Coordination behaviour of new open chain and macrocyclic peptidomimetic compounds with copper(ii)

Article abstract of DOI:10.1039/c5ra15852d

Two valine-derived bis(amino amides) ligands have been prepared and fully characterized. Both compounds contain additional functionalities that implement their basicity and their water solubility. Besides, compound 1 is an open chain ligand, while 2 is a macrocycle. Their protonation constants as well as their stability constants for the formation of the corresponding Cu²⁺ complexes have been determined potentiometrically. Important differences are associated to the macrocyclic effect and to the additional functionalities in the spacer. The presence of an additional amine group and/or the inclusion of a carboxylic side chain in this spacer increase the stabilities of the Cu²⁺complexes, suggesting its participation in the interaction with the metal. Thus, 2 is the first pseudopeptidic cyclophane of this family displaying the ability to form highly stable metal complexes in water. UV-Vis and ESI-MS were used for analyzing the complex species detected in the speciation diagram.

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Coordination behaviour of new open chain and macrocyclic
peptidomimetic compounds with copper (II)
Received 00th January 20xx,
Accepted 00th January 20xx
†
Prashant D. Wadhavane, Lingaraju Gorla, Armando Ferrer, Belén Altava, M. Isabel Burguete, M.
Ángeles Izquierdo and Santiago V. Luis*
DOI: 10.1039/x0xx00000x
Two valine-derived bis(amino amides) ligands have been prepared and fully characterized. Both compounds contain
additional functionalities that implement their basicity and their water solubility. Besides, compound 1 is an open chain
ligand, while 2 is a macrocycle. Their protonation constants as well as their stability constants for the formation of the
www.rsc.org/
2
+
corresponding Cu complexes have been determined potentiometrically. Important differences are associated to the
macrocyclic effect and to the additional functionalities in the spacer. The presence of an additional amine group and/or
2
+
the inclusion of a carboxylic side chain in this spacer increase the stabilities of the Cu complexes, suggesting its
participation in the interaction with the metal. Thus, 2 is the first pseudopeptidic cyclophane of this family displaying the
ability to form highly stable metal complexes in water. UV-Vis and ESI-MS were used for analyzing the complex species
detected in the speciation diagram.
coordination number the complexes can adopt structures that
can be described as either square pyramidal (sp) or trigonal
bipyramidal (tbp) depending mainly on the actual nature of the
Introduction
Enzymes are essential for biological processes. In many cases
their activity is associated to the presence of metalloenzymes
in which metal cofactors mediate the corresponding biological
ligand. One illustrative example is that of tripodal tetradentate
1
0
ligands that favour tbp coordination. In this regard, the
design and synthesis of ligands functionalized to achieve metal
complexation in a biomimetic approach is a challenge of
1
transformations. In particular, for enzymatic systems involving
transport of electrons and dioxygen, as well as oxidation and
2
oxygenation processes, the presence of copper is important.
1
1,12
current interest.
Although many model complexes use non amino acid ligands
mimicking various aspects of the ligand environment in
proteins, the development of new ligands based on the
presence of amino acid subunits is a fundamental approach in
The exact properties of those active centers are often related
to the coordination geometry of copper. As a diverse family of
1
d
proteins, copper proteins could be divided into several types.
One classical example is that of Azurins, which catalytic center
1
2,13
1
d,3
this area.
In this context, amino acid derived open-chain
harbors a type 1 copper site. In those systems a carboxylate
group, derived from the side chains of aspartate and
glutamate, is present. The involvement of a carboxyl group in
the coordination environment of the metal center also takes
place, moreover, in binuclear enzymes like the metalloenzyme
and macrocyclic compounds have recently drawn much
15
1
4
attention in very different fields like synthetic, bioorganic,
1
6
17
18
medicinal, supramolecular chemistry, and catalysis, and
recent contributions from our group have shown how
minimalistic pseudopeptides derived from simple natural
4
Zn-phosphorotriesterase.
1
9
amino acids can have important applications, for instance in
The most common geometries reported for copper complexes
6
2
the selective recognition of carboxylic acids and amino acid
0
5
include octahedral, tetrahedral, distorted tetragonal,
2
1
7
distorted trigonal pyramidal or bipyramidal, and square
derivatives,
and for developing new self-assembled
21a,23
2
materials or molecular rotors.
2
8
planar arrangements around the metal center. Copper
2
Previous studies have revealed the capacity of some C -
complexes with open-chain and macrocyclic polydentate
9
amine ligands are frequently pentacoordinate. With this
symmetric open-chain pseudopeptides to form stable metal
2
4
complexes in aqueous solution. However, the same capacity
is absent in most of the related pseudopeptidic cyclophanes
studied because of their higher lipophilic character, decreasing
their water solubility, and the structural strain developed for
a.
Departament of Inorganic and Organic Chemistry, Universitat Jaume I, Campus
del Riu Sec, Av. Sos Baynat, s/n, E-12071, Castellón (Spain). E-mail: luiss@uji.es.
†
Permanent address: Departamento de Química, Facultad de Ciencias Naturales, the coordination of several donor atoms in the macrocycle to
Universidad de Oriente, Avda. Patricio Lumumba s/n., 90500 Santiago de Cuba,
Cuba.
the same metal center. Taking all this into account, the design
Electronic Supplementary Information (ESI) available: Potential protonation and study of macrocyclic pseudopeptides incorporating
equilibria for 2 (S1). H NMR spectra of compound 1 at different pH values (S2),
Mass Spectra (S3) and computational models (S4). See DOI: 10.1039/x0xx00000x
1
carboxylate pendant groups coexisting with amino and amide
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groups is an important target. In such systems, besides the the pendant group is introduced at the central nitrogen atom
inclusion of additional donor groups, the presence of the of the spacer. In this regard, we followed successfully the
carboxylic group can provide an important contribution to the approach recently used for the preparation of related
water solubility of the resulting ligand, allowing its study under derivatives containing fluorescent subunits that can act as
2
6
aqueous conditions of biological relevance.
Here we present our results on the acid-base and coordination chain intermediate
properties of the macrocyclic pseudopeptide containing a in DMF at 50ºC for 24 h, using K
pendant carboxylic group. The structurally related open-chain the ester functionalized pendant arm. Then, deprotection of
pseudopeptide , lacking the carboxylic functionality has also the Cbz group was carried out by hydrogenolysis to provide
been studied for a better analysis of the effect of the the intermediate . Reaction of this compound with 1,3-
bis(bromomethyl)benzene provided the macrocyclic structure
from which, after hydrolysis of the ester group using lithium
hydroxide in a 2:1 THF-water mixture, the desired cyclic
pseudopeptide was obtained in 41% yield. The resulting
markers of acidic organelles within live cells. Thus, the open-
was reacted with methyl-2-bromoacetate
CO as the base, to introduce
6
2
2
3
1
8
carboxylic group and the macrocyclic nature present in 2.
9
Results and discussion
2
pseudopeptide is a highly functional macrocycle with a
relatively large and flexible cavity, which increases its potential
for the interaction with metal cations and its water solubility.
The structure of the ligands (
displayed in Chart 1. The synthesis of compounds
presented in Scheme 1 and follows the general synthetic
1
-
3
) considered in this work is
1
and is
2
2
5
approaches previously developed by us. The synthesis and
study of the open-chain ligand , containing a central spacer of
the same length than but lacking the additional amino group
has been reported elsewhere. The open-chain bis(amino
amide) compound can be easily prepared starting from N-
3
1
2
4
1
Cbz protected valine through the initial formation of the
corresponding activated N-hydroxysuccinimide ester, coupling
with diethylenetriamine and final N-deprotection. The N-Cbz
protected precursor of
a key intermediate for the synthesis of the macrocyclic
compound . Nevertheless this synthetic procedure is more
1 (Cbz-protected compound 6) was also
Chart 1 Bis(amino amide) ligands studied in this work.
2
complex as it requires a proper definition of the step at which
O
NH
NH
N-Hydroxysuccinimide
DCC, THF, rt, 3h
H2 / Pd, C
THF, 50 ºC;3h
Diethylenetriamine
DME, rt, 24h
O
O
NH
NH
O
O
NH
NH
O
OH
NH
N
NH
Cbz
O
Cbz
O
NH
NH
NH2
NH2
O
4
5
Cbz
6
Cbz
1
Methyl 2-bromoacetate
K2CO3, DMF, 50 ºC, 24h
O
O
O
O
N
N
H2 / Pd, C
THF, 50 ºC;3h
O
NH
NH
O
O
NH
NH
O
NH
NH
NH2
NH2
Cbz
Cbz
8
7
1,3-bis(bromomethyl)-benzene, K2CO3
CH3CN, reflux, 24 h
O
O
O
HN
HN
OH
LiOH,
THF / H2O (2:1)
rt, 24h
N
N
O
NH
NH
O
O
NH
HN
O
NH
HN
9
2
Scheme 1 Synthetic route for the preparation of the open chain (1) and macrocyclic (2) pseudopeptidic compounds
2
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Acid-base studies.
