H, H11), 8.61 (m, 2H), 8.16–8.06 (m, 4H), 7.63–7.57 (m, 6H), 6.52 (m, 1H, H9),

Molecules 2018, 23, 479
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4.38 (d, 2H, H6A, 2J = 16.0 Hz), 4.06 (d, 2H, H6B, 2J = 16.0 Hz), 3.45 (m), 2.82 (m, 2H). δC (solvent dmso-d6,
75.5 MHz, 298 K): 154.6, 154.5, 147.5, 146.9, 140.6, 125.1, 124.7, 124.4, 117.0, 56.8, 54.0, 34.5.
Procedure for the preparation of [CuL5](ClO4)2. A solution of hydrated Cu(ClO4)2 (0.150 mmol)
in acetonitrile (2 mL) was added to a solution of L5 (0.050 g, 0.150 mmol) in acetonitrile (5 mL).
The mixture was stirred at room temperature for 16 h and the solvent was removed under reduced
pressure. The solid was treated with diethyl ether (5 mL), filtrated and dried under vacuum.
[CuL5](ClO4)2·H2O ([CuL5](ClO4)2). Dark blue solid. Yield: 0.075 g, 82%. MS-ESI+, m/z (%BPI):
[CuL5(ClO4)]+, 495.1 (70%); [Cu(L5-H)]+, 395.1 (100%). Elem. Anal. Calcd for C19H20BCl2CuN3O10
·
H2O:
◦
−1
C, 37.2; H, 3.6; N, 6.9%. Found: C, 37.4; H, 3.8; N, 7.1%.
(2:1 electrolyte). IR: 3441 ν(O-H), 1613, 1576, 1487 ν(Ph/Py), 1346 ν(C-BO2), 1048 νa(Cl-O), 619 νa(O-Cl-O)
Λ
M (MeOH, 10−3 M, 25 C): 171.3 cm2·Ω−1
·
mol
cm−1. UV/vis diffuse reflectance spectroscopy: 603 nm.
3.3. X-ray Diffraction Studies
Crystals of [CuL4(SO4)] were obtained by evaporation of a solution of the isolated complex, as
described above. Crystals of [CuL4(H2PPi)] were grown by slow evaporation of an acetonitrile–methanol
(1:1) mixture containing stoichiometric amounts of [CuL4](ClO4)2 and sodium pyrophosphate.
Finally, [{CuL4}2( -PPi)] and [{ZnL4}2(
µ µ-PPi)] were crystallized by slow evaporation of solutions of
[CuL4](ClO4)2 (acetonitrile-methanol mixture) or [ZnL4](ClO4)2 (deuterium oxide) in the presence of
0.5 equiv. of sodium pyrophosphate.
Three-dimensional X-ray data were collected on Bruker X8 APEXII CCD ([{ZnL4}2(
µ
-PPi)],
-PPi)] and [CuL4(H2PPi)]) or BRUKER-NONIUS X8 APEX CCD ([CuL4(SO4)]) diffractometers.
The data of [{CuL4}2( -PPi)], [CuL4(H2PPi)]) and [CuL4(SO4)] were corrected for Lorentz and
polarization effects and for absorption by semiempirical methods based on symmetry-equivalent
reflections [79]. The crystal selected for [{ZnL4}2(
-PPi)] was a twin, so that the correction for absorption
[{CuL4}2(
µ
µ
µ
was made with TWINABS [80]. Two domains were found and two corrected hkl files were generated,
one for solving the structure (HKLF 4) and another for the final refinement (HKLF 5). Complex scattering
factors were taken from the program SHELX-2017 running under the WinGX program system as
implemented on a Pentium® computer [81
method with Superflip [83] (for complex [{ZnL4}2(
(for [{CuL4}2(
,
82]. The structures were solved by using the charge-flipping
-PPi)] or Patterson methods with DIRDIF2008 [84
-PPi)], [CuL4(H2PPi)] and [CuL4(SO4)]). All of the structures were refined by full-matrix
µ
]
µ
least-squares on F2 (SHELXL-2017) [85]. Hydrogen atoms were included in calculated positions and
refined in riding mode for all of the compounds with the following exceptions: hydrogen atoms of urea
NH moieties were refined freely in the final stages of refinement and hydrogen atoms of water molecules
(one molecule per unit cell in [CuL4(H2PPi)] and six molecules per unit cell in crystals of [{ZnL4}2(
µ-PPi)])
were located in a difference electron-density map and the H· · · H and O-H distances restrained.
In the final steps of refinement of the crystal structure of [{ZnL4}2(
µ-PPi)], the data were cleaned
up with SQUEEZE [86] to avoid the problems generated by disordered methanol and acetonitrile
molecules in two voids with volumes of 255 and 115 Å3 and electron counts of 82 and 35, respectively,
which agree with the presence of two acetonitrile and one methanol molecule, respectively.
