Cu2+ and AMP complexation of enlarged tripodal polyamines

Article abstract of DOI:10.1039/b605639c

The synthesis, characterization, Cu²⁺ coordination and interaction with AMP of three tripodal polyamines are reported. The polyamines are based on the structure of the tetraamine (tren) which has been enlarged with three propylamino functionali

Full text of DOI:10.1039/b605639c

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www.rsc.org/dalton | Dalton Transactions
Cu2+ and AMP complexation of enlarged tripodal polyamines†
M. Teresa Albelda,a Enrique Garc´ıa-Espan˜a,*a Hermas R. Jime´nez,b Jose´ M. Llinares,c Conxa Soriano,*c
Alejandra Sornosa-Tena and Begon˜a Verdejoa
Received 20th April 2006, Accepted 22nd June 2006
First published as an Advance Article on the web 10th July 2006
DOI: 10.1039/b605639c
The synthesis, characterization, Cu2+ coordination and interaction with AMP of three tripodal
polyamines are reported. The polyamines are based on the structure of the tetraamine (tren) which has
been enlarged with three propylamino functionalities (TAL), with a further anthrylmethyl fragment at
one of its terminal primary nitrogens (ATAL) or with naphthylmethyl fragments at its three ends
(N3TAL). The protonation constants of all three polyamines show that at pH 6, all six primary and
secondary nitrogen atoms in the arms are protonated. The interaction with Cu2+ and AMP
(adenosine-5ꢀ-monophosphate) has been studied by potentiometric, UV-Vis, ESI-MS spectroscopy and
NMR techniques. pH-Metric, NMR and ESI/MS techniques indicate that TAL and ATAL form with
AMP adducts of 1 : 1, 2 : 1 and 3 : 1 AMP : L stoichiometries in water. This is one of the first examples
for the formation of such complexes in aqueous solution. Formation of ternary complexes between
TAL, ATAL, N3TAL, Cu2+ and AMP is observed. Paramagnetic NMR techniques have been used to
obtain structural information on the binding mode of AMP to the Cu2+–TAL binuclear complexes.
electrostatic interactions when the pH is low enough for having
Introduction
a sufficiently high number of protonated amino groups within
Polyamines are water-soluble ambivalent receptors capable of
the receptor. Additionally, if the polyamine has a high number
interacting with metal ions and anionic species. Coordinative
of nitrogens appropriately distributed, intermediate situations in
interactions with metal ions will occur when the pH is high enough
which the polyamine is able to interact with metal ions and
to allow for a sufficiently large number of available electron pairs.
anions in separated compartments can also be envisaged. All
Interaction with anionic or polar species will be mainly driven by
these binding modes can be modulated by the length of the
hydrocarbon chains between the amino groups as well as by
the attachment of aromatic functionalities. The large structural
variability of polyamines and the pH regulation of their binding
aDepartament de Qu´ımica Inorga`nica, Institut de Cie`ncia Molecular (IC-
MOL), Universitat de Vale`ncia. Edificio de Institutos de Paterna, Apartado
modes allow for many applications such as selective metal or anion
complexation and detection.1–3 A particularly interesting point is
the development of ligands interfering selectively with structures
and functions of nucleic acids.4 The conformational changes of
such ligands induced by Cu2+ ions can lead to dramatic allosteric
effects in their association with double stranded DNA.5
Variation of the number and location of the protonated nitrogen
atoms allows for controlling the amount and density of positive
charges, which has a profound influence on the interactions with
nucleic acids. Introduction of condensed aromatic moieties into
such polyamines leads to stacking effects with nucleobases and in
particular to intercalation into double stranded nucleic acids.
A particularly high concentration of ammonium centers is
accessible with tripodal derivatives containing a large number
of nitrogen atoms. In this respect the tripodal polyamine tris(2-
aminoethyl)amine (tren) constitutes one of the most useful and
widely employed building block.6
Here, we report on the synthesis, characterization, Cu2+ coordi-
nation and interaction with adenosine-5ꢀ-monophosphate (AMP)
of three receptors of this class. In these receptors the polyamine
tren has been enlarged with three aminopropyl functionalities
(TAL), and further functionalised with an anthrylmethyl fragment
at one of its terminal primary nitrogens (ATAL) or with naphthyl-
methyl fragments at its three ends (N3TAL) (See Chart 1).7
de Correos 22085, 46071, Valencia, Spain. E-mail: enrique.garcia-es@uv.es;
Tel: 963544879
bDepartament de Qu´ımica Inorga`nica, Facultat de Qu´ımica, Universitat de
Vale`ncia, 46100, Burjassot, Valencia, Spain
cDepartament de Qu´ımica Orga`nica, Institut de Cie`ncia Molecular (IC-
MOL), Facultat de Farma`cia, Universitat de Vale`ncia, 46100, Burjassot,
Valencia, Spain
† Electronic supplementary information (ESI) available: Fig. S1: Dis-
tribution diagrams for the systems: (A) H+–TAL; (B) H+–ATAL; (C)
H+–N3TAL. Table S1: Observed chemical shifts for the interaction of
AMP with the tripodal ligands TAL and ATAL. Table S2: Logarithms of
the equilibrium constants for the interaction of AMP with the tripodal
polyamines TAL, ATAL and N3TAL. Table S3: 1H NMR hyperfine-
shifted resonances and temperature dependence of Cu2TAL, Cu2TAL–
AMP complexes in D2O at 25 ◦C and pH 8. Fig. S2: Distribution diagrams
for the system AMP–TAL: [TAL] = 1 × 10−3 mol dm−3, [AMP] = 1 ×
10−3 mol dm−3. Fig. S3: Distribution diagrams for the system AMP–
TAL: [TAL] = 1 × 10−3 mol dm−3, [AMP] = 3 × 10−3 mol dm−3. Fig.