The resulting distribution diagrams for the protonated species
of and are shown in Figure 1. For the open chain ligand the
diagram shows the predominance of non-protonated species
L) at pH values slightly higher than 9. For compound , the
1
2
The presence of the additional nitrogen atom in the central
spacer, in particular the tertiary one in
group present in the pendant arm of the macrocyclic
derivative must significantly affect the acid-base properties
of these compounds when compared to those of related
systems (i.e. ). Thus, the acid-base properties of and were
2, and the carboxylic
(
2
anionic species (L), corresponding to the presence of a
carboxylate group and non-protonated amino groups,
predominates in the very basic region (pH > 10), while the
neutral species (HL), is prevalent at the pH range 8 – 10. In
2
3
1
2
studied using potentiometric titrations. All the titrations were
carried out as is fully described in the Experimental Section, at
general, the different positively charged species of
slightly more acidic regions that those with the same charge in
, in particular for the fully protonated species (H L for and
L for ).
2 appear at
298.1 ± 0.1 K using NaCl 0.1 M to maintain a constant ionic
1
4
2
strength. The cumulative and stepwise stability constants for
the protonation of these pseudopeptidic valine derivates
obtained following this methodology are presented in Table 1,
H
3
1
Table
1 Logarithms of the stepwise protonation constants of pseudopeptidic
along with those for the related compound
3 previously compounds 1-3 determined in NaCl 0.1 M at 298.1+0.1 K.
2
4
determined. According to their structure, three protonation
constants are detected for and four for , while only two
protonation constants are present in the related compound
To facilitate the analysis of the data the monoanionic
carboxylate derived from has been considered as the L
a
b
Reaction
H + L
H + HL
1
2
3
1
2
c
HL
9.02(1)
10.01(1)
7.95(1)
6.45(1)
5.87(2)
8.11(4)
7.31(4)
3.
H
2
L
7.86(1)
6.93(1)
-
H + H
2
3
L
L
H
3
L
2
H + H
H
4
L
species (see Figure 1). Charges have been omitted for clarity in
the description of the different protonated and complex
species.
a
Charges omitted for clarity. b Taken from reference 24. c Values in parentheses
are the standard deviations in the last significant figure. Values lower than 2 have
not been considered. Charges omitted for clarity.
Regarding the values of the stepwise protonation constants,
data in Table 1 show that compound 2, having three basic
nitrogen atoms and one basic carboxylate, displays the higher
constant (log K = 10.01) for the formation of the [HL] species.
This first protonation can be assigned to the carboxylate group
or to one of the amine groups, leading to the formation of a
neutral or a zwitterionic species. In this regard, computational
calculations carried out at the PM3 level revealed a strong
1
00
H L
L
LH
3
3
8
0
L
HL
LH
LH
H L
2
2
60
4
2
0
0
0
stabilization of the zwitterionic structures for
neutral species. In the same way, as could be expected, this
first protonation is easier for the open-chain pseudopeptide
with three basic nitrogen atoms than for the reference dibasic
compound (log K = 9.02 for and 8.11 for ). However, the
second and third protonation constants are relatively similar
for and (log K = 7.86 and 7.95 for [H L], 6.93 and 6.45 for
H3L], respectively). Compound
protonation constant that is only slightly lower that the ones
obtained for and . This seems to suggest that the [H L]
species for and involves the preferential protonation at the
2 relative to the
1
2
6
10
pH
3
1
3
100
80
H
LH
L
LH
HL
L
L
4
4
1
2
H L
L2H
2
2
[
3
presents
a
second
6
4
2
0
0
0
0
LH
H
3
L
3
1
2
2
1
2
two more distant amine nitrogen atoms to reduce the
electrostatic repulsion. The higher conformational freedom of
the open chain compounds and the more favourable solvation
of the primary ammonium groups could partly compensate the
2
6
10
pH
Figure 1 Distribution diagrams for protonated species of 1 (top) and 2 (bottom) in 0.1
M NaCl at 298.1 K. Charges have been omitted for clarity.
effect in
2 of the presence of the carboxylate group. For the
The location of the protons in the protonated species is not a
macrocyclic ligand a fourth constant is identified, which can be
associated with its full protonation, including the acid group in
the pendant arm (log K = 5.87).
simple matter, in particular for 2, considering the presence of
several basic fragments, which leads to the existence of a very
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complex set of potential protonation equilibria at most nitrogen atoms, as the corresponding signals (AB doublets at >
1
protonation steps (see ESI for protonation equilibria). The H 3.6 ppm) are the ones experiencing a more significant shift
NMR spectra obtained at different pH values in D
agreement with the distribution diagrams obtained and corresponding to proton b located in
suggest that all the different nitrogen atoms are protonated, at amine group is also slightly shifted. On the other hand, the last
some extent, in the different [H L] species. Figure 2 displays protonation also seems to affect significantly to this position as
the H NMR spectra, at different pH values, for the macrocyclic shown by the additional changes observed at very acidic pH
compound (see ESI for data regarding compound ). The values. This is also in good agreement with the observation in
coexistence of several structures for each protonated species fluorescent systems related to that full protonation of the
is confirmed by the complexity of the spectra at intermediate benzylic amine groups, required for activating the
2
O are in when changing from pH 11.4 to pH 8.3, although the signal
α
relative to the tertiary
x
1
2
1
2
pH values. Interestingly, the formation of the zwitterionic fluorescence, takes place only at the acidic regions (pK
species seems to involve the protonation of one of the benzylic 4.83).
a
= 4.45-
L
H L
4
1
Figure 2 H NMR spectra in D
2
O for the different protonated species derived from compound 2 at different pH values. Major species present according to the distribution
diagrams: pH 11.4 (L); pH 8.3 (HL); pH 7.2 (H
2
L); pH 5.9 (H L); pH 2.7 (H L).
3
4
2
+
Determination of the Cu Complexes Formation Constants
2+
Table 2 Logarithms of the cumulative and stepwise formation constants for the Cu
2
+
The interaction of Cu and ligands
1
and
2
was systematically complexes of ligands 1 - 3 determined in 0.1 M NaCl at 298.1 ± 0.1 K.
studied by potentiometric titrations in aqueous solution over
the 2-11 pH range. The stability constants for the formation of
[
a]
b
Reaction
Cu+L
1
2
3
c
2
+
CuL
CuHL
CuH
11.03(1)
12.19(2)
6.76(1)
Cu complexes were determined in water for a 1:1 metal-
ligand ratio, using 0.1 M NaCl to maintain a constant ionic
strength and a temperature of 298.1 K. The results obtained
are presented in Table 2 and the corresponding distribution
diagrams are displayed in Figure 3.
Cu + L + H
Cu + L + 2H
Cu + L
16.46(1)
-
19.29(3) 12.51(4)
2
L
23.73(3)
2.44(1)
-7.05(1)
-
-
CuH-1L + H
CuH-2L + 2H
CuH-3L + 3H
CuHL
3.052(2)
-6.457(2)
-16.96(4)
5.43(1)
-
-0.23(2)
-9.89(3)
-
Cu + L
Cu + L
The presence of an additional nitrogen donor atom in the
central spacer linking the two amino acid subunits provides, as
could be expected, the formation of significantly more stable
complexes. This is clearly observed when comparing the values
CuL + H
CuHL + H
CuL
7.10(2)
4.44(3)
-9.75(1)
-9.49(1)
-
5.74(3)
-
CuH2L
CuH-1L + H
CuH-2L + H
-7.978(1)
-9.509(1)
-7.00(2)
-9.66(4)
-
CuH-1
L
CuH-2
L
CuH-3L + H -10.503(1)
of the constants for open chain pseudopeptides
shown in the table, these values are at least two orders of
magnitude higher for the ligand
for the formation of the [CuL] species. The value of this not been considered
constant for is higher than that reported for ethylenediamine
1 and 3. As
a
b
c
Charges omitted for clarity. Taken from reference 24. Values in parentheses
1. This is particularly relevant are the standard deviations in the last significant figure. Values lower than 2 have
.
1
4
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2
log K = 10.60) in spite of the more favourable chelating ring spacer. As this species starts to be present at pH values above
7
(
that can be achieved in the case of ethylenediamine, 8 (figure 3), this could be of interest in the development of
suggesting that more than two nitrogen atoms are involved in catalytic applications and biomimetic models, for instance as
2
8
the coordination to the metal. The existence of an additional simplified HCA mimics. For the macrocyclic ligand
carboxylate donor group in L for provides [CuL] formation not observed as the carboxylate group can coordinate to the
constants significantly higher for this ligand. However, if we copper center instead of the hydroxide anion.
compare the complex formation constants calculated for the The distribution diagrams for and displayed in Figure 3
neutral ligands (L for and HL for ) the situation is different. show that species with similar total charge are predominant at
Thus, the value of the constant for the formation of the [CuL] each pH region. In both cases, the monocationic and dicationic
dicationic species for (log K = 11.03) can be compared to that complexes ([CuH-1L] and [CuL] for and [CuL] and [CuHL] for
for the dicharged [CuHL] species for (HL + Cu CuHL, log K are the major species identified around neutral pH values.