Finally, refinement converged with anisotropic displacement parameters for all non-hydrogen
atoms for all four structures. Crystal data and details on data collection and refinement are summarized
in Table 5.

Molecules 2018, 23, 479
20 of 25
Table 5. Crystal data and refinement details.
[CuL4(SO4)]
[CuL4(H2PPi)]
[{CuL4}2(µ-PPi)]
[{ZnL4}2(µ-PPi)]
formula
MW
crystal system
space group
T/K
C21H22CuN6O7S
566.04
orthorhombic
Pbca
C23H29CuN7O11P2 C42H44Cu2N12O13P2 C42H56N12O19P2Zn
705.01
triclinic
P −1
1113.91
triclinic
P −1
1225.66
monoclinic
P 21/n
100(2)
100(2)
100(2)
100(2)
19.636(3)
8.913(1)
25.692(3)
90
7.606(2)
11.984(3)
17.360(4)
106.18(1)
95.12(1)
105.26(1)
1443.5(6)
726
13.5195(9)
13.6036(9)
14.5281(10)
76.346(4)
79.212(5)
76.738(4)
2502.0(3)
1144
10.2371(6)
17.6978(10)
28.5263(16)
90
95.104(3)
90
a/Å
b/Å
c/Å
α/deg
β/deg
γ/deg
V/Å3
90
90
4496.30(10)
2328
5147.7(5)
2536
F(000)
Z
8
2
2
4
0.71073
1.672
1.123
0.71073
1.622
0.939
0.71073
1.479
0.987
0.71073
1.581
1.081
λ, Å (Mo Kα)
−3
Dcalc/g·cm
−1
µ/mm
θ range/deg
Rint
2.969–25.149
0.0943
4016
1.855–30.706
0.0424
8880
1.457–26.389
0.0391
1.356–26.391
0.1223 c
13103
reflns obsd
GOF on F2
R1 a
10203
1.023
0.0320
0.0765
1.051
0.038
0.0980
1.013
0.0300
0.0746
1.011
0.0768
0.1789
wR2 (all data) b
Largest diff. peak
0.464 and −0.533
0.532 and −0.366
0.397 and −0.434
0.959 and −1.946
−3
and hole/e Å
[w(Fo4)]}1/2
.
Rint is based on
a
b
c
R1
=
Σ
||Fo|
−
|Fc||/
Σ
|Fo|. wR2 (all data) = {
Σ
[w(||Fo|2
−
|Fc|2)2]/
Σ
agreement between observed single and composite intensities and those calculated from refined unique intensities
and twin fractions for all 64,393 observations.
3.4. Computational Details
All of the calculations presented in this work were performed employing the Gaussian 09 package
(Revision B.01) [87]. Full geometry optimizations of [ZnL4(H2PPi)] and [{ZnL4}2(
µ-PPi)] complexes
were carried out in dmso solution employing DFT within the hybrid meta-GGA approximation
with the TPSSh exchange-correlation functional [88]. Input geometries were generated from the
crystallographic data of [CuL4(H2PPi)] and [{ZnL4}2(
µ-PPi)] by replacing the Cu(II) metal ion in the
first one. For geometry optimization purposes we used the standard Ahlrichs’ valence double- basis
ξ
set including polarization functions (SVP) [89]. No symmetry constraints have been imposed during
the optimizations. The stationary points found on the potential energy surfaces as a result of geometry
optimizations were tested to represent energy minima rather than saddle points via frequency analysis.
The default values for the integration grid (75 radial shells and 302 angular points) and the SCF energy
convergence criteria (10−8 a.u.) were used in all the calculations. The 31P NMR shielding tensors
were calculated using the GIAO [90] method at the TPSSh/TZVP [91] level. The isotropic shielding
constants (σ) were converted to chemical shifts with respect to 85% aqueous H3PO4 by using the
following expression [92,93]:
δ
calc = σcalc(PH3) − σcalc(complex) − 266.1
(1)
The shielding tensors of PH3 were calculated at the same computational level.
Throughout this work solvent effects (dmso) were included by using the polarizable continuum
model (PCM), in which the solute cavity is built as an envelope of spheres centered on atoms or
atomic groups with appropriate radii. In particular, we used the integral equation formalism (IEFPCM)
variant as implemented in Gaussian 09 [94].

Molecules 2018, 23, 479
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4. Conclusions
We have presented a series of Cu2+ and Zn2+ complexes containing dipicolylamine units for
metal ion coordination and urea groups. In the absence of phosphorylated anions, the urea group
coordinates to the metal ion through one of the nitrogen atoms. Addition of monophosphate anions
does not provoke the decoordination of the urea group, while the addition of PPi, ADP, or ATP
causes decoordination of the urea group and a change of the coordination environment from distorted
octahedral to trigonal bipyramidal. The coordination of the PPi and ADP anions to the metal ion
appears to be reinforced by the presence of hydrogen bonds with the urea unit, as also observed
in the solid state, while these interactions seem to be weaker in the case of ATP. The [CuL4]2+ and
[CuL5]2+ complexes show important selectivities towards PPi, ADP, and ATP over anions containing
a single phosphate group. The results indicate that the cooperative binding is established for the
complexes derived from L4 during the anion recognition and that it is necessary to consider the N-type
coordination mode of the urea moiety to prevent the receptor hydrolysis when designing urea based
receptors with different delocalized withdrawing groups in their backbones.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: The authors thank Xunta de Galicia (EM 2012/088) for generous financial support. I.C.-B. is
grateful to Ministerio de Educación y Ciencia (FPU program) for a predoctoral fellowship.
