Recognition of phosphopeptides by a dinuclear copper(II) macrocyclic complex in a water:methanol 50:50 v/v solution

Article abstract of DOI:10.1039/c7dt01724c

A new triethylbenzene-derived tetraazamacrocycle containing pyridyl spacers, L, was prepared and its dinuclear copper(ii) complex was used as a receptor for the recognition of phosphorylated peptides in aqueous solution. A detailed study of the acid-base

Full text of DOI:10.1039/c7dt01724c

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Dalton Trans., 2017, DOI: 10.1039/C7DT01724C.
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Recognition of Phosphopeptides by a Dinuclear Copper(II) Macrocyclic
Complex in Water:Methanol 50:50 v/v Solution
Lígia M. Mesquita,a Pedro Mateus,a Rui D. V. Fernandes,a Olga Iranzo,b Vânia André,c Filipe Tiago de
Oliveira,d Carlos Platas-Iglesias,e Rita Delgado*,a
a
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da
República, 2780–157 Oeiras, Portugal.
b Aix Marseille Univ., CNRS, Centrale Marseille, iSm2, Marseille, France
c Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais,
1049–001 Lisboa, Portugal.
d
Laboratório de Instrumentação, Engenharia Biomédica e Física da Radiação (LIBPhys-UNL),
Departamento de Física, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Monte
de Caparica, 2892–516 Caparica, Portugal.
e
Centro de Investigaciones Científicas Avanzadas (CICA) and Departamento de Química
Fundamental, Facultade de Ciencias, Universidade da Coruña, Campus da Zapateira-Rúa da Fraga
10, 15008 A Coruña, Spain.
Electronic supplementary information (ESI) available: Tables of overall protonation constants
of L and of the studied substrates, of overall stability constants of copper(II) with the studied substrates,
and overall association constants between the copper(II) complexes of L with the substrates. Figures of
species distribution of L, H2pST3 and of cascade species with peptides. Figure of the EPR spectra of
the copper(II) complexes at different pH values. Figures of 1D, 2D NMR and ESI mass spectra of the L
compound, of the phosphopeptides, and of some intermediate compounds: 2,4-bis(bromomethyl)-1,3,5-
triethylbenzene and (2,4,6-triethyl-1,3-phenylene)dimethanamine. DFT results.
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ABSTRACT
A new triethylbenzene-derived tetraazamacrocycle containing pyridyl spacers, L, was prepared and its
dinuclear copper(II) complex was used as receptor for the recognition of phosphorylated peptides in
aqueous solution. A detailed study of the acid-base behaviour of L and its copper(II) complexation
properties as well as of the cascade species with phosphorylated anions including two peptidic
substrates was carried out in H2O/MeOH (50:50 v/v) solution using different techniques, such as
potentiometry, X-band EPR and DFT calculations. The association constants of the dinuclear receptor
with the phosphorylated peptides and other anionic species revealed a clear preference towards
phenylic phosphorylated substrates, with values ranging 3.96–5.35 log units. Single-crystal X-ray
diffraction determination of the dicopper(II) complex of L showed the copper centres at a distance of
2–
5.812(1) Å from one another, with the phosphate group of the PhPO4 substrate well accommodated
between them. X-band EPR studies indicated a similar structure for this cascade complex and for the
other cascade complexes with the phosphorylated anions studied. DFT studies of the [Cu2L(μ-OH)]3+
complex revealed a different conformation of the ligand that brings the two copper centres at a very
short distance of 3.94 Å aided by the presence of a bridging hydroxide anion that provides a
Cu···O···Cu angle of 167.3º. This complex is EPR silent, in line with the singlet ground state obtained
using CASSCF(2,2) calculations and DFT calculations with the broken-symmetry approach. This
species coexists in solution with a complex in a different conformation, and having a Cu···Cu distance
of 6.63Å, in lower percentage.
INTRODUCTION
Protein phosphorylation is a key event in the signalling pathways that control many cell functions
including metabolism, transcription, cell cycle progression, apoptosis, and differentiation.1 Aberrant
protein phosphorylation/dephosphorylation is observed in several human pathologies, including
inflammatory, neurodegenerative and autoimmune diseases,2 and cancer.3 Phosphorylation of tyrosine,
threonine and serine residues on protein surfaces serves as a switch either by creating binding sites for
the establishment of protein-protein interactions or by promoting conformational changes that activate
enzymatic activity.4 Therefore, compounds able to bind phosphopeptides/phosphoproteins can
potentially be used as inhibitors of a particular deregulated signalling pathway or as analytical tools for
investigating phosphorylation-based cell signaling.5
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However, the recognition of phosphorylated peptides or proteins in the presence of other bio-
relevant anionic molecules is not straightforward. In fact, the binding of phosphorylated substrates with
high affinity and selectivity in aqueous media still remains a great research challenge.6 Notwithstanding
attempts have been made in order to develop suitable receptors for the recognition of
phosphopeptides/phosphoproteins. Among them, dinuclear metal complexes of ligands derived from
dipicolylamine or cyclen have been used for this purpose.7 Although the use of dinuclear metal
complexes for the binding of anionic substrates in aqueous medium is a proven strategy, achieving
selectivity through the formation of cascade species involves the location of the metal centres at a given
relatively rigid fixed distance,8 a requirement difficult to accomplish with non-cyclic ligands.
In this work, the dinuclear complexes of a ditopic hexaamine macrocycle are evaluated as a possible
alternative to non-cyclic ligands. Macrocyclic complexes were already used as receptors for the uptake
of phosphorylated substrates.9 However, to the best of our knowledge, studies reporting binding of
phosphorylated peptides to dinuclear complexes of macrocyclic ligands are scarce.10 In these receptors,
each metal centre is designed with one or two coordination sites occupied by water molecules or
weakly coordinated counterions easily replaced by the envisaged substrate, while the distance between
the two metal centres is modulated by rigid spacers.8
In the present study a new triethylbenzene-derived tetraazamacrocycle containing pyridyl spacers
(L, Scheme 1) is described and its dinuclear copper(II) complex evaluated as a receptor for
phosphorylated peptides. The compound was prepared under the expectation that while the pyridyl
heads would function as metal binding sites, the triethylbenzene groups might be engaged in π-π
interactions with the benzene group of phosphotyrosine. Peptides H3pST1 and H2pST3 (Scheme 1)
were selected as models of the phosphorylated sequences of signal transducer and activator of
transcription proteins STAT111 and STAT3.12 These sequences are involved in the homo and
heterodimerization of the latter proteins, a crucial event for nucleus translocation and subsequent up-
regulation of several genes involved in cell progression, differentiation, and survival.13
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Scheme 1 Ligand L and peptides studied in this work.
RESULTS AND DISCUSSION
Synthesis of compound L. The synthesis of L started with the preparation of 2,4-
bis(bromomethyl)-1,3,5-triethylbenzene from commercially available 1,3,5-triethylbenzene (Scheme 2,
steps a to c). Although this compound has been described previously,14 the product was not isolated and
instead used as a mixture of di- and tribromomethylated compounds. Herein, by using 3 equiv. of
paraformaldehyde and HBr relative to 1,3,5-triethylbenzene, and a precise control of the reaction
temperature at 80 ºC,15 the desired compound was obtained in 77% yield. The dibromide was then
converted to the diazide, followed by reduction with Ph3P in THF/H2O to afford the 2,4-
bis(aminomethyl)-1,3,5-triethylbenzene, using a procedure similar to that described for the preparation
of 2,4,6-tris(aminomethyl)-1,3,5-triethylbenzene.16 Compound L was then obtained in good yield by
reaction between 2,4-bis(aminomethyl)-1,3,5-triethylbenzene and 2,6-pyridinedicarbaldehyde in 2:2
ratio in MeCN, followed by addition of sodium borohydride for the reduction of imine groups to the
corresponding amines (Scheme 2, steps d and e). This compound is the macrocyclic version of a
cryptand reported by us.17
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Scheme 2 Synthetic procedure for the preparation of L. a) Zn, 3 equiv. (HCOH)n, 33% HBr in AcOH,
80 ºC, η = 77%; b) NaN3, DMF, r.t., η = 94%; c) Ph3P, THF:H2O (10:1), r.t., η = 73%; d) MeOH, r.t., η
= 77%; e) NaBH4, MeOH, reflux, η = 92%.
The H2pST3 peptide (Scheme 1) was obtained commercially and already used by us,10 and the
H3pST1 peptide was synthesized by using Fmoc chemistry,18 and was purified by preparative reversed-
phase HPLC.
Acid-base behaviour of L and H3pST1 and H2pST3 peptides, and copper(II)
complexation studies
The protonation constants of L and its stability constants with Cu2+ were determined at ionic strength
0.10 M in KNO3 and 298.2 K in H2O/MeOH (50:50 v/v). The studies were performed in a mixed
solvent system due to the precipitation of the ligand at pH 6.0 in pure water, preventing the
determination of the protonation constants in this medium. Furthermore, the copper(II) complex also
precipitates at pH > 7.0 at the concentrations required for potentiometric titrations. The corresponding
stepwise constants are collected in Table 1, and the overall constants are presented in Table S1 in the
ESI. The species distribution diagram for the protonation of L is shown in Fig. S1 in the ESI.
The four protonation constants determined for L correspond to the successive protonation of the
secondary amines. The pyridyl nitrogen atoms are not protonated in the working pH range (3.5–10.5).