S4: Distribution diagrams for the system AMP–ATAL: [ATAL] = 1 ×
10−3 mol dm−3, [AMP] = 2 × 10−3 mol dm−3. Fig. S5: Distribution
diagrams for the system AMP–ATAL: [ATAL] = 1 × 10−3 mol dm−3
,
[AMP] = 3 × 10−3 mol dm−3. Fig. S6: Distribution diagrams for the
ternary system Cu2+–TAL–AMP: (A) [Cu2+] = [TAL] = [AMP] = 1 ×
10−3 mol dm−3; (B) [Cu2+] = 2 × 10−3 mol dm−3, [TAL] = [AMP] = 1 ×
10−3 mol dm−3. Fig. S7: Distribution diagrams for the ternary system Cu2+
–
ATAL–AMP: (A) [Cu2+] = [ATAL] = [AMP] = 1 × 10−3 mol dm−3; (B)
[Cu2+] = 2 × 10−3 mol dm−3, [ATAL] = [AMP] = 1 × 10−3 mol dm−3
.
Fig. S8: Distribution diagrams for the ternary system Cu2+–N3TAL–
AMP: [Cu2+] = [N3TAL] = [AMP] = 1 × 10−3 mol dm−3. See DOI:
10.1039/b605639c
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of tris(tosylated) derivative (2) with N-(3-bromopropyl)-
phthalimide to give the enlarged compound (3). Compound
(3) is then treated with hydrazine to remove the protection
(compound (4)) and with an HBr–AcOH–PhOH mixture to give
the detosylated amine as its hydrobromide salt (TAL·6HBr).
The solid obtained was washed extensively with ethanol to yield
TAL·6HBr in a pure form as confirmed by elemental microanalysis
and spectroscopic data.
In order to obtain the terminally functionalized tripodal ligands,
we reacted TAL in its free amine form with the corresponding
anthracene or naphthalene carbaldehydes. For preparing ATAL,
in which the fluorophore was appended to just one of the arms of
TAL, a molar ratio anthracene-9-carbaldehyde : TAL of 1 : 3 was
used whereas for obtaining the tripodal compound N3TAL8 with
all its three arms functionalized with methylnaphthyl groups, a
reverse molar ratio naphthalene-1-carbaldehyde : TAL of 3 : 1 was
used. The overall yield is large enough to obtain the compounds
in a gram scale.
Chart 1
Protonation behaviour
Synthesis of polyamine compounds
Table 1 collects the stepwise protonation constants for the three
tripodal ligands determined at 298.1 K in 0.15 mol dm−3 NaCl and
Fig. S1 (ESI†) the distribution diagram for the species existing in
equilibrium.
TAL, ATAL and N3TAL were prepared following the general
synthetic strategy delineated in Scheme 1, which consists
of tosylation of tris(2-aminoethyl)amine (1) and reaction
All three ligands present six relatively high basicity constants
followed by a much lower one for the seventh protonation step
(Table 1 and Fig. S1, ESI†). On the other hand, the basicity
displayed by the non-substituted polyamine is clearly higher
than those of the substituted derivatives given the basicity order
TAL > ATAL > N3TAL. All these facts can be interpreted taking
into account repulsive interactions between same sign charges,
the inductive effects generated by the different substituents, the
different hydration of the compounds and possible hydrogen bond
formation.
It is well established that electrostatic repulsion between positive
charges separated by propylenic chains is considerably lower than
when the separation is by ethylenic chains.9 This is the reason
for the relatively small decrease in basicity observed in every one
of the six first protonations of all three ligands. The very low
seventh protonation constant would correspond to protonation
of the apical nitrogen atom. It is interesting to remark that
although these last constants are small, they can be determined
or estimated potentiometrically, in contrast with the polyamine
tren for which just three protonation constants could be measured
Table 1 Logarithms of the stepwise protonation constants for the ligands
TAL, ATAL and N3TAL determined at 298.0 0.1 K in 0.15 mol dm−3
NaCl
Reactiona
TAL
ATAL
N3TALc
L + H ↔ HL
10.34(7)b
10.26(2)
9.52(4)
8.68(4)
7.91(5)
7.37(4)
2.2(1)
10.41(3)
9.87(2)
9.17(3)
8.02(3)
7.2(3)
5.78(8)
<2.00
9.08(6)
8.70(5)
8.48(5)
7.76(4)
7.09(5)
6.80(4)
2.25(9)
HL + H ↔ H2L
H2L + H ↔ H3L
H3L + H ↔ H4L
H4L + H ↔ H5L
H5L + H ↔ H6L
H6L + H ↔ H7L
a Charges omitted. b Numbers in parenthesis are standard deviations in the
last significant figure. c Taken from ref. 8.