9.28). This seems to preclude a relevant contribution of the Overall, the macrocyclic ligand represents the first example
2, this is
2
1
2
1
2
1
1
2)
2
=
2
macrocyclic effect in this case, most likely for it is of this family of pseudopeptidic cyclophanes being able to
counterbalanced by the increase in structural stress associated display a significant capacity to form stable copper complexes
to the presence of the rigid aromatic ring. As has been in aqueous solutions and for a broad range of pH values.
observed for other C
2
symmetric pseudopeptides like 3,
ligands 1 and 2 can form stable copper complex through
mono- and bisdeprotonation of the N-H amide functional
groups, which can be easily monitored through spectroscopic
1
00
CuL
2
4
Cu
measurements. Both deprotonation steps are detected and
should correspond to the formation of the monocationic [CuH-
CuH-3L
8
0
CuH L
-
1
CuHL
CuH-2L
1
L] and neutral [CuH-2L] species for
and monoanionic [CuH-2L] species for
the case of a species like [CuH-1L] could also correspond to
1
and the neutral [CuH-1L]
60
2
. Although, formally, in
4
0
0
0
2
the complex formed by the ligand bisdeprotonated at both
amide groups and protonated at the carboxyl group, the large
difference between the pKa values for both functionalities
seems to preclude this possibility. The analysis of the
distribution diagrams is also consistent with this situation, as
2
2
6
10
pH
this species is only present for
2
at pH values around 10 at
100
CuHL
CuL
which the carboxyl group should not be protonated.
Cu
CuH L
-2
80
60
40
20
0
It is worth mentioning that the presence of an additional
nitrogen atom in the central spacer is not reflected in an
increased basicity of the [CuL] complex for
1, indicating a
CuH2L
strong participation of all the amino groups in the coordination
to the metal. Thus, the constants determined for the process
CuH L
-
1
CuL + H
CuHL are log K = 5.43 for 1 and log K = 5.74 for the
reference compound 3. A higher first protonation constant is
detected for 2 (log K = 7.10), but this must correspond to the
protonation of the carboxylate fragment in the monocationic
2
6
10
pH
2
+
2+
Figure 3 Distribution diagrams for the systems Cu -1 (top) and Cu -2 (bottom)
[CuL] species. As a matter of fact, a second protonation for this
determined in 0.1M NaCl. Charges omitted for clarity
complex, that should correspond to the protonation of a
nitrogen atom, is also detected (log K = 4.44). Interestingly, in
The use of ESI-MS has been revealed to be of great help for the
29
identification of supramolecular species. Nevertheless, it is
the case of
1, the formation of a monoanionic complex
species, [CuH-3L] is detected, which should correspond to the
deprotonation of a coordinated water molecule to form the
corresponding hydroxide. Such a complex has not been
detected for 3, revealing the important structural differences
associated to the presence of the third amino group in the
important to realize that the nature of the species present in
purely aqueous solutions can differ from those under the
conditions required for ESI-MS analysis, in particular taking
into account that ESI-MS experiments require the use of
methanolic solutions or water-methanol mixtures. In spite of
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this, its potential to detect species even at very low deprotonated amide groups, with the observation of the
3
0
-1 31
concentrations is a unique feature of this technique. On the corresponding amide bands at ca. 1670 cm . Taking
other hand, many different studies have shown how ESI-MS advantage of the distribution diagrams presented in Figure 3, a
allows properly detecting the complex species observed in UV-visible spectroscopic study was carried out, by selecting
solution. The identification of metal-ligand species is of preferentially those pH regions at which one of the complex
particular relevance as the exact nature of the complex can be species is dominant. A selection of the UV-visible spectra,
efficiently confirmed by the analysis of the corresponding obtained for
isotopic patterns. This has been used previously to confirm the Figure 5. The spectral window selected is the region
metal complex species formed by ligands related to corresponding to the d-d transitions, which provides useful
revealing that similar results are obtained in methanol and in information on the coordination geometries of the respective
1 and 2 at different pH values are gathered in
3,
2
4
9h,32
aqueous solutions containing some methanol. Accordingly, copper complexes.
the ESI technique in the positive mode of analysis was used to are presented in Table 3. Interestingly, the observed pH
confirm the species observed at the different pH regions in the dependence patterns of the molecular spectra for and are
distribution diagrams for and . Thus, for instance (Figure 4), very similar and clearly differ from those reported for
at basic pH values (pH range 9 - 11) a peak at 403 was clearly tetradentate ligands related to and having different central
detectable for ligand (Figure 4). This peak can be assigned to aliphatic spacers and amino acids side chains. In the case of
the solvated (H O) sodium ion adduct of the neutral species compounds related to , significant differences are observed in
CuH L], [CuH L + H O + Na ] . The base peak, however, the electronic spectra with changes in the pH that were
The respective observed values of
λ
max
1
2
1
2
3
2
4
1
3
2
+
+
[
-2
-2
2
appears at 363 corresponding to the monocationic species associated to the presence/absence of deprotonated
(2-x)+
CuH L + H] obtained from the neutral complex [CuH L], or complexes [CuH L]
[
. For regions involving the uncomplexed
-2
-2
-x
directly from the monocationic species [CuH L]. In addition, a cation or complexes with the fully protonated ligands
-1
+
peak at 385 can be assigned to [CuH L + Na ] , whose + H O absorption maxima were observed in the 640-690 range,
+
-
2
2
+
and + 2H O species are [CuH L + H O + Na ] (403) and [CuH- characteristic for octahedral copper complexes. On the
+
L + H O + Na ]+ (421), respectively. Complex species formed contrary, for regions involving the predominant formation of
+
2
-2
2
2
2
by the monodeprotonated or bisdeprotonated ligand complexes with deprotonated ligands, the maxima appear at
predominate in solution in this pH region. The second most the 490-560 range, corresponding to square planar to square-
intense peak appears at 302, corresponding to the protonated pyramidal geometries. In the case of ligands
1
and
2, however,
-
free ligand [HL]. The monoanionic [CuH L] species could not no significant shifts in the maxima are observed for most of
-3
be observed even with the use of the ESI in negative mode.
+ +2
The ESI MS spectra for the Cu complexes with
the pH values studied, although the intensity of the bands is
2
at pH=11.5 variable. Only at very acidic pH values, where the
2
+
presents a base peak at 545. Taking into account that L has uncomplexed Cu cation is the major species in solution, the
been defined for as monoanionic (see Figure 1), this peak can appearance of a new diffuse band at much larger wavelengths
be assigned to the monocationic [CuH L+H +Na ] species, and a strong decrease of the original band, accompanied by a
2
+
+ +
-2
while the peak at 561 should correspond to the monocationic shift in its maximum, are detected. In the case of
1
, the
+
+ +
CuH L+H +K ] species (see ESI), again in good agreement observed maxima range from 545 to 549 nm, except for the
[
-2
with the distribution diagram.
more acidic region, while for 2 the maxima of the spectra, for
the same pH range, appear at 579-590 nm.
Table 3 Observed values of λmax for the UV-visible spectra of Cu complexes with 1 and 2
at different pH values.
[CuH [- 2L + H
H
-2L1Cu+H
2
O+Na ]
+ +
+
O + Na ]
+
3
63.3
2
1
00
%
0
4
03.2
1
1
00
%
L
pH
λ
max
Major species from Distribution Diagrams
(abundance in %)
4
05.2
Simulated
(nm)
555
545
546
549
547
624
590
579
583
585
4
06.2
1
4.3
5.9
7.7
Cu (45), CuHL (45), CuL (10)
CuL (70) , CuHL (30),
0
00
4
03.4
3
65.4
Experimental
CuL (60) CuH-1L (40)
4
05.4
4
03.4
%
0
9
.0
1.0
3.7
CuH-1L (70), CuH-2L(10), CuL (10)
CuH-3L (90)
4
21.3
423.3
3
99.3 406.4
1
m/z
m/z
3
75 380 385 390 395 400 405 410 415 420 425 430 435 440
2
2
Cu (40), CuH L (40), CuHL (20)
4
05.4
3
42.5
5
8
.5
.4
CuHL (90)
CuL (90)
3
66.3
4
21.3
23.3
3
43.4
665.5 705.6
680 720
4
9.8
CuH-1L (30), CuH-2L (30), CuL (30)
3
60
400
440
480
520
2+
560
600
640
760
800
840
+
11.5
-2
CuH L (95)
Figure 4 ESI -MS spectra for Cu complexes with 1 at pH = 11. 0 in methanol.
