Tritopic phenanthroline and pyridine tail-tied aza-scorpiands

Article abstract of DOI:10.1039/b927418a

The synthesis of two new tritopic double-scorpiand receptors in which two equivalent 5-(2-aminoethyl)-2,5,8-triaza[9]-(2,6)-pyridinophane moieties have been linked with 2,6-dimethylpyridine (L1) or 2,9-dimethylphenanthroline (L2) units is reported for the

Full text of DOI:10.1039/b927418a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Tritopic phenanthroline and pyridine tail-tied aza-scorpiands†
Jorge Gonza´lez,a Jose´ M. Llinares,b Raquel Belda,a Javier Pitarch,a Concepcio´n Soriano,c Roberto Tejero,d
Begon˜a Verdejoa and Enrique Garc´ıa-Espan˜a*a
Received 7th January 2010, Accepted 9th March 2010
First published as an Advance Article on the web 24th March 2010
DOI: 10.1039/b927418a
The synthesis of two new tritopic double-scorpiand receptors in which two equivalent
5-(2-aminoethyl)-2,5,8-triaza[9]-(2,6)-pyridinophane moieties have been linked with
2,6-dimethylpyridine (L1) or 2,9-dimethylphenanthroline (L2) units is reported for the first time. Their
acid–base behaviour and Zn2+ coordination chemistry have been studied by pH-metric titrations,
molecular dynamic calculations, NMR, UV-Vis and steady-state fluorescence techniques. L1 and L2
behave, respectively, as hexaprotic and heptaprotic bases in the experimental conditions used (298.1
0.1 K, 0.15 mol dm-3 NaCl, pH range under study 2.0–11.0). These ligands are able to form mono-, bi-
and trinuclear Zn2+ complexes depending on the Zn2+-receptor molar ratio. Interaction of L1 and L2
with pyrophosphate (PPi), tripolyphosphate (TPP) and adenosine 5¢-triphosphate (ATP) has been
followed by pH-metric titrations, 1H and 31P NMR techniques and molecular dynamic analysis. Finally,
formation of mixed complexes Zn2+-L-PPi, Zn2+-L-TPP and Zn2+-L-ATP has been studied for both
receptors by potentiometric titrations.
TAR (trans activation responsive) element RNA that activates the
Introduction
synthesis of the full length HIV-mRNA.7
Multitopic ligands have received an increased interest in the
On the other hand, scorpiand molecules or lariat ethers are
last years. This interest stems from their potential applications
analogous terms describing compounds that have a central macro-
in fields such as molecular recognition, molecular devices, enzyme
cyclic core with a pendant arm including additional anchoring
mimicking and pharmaceutical chemistry.1–3
points.8–10 These molecules have attracted a lot of interest due to
the possibility of regulating the molecular reorganisation of their
flexible arm with respect to the macrocyclic core by coordination
to metal ions or/and changes in the hydrogen ion concentration
of the medium. Therefore, the preparation of new compounds
meeting features of double azamacrocycles and scorpiands is an
interesting goal due to their recognition capacity, molecular re-
arrangements and possible biomedical implications.
An elegant example of targeting biologically relevant anions
has been reported by J.-I. Hong, J. Yoon et al. These authors
designed a series of binuclear Zn2+ complexes that exhibited
selective fluorescence response when interacting with pyrophos-
phate (PPi).4 PPi is a biological important target because it is
the product of the hydrolysis of the nucleotide adenosine-5¢-
triphosphate (ATP) under cellular conditions and the detection of
PPi is being investigated as a real-time DNA sequencing method.5
Bicyclam, bicyclen and tricyclen molecules linked by different
alkyl and aryl groups display interesting activity as antiviral
drugs for the treatment of HIV-1 and HIV-2.6 Moreover, it has
been postulated that metal coordination can reinforce or decrease
the activity. In this sense, Kimura et al. reported that the Zn2+
complex of a tricyclen molecule, in which the tetraazamacrocycles
were connected by para-phenylene spacers, displayed the highest
inhibitory activity for the binding of the regulatory protein Tat to
Here we describe the synthesis, protonation, Zn2+ coordination
and interaction with PPi, tripolyphosphate (TPP) and ATP of
a couple of double-aza-scorpiand receptors in which two 5-
(2-aminoethyl)-2,5,8-triaza[9]-(2,6)-pyridinophane moieties have
been tied by their tails with 2,6-dimethylpyridine (L1) or 2,9-
dimethylphenanthroline (L2) linkers (Chart 1). We have addition-
ally studied by potentiometry the formation of mixed Zn2+-L-PPi,
Zn2+-L-TPP and Zn2+-L-ATP complexes with both receptors.
For means of clarity, with PPi4-, TPP5- and ATP4- we refer to the
fully deprotonated anions, while with PPi, TPP and ATP we will
be referring either to any of the species present in the multiprotic
systems independently of their protonation state or to the whole
polyprotic systems.
aInstituto de Ciencia Molecular (ICMOL), Departamento de Qu´ımica
Inorga´nica, Universidad de Valencia, Edificio de Institutos de Paterna,
Apartado de Correos 22085, 46071, Valencia, Spain. E-mail: enrique.garcia-
es@uv.es; Tel: 00 34 963544879
bInstituto de Ciencia Molecular (ICMOL), Departamento de Qu´ımica
Orga´nica, Fundacio´ General de la Universitat de Vale`ncia (FGUV), Edificio
de Institutos de Paterna, Apartado de Correos 22085, 46071, Valencia, Spain
Results and discussion
cDepartamento de Qu´ımica Orga´nica, Avda. Vicent Andre´s Estelle´s, S/n,
46100 Burjassot, Valencia, Spain
Synthesis of the receptors
dDepartamento de Qu´ımica-F´ısica, C/ Dr. Moliner 50, 46100 Burjassot,
Valencia, Spain
The synthesis of L1 and L2 has been carried out reacting two moles
of scorpiand 5-(2-aminoethyl)-2,5,8-triaza[9]-(2,6)-pyridinophane
(L3) that some of us have previously reported,10 with one
† Electronic supplementary information (ESI) available: Additional exper-
imental details and characterization data. See DOI: 10.1039/b927418a
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Table 1 Logarithms of the stepwise protonation constants for the proto-
nation of L1 and L2 determined in 0.15 mol dm-3 NaCl at 298.1 K
Reactiona
L1
L2
H + L = HL
9.96(2)b
9.52(2)
8.51(2)
7.64(2)
6.78(3)
5.94(4)
—
10.04(1)
9.80(1)
8.88(1)
8.15(1)
6.78(1)
5.95(1)
2.25(2)
51.84
H + HL = H2L
H + H2L = H3L
H + H3L = H4L
H + H4L = H5L
H + H5L = H6L
H + H6L = H7L
c
Log b
48.34
a Charges omitted for clarity. b Values in parentheses are standard deviation
in the last significant figure. c log b = R log K.