Author Contributions: David Esteban-Gómez and Israel Carreira-Barral conceived and designed the experiments;
Israel Carreira-Barral performed the experiments, analyzed the data and wrote a first draft of the manuscript;
Marta Mato-Iglesias performed the mass spectral analysis and characterization of some of the compounds; Isabel
Fernández-Pérez supervised the kinetic experiments; Carlos Platas-Iglesias supervised the computational study
and participated in analyzing the data; Andrés de Blas performed the X-ray crystal structures refinements; David
Esteban-Gómez and Carlos Platas-Iglesias supervised the research and participated in writing of the article.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
References
1.
Hargrove, A.E.; Nieto, S.; Zhang, T.; Sessler, J.L.; Anslyn, E.V. Artificial Receptors for the Recognition of
Phosphorylated Molecules. Chem. Rev. 2011, 111, 6603–6782. [CrossRef] [PubMed]
Knowles, J.R. Enzyme-Catalyzed Phosphoryl Transfer Reactions. Annu. Rev. Biochem. 1980, 49, 877–919.
[CrossRef] [PubMed]
2.
3.
4.
Burnstock, G. Purinergic cotransmission. Exp. Physiol. 2009, 94, 20–24. [CrossRef] [PubMed]
Hardie, D.G.; Hawley, S.A. AMP-activated protein kinase: The energy charge hypothesis revisited. BioEssay
2001, 23, 1112–1119. [CrossRef] [PubMed]
5.
6.
Suprenant, A.; Evans, R.J. ATP in synapses. Nature 1993, 362, 211–212. [CrossRef] [PubMed]
Murugappa, S.; Kunapuli, S.P. The role of ADP receptors in platelet function. Front. Biosci. 2006, 11,
1977–1986. [CrossRef] [PubMed]
7.
8.
9.
Rehamn, G.; Shehzad, A.; Khan, A.L.; Hamayun, M. Role of AMP-Activated Protein Kinase in Cancer
Therapy. Arch. Pharm. Chem. Life Sci. 2014, 347, 457–468. [CrossRef] [PubMed]
Rao, T.; Liang, L.; Zhang, L. Ratiometric Fluorescence Recognition for Pyrophosphate on the Basis of
Terpyridine Derivative. Anal. Sci. 2013, 29, 1165–1169. [CrossRef] [PubMed]
Shao, N.; Wang, H.; Gao, X.D.; Yang, R.H.; Chan, W.H. Spiropyran-based fluorescent anion probe and its
application for urinary pyrophosphate detection. Anal. Chem. 2010, 82, 4628–4636. [CrossRef] [PubMed]
10. Shin, I.-S.; Bae, S.W.; Kim, H.; Hong, J.-I. Electrogenerated chemiluminescent anion sensing: Selective
recognition and sensing of pyrophosphate. Anal. Chem. 2010, 82, 8259–8265. [CrossRef] [PubMed]
11. Lee, S.; Yuen, K.K.Y.; Jolliffe, K.A.; Yoon, J. Fluorescent and Colorimetric Chemosensors for Pyrophosphate.
Chem. Soc. Rev. 2015, 44, 1749–1762. [CrossRef] [PubMed]
12. Yu, C.-J.; Wu, S.-M.; Tseng, W.-L. Magnetite Nanoparticle-Induced Fluorescence Quenching of Adenosine
Triphosphate
−
BODIPY Conjugates: Application to Adenosine Triphosphate and Pyrophosphate Sensing.
Anal. Chem. 2013, 85, 8559–8565. [CrossRef] [PubMed]

Molecules 2018, 23, 479
22 of 25
13. McDonough, M.J.; Reynolds, A.J.; Lee, W.Y.G.; Jolliffe, K.A. Selective recognition of pyrophosphate in water
using a backbone modified cyclic peptide receptor. Chem. Commun. 2006, 2971–2973. [CrossRef] [PubMed]
14. Kumar, M.; Reddy, M.D.; Mishra, A.; George, S.J. The molecular recognition controlled stereomutation cycle
in a dynamic helical assembly. Org. Biomol. Chem. 2015, 13, 9938–9942. [CrossRef] [PubMed]
15. Francesconi, O.; Gentili, M.; Bartoli, F.; Bencini, A.; Conti, L.; Giorgi, C.; Roelens, S. Phosphate binding by a
novel Zn(II) complex featuring a trans-1,2-diaminocyclohexane ligand. Effective anion recognition in water.