The results agree well with those previously reported for the related macrobicyclic compound.17,19
The copper(II) complexation studies of L revealed its very high tendency to form dinuclear
complexes. Indeed, even at 1:1 Cu2+:L ratio (Fig. 1a), the dinuclear species start to form at pH 3.5
being the predominant species at pH > 5.8. The stepwise stability constant of the dinuclear complex
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[Cu2L]4+ (coordinated H2O molecules are omitted for simplicity), obtained by reaction of Cu2+ with
[CuL]2+, is lower than the stability constant of the mononuclear complex by 4.18 log units due to the
charge repulsion between the two closely located metal ions, besides the statistical factor. Nonetheless,
at 2:1 Cu2+:L ratio (Fig. 1b), the dinuclear species predominate above pH 4.5, and exist mainly in the
form of the monohydroxo complex, [Cu2L(OH)]3+. This hydroxo complex starts to form at a very
unusual low pH (pKa = 4.42), indicating that the hydroxide anion bridges the two copper centres that
thus must be located at short distance from one another (see discussion below). The value of the
association constant of OH– with the [Cu2L]4+ receptor takes the high value of 9.43 log units (see Table
1). Similar low pKa values for the hydrolysis of a coordinated water molecule were found in a few
dicopper(II) complexes of cryptands.20 However, the pKa value found in this work is, to the best of our
knowledge, the lowest value found for a dinuclear copper(II) complex of a macrocycle. In fact, the
deprotonation of one coordinated water molecule lowers the positive charge in the pocket formed
between the copper(II) centres at short distance, contributing to minimize the electrostatic repulsions.
Two other -hydroxo dicopper(II) complexes of macrocyclic ligands were found to have very short
Cu···Cu distances in their X-ray structures, but the pKa values of the water deprotonation were not
determined.21
Table 1 Stepwise protonation constants (KiH) of L and stepwise stability constants (KCu H L) of its
m
h
copper(II) complexes in H2O/MeOH (50:50 v/v).a
b
Equilibrium reaction
log KiH b Equilibrium reaction
log KCu
H L
m
h
[CuHL]3+ + H+ ⇌ [CuH2L]4+
[CuL]2+ + H+ ⇌ [CuHL]3+
Cu2+ + L ⇌ [CuL]2+
L + H+ ⇌ HL+
8.36(1)
7.52(1)
6.51(1)
5.79(1)
–
5.85(2)
7.10(2)
11.24(3)
8.38(2)
7.06(3)
4.42(1)
9.43(3)
HL+ + H+ ⇌ H2L2+
H2L2+ + H+ ⇌ H3L3+
H3L3+ + H+ ⇌ H4L4+
–
[CuL(OH)]+ + H+ ⇌ [CuL]2+
[CuL]2+ + Cu2+ ⇌ [Cu2L]4+
[Cu2L(OH)]3+ + H+ ⇌ [Cu2L]4+
[Cu2L]4+ + OH– ⇌ [Cu2L(OH)]3+
–
–
–
a T = 298.20.1 K; I = 0.100.01 M in KNO3. b Values in parenthesis are standard deviations in the last significant figures.
Precipitation of the [Cu2L(OH)]3+ complex (or [Cu2L(OH)2]2+ starting to be formed) occurred above
pH 8.00, which prevented further characterization of the system in solution, but at least it was possible
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to infer that one other possible coordinated water molecule only deprotonates at higher and more usual
pH, and so this hydroxide anion is likely not bridging the two copper centres.
(a)
(b)
Fig. 1 Species distribution diagrams calculated for the complexes of Cu2+ with L. (a) Cu:L 1:1 ratio,
CCu = CL = 2.010–3 M; and (b) Cu:L 2:1 ratio, CCu = 2CL = 2.010–3 M.
The protonation constants for the studied anions were also determined in the same experimental
conditions, including H2pST3,10 see Table S2 and Fig. S2 in the ESI. For the H3pST1 peptide the first
H
K1 value corresponds to the protonation of the lysine amine group in agreement with the value
H
obtained for H2pST3. The second constant, K2 , is mainly attributed to the protonation of the
phosphonate group of the phosphotyrosine (pY), agreeing well with the values obtained for the
2–
protonation of PhPO4 and of the pY of H2pST3. As a consequence, the HpST12– species, which
contains completely ionized anionic groups and a protonated lysine, dominates a very large pH range
(Fig. 2). In this peptide the K3H constant is mainly attributed to the protonation of the glutamate residue,
H
in agreement with the value obtained for acetate (log K1 = 5.24, see Table S2 in the ESI). These
considerations can be visualized by the speciation diagrams shown in Figs. 2 and S2. In all cases, due
to their high basicity, the amide centres remain protonated in solution within the pH range accessible
by potentiometry, and so it was impossible to determine their protonation constants.
The copper(II) complexation properties of the anions were also studied in the same conditions
(Table S3 in ESI). The peptidic substrates exhibit low affinity for copper(II), as expected of peptides
with blocked N-termini containing only weakly coordinating side chains.22 This means that the side
chains of the peptide, the pY in particular, do not participate in metal ion coordination. Due to the low
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affinity of H2pST1 for copper(II), determination of stability constants required using an excess of
peptide relative to the copper(II) ion and to perform back-titrations.
Fig. 2 Species distribution diagrams for the protonation of H3pST1. Cpeptide = 1.010–3 M.
Cascade species formed by the copper(II) complexes of L with phosphorylated
substrates. The very high association constant of the receptor to OH–, prompted us to study the
association of the dinuclear copper(II) complexes of L as receptor to p-cresol in order to test its binding
ability towards phenolic substrates. However, no association was detected within the working pH
range. The receptor was then used for the binding of phosphorylated anions. The corresponding
association constants were determined by potentiometry at the experimental conditions used before,
and the results are collected in Table 2 (see also Table S4 in the ESI). Experiments with one non-
phosphorylated peptide derivative of H2pST3, HST3,10c were also carried out. However, precipitation
during the titration prevented us from determining its stability constant with Cu2+ ion.
(3–n)–
(2–n)–
The receptor was found to form stable 1:1 associations with HnPO4
and with HnPhPO4
,
interacting with the anions in the form of [Cu2L]4+ as well as [Cu2L(OH)]3+ (see Tables 2 and Table S4
in ESI). The high affinity of the substrates for the hydroxocomplex [Cu2L(OH)]3+ suggests that the OH–
anion co-operates with the binding event. In fact, the hydroxide anion contributes to deprotonate the
–
–
phosphonate anions at a pH value lower than expected. Indeed, the pKa of H2PO4 is 7.51 (H2PO4 ⇌
2–
HPO4 + H+ pKa = 7.51) whereas when the same anion is coordinated to both copper centres forming
the cascade species its pKa is much lower of about 4 ([Cu2L(H2PO4)]3+ ⇌ [Cu2L(HPO4)]2+ + H+ pKa =
4.00). Furthermore, the formation of the [Cu2L(OH)]3+ species likely pre-organises the complex by
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bringing the two metal ions at a short distance, so that coordination of bridging phosphorylated anions
is favoured (see X-ray analysis below). Also noteworthy is the fact that PhPO42– is bound 0.64 log units
2–
stronger than HPO4 , in spite of its lower basicity, which suggests π-π interactions established between
the triethylbenzene units of the receptor and the phenyl group of the substrate may contribute to the
overall affinity.
2–
The high affinity of the receptor towards PhPO4 encouraged us to study the interaction of the
receptor with phosphorylated peptides, see Table 2. Indeed, high values of association constants were
determined for the binding of the receptor with the protonated forms of the phosphorylated peptides,
ranging 3.96–5.35 log units. Moreover, only species of 1:1 receptor:substrate stoichiometry were found
for all the cases, see Table 2 and the species distribution diagram in Fig. S3 in the ESI. The association
constants increase as the overall charge of the substrates decreases, as would be expected based on
electrostatic arguments. Accordingly, as the H3pST1 peptide has a positive charge one unit lower than
that of H2pST3 at any given pH value, the receptor shows a preference for the former peptide over
H2pST3 due to decreased electrostatic repulsions.
Table 2 Stepwise association constants (KCu (H L)S) for the indicated equilibria in H2O/MeOH (50:50
2
h
v/v).a
b
log KCu (H L)S
Equilibrium reaction
2
h
–
[Cu2L]4+ + H2PO4 ⇌ [Cu2L(H2PO4)]3+
4.09(3)
4.51(2)
[Cu2L(OH)]3+ + H2PO4 ⇌ [Cu2L(HPO4)]2+
–
[Cu2L(OH)]3+ + H(PhPO4)– ⇌ [Cu2L(PhPO4)]2+
5.16(1)
[Cu2L]4+ + H3pST3+ ⇌ [Cu2L(H3pST3)]5+
3.96(6)
4.87(1)
[Cu2L(OH)]3+ + H3pST3+ ⇌ [Cu2L(H2pST3)]4+
[Cu2L]4+ + H3pST1 ⇌ [Cu2L(H3pST1)]4+
[Cu2L(OH)]3+ + H3pST1 ⇌ [Cu2L(H2pST1)]3+
[Cu2L(OH)]3+ + H2pST1– ⇌ [Cu2L(HpST1)]2+
4.62(3)
5.05(1)
5.35(1)
b
a T = (298.20.1) K; I = (0.100.01) M in KNO3. Values in parenthesis are standard
deviations in the last significant figures.
In addition, it was found that the association constant values for the [Cu2L(OH)]3+ + H2pST1– ⇌
[Cu2L(HpST1)]2+ and [Cu2L(OH)]3+ + H3pST3+ ⇌ [Cu2L(H2pST3)]4+ equilibria are of the same
magnitude of that obtained for the [Cu2L(OH)]3+ + H(PhPO4)– ⇌ [Cu2L(PhPO4)]2+ equilibrium, which
suggests that both peptides are bound to the receptor through the phosphonate group of the
phosphotyrosine (pY residue).