Chart 1
Fig. 1 Molar fraction distribution diagram for the system L2-H+ and
steady-state fluorescence emission titration curve of L2 (lexc = 267 nm) for
a 1 ¥ 10-5 mol dm-3 aqueous solution in 0.15 mol dm-3 NaCl at 298.1
0.1 K. (ꢀ) monomer emission followed at 376 nm.
mole of pyridine-2,6-dicarboxaldehyde or 1,10-phenanthroline-
2,9-dicarboxaldehyde in dried ethanol, followed by in situ reduc-
tion with sodium borohydride (see Scheme 1 in the ESI,† and the
Experimental section). The compounds were finally precipitated as
their hydrochloride salts. Once optimized, this synthetic procedure
permits the new receptors L1 and L2 to be obtained on a gram
scale.
six protonation constants observed for both systems make the
hexaprotonated species H6L6+ prevail below pH 6 for L1, and in
a wide 2.5–6.0 pH range for L2.11 While the six first protonation
steps of both receptors should occur at the six secondary amino
groups present in both receptors, the seventh protonation step of
L2 should involve the connecting phenanthroline group as it has
been proved by NMR, UV-Vis and fluorescence emission spectra.
Acid–base studies
To obtain the constants for the interaction of L1 and L2 with
different guests in aqueous media, the previous determination
of the protonation constants of both hosts and guest species
in the same experimental conditions is required. Moreover, the
knowledge of which are the protonation sites in the receptors at the
studied pH values is essential to understand and rationalize their
coordinating ability towards either cationic or anionic substrates.
The protonation constants of L1 and L2 were obtained by
pH-metric titrations carried out at 298.1 K at 0.15 mol dm-3
NaCl (see Experimental section for further details). Although
chloride anions can interfere in the coordination of metal ions
and anions, the scarce solubility of L2 in perchlorate salts as
well as the biological relevance of NaCl led us to use this salt
as supporting electrolyte. Since all constants, namely protonation
constants of the receptors, formation constants of Zn2+ complexes,
binding constants with the polyphosphate anions and ATP, and
formation constants of mixed complexes have been conducted in
the same experimental conditions, the results presented here are
self-consistent.
1
The H NMR spectra show, below pH 3, clear downfield shifts
for signals of the phenanthroline protons labelled as P3, P4 and
P6 (for the labelling, see Chart 1), which supports protonation of
a phenanthroline nitrogen (see Fig. S2 in the ESI† in which the
variations of the 1H NMR signals with the pH are plotted for L2).
The UV-Vis titrations of L2 also show significant changes in the
200–400 nm region in correspondence with the protonation of the
phenanthroline ring (Fig. 2). Treatment of the spectrophotometric
data with the SPECFIT program12 allows for deriving a protona-
tion constant of 2.2(1) logarithmic units in good agreement with
the constant derived from the potentiometric titrations.
Table 1 gathers the stepwise protonation constants calculated
for the protonation of bis-scorpiands L1–L2. The distribution
diagrams are collected in Fig. S1 of the ESI† and in Fig. 1.
L1 presents in the pH range of study (pH = 2.0–11.0) just
six measurable protonation steps (Table 1, Fig. S1†) while L2
displays an additional seventh protonation step (Fig. 1). The high
Fig. 2 Spectrophotometric titration of L2 vs. pH recorded in H2O; T =
298.1 K; l = 1 cm; I = 1.0 mol dm-3 (NaCl); [L2] = 8.6 ¥ 10-5 mol dm-3;
(1) pH = 4.5; (2) pH = 1.0.
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The variation with the pH of the emission intensity at 376 nm
of L2 shows a bell-shaped curve with the maximum emission
corresponding to the pH range where the hexaprotonated species
(H6L26+) prevails in solution as can be seen in Fig. 1. Either
a protonation or a deprotonation process yields a decrease in
fluorescence. The lower emission of the H7L27+ species can be
ascribed to the fact that protonation of the phenanthroline can
stabilize the poorly emissive np* state with respect to the pp*
state, leading to inversion of states and thus to a decrease of the
fluorescence emission.13
the labelling, see Chart 1), suggests that the first two protonations
occur to a larger extent at the macrocyclic secondary nitrogen
atoms. The next two protons will be preferentially binding the
nitrogens of the arms as denoted by the larger downfield shifts
of protons labelled as 9 in the 7.0–8.5 pH range. In agreement
1
with the calculations, the variations in the H NMR data also
suggest the formation of different intramolecular hydrogen bonds
between protonated and non-protonated sites and the pyridine in
the intermediate protonation sites.
Molecular dynamic calculations14 suggest that these receptors
present two major different families of conformers, (i) a family
with more closed conformations that is observed for protonation
degrees from 0 to 5 and (ii) a family with more open confor-
mations for higher protonation degrees (Fig. 3). In the closed
conformations, intramolecular hydrogen bond networks involving
the protonated ammonium groups at each state as hydrogen bond
donors and the deprotonated amino groups and the pyridine
nitrogen atoms are observed. The composition and number of
hydrogen bonds would be changing from one protonation state
to another. p–p Stacking between the central pyridine ring and
the pyridine ring of one of the macrocycles is also observed in the
closed conformation. In the case of L2, the molecular dynamics
studies indicate that protonation of the phenanthroline ring in the
heptaprotonated species leads to an almost completely extended
conformation so that electrostatic repulsion between the positive
charges are minimized (Fig. 3).
Interaction with metal ions and anions
To check the coordination capabilities of these macrocycles, we
have carried out a preliminary study of the interaction of L1 and
L2 with Zn2+. Moreover, we have studied the interaction of L1 and
L2 with PPi, TPP and ATP. Finally, we have checked the formation
of mixed complexes in the systems Zn2+-L-PPi, Zn2+-L-TPP and
Zn2+-L-ATP (L = L1, L2).
Interaction with Zn2+
Potentiometric studies of the binary Zn2+-L1 and Zn2+-L2 sys-
tems show in both cases formation of mononuclear species of
[ZnHrL](2+r)+ stoichometries with r = 4–0, binuclear [Zn2HrL](4+r)+
species with r = 1, 0, -1 and trinuclear trihydroxylated species
for L1. In the case of L2 additional binuclear and trinuclear
dihydroxylated species have also been detected. The stability
constants for these complexes along with those formed by the
precursor scorpiand receptors L3 and L4 (Chart 1) are collected
in Table 2.10,15
As regards the mononuclear complexes, the first aspect that
deserves to be commented on is the significant difference in
stability between the ZnL2+ complexes of both receptors, L1
and L2. The stability constant of ZnL12+ is higher than those
obtained for the Zn2+ complexes of the precursor receptors L3 and
comparable to the receptor L4 (Table 2). Taking into account these
values and the crystal structures obtained for the Cu2+ complexes
of L3 and L4,10,15 it can be suggested that the binding of the
metal in the ZnL12+ species is likely involving the four nitrogens
of the macrocyclic unit, the secondary nitrogen of the tail and the
pyridine nitrogen of the linker (Fig. 4a).
Fig. 4 Possible coordination modes in complexes ZnL12+ (a) and Zn2L14+
(b).
Fig. 3 Minimum energy conformers for receptors H2L2 (A), H6L2 (B)
and H7L2 (C).
In the case of ZnL22+, however, the metal ion would be
pentacoordinated by the four nitrogen atoms of the macrocycle
and the secondary nitrogen atom of the linker.