Scheme 1 Synthesis pathway for the preparation of TAL, ATAL and
N3TAL.
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by potentiometry.10 The general decrease of basicity observed for
the compounds with the aromatic rings ATAL and N3TAL can be
ascribed to a poorer stabilization by hydration of the ammonium
groups in the more hydrophobic environments.
Therefore, in all the mononuclear Cu2+ complexes there are
non-coordinated nitrogen atoms that might permit the coordi-
nation with a further metal ion. However, in NaCl medium
addition of a further mole of Cu2+ ion leads, in all three cases,
to precipitation, and formation of binuclear complexes is not
detected. When changing the ionic strength to NaClO4 containing
Interaction with Cu2+ ions
the less coordinating ClO4 counter-anions,13 formation of the
−
binuclear Cu2+ complexes [Cu2(TAL)]4+, [Cu2(TAL)(OH)]3+ and
[Cu2(TAL)(OH)2]2+ is observed for TAL while for the other two
systems precipitation still persists. For a Cu2+–TAL mol ratio 2 : 1,
the binuclear species predominate in aqueous solution at pH values
over 7.0. The UV-vis spectra recorded for a 2 : 1 Cu2+ : TAL mol
ratio, although more intense, show similar characteristics to those
of the mononuclear species with the presence of a band at 740 nm
and an hypsochromically shifted shoulder at around 607 nm.
The equilibrium constants for the formation of Cu2+ com-
plexes with TAL, ATAL and N3TAL are included in Table 2.
TAL displays the highest stability constants followed by the
mono(anthrylmethyl) derivative ATAL while N3TAL is the
polyamine presenting the lowest stability constants with Cu2+.
[Cu(TAL)]2+ has three protonation constants from which the first
two are high and comparable to the protonation constants of
the free ligand while the third one is much lower. On the other
hand the stability constant for the [CuL]2+ complex of TAL
is clearly higher than that reported for the parent polyamine
tris(2-aminoethyl)amine (tren) (log KML = 19.58) in which its four
nitrogens are involved in the coordination to the metal ion.11,12
These data suggest a coordination number of five for the Cu2+–
TAL complexes. The visible spectra of the system Cu2+–TAL
were recorded at pH values where the different mononuclear
and binuclear species predominate in solution (see distribution
diagrams in ESI†). For 1 : 1 molar ratio the spectra of the two
main species in solution, [Cu(H2TAL)]4+ and [Cu(TAL)]2+, are
practically identical consisting of a wide band centred at 740 nm
(e = 270 M−1 cm−1) preceded by a shoulder at 600–620 nm.
These spectral features are characteristic of a trigonal bipyramidal
geometry.11 The absence of changes between the spectra of these
two species confirms that the protonation processes occur on
nitrogen atoms not involved in the coordination to the metal.
The analysis of the species formed in the system Cu2+–ATAL
reflects similar tendencies whereas the results are somewhat
different for the system Cu2+–N3TAL. First, a tetraprotonated
[Cu(H4N3TAL)]6+ complex has also been detected. In second
place, the constant for the third protonation step, [Cu(H2L)]4+
+ H+ = [Cu(H3L)]5+, is much higher for N3TAL than for the other
two polyamines. These along with the lower constant found for
the [Cu(N3TAL)]2+ complex suggest four coordinated nitrogens
in this system. Analogously to the system Cu2+–TAL the UV-Vis
spectra indicate a rhombic geometry for the system Cu2+–N3TAL
with a very broad band centered at around 740 nm and a shoulder
at ca. 620 nm.
Interaction with AMP
In order to explore the possibility to use the metal complexes
for simultaneous detection of nucleotides and to better understand
how the interaction with the nucleic acids occurs7 an analysis of
the interaction of the receptors with adenosine-5ꢀ-monophosphate
(AMP) was carried out.