Spectroscopic analyses also provided additional information on
the exact nature of the complex species. As for other ligands
This should be in good agreement with the maintenance, for
the different complex species formed, of a predominant
2
3, FT-IR spectra of the Cu complexes formed by 1
+
related to
square planar to square-pyramidal geometry (500-
h
00nm).Error! Bookmark not defined. This last geometry is
and 2 under basic condition revealed the participation of
6
6
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DOI: 10.1039/C5RA15852D
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ARTICLE
common to many complexes formed by pentadentate achieved involving the two deprotonated amide groups and
3
3
ligands, and many five-coordinate copper (II) complexes two amine nitrogen atoms. The additional coordination to the
adopt square pyramidal arrangements of the donor atoms third amino group, to the carboxylate in or to a water
around the metal center, in some cases with a clear distortion molecule could transform this square planar arrangement into
2
3
4
towards a trigonal pyramidal coordination. For 1, the square square pyramidal. However, the existence of different possible
planar base can be easily formed by the two amides and two structural possibilities for those arrangements and the low
amine groups from the amino acid moieties, while the energy differences calculated for many of them does not allow
additional nitrogen atom or, in basic media, a hydroxide anion an unambiguous definition of the coordination geometries in
could occupy the apical position.
A similar situation can be expected for the complexes formed Thus, different complex structures could coexist, for instance,
by ligand , although in this case we need to take into account for the [CuH-2L] species predominant at the basic region. The
the absence of additional data, in particular crystal structures.
2
the steric constrains imposed by the macrocyclic structure and same can apply for [CuL] and the related protonated complex
the presence of the carboxyl/carboxylate group that could act species for which an efficient coordination to the metal seems
as an additional donor group in the coordination to copper(II). to take place and can involve the amino groups and the
This can lead to distorted square pyramidal complexes or carbonyl oxygen atoms of the amide groups. Similar results are
distorted square planar complexes. In some cases, the obtained in the case of the ligand
transition from square pyramidal to trigonal bipyramidal has
1 (see ESI).
been observed to be associated to a bathochromic shift from
3
around 550 to ca. 600 nm, as is observed here.
3
Conclusions
The introduction of a nitrogen atom in the central spacer
linking the two amino acid subunits in C symmetric
pseudopeptides provides important changes in the properties
of the resulting derivatives (i.e. and ) relative to those of
the parent compounds like . The presence of this additional
2
1
2
3
nitrogen atom increases the basicity of the resulting system
and the number of donor atoms for coordination to metal ions
like Cu(II), but besides, facilitates the building of more
elaborated structures as is illustrated in the case of 2. Overall,
this modification significantly increases their solubility in water
and affords pentadentate ligands that are able to form more
stable Cu(II) complexes than the parent ligand
3. Copper
complex species dominate the distribution diagram for pH
values above 5. The geometry of these complexes, having
different protonation states, seems to be very similar, most
likely from square planar to square-pyramidal, a behaviour
clearly divergent from the one found for ligand
compounds. The existence of a large enough flexible cavity in
the case of the macrocyclic compound and the presence of
3 and related
2
the six potential donor groups in the macrocyclic structure
allows the formation of strong metal complexes for this ligand,
in spite of the important steric constraints often found for
3
5
structures of this class like polyaza [n] cyclophanes. Thus, the
macrocyclic ligand represents the first example of this family
2
of receptors with a significant capacity to form stable copper
complexes in aqueous solutions and for a broad range of pH
value.
Figure 5 UV-visible spectra obtained for 1:1 Cu:L mixtures for ligands 1 (top) and 2
bottom) at different pH values.
Acknowledgments
(
Financial support from the Spanish MINECO (CTQ2012-38543-
Computational models (MMFF) reveal, in good agreement with C03-01), Generalitat Valenciana (PROMETEO/2012/020) and
experimental data, that distorted square planar and square PPI-UJI (P1-1B-2013-38) is gratefully acknowledged. P.D.W.
pyramidal geometries can be easily achieved, for instance, for and L.G. thank GV for a Santiago Grisolía fellowship. The
complexes of both deprotonated ligands (see ESI). They authors are grateful to the SCIC of the Universitat Jaume I for
suggest, taking also into consideration former studies with the spectroscopic facilities.
related systems, that a square planar arrangement can be
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J. Name., 2013, 00, 1-3 | 7
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ARTICLE
Journal Name
-
1 1
2
d
8
360, 1736, 1685, 1644, 1532 cm ; H NMR (500 MHz, DMSO-
) (δ, ppm): 7.77 (s, 2H), 7.39 – 7.24 (m, 10H), 7.17 (d, 2H, J =
.8 Hz), 5.00 (m, 4H), 3.78 (t, 2H, J = 7.9 Hz), 3.58 (s, 3H), 3.37
Experimental Section
6
Materials and Reagents: All reagents were obtained from
commercial sources and used as received unless otherwise
(d, 2H), 3.10 (m, 4H), 2.63 (m, 4H), 1.91 (m, 4H), 0.82 (m, 12H);
stated. Dimethoxyethane (DME) was dried and distilled from 13
C NMR (126 MHz, DMSO-d
6
)(δ, ppm): 173.7, 171.5, 139.8,
molecular sieves (4 Å) and then stored over molecular sieves.
1
29.3, 128.6, 127.5, 67.5, 55.7, 55.0, 53.6, 51.5, 37.5, 31.2,
+
9.5, 18.0; HRMS (ESI-TOF) (m/z) Calcd. for C33
Deionised water was used from a Milli-Q water system by
1
Millipore.
+
(M+H )
H
47
N
5
O
8
6
42.3503, found 642.3508. Anal. Calcd. (%) for C33
1.8, H, 7.4, N, 10.9; found: C, 61.7, H, 7.5, N, 10.9.
47 5 8
H N O : C,
6
Synthesis and characterization of organic compounds.
Synthesis of 8. To a clear solution of compound
(25 g, mmol) in dry tetrahydrofuran (THF), 10 mol% of the catalyst (5
9.49 mmol) and N–hydroxysuccinimide (11.45 g, 99.49 mmol) wt% Pd on activated carbon) was added. The reaction mixture
at 0 ºC was added DCC (20.52 g, 99.49 mmol) in anhydrous was stirred under an H atmosphere (H balloon) at 50 °C for 4
7 (2.00 g, 3.11
Synthesis of 5. To a clear solution of N–Cbz–L–valine 4
9
2
2
THF in a dropwise manner, and the reaction mixture was h. The reaction was monitored by TLC, and after complete
stirred at 0–5 °C for 3 h. The dicyclohexylurea formed was consumption of the starting material the catalyst was filtered
filtered off and the filtrate was concentrated to dryness. The off through a Celite® bed and washed with THF. The crude
crude product was recrystallized from 2–propanol to obtain product was purified by column chromatography using
2
D
5
the pure product. Yield 87% (49 g); m.p. 119–120 ºC; [α]
= – MeOH/CH Cl /NH (1:15:0.01) as eluent. Evaporation of the
2 2 3
–1 1
2
0.1º (c = 0.1, CHCl
300 MHz, CDCl ) (δ, ppm): 1.02 (d, 6H, J = 7.4 Hz), 1.06 (d, 6H, yield): [α]D = 6.26º (c 0.1, CHCl ); IR (ATR) 3289, 3073, 2963,
J = 7.7 Hz), 2.31 (m, 1H), 2.77 (s, 4H), 4.66 (dd, 1H, J = 4.7 Hz), 2869, 1736, 1640, 1526 cm ; H NMR (500 MHz, CDCl ) (δ,
3
); IR (KBr) 3360, 1741, 1526 cm ; H NMR solvent under vacuum yielded the green oil
8 (0.462 g, 80%
2
5
(
3
3
-1
1
3
1
3
5
.13 (s, 2H), 5.43 (d, 1H, J = 9.5 Hz), 7.28–7.41 (m, 5H);
C
ppm): 7.50 (s, 2H), 3.26 (m, 4H), 3.09 (m, 2H), 2.66 (m, 4H),
): (δ, ppm): 17.2, 18.7, 25.5, 31.5, 57.4, 2.10 (m, 2H), 1.59 (m, 5H), 0.87 (d, 6H, J = 6.8 Hz), 0.74 (d, 6H, J
NMR (75 MHz, CDCl
3
1
3
6
7.2, 128.0, 128.1, 128.3, 135.8, 155.6, 167.5, 168.6; Anal. = 6.8 Hz); C NMR (75 MHz, CDCl ) (δ, ppm): 174.3, 171.8,
3
Calcd. (%) for C17
H, 5.9; N, 8.0.
H
20
N
2
O
6
: C, 58.6; H, 5.8; N, 7.7; found: C, 58.1; 68.2, 54.8, 54.1, 51.0, 43.4, 36.9, 31.2, 19.6, 17.9, 15.2; HRMS
+
+
(ESITOF) (m/z) Calcd. for C H N O (M+H ) 374.2767, found
1
7 35 5 4
Synthesis of 6. To a clear solution of the N-hydroxysuccinimide 374.2762. Anal. Calcd. (%) for C H N O : C, 49.9, H, 9.6, N,
1
7 35 5 4
ester of N-Cbz-L-valine
dimethoxyethane (DME, 250 mL) at 0 ºC, diethylenetriamine Synthesis of 9. A mixture of compound
4.46 g, 43.06 mmol) dissolved in dry DME (20 mL) was added anhydrous K2CO3 (1.85 g, 13.38 mmol) and 9,10-
5 (30.0 g, 86.12 mmol) in anhydrous 17.1; found: C, 49.9, H, 9.7, N, 17.0.