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In order to evaluate the possible impact of the direct binding of the studied anions with protonated
species of the ligand (HnLn+), the association constant of PhPO42– with the free ligand was determined.
It was verified that the obtained value of 2.20 in log units for the association constant corresponding to
–
3+
the equilibrium H4L4+ + HPhPO4 ⇄ H5LPhPO4 is very low. The percentage of this species in
presence of the copper(II) is ~ 10% at pH 3.0 and negligible at pH 4.0 (see the species distribution
shown in Fig. S3 in the ESI). As the association constants of (HnLn+) with the other studied
2–
phosphorylated anions are expected to be similar or even lower than the one determined for PhPO4
anion, it is possible to conclude that the association of the free ligand with the anion does not affect at
the determinations of the associations of the studied anions with the dicopper complex receptor.
Single Crystal X-Ray Diffraction Studies. The molecular structure of [Cu2L(μ-PhPO4)][Cu2L(μ-
PhPO4)(NO3)]3NO3·24H2O is shown in Fig. 3 along with the relevant atomic notation adopted.
Selected distances and angles are given in Table 3.
The asymmetric unit contains two independent dinuclear copper(II) complex cations: [Cu2L(μ-
PhPO4)]2+ (A) and [Cu2L(μ-PhPO4)(NO3)]+ (B) (see Fig. 3a). In complex cation A each copper centre
exhibits a distorted square planar geometry composed of three nitrogen donors from the 2,6-
bis(aminomethyl)pyridine moieties of the macrocycle and one oxygen atom of the bound substrate. The
2–
phosphate group of the PhPO4 substrate is well accommodated between the two copper centres,
placing them at a distance of 5.812(1) Å from one another. It bridges the two Cu centres in a syn-anti
mode which results in a non-collinearity of the two N8A–Cu1A–O1A and N26A–Cu2A–O3A axes and
an angle of 29.1(2)º between both CuN3O coordination planes. The dianion is strongly bound to the
metal centres, as evidenced by the short Cu–O distances (Cu1A–O1A and Cu2A–O3A distances of
1.872(4) and 1.862(6) Å, respectively).
The complex cation B presents very similar bond lengths and angles (see Table 3) and mainly
differs in the coordination of the Cu2 atom, which is pentacoordinate. In this case the basal plane is
defined by the three nitrogen donors from the macrocycle and one oxygen atom of the bound substrate
while an oxygen atom of one nitrate anion occupies the apical position. The trigonal distortion
calculated using the index structural parameter  (τ = 0 for a perfect square-pyramidal geometry and τ =
1 for an ideal trigonal-bipyramidal geometry)23 assumes a value of 0.19 which is consistent with a
distorted square pyramidal coordination sphere. As in complex cation A, the phosphate group of
2–
PhPO4 bridges the two copper centres in a syn-anti mode which results in a Cu···Cu distance of
5.811(1) Å.
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(a)
(b)
(c)
Fig. 3 (a) Molecular structure of [Cu2L(μ-PhPO4)]2+ and [Cu2L(μ-PhPO4)(NO3)]+ complex cations; (b)
and (c) detailed views of an asymmetrical half unit of the complex. Two close water molecules in each
complex cation involved in HOH∙∙∙OPO32- hydrogen bonds are also represented.
In addition to the above mentioned coordination features, in each complex cation two water-
mediated hydrogen bonds are found, established between two secondary amines of the macrocycle and
an uncoordinated oxygen atom of the dianion (O4A···O16w···N1A with distances of 2.649(8) and
2.916(8)
Å
and O4A···O15w···N28A with distances of 2.782(9) and 3.317(10) Å),
O4B···O17w···N28B with distances of 2.622(8) and 2.994(8) Å and O4B···O11w···N1B with
distances of 2.574(8) and 2.932(8) Å), see Table S5 in the ESI. Twenty other water molecules and three
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nitrate anions are found in the crystal structure but are not directly involved in the binding event (see
Fig. S4 in the ESI).
Bearing in mind that the chemistry of phenylphosphate is very rich especially with Cu, a Cambridge
Structural Database (CSD) survey was carried out to analyze the data retrieved for complexes in which
phosphate behaves either as monodentate or bidentate 2-1: 1 or -1 bridging ligand in copper(II)
complexes. This survey showed that in 40% of the cases the phosphate moiety plays a monodentate
role, in other 40% the phosphate works as a bridge between two Cu through coordination of two
oxygen atoms (2-1: 1), and only in 20% of the cases the phosphate acts as a bridge using only one O
atom (-1). Furthermore, all the bonds distances and angles reported for the structure disclosed herein
are in agreement with the data reported at CSD.
Table 3 Selected bond distances (Å) and angles (º) in the
coordination spheres of the [Cu2L(μ-PhPO4)]2+ (A) and [Cu2L(μ-
PhPO4)(NO3)] + (B) cation complexes
Complex
A
B
Distances / Å
N1–Cu1
N10–Cu1
N8–Cu1
O1–Cu1
O1C–Cu2
N19–Cu2
N26–Cu2
N28–Cu2
2.068(6)
2.092(8)
1.925(6)
1.872(4)
–
2.083(6)
1.929(6)
2.073(6)
1.862(6)
5.812(1)
2.072(7)
2.077(5)
1.921(6)
1.875(6)
2.393(8)
2.084(6)
1.924(6)
2.063(6)
1.859(6)
5.811(1)
O3–Cu2
Cu1···Cu2
Angles / º
164.4(2)
171.5(2)
162.3(2)
164.6(3)
94.1(3)
90.1(3)
86.2(3)
100.9(3)
162.1(2)
173.6(3)
N1–Cu1–N10
N8–Cu1–O1
O1C–Cu2–N19
O1C–Cu2–N28
O1C–Cu2–N26
O1C–Cu2–O3
N19–Cu2–N28
N26–Cu2–O3
–
–
–
–
160.9(2)
173.5(2)
Computational Studies. In spite of our efforts, it was not possible to obtain crystals of the [Cu2L(μ-
OH)]3+ complex suitable for X-ray diffraction studies. To overcome this problem, the structure of this
hydroxocomplex was investigated by using DFT calculations in aqueous solution (see computational
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details below). The minimum energy conformation obtained from these calculations presents a
conformation of the ligand with the ethyl groups at position 3 of each of the phenyl rings pointing to
the same side of the ligand. This is in contrast to the structure observed in the solid state for [Cu2L(μ-
PhPO4)(NO3)]+, in which these ethyl groups are pointing to opposite sides with respect to the
macrocycle main plane. This conformation of the ligand brings the two copper centres at a distance of
only 3.94 Å (5.81 Å for [Cu2L(μ-PhPO4)(NO3)]+ in the solid state) aided by the presence of a bridging
hydroxide anion that provides a Cu···O···Cu angle of 167.3º. Each copper centre presents a distorted
square planar coordination geometry composed of three nitrogen donors from the 2,6-
bis(aminomethyl)pyridine moieties of the macrocycle and one oxygen atom of the bridging anion, see
Fig. 4.
Fig. 4 Top: Structure of [Cu2L(μ-OH)]3+ obtained with DFT calculations (M06/TZVP, bulk solvent
effects considered using a polarized continuum) and bond distances of the metal coordination
environments (Å). Bottom: Active magnetic orbitals of [Cu2L(μ-OH)]3+ obtained with CASSCF(2,2)
calculations.
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The Cu···Cu distance in [Cu2L(μ-OH)]3+ is of the order of that found in the two dinuclear copper(II)
μ-OH complexes of macrocyclic ligands,20 and practically identical to the -hydroxo-dicopper(II)
polyazacryptate21 already studied. In the former structures involving macrocyclic ligands the Cu···Cu
distance are 3.284 Å20a and 3.642 Å,20b and the copper(II) centres are in approximate square-planar
20a
geometry. However, the Cu···O···Cu angles of around 132o
and 143o 20b, respectively, are
considerably smaller. Only one structure of a dinuclear copper(II) complex with polyazacryptand is
available in the literature.24 In this -hydroxo-dicopper(II) cryptate the copper centres were found to
have trigonal-bipyramidal geometry, with a Cu···Cu separation of 3.9 Å and an almost linear Cu–OH–
Cu motif (174.0º). Additionally, an effective antiferromagnetic interaction Jiso = 865 cm-1 was found
(JisoS1•S2, Jiso > 0 indicates antiferromagnetic interaction), due to the collinear disposition of bridge O
2pz and copper(II) magnetic orbitals that makes this hydroxo-bridged derivative diamagnetic.24
Complete active space self-consistent field (CASSCF) calculations were conducted to assess the
relative energies of the triplet and singlet states of [Cu2L(μ-OH)]3+. Our CASSCF calculations
considered the minimal active space (CASSCF(2,2)), where the two unpaired electrons occupy the two
magnetic orbitals.25 Inspection of the magnetically active orbitals (Fig. 4) shows that they correspond to
2
2
the symmetric and antisymmetric combinations of the Cu 3dx -y orbitals with tails on the bridging
hydroxide ligand and the nitrogen atoms of the macrocycle. At the CASSCF(2,2) level the [Cu2L(μ-
OH)]3+ complex presents antiferromagnetic coupling, as the singlet state is the ground state, with the
triplet state having an energy of 39.7 cm-1 with respect to the former. The inclusion of dynamic
correlation using the NEVPT2 method (see computational details bellow) increases this energy
difference to 66.1 cm-1. These results are in qualitative agreement with the EPR studies described
below.