Table 3 collects the corresponding data for the tripodal recep-
tors. An interesting feature is that the tripodal polyamines TAL
and ATAL are able to form AMP : L adduct complexes of 2 : 1
and 3 : 1 stoichiometries. Such stoichiometries have been checked
following the variations of the 31P NMR chemical shifts of AMP
when increasing amounts of TAL were added. The measurements
have been conducted in a pH region close to the first pKa of AMP.14
The effects of the AMP–L interaction will be an increase in the
apparent basicity of the polyamine and in the apparent acidity of
the anionic guest and thus the changes will be more noticeable in
Table 3 Logarithms of the equilibrium constants for the interaction of
AMP (AMP2− ≡ A) with the tripodal polyamines TAL, ATAL and N3TAL
determined at 298.0 0.1 K in 0.15 mol dm−3 NaCl
Reactiona
TAL
ATAL
N3TAL
A + HL ↔ HLA
5.17(5)b
5.02(5)
5.22(5)
5.28(5)
5.38(5)
5.36(4)
6.41(4)
3.78(5)
8.09(1)
8.25(9)
9.45(8)
8.40(8)
7.42(5)
2.74(5)
2.89(4)
11.83(6)
12.10(5)
3.74(6)
3.85(5)
12.31(5)
12.07(5)
10.87(4)
6.37(6)
6.56(7)
6.63(6)
6.80(6)
7.15(6)
7.65(7)
7.11(4)
6.28(1)
6.02(9)
6.30(1)
6.15(8)
6.42(9)
6.23(8)
6.71(8)
4.92(8)
A + H2L ↔ H2LA
A + H3L ↔ H3LA
A + H4L ↔ H4LA
A + H5L ↔ H5LA
A + H6L ↔ H6LA
HA + H5L ↔ H6LA
HA + H6L ↔ H7LA
2 A + H5L ↔ H5LA2
2 A + H6L ↔ H6LA2
2 HA + H5L ↔ H7LA2
A + HA + H5L ↔ H7LA2
2 HA + H6L ↔ H8LA2
A + H5LA ↔ H5LA2
A + H6LA ↔ H6LA2
3 A + H5L ↔ H5LA3
3 A + H6L ↔ H6LA3
A + H5LA2 ↔ H5LA3
A + H6LA2 ↔ H6LA3
2 A + HA + H6L ↔ H7LA3
A + 2 HA + H6L ↔ H8LA2
3 HA + H6L ↔ H9LA3
Table 2 Logarithms of the stability constants for the formation of Cu2+
complexes with the ligands TAL, ATAL and N3TAL determined at 298.0
0.1 K in 0.15 mol dm−3 NaCl
Reactiona
TAL
ATAL
N3TAL
8.01(1)
Cu + L ↔ CuL
22.91(4)b 19.08(4) 16.21(7)
CuL + H ↔ CuHL
9.52(3) 10.34(2)
8.37(4)
7.55(2)
6.60(3)
3.92(5)
CuHL + H ↔ CuH2L
CuH2L + H ↔ CuH3L
CuH3L + H ↔ CuH4L
2 Cu + L ↔ Cu2L
9.21(2)
3.96(2)
8.60(1)
4.81(2)
14.52(8)
30.75(6)c
Cu2L + H2O ↔ Cu2L(OH) + H
−9.82(3)
Cu2L(OH) + H2O ↔ Cu2L(OH)2 + H −10.17(6)
a Charges omitted. b Numbers in parentheses are standard deviations in
a Charges omitted. b Numbers in parentheses are standard deviations in
the last significant figure.
the last significant figure. c Values determined in 0.15 mol dm−3 NaClO4.
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such a region. The 31P NMR chemical shift of the system AMP–
TAL at pD = 6.5 shows significant changes for AMP : TAL molar
ratios 3 : 1, 2 : 1 and 1 : 1 with downfield shifts of 1.7, 2.1 and
3.1 ppm, respectively, with respect to the non-complexed AMP
(see Table S1, ESI†). For AMP : TAL molar ratios lower than
1 : 1, no further changes in chemical shift were observed. Similar
results are obtained for the system AMP : ATAL (Table S1, ESI†).
However, although in the case of ATAL formation of 2 : 1 and 3 : 1
AMP : L adduct species was also detected (see Table 3), the pH
range of formation of such species was narrower (see distribution
diagrams in Fig. S4 and S5, ESI†). Finally, in the case of the
polyamine with the three arms functionalised with naphthylmethyl
groups, the 2 : 1 and 3 : 1 AMP–L species were not detected either
by potentiometric or NMR methods.
ESI-MS spectra recorded in the positive mode for the sys-
tem AMP–TAL (Fig. 1) shows peaks at m/z 665.4, 1012.5
and 1359.5 attributable to the ionic species [H3(TAL)(AMP)]+,
[H5(TAL)(AMP)2]+ and [H7(TAL)(AMP)3]+, respectively support-
ing the formation of adduct species of 1 : 1, 2 : 1 and 3 : 1
AMP : L stoichiometries.
equilibrium and values of stability constants (Table 3). Since
both AMP and the receptors participate in overlapping proton
transfer processes, translating the cumulative stability constants
into representative stepwise constants requires to consider the
basicities of AMP14 and of the different ligands and assume that
the interaction will not affect much the pH range of existence
of the protonated species of AMP and L. If this is taken into
account, the stepwise constants shown in Table 3 can be deduced as
representative of the equilibria occurring in solution (the complete
set of cumulative constants is collected in Table S2, ESI†).
It deserves to be mentioned that the interactions presented by
all three polyamines are among the highest found for synthetic
receptors.15 It is interesting to note that in contrast to what
happens in the systems Cu2+–tripodal polyamines, N3TAL and
ATAL interact more strongly with AMP than TAL. p–p-Stacking
interactions could contribute to enhance the affinity of ATAL
and N3TAL for AMP with respect to TAL. Additionally, the
hydrophobic environment generated by the aryl moieties would
lower the local dielectric constant and thus reinforce electrostatic
interactions between the oppositely charged partners.