Org. Biomol. Chem. 2015, 13, 1860–1868. [CrossRef] [PubMed]
16. Deng, J.; Yu, P.; Yang, L.; Mao, L. Competitive Coordination of Cu2+ between Cysteine and Pyrophosphate
Ion: Toward Sensitive and Selective Sensing of Pyrophosphate Ion in Synovial Fluid of Arthritis Patients.
Anal. Chem. 2015, 85, 2516–2522. [CrossRef] [PubMed]
17. Niu, F.; Ying, Y.-L.; Hua, X.; Niu, Y.; Xu, Y.; Long, Y.-T. Electrochemically generated green-Fluorescent
N-doped carbon quantum dots for facile monitoring alkaline phosphatase activity based on the
Fe3+-mediating ON-OFF-ON-OFF fluorescence principle. Carbon 2018, 127, 340–348. [CrossRef]
18. Liu, Z.; Xiao, J.; Wu, X.; Lin, L.; Weng, S.; Chen, M.; Cai, X.; Lin, X. Switch-on fluorescent strategy based on N
and S co-doped graphene quantum dots (N-S/GQDs) for monitoring pyrophosphate ions in synovial fluid
of arthritis patients. Sens. Actuators B 2016, 229, 217–224. [CrossRef]
19. Smith, B.A.; Akers, W.J.; Leevy, W.M.; Lampkins, A.J.; Xiao, S.; Wolter, W.; Suckow, M.A.; Achilefu, S.;
Smith, B.D. Optical Imaging of Mammary and Prostate Tumors in Living Animals using Synthetic Near
Infrarred Zinc(II)-Dipicolylamine Probe for Anionic Cell Surfaces. J. Am. Chem. Soc. 2010, 132, 67–69.
[CrossRef] [PubMed]
20. Burke, C.R.; Lupták, A. DNA synthesis from diphosphate substrates by DNA polymerases. Proc. Natl. Acad.
Sci. USA 2018. [CrossRef] [PubMed]
21. Minami, T.; Emami, F.; Nishiyabu, R.; Kubo, Y.; Anzenbacher, P., Jr. Quantitative analysis of modeled ATP
hydrolysis in water by a colorimetric sensor array. Chem. Commun. 2016, 52, 7838–7841. [CrossRef] [PubMed]
22. Jolliffe, K.A. Pyrophosphate Recognition and Sensing in Water Using Bis[zinc(II)dipicolylamino]-
Functionalized Peptides. Acc. Chem. Res. 2017, 50, 2254–2263. [CrossRef] [PubMed]
23. Sigel, H.; Massoud, S.S.; Corfù, N.A. Comparison of the Extent of Macrochelate Formation in Complexes
of Divalent Metal Ions with Guanosine (GMP2−), Inosine (IMP2−), and Adenosine 50-Monophosphate
(AMP2−). The Crucial Role of N-7 Basicity in Metal Ion-Nucleic Base Recognition. J. Am. Chem. Soc. 1994
116, 2958–2971. [CrossRef]
,
24. Sigel, H.; Massoud, S.S.; Tribolet, R. Comparison of the Metal Ion Coordinating Properties of
Tubercidin 50-Monophosphate (7-Deaza-AMP) with Those of Adenosine 50-Monophosphate (AMP) ans
0
1,N6-Ethenoadenosine 5 -Monophosphate (
∈
-AMP). Definite Evidence for Metal Ion-Base Backbinding to N-7 and
Extent of Macrochelate Formation in M(AMP) and M(
∈-AMP). J. Am. Chem. Soc. 1988, 110, 6857–6865. [CrossRef]
25. Massoud, S.S.; Tribolet, R.; Sigel, H. Metal-ion-governed molecular recognition: extent of intramolecular
stack formation in mixed-ligand-copper(II) complexes containing a heteroaromatic N base and an adenosine
monophosphate (20AMP, 30AMP, or 50AMP) A structuring effect of the metal-ion bridge. Eur. J. Biochem.
1990, 187, 387–393. [CrossRef] [PubMed]
26. Sigel, H. Interactions of Metal Ions with Nucleotides and Nucleic Acids and their Constituents.
Chem. Soc. Rev. 1993, 22, 255–267. [CrossRef]
27. Massoud, S.S.; Sigel, H. Evaluation of the metal-ion-coordinating differences between the 20-, 30- and
0
5 -monophosphates of adenosine. Eur. J. Biochem. 1989, 179, 451–458. [CrossRef] [PubMed]
0
28. Massoud, S.S.; Sigel, H. Metal Ion Coordinating Properties of Pyrimidine-Nucleoside 5 -Monophosphates
(CMP, UMP, TMP) and of Simple Phosphate Monoesters, Including D-Ribose 50-Monophosphate.