8
(0.50 g, 1.33 mmol),
(
in a dropwise manner. The reaction mixture was stirred at bis(bromomethyl)-benzene (0.39 g, 1.47 mmol) in dry CH CN
3
room temperature for 18 h and then was heated to 40–50 °C (175mL) was stirred to reflux for 24 h under a nitrogen
for 6 h. The white solid formed was filtered and washed with atmosphere. The reaction was monitored by TLC. After
cold water and cold methanol. Yield 94% (23.27 g), mp = 175 complete consumption of the starting material, the reaction
2
D
5
ºC; [α]
= 11.39º (c = 0.1, CHCl
3
); IR (ATR) 3285, 2957, 1685, mixture was filtered and the solvent was evaporated under
-1 1
1
649, 1535, 1236 cm ; HNMR (500 MHz, DMSO-d ) (δ, ppm): vacuum. The crude product was purified by silica flash
6
7
.83 (s, 2H), 7.38–7.23 (m, 8H), 7.19 (d, 2H, J = 8.3 Hz), 5.07– chromatography using MeOH/CH Cl (1:25) as the eluent to
2
2
4
.94 (m, 4H), 3.83–3.68 (m, 4H), 3.10 (dd, 4H, J = 12.6, 6.1 Hz), yield the yellowish solid compound
9. Yield 72% (0.459 g), mp
1
3
25
1.98–1.84 (m, 2H), 0.82 (d, 12H, J = 6.1 Hz). CNMR (75 MHz, 106 ºC, [α]D = –24.36 (c = 0.1, CHC l ); IR (ATR) 3323, 2947,
3
-1 1
6
DMSO-d ) (δ, ppm): 174.7, 171.8, 156.8, 137.8, 129.0, 128.4, 2922, 2856, 1741, 1651, 1592, 1198 cm , H NMR (500 MHz,
1
28.3, 66.1, 61.1, 48.9, 30.9, 19.9, 18.9; HRMS (ESI-TOF)+ CDCl ) (δ, ppm): 7.68 (s, 2H), 7.38 (s, 1H), 7.26 – 7.07 (m, 5H),
3
+
43 5 6
Calcd. for C30H N O (M+H ) 570.3292. Found 570.3296 3.65 (dd, 4H, J = 30.0, 12.1 Hz), 3.53 (s, 3H), 3.42 (d, 2H, J =
+
M+H ); Anal. Calcd. for C30
(
H
43
N
5
O
6
H
2
O: C, 61.3, H, 7.7, N, 40.9 Hz), 3.30 (s, 2H), 3.19 (s, 2H), 2.85 (d, 2H, J = 3.4 Hz), 2.73
(dd, 4H, J = 41.5, 34.7 Hz), 2.06 (d, 2H, J = 5.3 Hz), 0.82 (dt,
11.9. Found. C, 61.4, H, 7.8, N, 11.9.
1
3
Synthesis of 7. A mixture of compound
anhydrous CO (24.25 g, 175.5 mmol) and methyl 174.2, 171.9, 140.1, 129.3, 128.5, 127.6, 67.8, 55.6, 53.6, 51.5,
bromoacetate (2.68 g, 19.30 mmol) was placed in a flask 37.4, 31.3, 19.5, 18.0. HRMS (ESI-TOF) Calcd. for C H N O
containing dry dimethylformamide (DMF, 10 mL) and heated (M+H ) 476.3237. Found 476.3235 (M+H ). Anal. Calcd. (%) for
to 50 ºC for 24 h under nitrogen atmosphere. The reaction was C H N O : C, 63.7, H, 8.7, N, 14.7; found. C, 63.5, H, 8.8, N,
6 (10 g, 17.55 mmol), 12H, J = 62.3, 31.1 Hz); C NMR (126 MHz, CDCl ) (δ, ppm):
3
K
2
3
+
2
5 41 5 4
+
+
2
5 41 5 4
monitored by TLC. After complete consumption of the starting 14.7.
material, distilled water was added (30 mL). This solution was Synthesis of 2. To a clear solution of
extracted by using ethyl acetate (EtOAc, 30 mL, x3). The mL of THF/H O (2:1), was added LiOH (0.07 g, 2.94 mmol) and
9
(0.2 g, 0.42 mmol) in 7
2
organic phase was dried over anhydrous MgSO
evaporated under vacuum. The product was purified by silica mL) The reaction was monitored by using TLC; after complete
flash chromatography using MeOH/CH Cl (1:50) to give the consumption of the starting material, the reaction mixture was
white solid (8.19 g, 75% yield): mp 156-158 ºC; [α]
0.45º (c 0.1, 55 CHCl ); IR (ATR) 3300, 3064, 3031, 2949, 2869, was added washed DOWEX ion exchange resin (4 g), and the
4
and the reaction was stirred at room temperature for 24 h. (2:1, 3
2
2
2
5
7
D
= – acidified (pH 6) by addition of 1:1 HBr/H O. To this solution
2
®
1
3
8
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5RA15852D
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ARTICLE
mixture was left for 18 h at room temperature. (The standard and the HySS program was used to obtain the distribution
®
38
procedure to wash the DOWEX ion exchange resin was as diagrams. The pH range investigated was 2.0-12.0 and the
follows: 50 mL deionized H O, 50 mL MeOH, 50 mL H 0, 50 mL concentration of the metal ions and of the ligands ranged from
THF, 50 mL H 0, 50 mL 10% NH in H O, H O till neutral pH, 50 1×10 to 5×10 M with Cu :L molar ratios as 1:1. The
mL 2M HCl, H O till neutral pH). The solvent of this mixture different titration curves for each system (at least two) were
2
2
-3
-3
2+
2
3
2
2
2
was evaporated under vacuum, and the resulting solid was put treated either as a single set or as separated curves without
®
in a column containing 20 g washed DOWEX ion exchange significant variations in the values of the stability constants.
resin and deionized H
purification were: 100 mL H
in H O. From this resin column purification the compound was NMR measurements. The H spectra were recorded on a
isolated by the elution with 5% NH
was evaporated under vacuum to yield the off-white solid
2
O. The eluents used for the resin column Finally, the sets of data were merged together and treated
2
O; 1:1 THF/H O 100 mL; 5 % NH simultaneously to give the final stability constants.
2
3
1
2
1
3
2
in H O. Then the water Varian INOVA 500 spectrometer (500 and 125 MHz for H and
1
3
2.
C NMR, respectively). The solvent signal was used as a
), reference standard. Adjustments to the desired pH were made
IR (ATR) 3285, 2957, 1685, 1649, 1535 cm ; H NMR (500 MHz, using drops of DCl or NaOD solutions. The pD was calculated
O) (δ, ppm): 7.46 – 7.27 (m, 3H), 7.14 (s, 1H), 3.83 (d, 2H, J = from the measured pH values using the correlation, pH = pD -
2
D
5
Yield 41% (0.80 g), mp 83 ºC, [α]
= –31.84º (c = 0.1, CHCl
3
-1 1
D
2
3
9
1
2
5
0
2.9 Hz), 3.68 (d, 2H, J = 13.0 Hz), 3.44 – 3.31 (m, 2H), 3.19 (dd, 0.4.
H, J = 18.6, 8.9 Hz), 3.13 (d, 2H, J = 19.2 Hz), 3.07 (d, 2H, J = Mass Spectrometry. Mass spectra were recorded on a hybrid
.9 Hz), 2.74 (d, 4H, J = 4.8 Hz), 1.85 (dt, 2H, J = 11.2, 5.6 Hz), QTOF I (quadrupole-hexapole-TOF) mass spectrometer with an
1
3
2
.83 (dd, 12H, J = 14.6, 6.8 Hz). C NMR (126 MHz, D O) (δ, orthogonal Z-spray-electrospray interface (Micromass,
ppm): 178.3, 173.7, 136.1, 131.1, 129.7, 128.8, 71.5, 65.8, Manchester, UK) either by electrospray positive mode (ES+) or
+
7.3, 53.9, 50.9, 37.1, 31.0, 18.4, 17.8. HRMS (ESI-TOF) Calcd. by electrospray negative mode (ES-). The desolvation gas as
5
+
+
39 5 4
for C24H N O (M+H ) 462.3080. Found 462.3082 (M+H ). well as nebulizing gas was nitrogen at a flow of 700L/h and 20
Anal. Calcd. (%) for C24
H
39
N
5
O
4
.H
2
O: C, 60.1, H, 8.6, N, 14.6; L/h respectively. The temperature of the source block was set
to 120ºC and the desolvation temperature to 150ºC. A
found. C, 60.0, H, 8.6, N, 14.9.