Perhaps, the most striking feature of the chemistry of these
ligands is the above mentioned formation of 2 : 1 and particularly,
3 : 1 AMP : L species by TAL and ATAL.
Interestingly, when one compares the stepwise constants for
the equilibria AMP2− + H6L6+ = [H6L(AMP)]4+, log K1 = 5.36;
AMP2− + [H6L(AMP)]4+ = [H6L(AMP)2]2+, log K2 = 2.89 and
AMP2− + [H6L(AMP)2]2+ = [H6L(AMP)3], log K3 = 3.85, the
trend K1 > K2 < K3 is found (Table 3). The same occurs when
analysing the constants for the successive addition of AMP to
the pentaprotonated receptor AMP2− + H5L5+ = [H5L(AMP)]3+,
log K1 = 5.38; AMP2− + [H5L(AMP)]3+ = [H5L(AMP)2]+, log K2 =
2.74 and AMP2− + [H5L(AMP)2]+ = [H5L(AMP)3]−, log K3 = 3.74
(Table 3). This trend can be explained taking into account that all
three arms are coinvolved in the binding of the first AMP molecule
(Scheme 2(A)). The entrance of the second AMP would imply an
opening of the receptor due to electrostatic repulsions between the
two anions (Scheme 2(B)). The third AMP will find an adequate
arrangement for binding and the constant will again increase (see
Scheme 2(C)).
Ternary complexes Cu2+–L–AMP
In order to shed light on how the preformed Cu2+ complexes might
interact with the phosphate groups of the nucleic acids,5,7 we have
analysed by potentiometry, UV-Vis and paramagnetic NMR the
formation of Cu2+–L–AMP mixed complexes. The relevant data
is presented in Table 4 and Fig. 2 shows the distribution diagram
for the system Cu2+–N3TAL–AMP in 2 : 1 : 1 molar ratio. The
other distribution diagrams are collected in Fig. S6, S7 and S8
of the ESI.† TAL, ATAL and N3TAL form mono- and dinuclear
Cu2+–L–AMP species in aqueous solution.
Formation of dinuclear complexes are favoured by the presence
of the nucleotide which provides additional binding sites for the
anchoring of the second metal ion. For instance, while no binuclear
complexes are observed in the binary system Cu2+–N3TAL, in the
presence of the nucleotide it has been possible to detect a very
stable [Cu2(N3TAL)(AMP)]2− complex (see Fig. 2).
Fig. 1 ESI (positive mode) mass spectra without fragmentation for
the system AMP–L (᭹, AMP). The peaks corresponding to the adduct
complexes are marked with arrows.
In order to compare the AMP·L adduct formation constants for
1H paramagnetic NMR measurements have been performed
in order to gain further insight about the interaction of
the different systems care must be taken in comparing the correct
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Fig. 2 Distribution diagram for the ternary system Cu2+–N3TAL–AMP:
[Cu2+] = 2 × 10−3 mol dm−3, [AMP] = [L] = 10−3 mol dm−3
.
Scheme 2 Schematic representation of the interaction between hexapro-
tonated TAL and AMP.
Table 4 Logarithms of the equilibrium constants for the interaction of
Cu2+ and AMP (AMP2− ≡ A) with the polyamines TAL, ATAL and
N3TAL determined at 298.0 0.1 K in 0.15 mol dm−3 NaCl
Fig. 3 400 MHz proton NMR spectra in D2O at 298 K of (A) Cu2–TAL
at pH = 8, (B) Cu2–TAL–AMP at pH = 8. The asterisks mark the residual
solvent (*H2O).
Reaction
TAL
ATAL
N3TAL
Cu + A + L ↔ CuALa
23.58(7) 21.7(1)
Cu + A + H +L ↔ CuHAL
Cu + A + 2 H + L ↔ CuH2AL
Cu + A + 3 H + L ↔ CuH3AL
Cu + A + 4 H + L ↔ CuH4AL
Cu + A + 5 H + L ↔ CuH5AL
2 Cu + A + L ↔ Cu2AL
36.00(9)b 33.71(7) 30.3(1)
45.37(6) 42.31(7) 37.9(1)
52.20(2) 43.39(2) 44.7(1)
(d). The values of the chemical shift, linewidths at half-height and
longitudinal relaxation times (T1) are reported in Table 5. The peak
linewidths measured at half-height are ca. ∼200 Hz for all signals,
except for signal (d) which has average linewidth of 1624 Hz. The
signals (b), (c) and (d) which appear at 2.4, 0.28 and −9.3 ppm
integrate twenty-four protons and exhibit very short T1 values
(<1 ms) can be assigned to the a-CH2 protons (see Scheme 3).