Establishment of Relations between Complex Stability and Phosphate Basicity. Inorg. Chem. 1988, 27,
1447–1453. [CrossRef]
29. Aoki, K. X-ray evidence for the metal ion bridged intra- and intermolecular stacking
interactions between nucleotide bases and aromatic heterocyclic rings within the ternary complex
0
[Cu(5 -AMP)(bpy)(H2O)]2(NO3)2·6H2O. J. Am. Chem. Soc. 1978, 100, 7106–7108. [CrossRef]
30. Pouessel, J.; Abada, S.; Le Bris, N.; Elhabiri, M.; Charbonnière, L.J.; Tripier, R. A new bis-tetraamine
ligand with a chromophoric 4-(9-anthracenyl)-2,6-dimethylpyridinyl linker for glyphosate and ATP sensing.
Dalton Trans. 2013, 42, 4859–4872. [CrossRef] [PubMed]

Molecules 2018, 23, 479
23 of 25
31. Pouessel, J.; Bazzicalipi, C.; Bencini, A.; Bernard, H.; Giorgi, C.; Handel, H.; Matera, I.; Le Bris, N.; Tripier, R.;
Valtancoli, B. Exploring New Molecular Architectures for Anion Recognition: Synthesis and ATP Binding
Properties of New Cyclam-Based Ditopic Polyammonium Receptors. Chem. Asian J. 2011, 6, 1582–1594.
[CrossRef] [PubMed]
32. Kubik, S. Receptors for Biologically Relevant Anions. In Anion Coordination Chemistry; Bowman-James, K., Bianchi, A.,
García-España, E., Eds.; John Wiley & Sons: Weinheim, Germany, 2012; pp. 363–464; ISBN 978-3-527-32370-8.
33. Bregier-Jarzebowska, R.; Gasowska, A.; Hoffman, S.K.; Lomozik, L. Interactions of diamines with
adenosine-50-triphosphate (ATP) in the systems including copper(II) ions. J. Inorg. Biochem. 2006, 162,
73–82. [CrossRef] [PubMed]
34. Kataev, E.A.; Müller, C.; Kolesnikov, G.V.; Khrustalev, V.N. Guanidinium-Based Artificial Receptors for
Binding Orthophosphate in Aqueous Solution. Eur. J. Org. Chem. 2014, 2747–2753. [CrossRef]
35. Xu, Z.; Singh, N.J.; Lim, J.; Pan, J.; Kim, H.-N.; Park, S.; Kim, K.-S.; Yoon, J. Unique Sandwich Stacking of
Pyrene-Adenine-Pyrene for Selective and Ratiometric Fluorescent Sensing of ATP at Physiological pH. J. Am.
Chem. Soc. 2009, 131, 15528–15533. [CrossRef] [PubMed]
36. Xuejian, L.; Smith, D.G.; Jolliffe, K.A. Are two better than one? Comparing intermolecular and intramolecular
indicator displacement assays in pyrophosphate sensors. Chem. Commun. 2016, 52, 8463–8466. [CrossRef]
37. Zwicker, V.E.; Liu, X.; Yuen, K.K.Y.; Jolliffe, K.A. Triazole-containing zinc(II)dipicolylamine-functionalised
peptides as highly selective pyrophosphate sensors in physiological media. Supramol. Chem. 2016, 28,
192–200. [CrossRef]
38. Zhao, R.R.; Xu, Q.L.; Yang, Y.; Cao, J.; Zhou, Y.; Xu, R.; Zhang, J.F. A coumarin-based terpyridine-zinc complex for
sensing pyrophosphate and its application in in vivo imaging. Tetrahedron Lett. 2016, 57, 5022–5025. [CrossRef]
39. Chao, D.; Ni, S. Nanomolar pyrophosphate detection and nucleus staining in living cells with simple
terpyridine-Zn(II) complexes. Sci. Rep. 2016, 6, 26477. [CrossRef] [PubMed]
40. Das, P.; Chandar, N.B.; Chourey, S.; Agarwalla, H.; Ganguly, B.; Das, A. Role of Metal Ion in Specific
Recognition of Pyrophosphate Ion under Physiological Conditions and Hydrolysis of the Phosphoester
Linkage by Alkaline Phosphatase. Inorg. Chem. 2013, 52, 11034–11041. [CrossRef] [PubMed]
41. Watchasit, S.; Suktanarak, P.; Suksai, C.; Ruangpornvisuti, V.; Tuntulani, T. Discriminate sensing of
pyrophosphate using a new tripodal tetramine-based dinuclear Zn(II) complex under an indicator
displacement assay approach. Dalton Trans. 2014, 43, 14701–14709. [CrossRef] [PubMed]
42. Huang, F.; Hao, G.; Wu, F.; Feng, G. Fluorescence sensing of ADP over ATP and PPi in 100% aqueous
solution. Analyst 2015, 140, 5873–5876. [CrossRef] [PubMed]