Synthesis of 1. To a clear solution of compound
7.55 mmol) in dry tetrahydrofuran (THF), 10 mol% of the negative scan mode, respectively. The cone voltage was
catalyst (5 wt% Pd on activated carbon) was added. The typically set to 20 V to control the extent of fragmentation of
reaction mixture was stirred under an atmosphere of H (H the identified ions. Sample solutions were infused via syringe
6 (10.0 g, capillary voltage of 3.5 and 3.3 KV was used in the positive and
1
2
2
balloon) at 50 °C for 4 h. The reaction was monitored by TLC, pump directly connected to the ESI source at a flow rate of 10
and after complete consumption of the starting material the mL/min. The observed isotopic pattern of each intermediate
catalyst was filtered off through a Celite® bed and washed with perfectly matched the theoretical isotopic pattern calculated
THF. The crude product was purified by column from their elemental composition using the MassLynx 4.0
4
0
chromatography using MeOH/CH
2 2
Cl (1:15:0.01) as eluent. program.
Evaporation of the solvent under vacuum yielded the white UV Spectroscopy: UV-Vis absorption spectra were recorded in
1
solid 1. Yield 88% (4.69 g). H NMR (300 MHz, CDCl ) (δ, ppm): MeOH, in a Hewlett–Packard 8453 apparatus, using solutions
3
-
3
7
2
6
.50 (s, 2H), 3.34 (d, 4H, J = 5.7 Hz), 3.20 (d, 2H, J = 3.6 Hz), (1x10 M) at different pH values containing 1:1 ligand to metal
.76 (t, 4H, J = 5.7 Hz), 2.30 – 2.19 (m, 2H), 1.52 (s, 3H), 0.96 (d, molar ratios. Additional experiments were carried in NaCl
1
3
H, J = 6.9 Hz), 0.82 (d, 6H, J = 6.8 Hz). C NMR (75 MHz, 0.1M solutions. Only minimal differences were observed in this
) (δ, ppm): 174.7, 77.5, 77.0, 76.6, 60.4, 48.7, 38.8, 31.0, case.
CDCl
9.7, 16.2; ESI–MS m/z = 302.2 (M+H ). Anal. Calcd. (%) for IR Spectroscopy: FTIR spectra were acquired on a JASCO 6200
3
+
1
C
14
H
31
N
5
O
2
: C, 55.8, H, 10.4, N, 23.3; found. C, 56.0, H, 10.5, N, instrument with a MIRacle single-reflection ATR diamond/ZnSe
accessory. The raw IR spectral data were processed with the
23.5.
-3
JASCO spectral manager software. Solutions (2x10 M) at
Electromotive Force Measurements
.
The potentiometric different pH values containing 1:1 ligand to metal molar ratios
titrations were carried out at 298.1 ± 0.1 K using NaCl 0.1 M as were used for those experiments.
supporting electrolyte. The experimental procedure (burette,
potentiometer, cell, stirrer, microcomputer, etc.) has been
6
3
fully described elsewhere. The acquisition of the emf data
was performed with the computer program CrisonCapture.
Notes and references
The reference electrode was an Ag/AgCl electrode in saturated 1. (a) Y. Lu, N. Yeung, Sieracki and N. M. Marshall, Nature,
KCl solution. The glass electrode was calibrated as a hydrogen-
ion concentration probe by titration of previously standardized
2009, 460, 855; (b) N. Mitic, S.J. Smith, A. Neves, L. W.
Guddat, L. R. Gahan and G. Shenk, Chem. Rev., 2006, 106,
3
338; (c) M. L. Zastrow and V. L. Pecoraro, Coord. Chem.
2
amounts of HCl with CO -free NaOH solutions and the
Rev., 2013, 257, 2565; (d) J. Liu, S. Chakraborty, P.
Hosseinzadeh, Y. Yu, S. Tian, I. Petrik, A. Bhagi and Y. Lu,
Chem. Rev., 2014, 114, 4366.
equivalent point determined by the Gran´s method, which
gives the standard potential, Eº', and the ionic product of
water [pKw=13.78(1)]. The computer program HYPERQUAD 2. (a) R. H Holm, P. Kennepohl and I. Solomon, Chem. Rev.,
3
7
1
996, 96, 2239; (b) N. Kitajima, and Y. Morooka, Chem. Rev.,
was used to calculate the protonation and stability constants,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Page 11 of 12
ARTICLE
View Article Online
DOI: 10.1039/C5RA15852D
Journal Name
1
2
994, 94, 737; (c) E. A. Lewis and W. B. Tolman, Chem. Rev.,
004, 104, 1047; (d) J. Reedijk and E. Bouwman, Bioinorganic
Pitarch-Jarque, R. Belda, F. Lloret, J. Ferrando-Soria, P.
Navarro, A. Lopera and E. García-España, Dalton Trans.,
2015, 44, 3378.
Catalysis, 2nd ed.; MarcelDekker: New York, 1999; (e) K. D.
Karlin, D.-H. Lee, V. Obias and K. J. Humphreys, Pure Appl. 10. A. G. Blackman, Polyhedron 2005, 24, 1.
Chem., 1998, 70, 855; (f) E. I. Solomon, F. Tuczek, D. E. Root 11. (a) J.-M. Lehn, Angew. Chem., Int. Ed., 1988, 27, 89; (b) K. D.
and C. A. Brown, Chem. Rev., 1994, 94, 827; (g) L. M. Mirica,
X. Ottenwaelder and T. D. P. Stack, Chem. Rev., 2004, 104,
Karlin, Science 1993, 261, 701; (c) R. M. Roat-Malone,
Bioinorganic Chemistry: A Short Course, 2nd ed.; John Wiley
& Sons, Inc.: Hoboken, NJ, 2007; (d) I. Bertini, I. H. B. Gray, E.
I. Stiefel and J. S. Valentine, Biological Inorganic Chemistry,
Structure and Reactivity, University Science Books, Sausalito,
CA, 2007; (e) R. Gramage-Doria, D. Armspach and D. Matt,
Coord. Chem. Rev., 2013, 257, 776; (f) O. Bistri and O.
Reinaud, Org. Biol. Chem., 2015, 13, 2849.
1
2
013; (h) L. Q. Hatcher and K. D. Karlin, Adv. Inorg. Chem.,
006, 58, 131; (i) W. Kaim and B. Schwederski,
Bioanorganische Chemie, 3 Aufl.; B. G.Teuber: Stuttgart,
Germany, 2004 (j) P. Haack and C. Limberg, Angew. Chem.
Int. Ed., 2014, 53, 4282.
3
4
.
.
(a) F. E. Dodd, J. Van Beeumen, R. R. Eady and S. S. Hasnain,
J. Mol. Biol., 1998, 282, 369; (b) K. Paraskevopoulos, M. 12. (a) H. Miyake and Y. Kojima, Coord. Chem. Rev., 1996, 148,
Sundararajan, R. Surendran, M. A. Hough, R. R. Eady, I. H.
Hillier and S. S. Hasnain, Dalton Trans., 2006, 3067; (c) A. C.
Merkle and N. Lehnert, Dalton Trans., 2012, 41, 3355.
301. (b) C. Bazzicalupi, A. Bianchi, E. García-España and E.
Delgado-Pinar, Inorg. Chim. Acta, 2014, 417, 3.
13. (a) F. Fache, E. Schulz, M. L. Tommasino and M. Lemaire,
Chem. Rev., 2000, 100, 2159; (b) M. Hechavarría-Fonseca
and B. König, Adv. Synth. Catal., 2003, 345, 1173; (c) J.-C.
Kizirian, Chem. Rev., 2008, 108, 140; (c) J. S. Prell, T. G. Flick,
J. Oomens, G. Berden and E. R. Williams, J. Phys. Chem. A.
2010, 114, 854; (d) S. K. Burke, Y. Xu and D. W. Margerum,
Inorg. Chem., 2003, 42, 5807; (e) B. J. Green, T. M. Tesfai and
D. W. Margerum, Inorg. Chem., 2004, 43, 1463; (f) B. J.
Green, T.M. Tesfai and D. W. Margerum, Dalton Trans., 2004,
3508; (g) T. M. Tesfai, B. J. Green and D. W. Margerum,
Inorg. Chem., 2004, 43, 6726; (h) V. N. Dokorou, C. J. Milios,
A. C. Tsipis, M. Haukka, P. G. Weidler, A. K. Powella and G. E.
Kostakis, Dalton Trans., 2012, 41, 12501; (i) C. R. Dennis, J. C.
Swarts and, D. W. Margerum, Reac. Kinet. Mech. Cat., 2012,
107, 27; (j) J. Dong, Y. Wang, Q. Xiang, X. Lv, W. Weng and Q.
Zeng, Adv. Synth. Catal., 2013, 355, 692.
(a) J. L. Vanhooke, M. M. Benning, F. M. Raushel and H. M.
Holden, Biochem. 1996, 35, 6020; (b) M. M. Benning, H.
Shim, F. M. Raushel and H. M. Holden, Biochem. 2001, 40,
2
712.
5
6
.
.
(a) N. Kitajima, K. Fujisawa and Y. Morooka, J. Am. Chem.