55.99(3)
50.72(5)
55.10(7)
31.89(7) 29.13(8)
2 Cu + A + H + L ↔ Cu2HAL
2 Cu + A + L + OH ↔ Cu2(OH)AL
CuL + A ↔ CuAL
43.06(3) 39.95(5)
22.61(7)
4.51(7)
5.60(6)
5.86(5)
5.90(4)
6.28(4)
5.66(2)
8.35(2)
6.12(3)
6.13(3)
CuHL + A ↔ CuHAL
3.46(4)
3.49(3)
4.30(1)
4.68(2)
4.17(2)
4.30(3)
4.29(3)
5.05(1)
CuH2L + A ↔ CuH2AL
CuH2L + HA ↔ CuH3AL
CuH3L + HA ↔ CuH4AL
CuH2L + H2A ↔ CuH4AL
CuH4L + HA ↔ CuH5AL
CuH3L + H2A ↔ CuH5AL
CuHAL + Cu ↔ Cu2HAL
CuAL + Cu ↔ Cu2AL
7.21(4)
7.52(1)
6.24(4)
8.31(4)
7.38(2)
7.4(1)
CuA + CuHL ↔ Cu2HAL
a Charges omitted. b Numbers in parentheses are standard deviations in
the last significant figure.
Scheme 3
1
Cu2+–TAL binuclear complexes with AMP. The H NMR spec-
trum of the system Cu2+–TAL in a 2 : 1 molar ratio recorded in
D2O at pH = 8 shows, in the downfield region, three well resolved
isotropically shifted signals (a), (b), (c) and one non-resolved signal
(B), Fig. 3(A). Additionally, it displays one upfield shifted signal
The signal that appears at 3.6 ppm (a) integrates for six protons
and exhibits a short T1 value (3.7 ms) can be assigned to the
remaining aliphatic protons of the tripodal polyamine (b-CH2).
The pattern of paramagnetic signals suggests a coordination for
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Table 5 1H NMR hyperfine-shifted resonances of Cu2TAL, Cu2TAL–AMP complexes in D2O at 25 ◦C and pH 8
Complex
Signal
d/ppm
No. of protons
6
Assignment
T1/ms
Dm1/2/Hz
T2a/ms
Cu2TAL
a
b
B
c
3.6
2.4
1.2
0.28
−9.3
b-CH2 (TAL)
3.7
150
2.1
b
b
<1
b
b
b
24
a-CH2 (TAL)
<1
<1
341
1624
0.93
0.20
d
Cu2TAL–AMP
aꢀ
bꢀ
cꢀ
dꢀ
eꢀ
gꢀ
fꢀ
3.6
2.5
0.27
−9.2
8.7
6.1
8.2
4.4
4.3
3.9
6
b-CH2 (TAL)
a-CH2 (TAL)
2.5
<1
<1
<1
11.2
48.7
45.4
36.8
35.3
21.9
195
1.6
b
b
24
361
1131
78
0.88
0.28
4.1
2 × 2
2 × 2
c-CH2 (AMP)
H2/8 CH (AMP)c
18
17.7
30
10.6
b
b
hꢀ
iꢀ
4 × 2
d–r-CH (AMP)c
b
b
jꢀ
24
13.3
a Measured from the line width at half-height. b Overlap prevents measurement of this value. c Tentative assignments.
two coppers ions such as that depicted in Scheme 3. As previously
described,16–18 spin-coupled binuclear Cu2+ complexes have similar
short T1 values and broad linewidths.
mode through the adenine group would present a different pattern
of chemical shifts with respect to the H2 and H8 AMP protons.
The integration of the signals in measurements performed with
different Cu2TAL : AMP molar ratios have allowed for establishing
the stoichiometries of the ternary complexes. The results show
formation of ternary 1 : 1 and 1 : 2 complexes.
In order to obtain the temperature dependence of the hyperfine-
shifted resonances, variable-temperature 1H NMR spectra of
the systems Cu2TAL and Cu2TAL:AMP in D2O were registered
from 283 to 323 K. All the signals of the two systems follow
an anti-Curie behavior excepting the broad signals (d and dꢀ)
that show a Curie behavior (see Table S3 of the ESI†). It is
important to note that the anti-Curie behavior is in accord with the
existence of antiferromagnetically coupled systems in both cases
studied.
In the 1H NMR spectrum of the ternary system Cu2TAL–AMP
in D2O at pH = 8, not only new signals (eꢀ–jꢀ) appear, Fig. 3(B),
but also the values of the proton longitudinal relaxation time are
relatively larger, ranging from 11.2 ms for the signal (eꢀ) to 48.7 ms
for the signal (gꢀ) (see Table 5).
The new group of signals can be assigned on the basis of
integrated protons and of the longitudinal relaxation times of
the hyperfine-shifted resonances. Signal (eꢀ), which integrates four
protons and exhibit the shortest T1 value of the new signal group
(11.2 ms) can be assigned to the c-CH2 AMP protons closest to
the dicopper site (see Scheme 4). For the other group of signals
(fꢀ–jꢀ), the specific assignment is given in Table 5.