43. Kumar, A.; Pandey, R.; Kumar, A.; Gupta, R.K.; Dubey, M.; Mohammed, A.; Mobin, S.M.; Pandey, D.S.
Self-assembled copper(II) metallacycles derived from asummetric Schiff base ligands: Efficient hosts for
ADP/ATP in phosphate buffer. Dalton Trans. 2015, 44, 17152–17165. [CrossRef] [PubMed]
44. Mesaquita, L.M.; Mateus, P.; Fernandez, R.D.V.; Iranzo, O.; André, V.; de Oliveira, F.T.; Platas-Iglesias, C.;
Delgado, R. Recognition of phosphopeptides by a dinuclear copper(II) macrocyclic complex in a water:
Methanol 50:50 v/v solution. Dalton Trans. 2017, 46, 9549–9564. [CrossRef] [PubMed]
45. Amendola, V.; Bergamaschi, G.; Guglielmo, L.; Izzo, L.; Mangano, C.; Mella, M.; Milanese, C.; Miljkovoc, A.
Dicopper(II) MozobilTM
: A dinuclear receptor for the pyrophosphate anion in aqueous solution.
Supramol. Chem. 2017, 29, 834–845. [CrossRef]
46. Amendola, V.; Bergamaschi, G.; Buttafava, A.; Fabbrizzi, L.; Monzani, E. Recognition and sensing of nucleoside
monophosphates by a dicopper(II) cryptate. J. Am. Chem. Soc. 2010, 132, 147–156. [CrossRef] [PubMed]
47. Pouessel, J.; Le Bris, N.; Bencini, A.; Giorgi, C.; Valtancoli, B.; Tripier, R. Glyphosate and ATP binding by
mononuclear Zn(II) complexes with non-symmetric ditopic polyamine ligands. Dalton Trans. 2012, 41,
10521–10532. [CrossRef] [PubMed]
48. Butler, S.J. Ratiometric Detection of Adenosine Triphosphate (ATP) in Water and Real-Time Monitoring of
Apyrase Activity with a Tripodal Zinc Complex. Chem. Eur. J. 2014, 20, 15768–15774. [CrossRef] [PubMed]
49. Carreira-Barral, I.; Rodríguez-Blas, T.; Platas-Iglesias, C.; de Blas, A.; Esteban-Gómez, D. Cooperative Anion
Recognition in Copper(II) and Zinc(II) Complexes with a Ditopic Tripodal Ligand Containing a Urea Group.
Inorg. Chem. 2014, 53, 2554–2568. [CrossRef] [PubMed]
50. Carreira-Barral, I.; Mato-Iglesias, M.; de Blas, A.; Platas-Iglesias, C.; Tasker, P.A.; Esteban-Gómez, D. Ditopic
receptors containing urea groups for solvent extraction of Cu(II) salts. Dalton Trans. 2017, 46, 3192–3206.
[CrossRef] [PubMed]

Molecules 2018, 23, 479
24 of 25
51. Djanashvili, K.; Frullano, L.; Peters, J.A. Molecular recognition of sialic acid end groups by phenylboronates.
Chem. Eur. J. 2005, 11, 4010–4018. [CrossRef] [PubMed]
52. Regueiro-Figueroa, M.; Djanashvili, K.; Esteban-Gómez, D.; de Blas, A.; Platas-Iglesias, C.; Rodríguez-Blas, T.
Towards Selective Recognition of Sialic Acid Through Simultaneous Binding to Its cis-Diol and Carboxylate
Functions. Eur. J. Org. Chem. 2010, 3237–3248. [CrossRef]
53. Regueiro-Figueroa, M.; Djanashvili, K.; Esteban-Gómez, D.; Chauvin, T.; Tóth, É.; de Blas, A.;
Rodríguez-Blas, T.; Platas-Iglesias, C. Molecular Recognition of Sialic Acid by Lanthanide(III) Complexes
through Cooperative Two-Site Binding. Inorg. Chem. 2010, 49, 4212–4223. [CrossRef] [PubMed]
54. Tandon, S.S.; McKee, V.J. Lead(II) and barium(II) complexes of a potentially octadentate macrocyclic ligand
(H4L), capable of providing endogenous alkoxy and phenoxy bridges. The X-ray crystal structures of
[Pb(H4L)][ClO4]2, [Pb(H2L)][ClO4]2, and [(H6L)(H2O)2][ClO4]2. J. Chem. Soc. Dalton Trans. 1989, 19–24.