The isotropic coupling constant characterizing the magnetic interaction in [Cu2L(µ-OH)]3+ was
further investigated using the DFT studies with the broken-symmetry approach. We found that
B3LYP/6-311G(d) calculations provide a calculated exchange coupling constant (Jiso = 898 cm-1) in
excellent agreement with the experimental value (Jiso = 865 ± 50 cm-1)24b for the cryptate reported by
Nelson (Table S6 in the ESI).24 The same computational approach gives an isotropic coupling constant
of Jiso = 443 cm-1 (JisoS1•S2, Jiso > 0 means anti-ferromagnetic coupling) for [Cu2L(µ-OH)]3+.
EPR Studies
In an effort to gain insight into the structures of the dinuclear copper(II) complexes and the
corresponding phosphonate cascade complexes in solution, EPR spectroscopic measurements were
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carried out in H2O:MeOH (50:50 v/v) frozen solutions at 110–135 K. Spectra of the dinuclear copper
2–
complexes of L were recorded at two different pH values, and the cascade complexes of PhPO4 and
HpST12– at the pH corresponding to the maximum percentage of the cascade species, which is of ~ 6.0
(see Fig. S3 in the ESI).
Several of the X-band EPR spectra collected are typical of dinuclear copper(II) complexes,
corresponding to the triplet state of a coupled dinuclear copper site with a total spin S = 1, see Figs. 5
and 6. Two different types of signals are observed: intense signals at around 3000 G (g ~ 2)
corresponding to ΔMs = ±1 transitions; and weak signals at ca. 1600 G (g ~ 4 region) from the ΔMs = 2
transition.26 In our case, only at certain conditions, very faint and lacking hyperfine splitting signals
were detected at the latter region (not shown). In some of our spectra (traces C in Figs. 5 and 6b), the
signal at g ~ 2 presents a well-resolved seven-line hyperfine pattern due to the interaction of both
copper nuclei (nuclear spin ICu = 3/2, 2ICu + 1 = 7).26
Fig. 5 X-band EPR spectra of (A) 1.94 mM Cu(NO3)2, (B) Cu2+:L (2:1) at pH 4.5 with CL = 5.04 mM,
(B-A) is the spectrum that results after subtracting spectrum A to spectrum B, and (C) Cu2+:L (2:1) at
pH 7.0 with CL = 4.39 mM (the signal intensity is multiplied by 5). The thinner line is a computer
simulation with spin Hamiltonian parameters mentioned in the text. All spectra recorded at 135 K, 2.0
mW microwave power, 1.0 mT modulation amplitude, and frequency (υ) of 9.5 GHz. All samples in
H2O:MeOH (50:50 v/v) solution.
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Trace B in Fig. 5 is the EPR spectrum of the Cu2+:L solution (2:1 ratio) at pH 4.5, and consists of
several species. The features in the range 2630–2970 G and the peak at 3280 G indicate the presence of
ca. 19% of Cu(NO3)2 (represented by trace A). Trace B-A is spectrum B after subtracting the
contribution of Cu(NO3)2, and reveals an EPR spectrum that suggests mono- and dinuclear copper(II)
complexes, in agreement with the species distribution diagram in Fig. 1b. Trace C represents the EPR
spectrum for a Cu2+:L (2:1) solution at pH 7.0. Under these conditions only the [Cu2L(µ-OH)]3+
complex is expected to exist in solution (Fig. 1b). Due to its large isotropic coupling constant of Jiso
=
443 cm-1 (JisoS1•S2, Jiso > 0 indicates anti-ferromagnetic coupling) obtained from DFT calculations and
expected for these type of complexes,27 this hydroxo-bridged species is essentially diamagnetic because
the ground state is S = 0 and the triplet state at X-band EPR conditions is not thermally accessible and
thus EPR silent, i.e. one should obtain no signal.24,27 Therefore, the EPR signal in trace C from Fig. 5
corresponds to an unknown species denoted by 1. This species may be attributed to a non-bridged
hydroxo complex, which cannot be distinguished from the bridged species using potentiometry, but the
formation of other species under the conditions used for EPR measurements (i.e. dinuclear species
containing nitrate or water ligands) cannot be ruled out. The EPR spectrum of species 1 could be
simulated28 assuming Jiso = 2.0 cm-1 (so that the triplet state is thermally accessible) and the parameters
listed in Table 4. According to these simulations species 1 represents about 46% of all possible
dicopper species present in solution. Therefore, about 54 % of all possible dicopper species in solution
are EPR silent. Since the hydroxo-bridged species should be EPR silent one can infer that 54 %
corresponds to [Cu2L(µ-OH)]3+. The relatively long Cu···Cu distance obtained for species 1 from EPR
spectral simulation (6.63(2) Å, Table 4) points to binuclear species lacking bridging ligands. Additional
evidence that the EPR signal tends to vanish at higher pH is provided by the evolution of the EPR
spectra of Cu2+:L (2:1) with pH (Fig. S5 in the ESI).
In the Fig. 6a is shown the X-band EPR spectrum of the cascade dicopper(II) complex of L with
2–
PhPO4 obtained at pH 5.8 and 110 K. The thinner trace is a computer simulation28 using the
parameters given in Table 4 for [Cu2L(µ-PhPO4)]2+, which represents about 95 % of all possible
dicopper complexes present in solution. Although the crystal structure shows two complexes with
different coordination environments at one of the copper sites, the EPR spectra suggest that in frozen
solution only one species is present. It is also worthwhile to note that the values for Jiso and Cu···Cu
distance obtained from the EPR simulation (-17 cm-1 and 5.45 Å, Table 4) are very similar to the ones
obtained from DFT calculations (B3LYP/6-311G(d)) for both structures: -18.7 cm-1 and 5.56 Å for
[Cu2L(µ-PhPO4)]2+ and -16.8 cm-1 and 5.66 Å for [Cu2L(µ-PhPO4)(NO3)]+. Thus, the analysis of the
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2-
EPR data provide solid evidence for the formation of dinuclear copper(II) species containing a PhPO4
anion bridging both metals.
Table 4 X-band EPR spectroscopic data for the copper(II) complexes in solution determined by
computer simulation.28
Parameter
Species 1
[Cu2L(µ-PhPO4)]2+
2.08(2)
[Cu2L(µ-HpST1)]2+
2.06(2)
gx1 = gx2
2.02(5)
gy2 = gy2
gz2 = gz2
2.09(4)
2.25(3)
38(2)
2.08(5)
2.24(3)
48(2)
2.08(5)
2.27(3)
45(2)
a
Ax1 = Ax2
Ay1 = Ay2
Az1 = Az2
a
39(2)
43(2)
41(2)
a
156(4)
2.0
155(4)
-17(1)
5.45(2)
~ 34
165(4)
-16(1)
5.94(6)
~ 35
Jiso (cm-1)
Cu···Cu (Å)
θ (°) b
6.63(2)
~ 42
a Values of A in 10-4 cm-1. b θ is the angle between the inter-copper coupling vector and the gz directions.
The cascade species formed in solution upon addition of HpST12– at pH 5.6 provide an EPR
spectrum at 127 K that can be simulated28 assuming the formation of three species (Fig. 6b): species 1
(trace A, 0.25 mM), Cu(NO3)2 (trace B, 0.62 mM) and [Cu2L(µ-HpST1)]2+ (trace C, 1.44 mM).
Therefore, ca. 85% of the binuclear copper(II) species correspond to the cascade complex of HpST12-
with a bridging phosphate moiety and the rest can be assigned to species 1. The similarity of all
parameters obtained from EPR simulated spectra for both cascade complexes support the idea that in
both cases the PhPO42– group is well accommodated between the Cu sites.
2–
The simulated EPR spectrum of the cascade complex species formed with PhPO4 indicates a
Cu···Cu separation of 5.45 Å, which is close to the 5.81 Å found in the corresponding structure
2–
determined by X-ray diffraction pointing to a very similar structure in solution with PhPO4 anion
bridging the two copper centres. The spectra with the studied peptides are both very similar and also
similar to the one with PhPO42– (see Fig. 6a and Table 4), pointing also to similar structures in solution.
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(a)
(b)
2–
Fig. 6 X-band EPR spectra for: (a) solution of the dicopper(II) cascade complex of L with PhPO4 at
pH 5.8 and 110 K for Cu2+:L (2:1) with CL = 2.0 mM, and PhPO42- concentration of 4 mM. The thinner
line is obtained by computer simulation using parameters in Table 4 for [Cu2L(µ-PhPO4)]2+ and
corresponds to ca. 1.9 mM. (b) Thicker trace D corresponds to the cascade complex solution with
HpST12– at pH 5.6 and 127 K for Cu2+:L (2:1) (2.0 mM) with CL = 2.0 mM. The thinner trace D is the
sum of three species represented by traces A to C obtained by computer simulation with parameters on
Table 4. Trace A represents 0.25 mM of 1, trace B represents 0.62 mM of Cu(NO3)2 and 1.44 mM of
[Cu2L(µ-HpST1)]2+ corresponds to trace C. All spectra recorded at 2.0 mW microwave power, 1.0 mT
modulation amplitude, and frequency (υ) of 9.5 GHz. All samples in H2O:MeOH (50:50 v/v) solution.
CONCLUSIONS
A new triethylbenzene-derived tetraazamacrocycle containing pyridyl spacers was prepared in good
yield by a “one-pot” procedure. The corresponding dinuclear copper(II) complexes were studied as
receptors for the recognition of phosphorylated peptides in aqueous solution.
Single-crystal X-ray diffraction determination of the dicopper(II) complex of L showed the copper
2–
centres at a distance of 5.81 Å from one another, with the phosphate group of the PhPO4 substrate
well accommodated between them. A similar structure for this cascade complex, and for the other
cascade complexes with all the phosphorylated anions studied, is found in solution by EPR studies.