Conclusions
The synthesis of three tripodal polyamines is reported. The
ligands are based on the well-known structure of the polyamine
tris(2-aminoethyl)amine (tren) which has been enlarged with
aminopropyl groups (TAL) and further functionalized at one of
his termini with an anthrylmethyl group (ATAL) or at its three
termini with naphthylmethyl groups (N3TAL). These polyamines
interact in aqueous solution with the nucleotide AMP displaying
among the highest constants reported in the literature. On the
other hand, polyamines ATAL and N3TAL with aryl groups
show higher constants than TAL. For TAL and ATAL, pH-
metric titration, 31P NMR measurements and mass spectroscopy
evidence the formation of AMP–L species of 2 : 1 and 3 : 1
stoichiometries.
Formation of Cu2+ dinuclear complexes are favoured by the
presence of the nucleotide which provides additional binding sites
for the anchoring of the second metal ion. 1H NMR paramagnetic
studies have confirmed the formation of ternary Cu2+–AMP–TAL
complexes in which AMP behaves as a bridging didentate ligand
through its phosphate group.
Scheme 4
These results are in accordance with a binding occurring
through the phosphate group and support an Cu–O–O–Cu
ligation mode for AMP (see Scheme 4). A Cu–N–N–Cu ligation
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dC (ppm): 131.1, 129.9, 128.3, 126.0, 123.0, 49.1, 45.2, 45.1, 44.9,
43.6, 36.9, 24.1, 23.0.
Experimental
N,Nꢀ-{2-Bis[2-(3-aminopropylamino)ethyl]aminoethyl}-1,3-
propanediamine hexahydrobromide (TAL·6HBr)
emf Measurements
N,Nꢀ-Bis(p-tolylsulfonyl-2-aminoethyl)ethane-1,2-diamine
(2)
(6.0 g. 9.9 mmol) and K2CO3 (10.9 g, 78.8 mmol) was suspended
with 300 mL of CH3CN. To this mixture was added N-(3-
bromopropyl)phthalimide (10.9 g. 39.4 mmol) in 150 mL of
CH3CN, then was refluxed for 48 h and filtered off. The solution
was vacuum evaporated to dryness and the residue suspended
in refluxing ethanol to give 3 as a white solid (yield 89%). Mp:
59–61 C. H NMR (CDCl3): dH (ppm) 7.60–7.72 (m, 6H), 7.25
(d, J = 8 Hz, 2H), 3.66 (t, J = 7 Hz, 2H), 3.16 (t, J = 7 Hz, 2H),
3.19 (t, J = 7 Hz, 2H), 2.84 (t, J = 7 Hz, 2H), 2.37 (s, 3H), 1.90
(dd, J1 = 7 Hz, J2 = 7 Hz, 2H). 13C NMR (CDCl3), dC (ppm):
168.0, 143.0, 133.8, 131.9, 129.7, 127.2, 123.5, 123.0, 53.9, 47.0,
46.8, 35.5, 27.4, 21.4.
The potentiometric titrations were carried out at 298.1 0.1 K in
0.15 mol dm−3 NaCl. The experimental procedure used (burette,
potentiometer, cell, stirrer, microcomputer, etc.) was the same
that has been fully described elsewhere.19 The acquisition of the
emf data was performed with the computer program PASAT.20
The reference electrode was an Ag/AgCl electrode in saturated
KCl solution. The glass electrode was calibrated as an hydrogen-
ion concentration probe by titration of previously standardised
amounts of HCl with CO2-free NaOH solutions and determining
the equivalent point by the Gran’s method,21 which gives the
◦
1
◦
ꢀ
standard potential, E , and the ionic product of water (pKw
13.73(1)). The concentrations of the different metal ions employed
were determined gravimetrically by standard methods.
=
Compound 3 (5.0 g, 4.26 mmol) was treated with hydrazine
hydrate 85% (3 mL) and ethanol (500 mL) and the mixture was
refluxed for 24 h, then the resulting solid filtered off. The solution
was vacuum evaporated to give (4) as an oil (yield 79%). 1H NMR
(CDCl3): dH (ppm) 7.69 (d, J = 8 Hz, 2H), 7.29 (d, J = 8 Hz, 2H),
3.11–3.19 (m, 4H), 2.76 (t, J = 7 Hz, 2H), 2.72 (t, J = 7 Hz, 2H),
2.41 (s, 3H), 1.64–1.71 (m, 2H). 13C NMR (CDCl3), dC (ppm):
143.3, 136.2, 129.6, 127.1, 54.4, 47.2, 47.0, 39.0, 32.3, 21.5.
Compound 4 (2.5 g, 3.19 mmol) was suspended with phenol
(11.44 g, 121.5 mmol) and HBr–acetic acid 33% (125 mL). The
mixture was stirred at 90 ◦C for 24 h. Then was cooled and
the resulting solid was filtered off and washed with ethanol to
give N,Nꢀ-{2-bis[2-(3-aminopropylamino)ethyl]aminoethyl}-1,3-
propanediamine hexahydrobromide (TAL·6HBr) (yield 52%).