In both systems, formation of binuclear complexes is observed
above pH 4 for Zn2+:L 2 : 1 molar ratio (see distribution diagrams
in ESI, Fig S3b and Fig. S4b†). The stepwise constants for the
formation of the binuclear complexes (ZnL2+ + Zn2+ = Zn2L4+; log
1H NMR experiments performed at variable pH for both
receptors show that all 1H signals experience downfield shifts
upon protonation indicating that protons are shared between the
different sites (ESI, Fig. S1 and S2†). However, for L1 the slightly
larger downfield shift (Dd = 0.87 ppm, Fig. S1†) observed in the
8.5-10.0 pH range for the singlet signal of pyridyl protons 1 (for
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Table 2 Logarithms of the stability constants for the formation of mononuclear, binuclear and trinuclear complexes of Zn2+:L1, L2, L3 and L4 calculated
in 0.15 mol dm-3 NaCl at 298.1 0.1 K
Reactiona
L1
L2
L3
L4
ZnH3L + H = ZnH4L
4.24(5)b
4.23(5)
7.7(1)
9.3(1)
19.8(1)
—
4.83(2)
32.16(2)
12.4(1)
-10.6(1)
—
3.61(1)
6.28(4)
8.48(4)
10.2(1)
16.9(1)
—
4.37(1)
33.12(1)
16.2(1)
-10.05(2)
-11.9(1)
20.46(2)
11.50(3)
—
—
3.95(4)
5.23(2)
17.42(4)
—
—
—
—
—
—
—
—
3.34(2)
18.97(1)
-11.3(3)
—
—
—
—
—
—
—
ZnH2L + H = ZnH3L
ZnHL + H = ZnH2L
ZnL + H = ZnHL
Zn + L = ZnL
ZnL +H2O = ZnL(OH) + H
Zn2L + H = Zn2HL
2Zn + L = Zn2L
ZnL + Zn = Zn2L
Zn2L + H2O = Zn2L(OH) + H
Zn2L(OH) + H2O = Zn2L(OH)2 + H
3Zn + L + 2H2O = Zn3L(OH)2 + 2H
3Zn + L + 3H2O = Zn3L(OH)3 + 3H
—
—
—
—
11.5(1)
a Charges omitted. b Values in parenthesis show standard deviation in the last significant figure.
K = 12.4 for L1 and log K = 16.2 for L2, (Table 2) show the absence
of positive cooperativity in this process. Moreover, the much lower
stepwise value obtained for the formation of Zn2L14+ indicates
that the binding of the second metal ion will either involve a lower
number of nitrogens than in L2 or will require of a molecular
reorganisation of the coordination sphere, implying breaking and
bond formation (Fig. 4b).
However, when passing from 2 : 1 to 3 : 1 molar ratios, the largest
changes are seen for the phenanthroline signals which shift
downfield and broaden considerably, suggesting that the third
metal ion is bound at this region of the molecule. The lower number
of coordinated nitrogens involved in the coordination sphere of the
last metal ion would be favouring the formation of hydroxylated
species.
The distribution diagrams (ESI, Fig. S3 and S4†) show that the
nuclearity of the species formed depends very much on the Zn2+-
receptor molar ratio. Trinuclear complexes are observed above pH
ca. 8 for 3 : 1 Zn2+ receptor molar ratios for L1 and pH ca. 9 for L2
(ESI, Fig. S3c and S4c†). Although binuclear complexes have been
reported in many systems consisting of two macrocycles linked by
different alkyl or aryl bridges, to our knowledge, the number of
systems in which trinuclear species has been evidenced is much
more scarce.16,17
Fig. 5 collects the aromatic zone of the 1H NMR spectra
recorded at pD = 10 for the system Zn2+:L2, where formation
of mono-, bi- and trinuclear species is observed (see ESI, Fig. S3
and S4†). The 1H spectra show significant changes in the pyridine
signals when the molar ratio is increased from 1 : 1 to 2 : 1.
Interaction with PPi, TPP and ATP
Interaction of L1 and L2 with PPi, TPP and ATP has been
followed by potentiometric studies, 1H and 31P NMR spectroscopy,
and molecular dynamic analysis. The analysis of the pH-metric
titrations with the HYPERQUAD set of programs18 gave the
model and values of the cumulative constants collected in Table
S2 of the ESI.†
For all the studied systems only anion-receptor adducts of 1 : 1
stoichiometry have been found. The protonation degrees ranged
from 1 to 7 for the systems L1-PPi and L1-ATP, from 4 to 7
for the system L1-TPP, from 1 to 8 for L2-PPi and L2-ATP, and
from 2 to 8 for L2-TPP. As shown by the distribution diagrams
collected in the ESI (Fig. S5 and Fig. S6†), the adducts prevail in a
wide pH range for all the studied systems. The 1 : 1 anion-receptor
stoichiometry was also checked by 31P NMR spectroscopy for the
systems L1-PPi, L1-TPP, L2-PPi and L2-TPP. 31P NMR spectra
of D2O solutions in which increasing amounts of L1 or L2 were
added either to PPi or to TPP solutions were registered at pD =
5.9 and 7.5 indicating a 1 : 1 anion-receptor stoichiometry (Fig. 6,
ESI, Fig. S7, S8 and S9†).
Fig. 5 1H NMR of Zn2+:L2 in different molar ratios at pD = 10.0.
2370 | Org. Biomol. Chem., 2010, 8, 2367–2376
Fig. 6 Plot of Dd 31P signal of PPi versus [PPi]/[L1] at pD = 5.9 in D2O.
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Table 3 Logarithms of the stepwise constants for the interaction of L1
Similar results were obtained for ATP, the spectra were recorded
in this case at pD = 7.0. The downfield shifts observed for
the signals of Pg and Pb of ATP (Dd = 3.9 ppm and Dd =
1.6 ppm, respectively) in the 31P NMR spectra of the system
L1-ATP confirm the interaction (Fig. 7). As usually happens for
these systems, the shift observed for the chemical signal of Pa is
more reduced Dd = 0.1 ppm.19 Interestingly enough, the 31P NMR
spectrum of the L1-ATP system at pH = 7.0 is coincident with the
spectrum of free ATP at pD = 9.0, denoting that the interaction
with the polyamine makes ATP more acidic. Moreover, the 31P
NMR spectrum at pD = 9.0 of the L1-ATP system does not
present any difference with the spectrum of free ATP at the same
pD. In fact, at this pD, ATP is already in its fully deprotonated
ATP4- form and therefore, interaction with the polyammonium
receptor cannot produce any change in its protonation degree
(Fig. 7). Similar results are obtained for the system L2-ATP (ESI,
Fig. S10†).
with PPi, TPP and ATP calculated in 0.15 mol dm-3 NaCl at 298.1 0.1 K
Reactiona
A = PPi4-
A = TPP5-
A = ATP4-
HL + A = HLA
3.12(4)b
3.90(2)
4.07(5)
5.03(4)
4.54(4)
6.49(3)
5.13(3)
5.52(3)
5.51(3)
5.83(3)
—
—
—
4.04(4)
4.13(5)
5.84(1)
5.07(1)
5.09(3)
3.9(1)
4.5(1)
3.63(4)
3.40(3)
3.48(4)
4.36(2)
—
5.54(1)
5.65(1)
5.39(1)
3.84(2)
—
H2L + A = H2LA
H3L + A = H3LA
H4L + A = H4LA
H3L + HA = H4LA
H5L + A = H5LA
H4L + HA = H5LA
H5L + HA = H6LA
H6L + HA = H7LA
H6L + H2A = H8LA
a Charges omitted. b Values in parenthesis show standard deviation in the
last significant figure.