DFT studies of the [Cu2L(μ-OH)]3+ complex revealed a conformation of the ligand that brings the
two copper centres at a very short distance of 3.94 Å aided by the presence of a bridging hydroxide
anion that provides a Cu···O···Cu angle of 167.3º. This complex is diamagnetic, as proved by EPR
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experiments and CASSCF calculations, and it coexists in solution with a complex in a different
conformation, in lower percentage, with a Cu···Cu distance of 6.63 Å.
It was found that the receptor forms very stable associations with the several protonated forms of the
phosphorylated peptides, with constant values ranging 3.96–5.35 log units, with the affinity of the
receptor towards the H3pST1 peptide being slightly higher than towards the H2pST3 one, in
water:methanol 50:50 v/v. These promising results suggest the potential use of this complex as a binder
of phosphorylated STAT proteins upon slight modifications of the ligand in order to become it soluble
in water. Such studies will be described in due course.
EXPERIMENTAL SECTION
General considerations. All solvents and reagents used were purchased from commercial sources,
had reagent grade quality and were used as supplied without further purification, except 2,6-
pyridinedicarbaldehyde, which was prepared according to literature methods.29 The H2pST3 peptide
was obtained commercially through CASLO ApS (mass spectrometry certificates can be found in Fig.
S6, and NMR spectra made by us in Figs. S7–S8 in the ESI).10 All NMR spectra were recorded on a
13
Bruker Avance III 400 (1H at 400.13 MHz and C at 100.61 MHz). TMS was used as a reference for
1
the H NMR measurements in CDCl3. Peak assignments were based in peak integration and
multiplicity for 1D 1H spectra, and 2D COSY, NOESY, HMQC and HMBC experiments.
Microanalyses were carried out by the ITQB Microanalytical Service. Mass spectra have been acquired
in the positive mode, after direct injection of the sample solutions with a syringe pump into a Bruker
Daltonics Esquire 3000plus mass spectrometer equipped with an ESI source.
Caution! Although no problems were encountered during this work with the organic and inorganic
azides, these compounds should be considered potentially explosive.
Syntheses
Synthesis of the peptide H3pST1. The H3pST1 peptide (Scheme 1) was synthesized by using Fmoc
chemistry.18 The peptide was assembled on a rink amide MBHA (4-methylbenzhydrylamine) resin
using
the
HBTU–HOBt–DIEA
[N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate (1-hydroxybenzotriazole) – N,N-diisopropylethylamine] activation method and
was acetylated in the N-terminal using 10% acetic anhydride in DMF. Cleavage and removal of the
protecting groups were simultaneously performed by treatment with a mixture of TFA–TIS–water
(95:2.5:2.5 % v/v) for 2 h at r.t. and under nitrogen. The solution was filtered to remove the resin and
then evaporated under a nitrogen stream. Cold diethyl ether was added to precipitate the crude peptide
which was purified by preparative reversed-phase HPLC in a Phenomenex Jupiter Proteo column
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(250×21.20 mm, 4 μm, 90 Å) using solvent A (99.9% H2O–0.1% TFA) and solvent B (90% CH3CN–
9.9% H2O–0.1% TFA) mixtures. H3pST1 was eluted from the column with a linear gradient from 0 to
25 % B in 12 min at a flow rate of 10 mL min−1 (Rt = 18 min). The purity of the peptide was checked
by analytical reversed-phase HPLC (Phenomenex Jupiter Proteo column, 250×4.6 mm, 4 μm, 90 Å)
and it was higher than 95%. The peptide H3pST1 was obtained as a white fluffy solid (0.200 g) with a
yield of 19%. 1H NMR (400 MHz, D2O) δ 0.81–0.87 (6 H, m, 2×CH3 Ile), 1.12–1.20 (1 H, m, γ-CHH
Ile), 1.19 (3 H, d, J = 6.9 Hz, CH3 Thr), 1.29–1.48 (5 H, m, 2×CH2 Lys + γ-CHH Ile), 1.73–1.82 (3 H,
m, CH2 Lys + β-CH Ile), 1.89–1.95 (1 H, m, β-CHH Glu), 1.93 (3 H, s, CH3CO), 2.03–2.08 (1 H, m, β-
CHH Glu), 2.21–2.26 (2 H, m, γ-CH2 Glu), 2.57 (2 H, t, J = 6.7 Hz, ε-CH2 Lys), 2.85–3.07 (2 H, m, β-
CH2 Tyr), 4.13–4.20 (2 H, m, β-CH Thr + α-H Glu), 4.24–4.28 (2 H, m, α-H Thr + α-H Ile), 4.33–4.37
(1 H, m, α-H Lys), 4.51–4.55 (1 H, m, α-H Tyr), 7.13 (4 H, q, J = 8.7Hz, 4×H-bz) ppm. 13C NMR (101
MHz, D2O) δ 9.81 (CH3 Ile), 14.67 (CH3 Ile), 18.74 (γ-CH2 Ile), 18.82 (CH3 Thr), 21.52 (CH3CO),
22.39 (CH2 Lys), 30.74 (CH2 Lys), 31.32 (CH2 Lys), 33.60 (γ-CH2 Glu), 34.00 (β-CH2 Glu), 35.98 (β-
CH Ile), 36.18 (β-CH2 Tyr), 40.29 (ε-CH2 Lys), 53.53 (α-C Ile), 53.82 (α-C Lys), 55.14 (α-C Tyr),
55.26 (α-C Glu), 57.89 (α-C Thr), 66.92 (β-CH Thr), 120.40 (C3/C5-bz), 129.74 (C2/C6-bz), 130.04
(C1-bz), 153.02 (C4-bz), 170.94 (CH3CO), 171.81 (C=O Ile), 173.06 (C=O Tyr), 173.33 (C=O Thr),
174.03 (C=O Glu), 181.37 (C=O Lys), 182.08 (CO2H Glu) ppm. ESI−MS (MeOH) m/z 398.6 [M + H
+ Na]2+; 774.2 [M + H]+; 796.1 [M + Na]+, see Figs. S9–S13 in the ESI.
Syntheses of L and intermediate compounds
2,4-bis(bromomethyl)-1,3,5-triethylbenzene.30 To a solution of 1,3,5-triethylbenzene (9.41 mL, 0.05
mol), paraformaldehyde (4.50 g, 0.15 mol) and glacial acetic acid (25 mL) were added 3 equiv. of
HBr/AcOH 33% (26 mL) under stirring. The solution was stirred at 80 ºC under nitrogen for 8 h.
Afterwards, the solution was poured into distilled water (50 mL) and the precipitate formed was filtered
off. The solid residue was transferred to a separation funnel with water (50 mL) and extracted with
CHCl3 (3×40 mL). The organic layer was dried with anhydrous Na2SO4, filtered and evaporated to
dryness. The product was purified by column chromatography in silica gel using n-hexane as eluent to
1
give a white powder. Yield: 13.4 g, 77%. H NMR (400 MHz, CDCl3) δ (ppm): 1.28–1.36 (9 H, m,
CH3 C1,5-ethyl and C3-ethyl), 2.77 (4 H, q, J = 7.5 Hz, CH2 C1,5-ethyl), 2.94 (2 H, q, J = 7.7 Hz, CH2
C3-ethyl), 4.61 (4 H, s, CH2Br), 6.98 (1 H, s, H6 bz), see Fig. S14 in the ESI.
2,4-bis(azidomethyl)-1,3,5-triethylbenzene. To
a
solution of 2,4-bis(bromomethyl)-1,3,5-
triethylbenzene (4.25 g, 12.2 mmol) in DMF (125 mL) were added small portions of NaN3 (3.17 g,
48.8 mmol) during 30 min under stirring. The solution was stirred under nitrogen for 24 h. The NaBr
that precipitated was filtered through Celite and the solution was evaporated to dryness. The residue
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was dissolved in CH2Cl2 (100 mL), transferred to a separation funnel and washed with water (2×100
mL) and with a NaCl saturated solution (100 mL). The organic layer was dried with anhydrous
1
Na2SO4, filtered and evaporated to dryness to give an orange oil. Yield: 3.14 g, 94%. H NMR (400
MHz, CDCl3) δ (ppm): 1.21–1.29 (9 H, m, CH3 C1,5-ethyl and C3-ethyl), 2.74 (4 H, q, J = 7.4 Hz, CH2
13
C1,5-ethyl), 2.84 (2 H, q, J = 7.7 Hz, CH2 C3-ethyl), 4.46 (4 H, s, CH2N3), 7.05 (1 H, s, H6 bz). C
NMR (100 MHz, CDCl3) δ (ppm): 15.66 (CH3 C1,5-ethyl), 16.12 (CH3 C3-ethyl), 22.87 (CH2 C3-ethyl),
26.47 (CH2 C1,5-ethyl), 47.77 (CH2N3), 127.77 (C6), 128.78 (C2 and C4), 143.89 (C3), 144.78 (C1 and
C5), see Figs. S15–S20 in the ESI.