Soc., 1990, 112, 3210.; (b) R. C. Elder and M. C. Hill, Inorg.
Chem., 1979, 18, 729.
(a) P. Comba, T. W. Hambley, M. A. Hitchman and H.
Stratemeier, Inorg. Chem. 1995, 34, 3903; (b) B. J. Hathaway,
J. Chem. Soc., Dalton Trans., 1972, 1196; (c) B. J. Hathaway
and D. E. Billing, Coord. Chem. Rev., 1970, 5, 143; (d) M.
Kalanithi, M. Rajarajan, P. Tharmaraj and S. Johnson Raja,
Med. Chem. Res., 2015, 24,1578.
7
.
(a) P. Comba, A. M. Sargeson, L. M. Engelhardt, J. M.
Harrowfield, A. H. White, E. Horn and M. R. Snow, Inorg.
Chem. 1985, 24, 2325; (b) P. V. Bernhardt, R. Bramley, L. M. 14. (a) S. E. Gibson and C. Lecci, Angew. Chem., Int. Ed. 2006,
Engelhardt, J. M. Harrowfield, D. C. R. Hockless, B. R.
Korybut- Daszkiewicz, E. R. Krausz, T. Morgan and A. M.
Sargeson, Inorg. Chem. 1995, 34, 3589; (c) P. Comba, C. Haaf
and H. Wadepohl, Inorg. Chem., 2009, 48, 6604; (d) I.
Castillo, A. C. Neira, E. Nordlander and E. Zeglio, Inorg. Chim.
Acta, 2014, 422, 152; (e) A. Kaur, T. G. Ribelli, K. Schröder, K.
Matyjaszewski and T. Pintauer, Inorg. Chem., 2015, 54, 1474.
(a) E. V. Rybak-Akimova, A. Y. Nazarenko, L. Chen, P. W.
Krieger, A. M. Herrera, V. V. Tarasov and P. D. Robinson,
Inorg. Chim. Acta, 2001, 324, 1; (b) J. P. Schneider and J. W.
Kelly, J. Am. Chem. Soc., 1995, 117, 2533; (c) R. Kannappan,
S. Tanase, I. Mutikainen, U. Turpeinen and J. Reedijk, Inorg.
Chim. Acta, 2005, 358, 383; (d) M. R. Bryant, A. D. Burrows,
45, 1364; (b) J. F. Billing and U. J. Nilsson, J. Org. Chem. 2005,
70, 4847; (c) S. Punna, J. Kuzelka, Q. Wang and M. G. Finn,
Angew. Chem., Int. Ed. 2005, 44, 2215; (d) P. P. Cristau, M.-T.
Martin, M.-E. Tran Hu Dau, J.-P. Vors and Zhu, J. Org. Lett.,
2004, 6, 3183; (e) J. E. Redman, K. M. Wilcoxen and M. R.
Ghadiri, J. Comb. Chem., 2003, 5, 33; (f) E. Locardi, M.
Stöckle, S. Garner and H. Kessler, J. Am. Chem. Soc., 2001,
123, 8189. (g) C. J White and A. K Yudin, Nat. Chem., 2011, 3,
509. (h) S. E. Gibson and C. Lecci, Angew. Chem., Int. Ed.
2006, 45, 1364. (i) B. Lewandowski, G. De Bo, J. W. Ward, M.
Papmeyer, S. Kuschel, M. J. Aldegunde, P. M. E. Gramlich, D.
Heckmann, S. M. Goldup, D. M. D'Souza, A. E. Fernandes and
D. A. Leigh, Science, 2013, 339, 189.
8
.
C. M. Fitchett, C. S. Hawes, S. O. Hunter, L. L. Keenan, D. J. 15. (a) S. Fernández-López, H.-S. Kim, E. C. Choi, M. Delgado, J. R.
Kelly, P. E. Kruger, M. F. Mahonb and C. Richardson, Dalton
Trans., 2015, 44, 9269.
Granja, A. Khasanov, K. Kraehenbuehl, G. Long, D. A.
Weinberger, K. M. Wilcoxen and M. R. Ghadiri, Nature,
2001, 412, 452; (b) S. Kubik, Chem. Soc. Rev., 2009, 38, 585.
(c) A. V. Gulevich, L. S. Koroleva, O. V. Morozova, V. N.
Bakhvalova, V. N. Silnikov and V. G. Nenajdenko, J. Org.
Chem., 2011, 7, 1135. (d) K. Klemm, M. R. Stojkovic, G.
Horvat, V. Tomisic, I. Piantanida and C. Schmuck, Chem.;Eur.
J. 2012, 18, 1352. (e) W.-C. Lin, Y.-P. Tseng, C.-Y. Lin and Y.-P.
Ye, Org. Biomol. Chem. 2011, 9, 5547.
9
.
(a) S. R. Batten, B. F. Hoskins, B. Moubaraki, K. S. Murray and
R. Robson, Chem. Commun., 2000, 1095; (b) T. Osako, K. D.
Karlin and S. Itoh, Inorg. Chem., 2005, 44, 410; (c) M. Meyer,
L. Fremond, E. Espinosa, R. Guilard, Z. P. Ou and K. M. Kadish,
Inorg. Chem., 2004, 43, 5572; (d) K. Miyoshi, H. Tanaka, E.
Kimura, S. Tsuboyama, S. Murata, H. Shimizu and K. Ishizu,
Inorg. Chim. Acta ,1983, 78, 23; (e) R. M. Nunes, R. Delgado,
M. F. Cabral, J. Costa, P. Brandao, V. Felix and B. J. 16. a) W. A. Loughlin, J. D.A. Tyndall, M. P. Glenn and D. P.
Goodfellow, Dalton Trans., 2007, 4536; (f) H. Nagao, N.
Komeda, M. Mukaida, M. Suzuki and K. Tanaka, Inorg.
Chem., 1996, 35, 6809; (g) E. J. Laskowski, D. M. Duggan and
D. N. Hendrickson, Inorg. Chem., 1975, 14, 2449; (h) A.
Algarra, M. G. Basallote, C. E. Castillo, M. Paz, M.; A. Ferrer,
E. García-España, J. M. Llinares, M. A. Máñez and C. Soriano,
Inorg. Chem., 2009, 48, 902; (i) C. E. Castillo, A. G. Algarra, M.
Á. Máñez, C. Duboc and M. G. Basallote, Eur. J. Inorg. Chem.,
Fairlie, Chem. Rev., 2004, 104, 6085; (b) R. C. Reid, L. K.
Pattenden, J. D. A.Tyndall, J. L. Martin, T. Walsh and D. P.
Fairlie, J. Med. Chem., 2004, 47, 1641; (c) R. C. Reid, G.
Abbenante, S. M. Taylor and D. P. Fairlie, J. Org. Chem., 2003,
68, 4464; (d) X. Hu, K. T. Nguyen, C. L. M. J. Verlinde, W. G. J.
Hol and D. Pei, J. Med. Chem. 2003, 46, 3771. (e) P. T.
Lansbury and H. A. Lashuel, Nature, 2006, 443, 774. (f) G. A.
Silva, C. Czeisler, K. L. Niece,. E. Beniash, D. A. Harrington, J.
A. Kessler and S. I. Stupp, Science, 2004, 303, 1352.
2
012, 2514; (j) L. Acosta-Rueda, E. Delgado-Pinar, J. Pitarch-
Jarque, A. Rodríguez, S. Blasco, J. González, M. G. Basallote 17. (a) For some examples on related systems: K. Choi and A. D.
and E. García-España, Dalton Trans., 2015, 44, 8255; (k) J.
Hamilton, Coord. Chem. Rev., 2003, 240, 101; (b) K. Choi and
1
0 | J. Name., 2012, 00, 1-3
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Page 12 of 12
View Article Online
DOI: 10.1039/C5RA15852D
Journal Name
ARTICLE
A.D. Hamilton, J. Am. Chem. Soc., 2003, 125, 10241; (c) K.
Choi and A.D. Hamilton, J. Am. Chem. Soc., 2001, 123, 2456;
Chem., 2011, 83, 9251; (c) L. Cera and C. A. Schalley, Chem.
Soc. Rev., 2014, 43, 1800.
d) L. Somogyi, G. Haberhauer and J. Rebek, Tetrahedron, 30. (a) K. Schug, P. Fryčák, N. M. Maier and W. Lindner, Anal.
(
2
1
001, 57, 1699; (e) M. M. Conn and J. Rebek, Chem. Rev.
997, 97, 1647. (f) R. J. Brea, C. Reiriz and J. R. Granja, Chem.
Chem., 2005, 77, 3660; (b) A. Di Tullio, S. Reale and F. J. De
Angelis, Mass Spectrom., 2005, 40, 845; (c) T. Becherer, D.