Table 4 Logarithms of the stepwise constants for the interaction of L2
with PPi, TPP and ATP calculated in 0.15 mol dm-3 NaCl at 298.1 0.1 K
Reactiona
A = PPi4-
A = TPP5-
A = ATP4-
HL + A = HLA
3.3(1)b
3.58(4)
3.91(6)
4.60(3)
4.61(4)
5.98(4)
4.62(4)
5.43(3)
5.88(3)
5.32(4)
—
4.52(3)
4.95(3)
5.34(3)
5.86(2)
—
6.88(2)
6.99(2)
6.88(1)
7.07(2)
6.64(3)
H2L + A = H2LA
H3L + A = H3LA
H4L + A = H4LA
H3L + HA = H4LA
H5L + A = H5LA
H4L + HA = H5LA
H5L + HA = H6LA
H6L + HA = H7LA
H6L + H2A = H8LA
4.11(1)
4.09(3)
5.05(2)
5.65(2)
6.26(2)
5.49(2)
6.32(2)
6.66(2)
5.65(3)
a Charges omitted. b Values in parenthesis show standard deviation in the
last significant figure.
different systems and to establish selectivity ratios is to use effective
constants.19 The effective constants Keff are calculated at every pH
value as the quotient between the overall amount of complexed
species and the overall amounts of free receptor and substrate
independently of their protonation degree.
Fig. 8 and 9 represent the plot of the logarithms of the effective
conditional constant vs. pH for the studied systems.
Fig. 7 31P NMR spectra of L1 at pD = 7.0 and 9.0 in D2O.
To translate the values of the cumulative constants into stepwise
constants representative of the actual equilibrium occurring in
solution, the protonation degrees of substrate and receptors at
every equilibrium need to be known and therefore, the basicity
constants of substrates and receptors have to be taken into
account. By doing this, the stepwise constants collected in Table 3
and 4 have been calculated. These constants clearly show that L2
interacts stronger with TPP and ATP than L1, while PPi displays
slightly higher constants with L1 than with L2.
Fig. 8 Plot of the logarithms of the effective constants for the systems
L1-PPi, L1-TPP and L1-ATP.
Nevertheless, there is a number of equilibria in which the
substrates and receptors have very close basicities and thus, it
is difficult to decide if the proton will be in the substrate or
in the guest species or even shared between them. The most
unambiguous way to compare the relative stabilities of the
These plots show for L1 the stability trend PPi ª ATP > TPP
while for L2 it would be ATP > TPP >PPi.
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much disrupted by the interaction with ATP and the stacking of
adenine will occur with the macrocyclic pyridines. This is further
confirmed by 1D selective NOE NMR experiments that show
intense effects between A2 of adenine and the pyridyl protons of
the macrocycles (see ESI, Fig. S12†).
For L2, the anomeric and aromatic signals of ATP and the
signals from the phenanthroline ring show an important upfield
shift, in contrast with the pyridine ring signals that experiences
a downfield shift (Fig. 11). Moreover, 1D selective NOE NMR
experiments show that irradiation of A2 adenine affects protons
of phenanthroline labeled as P3 and P4 (see ESI, Fig. S13†).
These shifts indicate that while p–p stacking between adenine and
phenanthroline is produced following the formation of the adduct
species, the stacking between pyridine and phenanthroline that
occurred in the free ligand is being disrupted.
Molecular dynamic calculations for the interaction of H6L22+
and ATP4-, which are the species predominating in solution at
neutral pH, show that effectively in the family of minimum energy
conformers the adenine ring is stacked with the phenanthroline
moiety while no internal stacking is produced in the receptor. The
phosphate chain is placed between the macrocyclic rings so that
maximum electrostatic interaction is achieved with the positive
charges of the receptor (Fig. 12). Fig S14† collects the family of ten
minimum energy conformers, which show that this arrangement
is conserved in all of them.
Fig. 9 Plot of the logarithms of the effective constants for the systems
L2-PPi, L2-TPP and L2-ATP.
1H NMR spectra coupled with molecular dynamic studies
provide interesting structural insights about the interaction of ATP
with L1 and L2. The 1H NMR spectra of the systems L1-ATP and
L2-ATP recorded at pH 7.0 and 9.0 show upfield shifts of the
aromatic and anomeric signals of ATP (Fig. 10 and 11, and ESI,
Fig. S11†) and of selected signals of the aromatic regions of the
receptors denoting that stacking occurs in both ligands. For L2,
the observed shifts are, however, larger than in the case of L1 in
correspondence with the presence of the extended phenanthroline
ring.
Fig. 10 Upfield region including aromatic and anomeric signals of the
1H NMR spectra of ATP, L1 and L1-ATP recorded in D2O at pD = 7.0.
Fig. 12 Minimum energy conformer calculated for the interaction of
H6L26+ with ATP.
Formation of mixed complexes
It is well known that metal complexes either with coordinatively
unsaturated sites or with ancillary ligand in its coordination
sphere can interact with anionic species through coordinative
bonds. In our case, although the mononuclear and binuclear
complexes seem to be penta or hexacoordinated, the formation
of trinuclear species should provide vacant coordination sites for
anion interaction. Moreover, even pentacoordinated Zn2+ sites
might in principle interact with further ligands due to the facility
of this 3d10 metal ion to increase its coordination number to
six. Additionally, in the mononuclear complexes the presence
of a metal-free macrocyclic unit that can bear protonation may
give rise to mixed binding modes. The metal site can provide
coordinative bonds while the metal-free macrocycle can interact
Fig. 11 Upfield region including aromatic and anomeric signals of the
1H NMR spectra of ATP, L2 and L2-ATP recorded in D2O at pD = 7.0.