2,4-bis(aminomethyl)-1,3,5-triethylbenzene. To a round bottom flask with 2,4-bis(azidomethyl)-
1,3,5-triethylbenzene (3.14 g, 11.5 mmol) were added small portions of triphenylphosphine (12.11 g,
46.2 mmol) followed by a 10:1 THF/H2O solution (29 mL). The mixture was stirred under nitrogen for
24 h and then it was evaporated to dryness. The residue was dissolved in CH2Cl2 (20 mL), transferred
to a separating funnel and washed with a 1 M HCl solution (3×20 mL). The aqueous phases were
combined, extracted with ethyl acetate (4×25 mL) and 3 M NaOH was added until pH 12. After that,
the aqueous phase was extracted with CHCl3 (3×40 mL). The organic layer was dried with anhydrous
1
Na2SO4, filtered and evaporated to dryness to give an orange oil. Yield: 1.84 g, 73%. H NMR (400
MHz, CDCl3) δ (ppm): 1.21–1.27 (9 H, m, CH3 C4,6-ethyl and C2-ethyl), 2.71 (4 H, q, J = 7.4 Hz, CH2
13
C4,6-ethyl), 2.85 (2 H, q, J = 7.7 Hz, CH2 C2-ethyl), 3.87 (4 H, s, CH2NH2), 6.95 (1 H, s, H5 bz). C
NMR (100 MHz, CDCl3) δ (ppm): 16.27 (CH3 C4,6-ethyl), 17.11 (CH3 C2-ethyl), 22.49 (CH2 C2-ethyl),
26.12 (CH2 C4,6-ethyl), 39.49 (CH2NH2), 127.71 (C5), 136.78 (C1 and C3), 141.01 (C2), 141.68 (C4
and C6), see Figs. S21–26 in the ESI.
L. To a solution of 2,4-bis(aminomethyl)-1,3,5-triethylbenzene (0.80 g, 3.7 mmol) in CH2Cl2 (71 mL)
and MeOH (35 mL) was added 2,6-pyridinedicarboxaldehyde (0.50 g, 3.7 mmol) under stirring. The
solution was left overnight with stirring and under nitrogen. The solution was evaporated to dryness at
r.t. and MeCN was added (60 mL). The precipitate formed was isolated by filtration and washed with
MeCN (2×40 mL) (0.89 g, 77%).1H NMR (400MHz, CDCl3) δ (ppm): 1.18–1.22 (18 H, m, CH3 C4,6-
ethyl and C2-ethyl), 2.30 (4 H, q, J = 7.0 Hz, CH2 C2-ethyl), 2.50 (8 H, m, CH2 C4,6-ethyl), 5.09 (8 H, s,
bz-CH2N), 7.05 (2 H, s, H5 bz), 7.77 (2 H, t, J = 7.85 Hz, H4 pyr), 7.83 (4 H, s, pyr-CHN), 8.12 (4 H,
d, J = 7.83 Hz, H3 and H5 pyr), see Fig. S27 in the ESI. The precipitate (0.89 g, 1.4 mmol) was then
dissolved in MeOH (80 mL) and solid NaBH4 (0.42 g, 11.1 mmol) was added in small portions to avoid
excessive foaming. Upon this addition, the mixture was left under stirring at r.t. until the bubbling
ceased, and then refluxed for 4 h. The solution was filtered and evaporated under vacuum almost to
dryness, then water was added (20 mL) and the remaining MeOH was evaporated. Afterwards, the
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solution was made strongly basic with 6 M KOH and extracted with CHCl3 (3×50 mL). The organic
layer was dried with anhydrous Na2SO4, filtered and evaporated to give a white powder. Yield: 0.83 g
(70%). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.05 (6 H, t, J = 7.52 Hz, CH3 C2-ethyl), 1.23 (12 H, t, J
= 7.45 Hz, CH3 C4,6-ethyl), 1.88 (4 H, s, NH), 2.72–2.83 (12 H, m, CH2 C4,6-ethyl and C2-ethyl), 3.72
(8 H, s, bz-CH2N), 3.97 (8 H, s, pyr-CH2N), 6.92 (2 H, s, H5 bz), 7.12 (4 H, d, J = 7.30 Hz, H3 and H5
13
pyr), 7.58 (2 H, t, J = 7.73 Hz, H4 pyr). C NMR (100MHz, CDCl3) δ (ppm): 15.99 (CH3 C4,6-ethyl),
17.45 (CH3 C2-ethyl), 22.45 (CH2 C2-ethyl), 26.19 (CH2 C4,6-ethyl), 46.78 (bz-CH2N), 55.74 (pyr-
CH2N), 121.03 (C3 and C5 pyr), 127.28 (C5 bz), 133.49 (C1 and C3 bz), 136.76 (C4 pyr), 142.07 (C2
pyr), 142.86 (C4 and C6 bz), 159.34 (C2 and C4 pyr). Elem. Anal. found: C, 73.00; H, 8.17; N, 12.33.
Calc. for C42H58N6·0.4CHCl3: C, 73.31; H, 8.17; N, 12.10%; ESI−MS (MeOH) m/z 647.5 [M + H]+,
see Figs. S28–S34 in the ESI.
Synthesis of dicopper(II) complexes. The ligand L (2.0 mmol) and the Cu(NO3)2 (4 mmol), from
standard titrated solutions, were dissolved in MeOH:H2O (50:50, 10.0 mL) and 0.10 M KOH was
added to adjust the pH value to 6.5. The solution was stirred overnight. ESI−MS (H2O/MeOH, pH =
6.5) m/z 913.3 [Cu2L(OH)(NO3)2]+; 385.2 [Cu2L-2H]2+ (see Fig. S35 in the ESI). The UV-vis of the
complex at pH 7.0 is shown at Fig. S37 (left) in the ESI.
Synthesis of [Cu2L(μ-PhPO4)][Cu2L(μ-PhPO4)(NO3)]3NO3·24H2O. The ligand L (3.3 mg, 4 μmol)
was dissolved in MeOH:H2O (50:50, 4 mL) and 0.002 M Cu(NO3)2 (8 μmol), and 0.001 M
phenylphosphoric acid (4 μmol) solutions were added. The pH was adjusted to 5.1 with KOH solution.
The solution was allowed to slowly evaporate at r.t. Dark blue single crystals suitable for X-ray
crystallographic determination were obtained within 7 days. ESI−MS (MeOH) m/z 472.1
[Cu2L(PhPO4)]2+; 385.2 [Cu2L- 2H]2+ (see Fig. S36 in the ESI). The UV-vis of this cascade complex at
–
pH 5.8 and Cu2+:L:HPhPO4 2:1:1 ratio is shown at Fig. S37 (right) in the ESI.
Potentiometric measurements. Reagents and solutions. All the solutions were prepared in a
H2O/MeOH (50:50 v/v) mixed solvent. A stock solution of L was prepared at ca. 2.010–3 M with
0.0225 M HNO3 maintaining the 50:50 proportion of water/methanol. Phosphate and phenyl phosphate
solutions were prepared from the corresponding acids. A stock solution of Cu(NO3)2 (analytical grade)
was prepared at about 2.5×10–2 M and the exact concentrations checked by titration with K2H2edta
following standard methods.31 Carbonate-free solutions of the KOH titrant were prepared from a Merck
ampoule diluted to 500 mL of water (freshly boiled for about 2 h and allowed to cool under nitrogen) to
which 500 mL of MeOH were added. These solutions were discarded every time carbonate
concentration was about 0.5% of the total amount of base. The titrant solutions were standardized by
Gran’s method.32
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Equipment and working conditions. The equipment used was described before.10,33 A Metrohm
6.0123.100 glass electrode and a Metrohm 6.0733.100 Ag/AgCl reference electrode were used for the
measurements. The ionic strength of the experimental solutions was kept at 0.10±0.01 M with KNO3,
the temperature was maintained at 298.2±0.1 K. Atmospheric CO2 was excluded from the titration cell
during experiments by passing purified nitrogen across the top of the experimental solution.
Measurements. The [H+] of the solutions was determined by the measurement of the electromotive
force of the cell, E = Eºʹ + Q×log [H+] + Ej. The term pH is defined as –log [H+]. Eºʹ, Q, Ej and Kw were
determined by titration of a solution of known hydrogen-ion concentration at the same ionic strength,
using the acid pH range of the titration. The liquid-junction potential, Ej, was found to be negligible
under the experimental conditions used. The value of Kw was determined from data obtained in the
alkaline range of the titration, considering Eºʹ and Q valid for the entire pH range and found to be equal
to 10–13.86 in our experimental conditions. Before and after each set of titrations the glass electrode was
calibrated as a [H+] probe by titration of a 1.00010–3 M standard HNO3 solution with standard KOH.
Every measurement was carried out with 0.040 mmol of ligand L in a total volume of 40.00 mL, except
for the measurements involving peptides (H2pST3 and H3pST1), where 0.010 mmol of the peptide in a
total volume of 15.00 mL was used. The exact concentration of L and of the peptidic substrates was
obtained by titration with a standard KOH solution. Copper(II) complexation experiments were
performed in the presence of Cu(NO3)2 in 1:1 and 2:1 CCu:CL ratios. The ternary systems measurements
were carried out in the simultaneous presence of L, Cu(NO3)2 and each anion at 2:1:1 M:L:S ratios (L =
ligand, M = metal and S = substrate). In each titration 85 to 120 points were collected, and a minimum
of two titration curves were performed. All the substrates were independently titrated alone and in the
presence of copper(II) ion at 0.5:1, 1:1 and 2:1 CCu/CS ratios and the respective equilibrium constants
used in the calculations. Backtitrations with standard HNO3 solution were performed to confirm the
values of the final Eºʹ readings.