[CrossRef]
55. Habata, Y.; Akabori, S. Molecular structure of novel polymer-like complexes of armed-azacrown ethers with
alkali-metal cations. J. Chem. Soc. Dalton Trans. 1996, 3871–3882. [CrossRef]
56. Geary, W.J. The use of conductivity measurements in organic solvents fot characterization of coordination
compounds. Coord. Chem. Rev. 1971, 7, 81–122. [CrossRef]
57. Halcrow, M.A. Jahn-Teller distortions in transition metal compounds, and their importance in functional
molecular materials. Coord. Chem. Rev. 2013, 42, 1784–1795. [CrossRef] [PubMed]
58. Hay, B.P.; Dixon, D.A.; Bryan, J.C.; Moyer, B.A. Crystallographic evidence for oxygen acceptor directionality
in oxyanion hydrogen bonds. J. Am. Chem. Soc. 2002, 124, 182–183. [CrossRef] [PubMed]
59. Ikotun, O.F.; Ouellette, W.; Lloret, F.; Kruger, P.E.; Julve, M.; Doyle, R.P. Synthesis, structural, thermal
and magnetic characterization of a pyrophosphate-bridged cobalt(II) complex. Eur. J. Inorg. Chem. 2008
,
2691–2697. [CrossRef]
60. Sartoris, R.P.; Nascimento, O.R.; Santana, R.C.; Perec, M.; Baggio, R.F.; Calvo, R. Structure and Magnetism
of a binuclear Cu(II) pyrophosphate: Transition to a 3D magnetic behaviour studied by single cristal EPR.
Dalton Trans. 2015, 44, 4732–4743. [CrossRef] [PubMed]
61. Lee, J.H.; Park, J.; Lah, M.S.; Chin, J.; Hong, J.-I. High affinity pyrophosphate receptor by a synergistic effect
between metal coordination and hydrogen bonding in water. Org. Lett. 2007, 9, 3729–3731. [CrossRef] [PubMed]
62. Kim, S.K.; Lee, D.H.; Hong, J.-I.; Yoon, J. Chemosensors for pyrophosphate. Acc. Chem. Res. 2009, 42, 23–31.
[CrossRef] [PubMed]
63. Addison, A.W.; Rao, T.N.; Reedijk, J.; van Rijn, J.; Verschoor, G.C. Synthesis, structure, and spectroscopic
properties of copper(II) compounds containing nitrogen–sulphur donor ligands; the crystal and molecular
structure of aqua[1,7-bis(N-methylbenzimidazol-2’-yl)-2,6-dithiaheptane]copper(II) perchlorate. J. Chem. Soc.
Dalton Trans. 1984, 1349–1356. [CrossRef]
64. Evans, R.; Deng, Z.; Rogerson, A.K.; McLachlan, A.S.; Richards, J.J.; Nilsson, M.; Morris, G.A. Quantitative
Interpretation of Diffusion-Ordered NMR Spectra: Can We Rationalize Small Molecule Diffusion Coefficients?
Angew. Chem. Int. Ed. 2013, 52, 3199–3202. [CrossRef] [PubMed]
65. Salvestrini, S.; Di Cerbo, P.; Capasso, S. Kinetics of the chemical degradation of diuron. Chemosphere 2002, 48,
69–73. [CrossRef]
66. Zambelli, B.; Musiani, F.; Benini, S.; Ciurli, S. Chemistry of Ni2+ in Urease: Sensing, Trafficking, and Catalysis.
Acc. Chem. Res. 2011, 44, 520–530. [CrossRef] [PubMed]
67. Ciurli, S.; Benini, S.; Rypniewski, W.R.; Wilson, K.S.; Miletti, S.; Mangani, S. Structural properties of the
nickel ions in urease: Novel insights into the catalytic and inhibition mechanisms. Coord. Chem. Rev. 1999
190–192, 331–335. [CrossRef]
,
68. Zimmer, M. Molecular Mechanics Evaluation of the Proposed Mechanisms for the Degradation of Urea by
Urease. J. Biomol. Struct. Dyn. 2000, 17, 787–797. [CrossRef] [PubMed]
69. Kistiakowsky, G.B.; Rosenberg, A.J. The Kinetics of Urea Hydrolysis by Urease. J. Am. Chem. Soc. 1952, 74,
5020–5025. [CrossRef]
70. Krajewska, B. Ureases I. Functional, kinetic and catalytic properties: A review. J. Mol. Catal. B Enzym. 2009,
59, 9–21. [CrossRef]
71. Dixon, N.E.; Riddles, P.W.; Gazzola, C.; Blakeley, R.L.; Zerner, B. Jack bean urease (EC 3.5.1.5). V. On the
mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds.
Can. J. Biochem. 1980, 58, 1335–1344. [CrossRef] [PubMed]

Molecules 2018, 23, 479
25 of 25
72. Callahan, B.P.; Yunan, Y.; Wolfenden, R. The Burden Borne by Urease. J. Am. Chem. Soc. 2005, 127,
10828–10829. [CrossRef] [PubMed]
73. Choi, K.-Y.; Jeon, Y.-M.; Ryu, H.; Oh, J.-J.; Lim, H.-H.; Kim, M.W. Synthesis and characterization of
syn-anti carboxylate-bridged one-dimensional copper(II) complexes with bis(2-pyridylmethyl)amino acids.