Meshcheryakov, A. Springer, V. Böhmerb and C. A. Schalley,
J. Mass. Spectrom. 2009, 44, 1338; (d) W. Jiang, A. Schäfer, P.
C. Mohr, and C. A. Schalley, J. Am. Chem. Soc., 2010, 132,
2309; (e) V. Lemaur, G. Carroy, F. Poussigue, F. Chirot, J. De
Winter, L. Isaacs, P. Dugourd, J. Cornil and P. Gerbaux,
ChemPlusChem, 2013, 78, 959; (f) D. Cubrilovic, W. Haap, K.
Barylyuk, A. Ruf, M. Badertscher, M. Gubler, T. Tetaz, C.
Joseph, J. Benz and R. Zenobi; ACS Chem. Biol., 2014, 9, 218.
Soc. Rev., 2010, 39, 1448. (g) Y.-D Wu and S. Gellman, Acc.
Chem. Res. 2008, 41, 1231. (h) J. S. Nowick, Acc. Chem. Res.,
2
Angew. Chem., Int. Ed., 2011, 50, 3715. (j) M. Suzuki and K.
Hanabusa, Chem. Soc. Rev., 2009, 38, 967. (k) D. Das, S.
Maiti, S. Brahmachari and P. K. Das, Soft Matter 2011, 7,
7
8. (a) A. Arbaoui, C. Redshaw, N. M. Sanchez-Ballester, M. R. J.
008, 41, 1319. (i) D. Ke, C. Zhan, A. D. Q Li and J. Yao,
291.
1
Elsegood and D. L. Hughes, Inorg. Chim. Acta, 2011, 365, 96. 31. (a) T. R. Wagler, Y. Fang and C. J. Burrows, J. Org. Chem.
(
7
b) M. Merschky and C. Schmuck, Org. Biomol. Chem., 2009,
, 4895. c) L. Weselinki, E. Szyk and J. Jurczak, Tetrahedron
1989, 54, 1584; (b) B. Dangel, M. Clarke, J. Haley, D. Sames
and R. Polt, J. Am. Chem. Soc., 1997, 119, 10865; (c) C. L.
Weeks, P, Turner, R. R. Fenton and P. A. Lay, J. Chem. Soc.,
Dalton Trans., 2002, 931; (d) M. I. Burguete, F. Galindo, S. V.
Luis and L. Vigara, Dalton Trans., 2007, 4027; (e) J. Singh, G.
Hundal and R. Gupta, Eur. J. Inorg. Chem., 2008, 2052; f) R.
Shakya, A. Jozwiuk, D. R. Powell and R. P. Houser, Inorg.
Chem., 2009, 48, 4083.
Lett., 2011, 52, 381. (d) J. Paradowska, M. Pasternak, B. Gut,
B. Gryzzo and J. Mlynarski, J. Org. Chem., 2012, 77, 173. (e) F.
Adrian, M. I. Burguete, J. M. Fraile, J. I. García, E. García-
España, S. V. Luis, J. A. Mayoral, A. J. Royo, Eur. J. Inorg.
Chem., 1999, 2347.
1
2
9. S. V. Luis and I. Alfonso, Acc. Chem. Res., 2014, 47, 112.
0. (a) F. Galindo, J. Becerril, M. I. Burguete, S. V. Luis and L. 32. (a) R. Polt, B. D. Kelly, B. D. Dangel, U. B. Tadikonda, R. E.
Vigara, Tetrahedron Lett., 2004, 45, 1659; (b) B. Altava, M. I.
Burguete, N. Carbó, J. Escorihuela and S. V. Luis, Tetrahedron
Asymm., 2010, 21, 982.
Ross, A. M. Raitsimring and A. V. Astashkin, Inorg. Chem.,
2003, 42, 566; (b) S. Autzen, H.-G. Korth, R. Boese, H. De
Groot and R. Sustmann, Eur. J. Inorg. Chem., 2003, 1401.
2
1. (a) I. Alfonso, M. I. Burguete, F. Galindo, S. V. Luis and L. 33. G. A. McLachlan, G. D. Fallon, R. L. Martin and Leone Spiccia,
Vigara, J. Org. Chem., 2009, 74, 6130; (b) S. V. Luis, F. Inorg. Chem., 1995, 34, 254.
Galindo, M. I. Burguete and L. Vigara, J. Photobiol. 34. E. C. Constable, C. E. Housecroft, J. R. Price and J. A.
Photochem. A, 2010, 209, 61; (c) B. Altava, M. I. Burguete, N. Zampese, Cryst. Eng. Comm., 2010, 12, 3163.
Carbó, S. V. Luis, V. Martí and C. Vicent, Tetrahedron Lett., 35. E. García-España and S. V. Luis, Supramol. Chem., 1996, 6,
013, 54, 72. (d) V. Martí-Centelles, M. A. Izquierdo, M. I. 257.
Burguete, F. Galindo and S. V. Luis, Chem. Eur. J., 2014, 20, 36. (a) E. García-España, M.-J. Ballester, F. Lloret, J. M. Moratal,
2
7
and I. Alfonso, J. Org. Chem., 2014, 79, 4590.
2. (a) J. Becerril, B. Escuder, R. Gavara, S. V. Luis, J. F. Miravet,
Eur. J. Org. Chem. 2005, 481; (b) I. Alfonso, M. Bru, M. I.
465. (e) E. Faggi, A. Moure, M. Bolte, C. Vicent, S. V. Luis
J. Faus and A. Bianchi, J. Chem. Soc., Dalton Trans., 1988,
101; (b) M. I. Burguete, E. García-España, L. López-Diago, S.
V. Luis, J. F. Miravet, D. Sroczynski, Org. Biomol. Chem.,
2007, 5, 1935.
2
Burguete, E. García-Verdugo and S. V. Luis, Chem. Eur. J., 37. P. Gans, A. Sabatini, A. Vacca, Talanta, 1996, 43, 1739.
010, 16, 1246; (c) J. Rubio, I. Alfonso, M. I. Burguete and S. 38. L. Alderighi, P. Gans, A. Ienco, D. Peters, A. and Vacca, Coord.
V. Luis, Soft Matter, 2011, 7, 10737; (d) J. Rubio, I. Alfonso, Chem. Rev., 1999, 184, 311.
M. I. Burguete and S. V. Luis, Chem. Commun., 2012, 48, 39. (a) P. K. Glasoe, F. A. Long, J. Phys. Chem., 1960, 64, 188; (b)
2
2
Luis, Tetrahedron 2013, 69, 2302.
210; (e) J. Rubio, V. Martí-Centelles, M. I. Burguete and S. V.
A. K. Covington, M. Paabo, R. A. Robinson and R. G. Bates,
Anal. Chem., 1968, 40, 700.
3. (a) I. Alfonso, M. I. Burguete and S. V. Luis, J. Org. Chem. 40. a) MassLynx Software, version 4.0. b) Moco, S., Bino, R.J.,
2
2
2
2
006, 71, 2242; (b) I. Alfonso, M. I. Burguete, F. Galindo, S. V.
Vorst, O., Verhoeven, H.a., de Groot, J., van Beek, T. A.,
Vervoot, J. and R. de Vos, C. H., Plant Physiolgy, 2006, 41,
1205.
Luis and L. Vigara, J. Org. Chem. 2007, 72, 7947; (c) E. Faggi,
S. V. Luis and I. Alfonso, RSC Advances, 2013, 3, 11556.
4. (a) S. Blasco, M. I. Burguete, M. P. Clares, E. García-España,
J. Escorihuela and S. V. Luis, Inorg. Chem., 2010, 490, 7841;
(b) I. Martí, A. Ferrer, J. Escorihuela, M. I. Burguete and S. V.
Luis, Dalton Trans., 2012, 41, 6764.
5. (a) J. Becerril, M. Bolte, M. I. Burguete, F. Galindo, E. García-
España, S. V. Luis and J. F. Miravet, J. Am. Chem. Soc., 2003,
1
25, 6677; (b) S. Blasco, M. I. Burguete, M. P. Clares, E.
García-España, J. Escorihuela and S. V. Luis, Inorg. Chem.
010, 49, 7841; (c) I. Martí, A. Ferrer, J. Escorihuela, M. I.
2
Burguete and S. V. Luis, Dalton Trans., 2012, 41, 6764.
6. P. D. Wadhavane, M. A. Izquierdo, D. Lutters, M. I. Burguete,
M. J. Marin, D. A. Russell, F. Galindo and S. V. Luis, Org.
Biomol. Chem., 2014, 12, 823.
2
2
2
7. P. Paoletti, Pure Appl. Chem., 1984, 56, 491.
8. (a) B. Altava, M. I. Burguete, S. V. Luis, J. F. Miravet, E.
García-España, V. Marcelino and C. Soriano, Tetrahedron,
1
997, 53, 4751; (b) S. Andrés, B. Escuder, A. Domènech, E.
García-España, S. V. Luis, V. Marcelino, J. M. Llinares, J. A.
Ramírez and C. Soriano, J. Phys. Org. Chem., 2001, 14, 495.
9. a) F. Rosu, E. De Pauw and V. Gabelica, Biochimie, 2008, 90,
2
1
074; (b) E. B. Erba, K. Barylyuk, Y. Yang and R. Zenobi, Anal.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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