Calculation of equilibrium constants. Overall protonation constants, βiH, of the free ligand L and of the
studied substrates, the overall stability constants of complexes, βM
and the overall association
H L
m h l
constants of the complexes of L with the substrates, βM
were calculated by fitting the
,
H L S
m h l s
potentiometric data obtained for all the performed titrations in the same experimental conditions with
the Hyperquad program.34 The hydrolysis constants for copper(II) were held constant during data
refinement. The initial computations were obtained in the form of overall constants, βH L
=
h
[HhLl]/[H]h[L]l, βM
= [MmHhLl]/[M]m[H]h[L]l or βM
= [MmHhLlSs]/[M]m[H]h[L]l[S]s. The
H L S
h l s
H L
m h l
m
errors quoted are the standard deviations of the overall constants given directly by the program for the
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input data, which include all the experimental points of all titration curves. The errors quoted for the
stepwise constant values were calculated using the propagation rules. The HySS program35 was used to
calculate the concentration of equilibrium species from the calculated constants from which distribution
diagrams were plotted. The species considered in a particular model were those that could be justified
by the principles of coordination and supramolecular chemistry.
EPR Spectroscopy. The X-band EPR spectra were recorded in perpendicular-mode with a Bruker
EMX 8/2.7 spectrometer equipped with continuous-flow cryostat for liquid nitrogen. EPR spectra
simulations and species concentrations were determined with the software SpinCount developed by M.
P. Hendrich.28 EPR signals were quantified by relative to a 5 mM Cu(NO3)2 in H2O:MeOH (50:50 v/v)
solution. The spin Hamiltonian used for computer simulations is as follows,
H = βB•g1•S1 + βB•g2•S2 + S1•A1•I1 + S2•A2•I2 + S1•J•S2
where 1 and 2 denote the two copper ions (S1 = S2 = ½). The first two terms represent the electronic
Zeeman interactions, and the third and fourth terms the hyperfine interactions. The term S1•J•S2
contains both the isotropic (exchange, JisoS1•S2, Jiso > 0 means anti-ferromagnetic coupling) and
anisotropic (dipolar) spin-spin interactions. This term, is calculated from values of Jiso, the inter-copper
distances (Cu···Cu ), and the angles describing the relative orientations of the two g tensors and the
inter-copper vector.36,37
EPR samples were prepared in H2O/MeOH (50:50 v/v) using stock solutions of L (8.9×10−3 M),
Cu(NO3)2 (5.0×10−2 M), H2PhPO4 (5.0×10−2 M) and H3pST1 (2.3×10−3 M). The copper(II) complexes
and cascade complex solutions were prepared from the stock solutions at the different pH by addition
of standard KOH solutions (0.1 and 0.01 M). Sample concentrations are mentioned in the text.
Single Crystal X-ray Diffraction. Crystals of [Cu2L(μ-PhPO4)][Cu2L(μ-PhPO4)(NO3)]3NO3·24H2O
suitable for X-ray diffraction study were mounted with Fomblin© in a cryoloop. Data were collected
on a Bruker AXS-KAPPA APEX II diffractometer with graphite-monochromated radiation (MoK, 
= 0.71073 Å) at 150 K. The X-ray generator was operated at 50 kV and 30 mA and the X-ray data
collection was monitored by the APEX238 program. All data were corrected for Lorentzian,
polarization and absorption effects using the SAINT38 and SADABS38 programs. SIR9739 and
SHELXS-9740 were used for structure solution and SHELXL-9740 for full matrix least-squares
refinement on F2. These three programs are included in the package of programs WINGX-Version
2014.1.41 The crystals quality and their diffraction were very poor. Even though many crystals were
tested, the presented data set are the best data possible for these crystals. The low ratio of observed
reflections is due to very weak crystals. Yet, the anisotropic refinement was possible. Non-hydrogen
atoms were refined anisotropically. A full-matrix least-squares refinement was used for the non-
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hydrogen atoms with anisotropic thermal parameters. All the hydrogen atoms were inserted in idealized
positions and allowed to refine in the parent carbon atom. There is one disordered nitrate in the
structure, for which a model was determined (to better represent this model, coordinates do not form a
properly connected set - alert B at checkcif). Hydrogen atoms of water molecules have not been added
as they were not possible to locate from the electron density map. Furthermore, some water molecules
are disordered but it was not possible to accurately model that disorder, what is reflected in the checkcif
generating alerts B regarding short distances between atoms.
43
Molecular diagrams presented are drawn with PyMOL.42 PLATON was used to calculate bond
distances and angles as well as hydrogen bond interactions. In Table 6 the data collection and
refinement details are summarized. CCDC 1499385 contains the supplementary crystallographic data
for this article.
Table 6 Crystallographic data and structure refinement details for [Cu2L(μ-
PhPO4)][Cu2L(μ-PhPO4)(NO3)]3NO3·24H2O
chemical formula
formula weight
C96H126O44Cu4N16P2
2524.22
temperature, K
150(2)
wavelength, Å
0.71073
crystal form, colour
crystal size, mm
crystal system
Block, blue
0.170.070.03
Triclinic
space group
P-1
a, Å
b, Å
c, Å
 ()
12.6536(6)
18.7909(9)
26.3285(12)
81.480(2)
79.723(2)
88.933(2)
6091.8(5)
2
 ()
 ()
V, Å3
Z
F(000)
2624
1.376
0.803
d (mg.cm–3)
µ (mm–1)
1.941 – 25.242
24795/11252
0.1095
 range ()
reflections collected/unique
Rint
GoF
1.048
R1= 0.0926,a wR2 = 0.2086b
Final R indices [I >2(I)]
2 2
a R1 = ||Fo| – |Fc||/|Fo|. b wR2 = [[w(Fo2 – Fc2)2]/[w(Fo ) ]]1/2
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Computational methods. Full geometry optimizations of the [Cu2L(OH)]3+ and [Cu2L(µ-PhPO4)]2+
complexes were performed in aqueous solution employing DFT calculations within the hybrid meta-
GGA approximation with the M06 exchange-correlation functional44 and the Gaussian 09 package
(Revision D.01).45 In these calculations we used the standard Ahlrichs’ split valence (SVP)46 and
triple- (TZVP)47 basis sets including polarization functions. Both basis sets provide very similar
optimized structures. Solvent effects (water) were included by using the polarizable continuum model
(PCM). In particular we employed the integral equation formalism (IEFPCM) variant as implemented
in Gaussian 09.48 No symmetry constraints have been imposed during the optimizations. The nature of
the optimized geometries as true energy minima was confirmed by using frequency analysis (0
imaginary frequencies). The default values for the integration grid (75 radial shells and 302 angular
points) and the SCF energy convergence criteria (10-8) were used in all calculations.
Nonrelativistic energy levels and wave functions were computed using the Complete Active Space
Self-Consistent Field (CASSCF) method49 and the ORCA program package (Version 3.0.3)50 along
with the TZVP basis set. CASSCF calculations were performed by using an active space including two
electrons distributed into the two magnetically active Cu 3d-based molecular orbitals (CASSCF(2,2)).
The RIJCOSX approximation51 was used to speed up the calculations using the Def2-TZVPP/J
auxiliary basis set as constructed automatically by ORCA. The CASSCF wavefunctions were
subsequently analyzed using N-electron valence perturbation theory to second order (NEVPT2).52
Solvent effects (water) were taken into account by using the conductor-like screening model
(COSMO)53 as implemented in ORCA.
DFT calculations were employed to estimate isotropic exchange coupling constants using the
broken-symmetry approach originally introduced by Noodleman.54 Since the value for the exchange
coupling constants for the various complexes studied have a wide range, it is advised to use a
modification of Noodleman’s approach that was carry out by Yamaguchi and co-workers,55 who
obtained
Jiso = 2 (ESCF(HS) – ESCF(BS))/(<S2>HS – <S2>BS)
where <S2>HS, <S2>BS and ESCF(HS), ESCF(BS) are expectation values of the spin-squared operator
determinants and the energies for the HS and BS, respectively. The Yamaguchi expression is valid for
the whole range of coupling strengths and reduces to the Noodleman equation in the weak coupling
limit.54 In these calculations we tested Becke’s three parameter hybrid functional (B3LYP) besides the
M06 functional, in combination with either TZVP or 6-311G(d) basis sets. Both ORCA and Gaussian
were used for BS calculations, providing virtually identical results. Since the B3LYP/6-311G(d)
method provided the best results for the reference compound reported by Nelson,24b subsequent
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calculations employed this method to estimate the coupling constants of [Cu2L(µ-OH)]3+, [Cu2L(µ-
PhPO4)]2+ and [Cu2L(µ-PhPO4)(NO3)]+, see Tables S7–S11 in the ESI. The ferromagnetic (HS, for
High Spin) and broken-symmetry (BS) optimizations were carried out by utilizing the Gaussian 09
built-in “fragments” module, and an initial guess wavefunction was generated and used as a starting
point for the optimization calculations. The geometry optimizations were terminated upon reaching the
default convergence criteria. The optimizations did not impose any symmetry. The frequencies for the
equilibrium conformation obtained with the FREQ keyword were all positive, indicating the
identification of a minimum.
Conflict of interest
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
The authors acknowledge Fundação para a Ciꢀncia e a Tecnologia (FCT) for the financial support and
the fellowships of L. Mesquita and of R. Fernandes under Project PTDC/QEQ-SUP/2718/2012. The
authors also acknowledge support by FCT under Project PTDC/BIA-PRO/101837/2008, and under
project RECI/BBB-BQB/0230/2012 for the NMR spectrometers as part of the National NMR Facility.
The X-ray facilities thank FCT for funding (RECI/QEQ-QIN/0189/2012). M. C. Almeida from the
ITQB Analytical Services Unit is acknowledged for providing elemental analysis and ESI-MS data. P.
Mateus and V. André acknowledge FCT for the fellowships SFRH/BPD/79518/2011 and
SFRH/BPD/78854/2011, respectively. C. P.-I. thanks Centro de Supercomputación de Galicia for
providing the computer facilities.