Inspection of the shifts of the receptor signals permit some
conclusions to be drawn about the structure of the adducts. In
the case of L1, while the NMR signals of the protons of the
macrocyclic pyridine rings shift downfield, those of the protons
of the pyridine ring in the central bridge practically do not move,
which means that the conformation of the macrocycle is not very
2372 | Org. Biomol. Chem., 2010, 8, 2367–2376
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Table 5 Logarithms of the stability constants for the formation of the
mixed complexes of Zn2+ and PPi, TPP and ATP with L1 calculated in
0.15 mol dm-3 NaCl at 298.1 0.1 K
Table 6 Logarithms of the stability constants for the formation of the
mixed complexes of Zn2+ and PPi, TPP and ATP with L2 calculated in
0.15 mol dm-3 NaCl at 298.1 0.1 K
Reactiona
A = PPi4- A = TPP5- A = ATP4-
Reactiona
A = PPi4- A = TPP5- A = ATP4-
6H + Zn + L + A = ZnH6LA
5H + Zn + L + A = ZnH5LA
4H + Zn + L + A = ZnH4LA
3H + Zn + L + A = ZnH3LA
2H + Zn + L + A = ZnH2LA
H + Zn + L + A = ZnHLA
—
—
59.78(4)b
55.72(8)
52.61(3)
47.44(4)
40.71(4)
—
48.23(2)
43.48(2)
37.09(3)
27.50(4)
6H + Zn + L + A = ZnH6LA
5H + Zn + L + A = ZnH5LA
4H + Zn + L + A = ZnH4LA
3H + Zn + L + A = ZnH3LA
2H + Zn + L + A = ZnH2LA
H + Zn + L + A = ZnHLA
Zn + L + A = ZnLA
—
64.86(6)
61.70(1)
56.12(8)
49.88((5)
42.34(5)
33.90(5)
24.50(5)
53.21(3)
47.79(4)
40.00(6)
29.97(6)
63.73(3)b
60.54(1)
55.56(6)
49.15(5)
41.55(5)
32.26(6)
21.74(8)
52.09(1)
46.19(2)
36.31(6)
27.50(4)
59.1(1)
54.90(4)
48.69(6)
41.13(6)
32.40(8)
58.01(8)
54.10(3)
48.30(4)
40.92(4)
32.22(9)
50.16(3)
44.77(4)
36.98(5)
—
62.50(1)
57.20(5)
49.54(5)
42.21(6)
33.45(4)
23.50(7)
54.52(2)
49.79(3)
41.59(5)
31.45(5)
2H + 2Zn + L + A = Zn2H2LA 50.29(4)
H + 2Zn + L + A = Zn2HLA
2Zn + L + A = Zn2LA
H2O + 2Zn + L + A =
Zn2LA(OH) + H
3Zn + L + A = Zn3LA
H2O + 3Zn + L + A =
Zn3LA(OH) + H
44.58(7)
36.43(4)
—
2H + 2Zn + L + A = Zn2H6LA
H + 2Zn + L + A = Zn2HLA
2Zn + L + A = Zn2LA
H2O + 2Zn + L + A =
—
—
43.56(2)
35.16(3)
41.73(2)
33.37(3)
Zn2LA(OH) + H
2H + 3Zn + L + A = Zn3H2LA
H + 3Zn + L + A = Zn3HLA
3Zn + L + A = Zn3LA
—
—
55.66(3)
50.99(4)
42.8(1)
53.99(4)
49.27(4)
41.11(4)
52.71(4)
47.76(4)
39.24(7)
a Charges omitted. b Values in parenthesis show standard deviation in the
last significant figure.
H2O + 3Zn + L + A =
34.82(4)
Zn3LA(OH) + H
2H2O + 3Zn + L + A =
31.50(6)
29.48(6)
25.3(1)
Zn3LA(OH)2 + 2H
a Charges omitted. b Values in parenthesis show standard deviation in the
last significant figure.
through hydrogen bonds and when protonated also through
charge-charge interactions with the anion.
We have studied the interaction of the Zn2+ complexes of L1 and
L2 with PPi, TPP and ATP. For TPP, complexes of stoichiometries
[ZnHrLTPP](r-3)+ (L1, r = 1–5; L2, r = 0–6), [Zn2HrLTPP](r+1)+
(L1, r = 0–2; L2, r = -1–2) and [Zn3LTPP](2+r)+ (L1, r = -1–
0; L2, r = -2–1), and for ATP the complexes of stoichiometries
[ZnHrLATP](r-2)+ (L1, r = 2–6; L2, r = 0–6), [Zn2HrLATP]r+ (L1,
r = -1–2; L2, r = -1–2) and [Zn3L1ATP](2+r)+ (L1, r = -1–0;
L2, r = -2–2) have been detected. In the case of PPi complexes
of stoichiometries [ZnHrLPPi](r-2)+ (L1, r = 1–5; L2, r = 0–5),
[Zn2HrLPPi]r+ (L1, r = 0–2; L2, r = -1–2) while for L2 complexes
of [Zn3HrL2PPi](r+2)+ (r = -2–1) were also detected. The occurrence
of precipitation when working in Zn2+ : L1 : PPi 3 : 1 : 1 molar ratios
made the data not amenable to analysis.
The cumulative binding constants are presented in Table 5 and
6. Fig. 13 and 14 collect, respectively, the distribution diagram
for the systems Zn2+-L1-ATP and Zn2+-L2-ATP for molar ratios
1 : 1 : 1, 2 : 1 : 1 and 3 : 1 : 1.
The distribution diagrams in Fig. 13 and 14, and the distribution
diagrams for the other studied systems, which are collected in the
ESI (Fig. S15–S18†) show that the mixed complexes predominate
throughout a wide pH range. Binary anion-receptor complexes
are only observed at acidic pH values where the zinc complexes
are not yet formed. Also disruption of the ternary complexes is
observed for Zn2+ : L 3 : 1 molar ratio at basic pH values, where
the hydroxo anions compete with ATP for binding to the anion.
Decomposition of the cumulative constants into representative
stepwise constants is rather cumbersome due to the multiple
overlapped equilibria occurring in solution. However, one way
of comparing the efficiency of the Zn2+-L1 and Zn2+-L2 to bind
ATP is to calculate the distribution diagrams for the Zn2+-L1-L2-
ATP system in appropriate molar ratios and represent the amount
of ATP coordinated to Zn2+-L1 or to Zn2+-L2 against the pH. As
an example, Fig. 15, which collects such representations for the
Zn2+-L1-L2-ATP system for molar ratios 2 : 1 : 1 : 1, 4 : 1 : 1 : 1 and
6 : 1 : 1, puts into evidence that, in general and similarly to what
happened in the binary systems, ATP is bound preferentially by
Fig. 13 Distribution diagrams of the species for the Zn2+:L1:ATP systems
as a function of pH in aqueous solution in 0.15 mol dm-3 at 298.1 K; [L1] =
1 ¥ 10-3 mol dm-3, [ATP] = 1 ¥ 10-3 mol dm-3. (a) [Zn+2] = 1 ¥ 10-3 mol dm-3,
(b) [Zn+2] = 2 ¥ 10-3 mol dm-3 and (c) [Zn+2] = 3 ¥ 10-3 mol dm-3.
the ZnL2 complexes. Similar conclusions can be derived for the
other two anions (ESI, Fig. S19–S20†).
A question that remains open is whether the introduction of
the metal ions leads to greater amounts of complexation of the
anions with respect to the systems in the absence of metal ions.
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Fig. 14 Distribution diagrams of the species for the Zn2+:L2:ATP systems
as a function of pH in aqueous solution in 0.15 mol dm-3 at 298.1 K; [L2] =
1 ¥ 10-3 mol dm-3, [ATP] = 1 ¥ 10-3 mol dm-3. (a) [Zn+2] = 1 ¥ 10-3 mol dm-3,
(b) [Zn+2] = 2 ¥ 10-3 mol dm-3 and (c) [Zn+2] = 3 ¥ 10-3 mol dm-3.
Fig. 15 Representation of the percentages of complexed ATP to L1, L2
and to Zn2+-L1 and to Zn2+-L2 versus pH calculated from distribution
diagrams of the species for the Zn2+:L1:L2:ATP calculated for concentra-
tions dm-3 at 298.1 K; [L1] = [L2] [ATP] = 1 ¥ 10-3 mol dm-3. (a) [Zn+2] =
2 ¥ 10-3 mol dm-3, (b) [Zn+2] = 4 ¥ 10-3 mol dm-3 and (c) [Zn+2] = 6 ¥
10-3 mol dm-3.