Polyhedron 2004, 23, 903–911. [CrossRef]
74. Caneda-Martínez, L.; Valencia, L.; Fernández-Pérez, I.; Regueiro-Figueroa, M.; Angelovski, G.; Brandariz, I.;
Esteban-Gómez, D.; Platas-Iglesias, C. Toward inert paramagnetic Ni(II)-based chemical exchange saturation
transfer MRI agents. Dalton Trans. 2017, 46, 15095–15106. [CrossRef] [PubMed]
75. Amendola, V.; Boiocchi, M.; Esteban-Gómez, D.; Fabbrizzi, L.; Monzani, E. Chiral receptors for phosphate
ions. Org. Biomol.Chem. 2005, 3, 2632–2639. [CrossRef] [PubMed]
76. Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in solution. Determination of equilibrium constants
with the HYPERQUAD suite of programs. Talanta 1996, 43, 1739–1753. [CrossRef]
77. Wolsey, W.C. Perchlorate salts, their uses and alternatives. J. Chem. Educ. 1973, 50, A335–A337. [CrossRef]
78. Shoji, A.; Kuwahara, M.; Ozaki, H.; Sawai, H. Modified DNA Aptamer That Binds the (R)-Isomer of a Thalidomide
Derivative with High Enantioselectivity. J. Am. Chem. Soc. 2007, 129, 1456–1464. [CrossRef] [PubMed]
79. Krause, L.; Herbst-Irmer, R.; Sheldrick, G.M.; Stalke, D. Comparition of silver and molybdenum microfocus
X-ray sources for single-crystal structure determination. J. Appl. Cryst. 2015, 48, 3–10. [CrossRef] [PubMed]
80. Sheldrick, G.M. TWINABS—Bruker AXS Scaling for Twinned Crystals—Version 2012/1; Georg-August-
Universität Göttingen: Göttingen, Germany, 2012.
81. Sheldrick, G.M. A short history of SHELX. Acta Cryst. 2008, A64, 112–122. [CrossRef] [PubMed]
82. Farrugia, L.J. WinGX MS-Windows system of programs for solving, refining and analysing single crystal
X-ray diffraction data for small molecules. J. Appl. Cryst. 1999, 32, 837–838. [CrossRef]
83. Palatinus, L.; Chapuis, G. SUPERFLIP—A computer program for the solution of crystal structures by charge
flipping in arbitrary dimensions. J. Appl. Cryst. 2007, 40, 786–790. [CrossRef]
84. Beurskens, P.T.; Beurskens, G.; de Gelder, R.; Garcia-Granda, S.; Gould, R.O.; Smits, J.M.M. The DIRDIF2008
Program System; Crystallography Laboratory, University of Nijmegen: Nijmegen, The Netherlands, 2008.
85. Sheldrick, G.M. Crystal structure refinement with SHELXL. Acta Cryst. 2015, C71, 3–8. [CrossRef]
86. Spek, A.L. PLATON SQUEEZE: A tool for the calculation of the disordered solvent contribution to the
calculated structure factors. Acta Crystallogr. Sect. C 2015, 71, 9–18. [CrossRef] [PubMed]
87. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.;
Mennucci, B.; Petersson, G.A.; et al. Gaussian 09, Revision A.01; Gaussian, Inc.: Wallingford, CT, USA, 2009.
88. Tao, J.M.; Perdew, J.P.; Staroverov, V.N.; Scuseria, G.E. Climbing the density functional ladder: Nonempirical
meta-generalizated gradient approximation designed for molecules and solids. Phys. Rev. Lett. 2003, 91,
146401–146405. [CrossRef] [PubMed]
89. Schaefer, A.; Horn, H.; Ahlrichs, R. Fully optimized contracted Gaussian basis sets for atoms Li to Kr.
J. Chem. Phys. 1992, 97, 2571–2577. [CrossRef]
90. Ditchfield, R. Self-consistent perturbation theory of diamagnetism. Mol. Phys. 1974, 27, 789–807. [CrossRef]
91. Schaefer, A.; Huber, C.; Ahlrichs, R. Fully optimized contracted Gaussian basis sets of triple zeta valence
quality for atoms Li to Kr. J. Chem. Phys. 1994, 100, 5829–5835. [CrossRef]
92. Wüllen, C.V. A comparison of density functional methods for the calculation of phosphorous-31 NMR
chemical shifts. Phys. Chem. Chem. Phys. 2000, 2, 2137–2144. [CrossRef]
93. Rezaei-Sameti, M. Ab initio calculations of 31P NMR chemical shielding tensors in alkyl phosphorus compounds
and comparison with experimental values. J. Mol. Struct. (Theochem) 2008, 867, 122–124. [CrossRef]
94. Tomasi, J.; Mennucci, B.; Cammi, R. Quantum Mechanical Continuum Solvation Models. Chem. Rev. 2005
105, 2999–3093. [CrossRef] [PubMed]
,
Sample Availability: Samples of the compounds are not available from the authors.
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