REFERENCES
1 G. Manning, D. B. Whyte, R. Martinez, T. Hunter and S. Sudarsanam, Science, 2002, 298, 1912–
1934.
2 a) H. Patterson, R. Nibbs, I. McInnes and S. Siebert, Clin. Exp. Immunol., 2013, 176, 1–10; b) M. J.
Jembrek, M. Babić, N. Pivac, P. R. Hof and G. Šimić, Transl. Neurosci., 2013, 4, 466–476.
3 J. Brognard and T. Hunter, Curr. Opin. Genet. Dev., 2011, 21, 4–11.
4 J. A. Ubersax and J. E. Ferrell Jr, Nat. Rev. Mol. Cell Biol., 2007, 8, 530–541.
5 a) A. Ojida, Y. Miyahara, T. Kohira and I. Hamachi, Biopolymers, 2004, 76, 177–184; b) A. Ojida
and I. Hamachi, Bull. Chem. Soc. Jpn., 2006, 79, 35–46; c) H. T. Ngo, X. Liu and K. A. Jolliffe,
27

Dalton Transactions
Page 28 of 32
View Article Online
DOI: 10.1039/C7DT01724C
Chem. Soc. Rev. 2012, 41, 4928–4965; d) R. Kubota and I. Hamachi, Chem. Soc. Rev., 2015, 44,
4454–4471.
6 A. E. Hargrove, S. Nieto, T. Zhang, J. L. Sessler and E. V. Anslyn, Chem. Rev., 2011, 111, 6603–
6782.
7 a) A. Ojida, Y. Mito-oka, M.-a. Inoue and I. Hamachi, J. Am. Chem. Soc., 2002, 124, 6256–6258; b)
A. Ojida, Y. Mito-oka, M.-a. Inoue and I. Hamachi, J. Am. Chem. Soc. 2003, 125, 10184–10185; c)
A. Ojida, Y. Mito-oka, K. Sada and I. Hamachi, J. Am. Chem. Soc., 2004, 126, 2454–2463; d) A.
Ojida, M.-a. Inoue, Y. Mito-oka, H. Tsutsumi, K. Sada and I. Hamachi, J. Am. Chem. Soc., 2006,
128, 2052–2058; e) A. Grauer, A. Riechers, S. Ritter and B. Konig, Chem. Eur. J., 2008, 14, 8922–
8927; f) T. Sakamoto, M.-a. Inoue, A. Ojida and I. Hamachi, Bioorg. Med. Chem. Lett., 2009, 19,
4175–4177; g) Y. Ishida, M.-a. Inoue, T. Inoue, A. Ojida and I. Hamachi, Chem. Commun., 2009,
2848–2850; h) A. Ojida, T. Sakamoto, M.-a. Inoue, S.-h. Fujishima, G. Lippens and I. Hamachi, J.
Am. Chem. Soc., 2009, 131, 6543–6548; i) J. A. Drewry, S. Fletcher, P. Yue, D. Marushchak, W.
Zhao, S. Sharmeen, X. Zhang, A. D. Schimmer, C. Gradinaru, J. Turkson and P. T. Gunning, Chem.
Commun., 2010, 46, 892–894; j) A. Riechers, A. Grauer, S. Ritter, B. Sperl, T. Berg and B. Konig, J.
Mol. Recognit., 2010, 23, 329–334; k) H. Akiba, J. Sumaoka and M. Komiyama, Chem. Eur. J.,
2010, 16, 5018–5025; l) M. Bhuyan, E. Katayev, S. Stadlbauer, H. Nonaka, A. Ojida, I. Hamachi
and B. König, Eur. J. Org. Chem., 2011, 2807–2817; m) S. Zhang, L. Han, C. Li, J. Wang, W.
Wang, Z. Yuan and X. Gao, Tetrahedron, 2012, 68, 2357–2362; n) J. A. Drewry, E. Duodu, A.
Mazouchi, P. Spagnuolo, S. Burger, C. C. Gradinaru, P. Ayers, A. D. Schimmer and P. T. Gunning,
Inorg. Chem., 2012, 51, 8284−8291; o) J. H. Kang, H. J. Kim, T.-H. Kwon and J.-I. Hong, J. Org.
Chem., 2014, 79, 6000−6005; p) H. Akiba, J. Sumaoka, T. Hamakubo and M. Komiyama, Anal.
Bioanal. Chem., 2014, 406, 2957–2964; q) D. Kraskouskaya, M. Bancerz, H. S. Soor, J. E. Gardiner
and P. T. Gunning, J. Am. Chem. Soc., 2014, 136, 1234−1237.
8 P. Mateus, L. M. P. Lima and R. Delgado, Polyhedron, 2013, 52, 25–42.
9 a) R. J. Motekaitis and A. E. Martell, Inorg. Chem., 1992, 31, 5534–5542; b) R. J. Motekaitis and A.
E. Martell, Inorg. Chem., 1994, 33, 1032–1037; c) P. E. Jurek, A. E. Martell, R. J. Motekaitis and R.
D. Hancock, Inorg. Chem., 1995, 34, 1823–1829; d) Q. Lu, J. J. Reibenspies, A. E. Martell and R. J.
Motekaitis, Inorg. Chem., 1996, 35, 2630–2636; e) D. A. Nation, A. E. Martell, R. I. Carroll and A.
Clearfield, Inorg. Chem., 1996, 35, 7246–7252; f) J. B. English, A. E. Martell, R. J. Motekaitis and
I. Murase, Inorg. Chim. Acta, 1997, 258, 183–192; g) D. A. Nation, Q. Lu and A. E. Martell, Inorg.
Chim. Acta, 1997, 263, 209–217; h) Q. Lu, J. H. Reibenspies, R. I. Carroll, A. E. Martell and A.
Clearfiled, Inorg. Chim. Acta, 1998, 270, 207–215; i) T. F. Pauwels, W. Lippens, G. G. Herman and
28

Page 29 of 32
Dalton Transactions
View Article Online
DOI: 10.1039/C7DT01724C
A. M. Goeminne, Polyhedron, 1998, 17, 1715–1723; j) A. E. Martell, R. J. Motekaitis, Q. Lu and D.
A. Nation, Polyhedron, 1999, 18, 3203–3218; k) L. Fabbrizzi, N. Marcotte, F. Stomeo and A.
Taglietti, Angew. Chem., Int. Ed., 2002, 41, 3811–3814; l) N. Marcotte and A. Taglietti, Supramol.
Chem., 2003, 15, 617–625; m) J. E. Barker, Y. Liu, N. D. Martin and T. Ren, J. Am. Chem. Soc.,
2003, 125, 13332–13333; n) M. A. Saeed, D. R. Powell and M. A. Hossain, Tetrahedron Lett., 2010,
51, 4904–4907; o) V. Amendola, G. Bergamaschi, A. Buttafava, L. Fabbrizzi and E. Monzani, J.
Am. Chem. Soc., 2010, 132, 147–156.
10 (a) H. Akiba, J. Sumaoka, and M. Komiyama, Chem. Eur. J., 2010, 16, 5018–5025; b) J. Sumaoka,
H. Akiba, and M. Komiyama, Int. J. Anal. Chem., vol. 2016, Article ID 3216523, 14 pages.
doi:10.1155/2016/3216523; c) L. M. Mesquita, F. Herrera, C. V. Esteves, P. Lamosa, V. André, P.
Mateus and R. Delgado, Inorg. Chem., 2016, 55, 3589–3598.
11 X. Chen, U. Vinkemeier, Ya. Zhao, D. Jeruzalmi, J. E. Darnell Jr. and J. Kuriyan, Cell, 1998, 93,
827–839.
12 S. Becker, B. Groner and C. W. Müller, Nature, 1998, 394, 145–151.
13 J. E. Darnell Jr., Science, 1997, 277, 1630–1635.
14 L. A. Cabell, M. D. Best, J. J. Lavigne, S. E. Schneider, D. M. Perreault, M.-K. Monahan and E. V.
Anslyn, J. Chem. Soc., Perkin Trans. 2, 2001, 315–323.
15 A. W. van der Made and R. H. van der Made, J. Org. Chem., 1993, 58, 1262–1263.
16 K. J. Wallace, R. Hanes, E. V. Anslyn, J. Morey, K. V. Kilway and J. Siegel, Synthesis, 2005, 2080–
2083.
17 P. Mateus, R. Delgado, P. Brandão and V. Félix, J. Org. Chem., 2009, 74, 8638–8646.
18 Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Eds.: W. C. Chan, P. D. White, Oxford
University Press, New York, 2000.
19 P. Mateus, R. Delgado, F. Lloret, J. Cano, P. Brandão and V. Félix, Chem. Eur. J., 2011, 17, 11193–
11203.
20 a) R. J. Motekaitis, A. E. Martell, J.-M. Lehn and E.-I. Watanabe, Inorg. Chem., 1982, 21, 4253–
4251; b) R. Menif, J. Reibenspies and A. E. Martell, Inorg. Chem., 1991, 30, 3446–3454; c) F.
Arnaud-Neu, S. Fuangswasdi, B. Maubert, J. Nelson and V. McKee, Inorg. Chem., 2000, 39, 573–
579.
21 a) P. L. Burk, J. A. Osborn, M.-T. Youinou, Y. Agnus, R. Louis and R. Weiss, J. Am. Chem. Soc.,
1981, 103, 1273–1274; b) P. K. Coughlin and S. J. Lippard, J. Am. Chem. Soc., 1981, 103, 3228–
3231.
22 I. Sꢁvꢂgꢁ, C. Kꢂllay and K. Vꢂrnagy, Coord. Chem. Rev., 2012, 256, 2225−2233.
29