To address this point, we have plotted the amounts of complexed
anionic substrates either in the absence of metal or in the presence
of one, two and three equivalents of Zn2+ (Fig. 16 and ESI, S21–
22†). Fig. 16 shows this representation for ATP. It can be seen that
although the addition of the metal ion favours the complexation of
the anion at basic pH values, the percentages of complexed anions
are quite close in the remaining pH window. As above mentioned,
only at the acidic pH values in which formation of Zn2+ complexes
does not occur, the binary species anion-L predominate.
These results support again the high coordination numbers
inferred from the speciation studies for the Zn2+ complexes. In
these complexes, the nitrogen atoms will either fully or almost fully
occupy the coordination spheres and there is no available room for
new ligands to get close to the metal and therefore, the interaction
with the anion should mainly be of charge to charge nature. It
is only for the trinuclear complexes where significant differences
in the amounts of complexed anionic species are found for the
free and complexed systems. This can be ascribed to the fact that
there are not enough nitrogen atoms to saturate the coordination
sphere of the third metal ion. Nevertheless, as the metal complex
almost quantitatively complex the anions in the form of mixed
species, this should represent a new binding mode that can be of
relevance in various respects, one of them the likely interaction of
these systems with nucleic acids, which we are currently exploring.
Fig. 16 Overall amounts of complexed anions in the binary and ternary
systems of ATP for (a) L1 and (b) L2.
dimethylphenanthroline linkages is achieved through a straight
through synthetic procedure that permits the compounds to be
obtained on a gram scale. The number of nitrogen atoms is
sufficient to provide penta- or even hexacoordinated mononuclear
and binuclear complexes. Both ligands interact strongly with PPi,
TPP and ATP as proved by pH-metric and NMR techniques.
Conclusions
The synthesis of two new tail-tied aza macrocycles in which
two pyridinophane scorpiand equivalent units have been
covalently connected through 2,6-dimethylpyridine or 2,9-
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Stacking interactions are clearly evidenced for the interaction of
ATP with L2 containing condensed aromatic rings. Although
formation of mixed complexes Zn2+-L-anion occurs at a large
extent, the amounts of complexed anion by the Zn2+-L complexes
are only clearly higher than those attained by the free ligand at
basic pHs. The versatility and different binding modes for anionic
species displayed by these tail-tied receptors make them appealing
candidates for interaction and activation of nucleic acids. We are
currently exploring this point.
etc.) has been fully described elsewhere.21 The acquisition of the
EMF data was performed with the computer program PASAT.22
The reference electrode was an Ag/AgCl electrode in saturated
KCl solution. The glass electrode was calibrated as a hydrogen
ion concentration probe by titration of previously standardized
amounts of HCl with CO2-free NaOH solutions and the equivalent
point determined by the Gran’s method,23 which gives the standard
potential, E◦¢, and the ionic product of water (pKw = 13.73(1)).
The computer program HYPERQUAD was used to calculate
the protonation and stability constants.18 The HYSS24 program
was used to obtain the distribution diagrams The pH range
investigated was 2.5-11.0. In the binary Zn2+-L1 and Zn2+-L2
systems, the concentration of Zn2+ and of the ligands ranged from
1 ¥ 10-3 to 5 ¥ 10-3 mol dm-3 with Zn2+ : L molar ratios varying
from 2 : 1 to 1 : 2. In the binary PPi-L, TPP-L and ATP-L systems
concentrations of the anion and of the receptors go from 1 ¥
10-3 mol dm-3 to 5 ¥ 10-3 mol dm-3. The protonation constants
for ATP, PPi and TPP were either taken from ref. 19 (ATP) or
redetermined in our experimental conditions.
For the ternary systems, aqueous solutions at acidic pH
containing Zn2+ : L : anion in 1 : 1 : 1, 2 : 1 : 1 or 3 : 1 : 1 molar ratios
were titrated with NaOH solutions. The formation constants of
Zn2+-PPi, Zn2+-TPP and Zn2+-ATP were taken from the literature25
or re-determined under our experimental conditions (PPi, log
bZnL = 9.52(1); TPP, log bZnL = 6.68(1), log bZnHL = 11.78(2),
log bZnL(OH) = -2.21(2). The titration curves for each system (at
least two titrations, ca. 200 experimental) were treated either as a
single set or as separated curves without significant variations in
the values of the stability constants.
Experimental section
Synthesis of L1
5-(2-Aminoethyl)-2,5,8-triaza[12]-2,6-pyridinophane10 (1.04 g,
4.16 mmol) and 2,6-pyridinedicarboxaldehyde (0.28 g, 2.08 mmol)
were dissolved in 40 mL anhydrous ethanol and the mixture was
stirred for 2 h at room temperature. NaBH4 (0.78 g, 20.70 mmol)
was then added and the resulting solution stirred for 2 h at room
temperature. The ethanol was removed under reduced pressure.
The resulting residue was treated with H2O (10 mL) and extracted
with CH2Cl2 (3 ¥ 20 mL). The organic phase was removed
at reduced pressure, and the resulting residue was dissolved in
ethanol and precipitated as hydrochloride salt of L1 in 63% yield
(see Scheme 1 in the ESI†). mp: 223–225 ◦C. 1H NMR (300 MHz,
D2O): d (ppm) = 7.64 (t, 2H, J = 8 Hz), 7.63 (t, 1H, J = 8 Hz),
7.18 (d, 2H, J = 8 Hz), 7.13 (d, 4H, J = 8 Hz), 4.32 (s, 8H), 4.18 (s,
4H), 3.11-3.16 (m, 4H), 2.85-2.96 (m, 12H), 2.62-2.65 (m, 8H). 13
C
NMR (75.43 MHz, D2O): d (ppm) = 140.1, 139.7, 123.5, 122.5,
51.4, 51.2, 50.9, 49.8, 46.3, 43.5.
When more than one model could fit the experimental data, the
most reliable chemical model was chosen by performing F tests at
the 0.05 confidence level.26,27
Calc for C33H51N11·6HCl 4H2O: C, 44.4; H, 7.3; N, 17.3. Found:
C, 44.6; H, 7.7; N, 16.5. MS (FAB) m/z 601 [M]+
Synthesis of L2
NMR measurements
5-(2-Aminoethyl)-2,5,8-triaza[12]-2,6-pyridinophane10 (1.04 g,
4.16 mmol) and 1,10-phenanthroline-2,9-dicarboxaldehyde20
(0.49 g, 2.08 mmol) were dissolved in 40 mL anhydrous ethanol
and the mixture was stirred for 2 h at room temperature. NaBH4
(0.78 g, 20.70 mmol) was then added and the resulting solution
stirred for 2 h at room temperature. The ethanol was removed
under reduced pressure. The resulting residue was treated with
H2O (10 mL) and extracted with CH2Cl2 (3 ¥ 20 mL). The organic
phase was removed at reduced pressure and the resulting residue
was dissolved in ethanol and preci◦pitated as hydrochloride salt of
L2 in 50% yield. mp: decomp. 345 C. 1H NMR (300 MHz, D2O):
d (ppm) = 8.73 (d, J = 8 Hz, 2H), 8.12 (s, 2H), 8.03 (d, J = 8 Hz,
2H), 7.95 (t, J = 7 Hz, 2H), 7.44 (d, J = 7 Hz, 4H), 4.87 (s, 4H),
4.62 (s, 8H), 3.60-3.53 (m, 4H), 3.31-3.20 (m, 12H), 3.00-2.90 (m,
8H). 13C NMR (75.43 MHz, D2O): d (ppm) = 150.6, 149.0, 141.7,
141.3, 139.9, 129.8, 127.8, 124.6, 122.3, 51.4, 51.2, 50.8, 49.6, 46.1,
43.8.
1
The H and 13C spectra were recorded on a Bruker 300 DRX
spectrometer at 300 MHz for H and 75.43 MHz for 13C. The
1
NMR experiments involving 31P were recorded on a Varian Unity
300 spectrometer equipped with a switchable probe. The chemical
shifts were recorded in ppm. All spectra were recorded at room
temperature and the concentration of L1·6HCl, L2·6HCl, PPi,
TPP and ATP was 2 mM in D2O and the [Zn2+] was kept between
2-6 mM. The pD was adjusted with a concentrated solution of
DCl or NaOD in D2O. 1D-NMR experiments were recorded in a
Bruker AV 500.
Molecular dynamics simulations
Molecular dynamics simulations were carried out using AMBER8
and GAFF potentials at 325 K.14 The process comprised a
convenient heating stage (in several steps until a final temperature
of 325 K is reached) after which the simulation was done. The
heating stage comprised 6 steps along which the temperature was
slowly increased for a period of 30 picoseconds plus 2 steps,
of 20 picosecond each, at the end until the target temperature
was reached, namely 325 K. Then the system was ready for a
production stage of 77 nanoseconds along which energies and data
were saved every 500 steps (each step 0.2 femtoseconds). Minimum
energy conformers were extracted from the trajectories. Files with
Calc for C40H54 N12·6HCl: C, 52.5; H, 5.9; N, 18.4. Found: C,
52.8; H, 8.0; N, 18.2. MS (FAB) m/z 704 [M+H]+
EMF measurements
The potentiometric titrations were carried out at 298.1 0.1 K
using NaCl 0.15 M as supporting electrolyte. The experimental
procedure (burette, potentiometer, cell, stirrer, microcomputer,
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trajectories in AMBER8 format and GAFF potentials at 325 K
are available on request.14
11 A. Bencini, A. Bianchi, E. Garc´ıa-Espan˜a, M. Micheloni and J. A.
Ram´ırez, Coord. Chem. Rev., 1999, 188, 97.
12 (a) SPECFIT, A Program for Global Least Square Fitting of Equilibrium
and Kinetic Systems using Factor Analysis and Marquardt Minimization,
Version 1-26; 4 Spectrum Software Associates, Chapel Hill, NC, 1996;
(b) H. Gampp, M. Maeder, C. J. Meyer and A. D. Zuberbuhler, Talanta,
1985, 32, 95; (c) H. Gampp, M. Maeder, C. J. Meyer and A. D.
Zuberbuhler, Talanta, 1985, 32, 257; (d) H. Gampp, M. Maeder, C. J.
Meyer and A. D. Zuberbuhler, Talanta, 1986, 33, 943.
13 (a) C. Bazzicalupi, A. Bencini, A. Bianchi, C. Giorgi, V. Fusi, B.
Valtancoli, M. A. Bernardo and F. Pina, Inorg. Chem., 1999, 38, 3806;
(b) A. Bencini, M. A. Bernardo, E. Garc´ıa-Espan˜a, C. Giorgi, S. V.
Luis, F. Pina and B. Valtancoli, Advances in Supramolecular Chemistry,
ed. G. Gokel, 2002, 8, 79.
Acknowledgements
Financial support from Ministerio de Ciencia e Innovacio´n
(CTQ2006-15672-CO5-01). J.G. wants to thank Ministerio de
Educacio´n y Ciencia (Spain) for his FPU fellowship. One of us (J.
P.) wants to thank Generalitat Valenciana for a Geronimo Forteza
formation contract.
14 Molecular dynamics calculations were performed using AMBER8:
D. A. Case, T. A. Darden, T. E. Cheatham III, C. L. Simmerling, J.
Wang, R. E. Duke, R. Luo, K. M. Merz, B. Wang, D. A. Pearlman,
M. Crowley, S. Brozell, V. Tsui, H. Gohlke, J. Mongan, V. Hornak, G.
Cui, P. Beroza, C. Schafmeister, J. W. Caldwell, W. S. Ross and P. A.
Kollman, AMBER 8, 2004, University of California, San Francisco,
and AFF potentials; J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kolman
and D. A. Case, J. Comput. Chem., 2004, 25, 1157 at 325 K.
Notes and references
1 (a) J. M. Lehn, Supramolecular Chemistry. Concepts and Perspectives,
VCH, Weinheim, 1995; (b) H. J. Schneider, Principles and Methods in
Supramolecular Chemistry, John Wiley & Sons, Chichester, UK, 2000;
(c) Supramolecular Chemistry of Anions, ed. A. Bianchi, K. Bowman-
James and E. Garc´ıa-Espan˜a, John Wiley & Sons, Chichester, UK,
1997; (d) J. L. Sessler, P. A. Gale and W. S. Cho, Anion Receptor
Chemistry, RSC Publishing, Cambridge, UK, 2006; (e) C. Caltagirone
and P. A. Gale, Chem. Soc. Rev., 2009, 38, 520; (f) J. W. Steed, Chem. Soc.
Rev., 2009, 38, 506; (g) S. Kubik, C. Reyheller and S. Stuwe, J. Inclusion
Phenom. Macrocyclic Chem., 2005, 52, 137; (h) H. J. Schneider and
A. K. Yatsimirsky, Chem. Soc. Rev., 2008, 37, 263.
2 (a) C. Bazzicalupi, A. Bencini, A. Bianchi, A. Danesi, C. Giorgi, C.
Lodeiro, F. Pina, S. Santarelli and B. Valtancoli, Chem. Commun., 2005,
2630; (b) Q. X. Xiang, J. Zhang, P. Y. Liu, C. Q. Xia, Z. Y. Zhou, R. G.
Xie and X. Q. Yu, J. Inorg. Biochem., 2005, 99, 1661; (c) E. Garc´ıa-
Espan˜a, P. Gavin˜a, J. Latorre, C. Soriano and B. Verdejo, J. Am. Chem.
Soc., 2004, 126, 5082.
15 L4 has been obtained by reacting L3 with 2-pyridinecarbaldehyde. 1H
NMR (D2O, 300 MHz): dH 2.93 (t, J = 5 Hz, 4H), 3.13 (t, J = 8 Hz, 2H),
3.28 (t, J = 5 Hz, 4H), 3.45 (t, J = 8 Hz, 2H), 4.57 (s, 2H), 4.64 (s, 4H),
7.45 (d, J = 8 Hz, 2H), 7.75 (ddd, J1 = J2 = 8 Hz, J3 = 1 Hz, 1H), 7.83
(d, J = 8 Hz, 1H), 7.96 (t, J = 8.0 Hz, 1H), 8.25 (ddd, J1 = J2 = 8 Hz,
J2 = 2 Hz, 1H), 8.72 (dd, J1 = 5 Hz, J2 = 1 Hz, 1H).13C NMR (D2O,
75.43 MHz): dC 43.9, 46.2, 49.8, 50.0, 50.8, 51.2, 122.5, 126.5, 126.6,
140.1, 143.3, 146.8, 147.6, 149.2. Anal Calcd. For C19H28N6·3HCl:C,
50.68; H, 6.94; N, 18.75. Found: C, 50.3, H, 7.1; N 18.5. S. Blasco, B.
Verdejo, M. P. Clares, C. E. Castilllo, A. Algarra, J. Latorre, M. A.
Manez, M. Garcıa-Basallote, C. Soriano and E. Garcıa-Espana, work
˜
´
´
˜
3 (a) S. K. Kim, D. H. Lee, J.-I. Hong and J. Yoon, Acc. Chem. Res.,
2009, 42, 23; (b) S. Develay, R. Tripier, M. Le Baccon, V. Patinec, G.
Serratrice and H. Handel, Dalton Trans., 2006, 3418; (c) S. Aoki, H.
Kawatani, T. Goto, E. Kimura and M. Shiro, J. Am. Chem. Soc., 2001,
123, 1123; (d) M. T. Albelda, J. C. Frias, E. Garc´ıa-Espan˜a and S. V.
Luis, Org. Biomol. Chem., 2004, 2, 816.
4 (a) A. S. Dele´pine, R. Tripier and H. Handel, Org. Biomol. Chem., 2008,
6, 1743; (b) D. H. Lee, J. H. Im, S. U. Son, Y. K. Chung and J.-I. Hong,
J. Am. Chem. Soc., 2003, 125, 7752; (c) D. H. Lee, S. Y. Kim and J.-I.
Hong, Angew. Chem., Int. Ed., 2004, 43, 4777; (d) H. N. Lee, Z. Xu,
S. K. Kim, K. M. Swamy, Y. Kim, S.-J. Kim and J. Yoon, J. Am. Chem.
Soc., 2007, 129, 3828.
5 (a) M. Ronaghi, S. Karamohamed, B. Pettersson, M. Uhle´n and P.
Nyre´n, Anal. Biochem., 1996, 242, 84; (b) S. Xu, M. He, H. Yu, X. Cai,
X. Tan, B. Lu and B. Shu, Anal. Biochem., 2001, 299, 188.
6 (a) G. J. Bridger, R. T. Skerlj, S. Padmanabhan, S. A. Martelluci, G. W.
Henson, M. J. Abrams, H. C. Joao, M. Witvrouw, K. de Vreese, R.
Pauwels and E. de Clercq, J. Med. Chem., 1996, 39(1), 109; (b) Z. Guo
and P. J. Sadler, Adv. Inorg. Chem., 2000, 48, 183.
in preparation.
16 (a) E. Kimura, M. Kikuchi, H. Kitamura and T. Koike, Chem.–Eur. J.,
1999, 5, 3113; (b) G. Ambrosi, M. Formica, V. Fusi, L. Giorgi, E.
Macedi, M. Micheloni, P. Paoli and P. Rossi, Inorg. Chem., 2009, 48,
10424.
17 (a) M. Arca, A. Bencini, E. Berni, C. Caltagirone, F. A. Devillanova,
F. Isaia, A. Garau, C. Giorgi, V. Lippolis, A. Perra, L. Tei and B.
Valtancoli, Inorg. Chem., 2003, 42, 6929; (b) A. Bencini, E. Berni, A.
Bianchi, C. Giorgi, B. Valtanacoli, D. K. Chand and H. J. Schneider,
Dalton Trans., 2003, 793; (c) A. Bencini, S. Biagini, C. Giorgi, H.
Handel, M. L. Baccon, P. Mariani, P. Paoletti, P. Paoli, P. Rossi, R.
Tripier and B. Valtancoli, Eur. J. Org. Chem., 2009, 5610.
18 P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43, 1739.
19 (a) M. T. Albelda, J. Aguilar, S. Alves, R. Aucejo, P. Diaz, C. Lodeiro,
J. C. Lima, E. Garcıa-Espana, F. Pina and C. Soriano, Helv. Chim.
´
˜
Acta, 2003, 86, 3118; (b) A. Bencini, A. Bianchi, E. Garcıa-Espana,
E. C. Scott, L. Morales, B. Wang, T. Deffo, F. Takusagawa, M. P.
Mertes, K. B. Mertes and P. Paoletti, Bioorg. Chem., 1992, 20, 8.
20 C. J. Chandler, L. W. Deady and J. A. Reiss, J. Heterocycl. Chem., 1981,
18, 599.
´
˜
7 E. Kikuta, S. Aoki and E. Kimura, J. Am. Chem. Soc., 2001, 123, 7911.
8 (a) T. J. Lotz and T. A. Kaden, J. Chem. Soc., Chem. Commun., 1977, 1;
(b) V. Amendola, L. Fabbrizzi, M. Lichelli, C. Mangano, P. Pallavicini,
L. Parodi and A. Poggi, A., Coord. Chem. Rev., 1999, 190–192, 649;
(c) G. W. Gokel, Chem. Soc. Rev., 1992, 21, 39; (d) G. W. Gokel, L. J.
Barbour, S. L. De Wall and E. S. Meadows, Coord. Chem. Rev., 2001,
222, 127.
21 E. Garcıa-Espana, M. J. Ballester, F. Lloret, J. M. Moratal, J. Faus and
´
˜
A. Bianchi, J. Chem. Soc., Dalton Trans., 1988, 101.
22 M. Fontanelli and M. Micheloni, Proceedings of the I Spanish-Italian
Congress on Thermodinamics of Metal Complexes, Penıscola, Castellon,
1990. Program for the automatic control of the microburette and the
acquisition of the electromotive force readings.
˜´
´
9 (a) V. Amendola, D. Esteban-Go´mez, L. Fabbrizzi, M. Licchelli, E.
Monzani and F. Sanceno´n, Inorg. Chem., 2005, 44, 8690; (b) L.
Fabbrizzi, M. Licchelli, P. Pallavicini and L. Parodi, Angew. Chem.,
Int. Ed., 1998, 37, 800; (c) S. Aoki, D. Kagata, M. Shiro, K. Takeda
and E. Kimura, J. Am. Chem. Soc., 2004, 126, 13377; (d) S. Aoki, K.
Iwaida, N. Hanamoto, M. Shiro and E. Kimura, J. Am. Chem. Soc.,
2002, 124, 5256.
10 B. Verdejo, A. Ferrer, S. Blasco, C. E. Castillo, J. Gonza´lez, J. Latorre,
M. A. Ma´n˜ez, M. G. Basallote, C. Soriano and E. Garc´ıa-Espan˜a,
Inorg. Chem., 2007, 46, 5707.
23 (a) G. Gran, Analyst, 1952, 77, 661; (b) F. J. Rossotti and H. Rossotti,
J. Chem. Educ., 1965, 42, 375.
24 P. Gans, Programs to determinethe distribution of Species in multiequi-
libria systems from the stability constants and mass balance equations..
25 R. M. Smith and A. E. Martell, NIST Stability Constants Database, ver-
sion 4.0, National Institute of Standards and Technology, Washington
D.C., 1997.
26 W. C. Hamilton, Statistics in Physical chemistry, The Ronald Press Co.,
New York, 1964.
27 L. Bologni, A. Sabatini and A. Vacca, Inorg. Chim. Acta, 1983, 69, 71.
2376 | Org. Biomol. Chem., 2010, 8, 2367–2376
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