Mp: 267–271 ◦C. Anal. Calc. for C15H45N7Br6: C, 22.44, H, 5.65,
The computer program HYPERQUAD,22 was used to calculate
the protonation and stability constants. At least three titration
curves were performed for each system (ca. 100 experimental
points). The pH range investigated was 2.5–10.5 and the con-
centration of AMP and receptors ranged from 3 × 10−4 to a
maximun value of 1.5 × 10−3 mol dm−3). The different titration
curves for each system were treated either as a single set or as
separated curves without significant variations in the values of
the stability constants. Several measurements were made both in
formation and in dissociation (from acid to alkaline pH and vice
versa) to check the reversibility of the reactions. The sets of data
were merged together and treated simultaneously to give the final
stability constants.
In the case of the ternary systems we have performed the
following sequence. First, in the data treatment of the ternary
systems Cu2+–L–AMP we have included as fixed parameters the
protonation constants of AMP,14 the constants for the formation
of Cu2+–AMP complexes23 and the previously discussed proto-
nation constants of TAL, ATAL and N3TAL (Table 1), those
for the system AMP–L (Table 3) and those for the formation of
binary Cu2+ complexes (Table 2). In this way we have obtained
the equilibrium constant shown in Table 4. Secondly, in order to
check the consistency of the model, we have then fitted conjointly
the titrations of the binary system Cu2+–L and those of the ternary
systems Cu2+–L–AMP, leaving as parameters to be refined all
the constants for the Cu2+–L and for the Cu2+–L–AMP mixed
complexes. The model system obtained was the same in both
fittings and the differences between the values of the constants
obtained fell within the limits of the standard deviations.
1
N, 12.21. Found: C, 22.5, H, 5.4, N, 12.4%. H NMR (D2O): dH
(ppm) 3.20–3.31 (m, 4H), 3.12 (t, J = 8 Hz, 2H), 2.95 (t, J = 7 Hz,
2H), 2.10–2.20 (m, 2H). 13C NMR (D2O), dC (ppm): 49.1, 45.3,
44.9, 36.9, 24.1.
N-9-Anthrylmethyl-Nꢀ-(2-bis{2-[3-aminopropylamino]ethyl}-
aminoethyl)-1,3-propanediamine hexahydrochloride (ATAL·6HCl)
N,Nꢀ-{2-Bis[2-(3-aminopropylamino)ethyl]aminoethyl}1,3-pro-
panediamine (TAL) (1.5 g, 4.8 mmol) and anthracene-9-
carbaldehyde (0.8 g, 3.8 mmol) were stirred for 3 h in 75 mL
of dry ethanol. NaBH4 (1.8 g, 47.8 mmol) was then added
and the resulting solution stirred for 1 h at room temperature.
The ethanol was removed under reduced pressure. The resulting
residue was treated with water and dichloromethane. The organic
phase was removed at reduced pressure and the resulting residue
was dissolved in ethanol and precipitated as its hydrochlo-
ride salt of N-9-anthrylmethyl-N-(2-bis{2-[3-aminopropylamino]-
ethyl}aminoethyl)-1,3-propanediamine (ATAL) (yield 70%). Mp:
217–220 ◦C
Anal. Calc. for C30H49N7·6HCl: C, 49.60, H, 7.63, N, 13.50.
Found: C, 49.4, H, 7.8, N, 13.3%. 1H NMR (D2O): dH (ppm) 8.72
(s, 1H), 8.29 (d, J = 8 Hz, 2H), 8.18 (d, J = 8 Hz, 2H), 7.74 (dd,
J1 = J2 = 8 Hz, 2H), 7.63 (dd, J1 = J2 = 8 Hz, 2H), 5.31 (s, 2H),
3.39 (t, J = 8 Hz, 2H), 3.15–3.27 (m, 10H), 3.10 (t, J = 8 Hz,
6H), 2.92 (t, J = 6 Hz, 6H), 2.12–2.22 (m, 6H). 13C NMR (D2O),
NMR Measurements
1
The H and 13C NMR spectra were recorded on Bruker Avance
DPX 300 MHz spectrometer operating at 299.95 MHz for 1H and
at 75.43 for 13C. For the 13C NMR spectra, dioxane was used as
1
a reference standard (d = 67.4 ppm) and for the H spectra, the
solvent signal. The concentrations of AMP and of the receptors
employed in the NMR measurements were in the range 2.5 ×
10−4–10−3 mol dm−3. Within the concentration range used, dilution
experiments show maximum shifts of the aromatic signals of ca.
4480 | Dalton Trans., 2006, 4474–4481
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0.005 ppm discarding significant self-aggregation processes in any
of the studied systems.
95, 2529; (e) A. Bianchi, E. Garc´ıa-Espan˜a and K. Bowman-James,
Supramolecular Chemistry of Anions, Wiley-VCH, New York, 1997.
2 (a) P. A. Gale, Coord. Chem. Rev., 2003, 240, 191; (b) J. M. Llinares,
D. Powell and K. Bowman-James, Coord. Chem. Rev., 2003, 240, 57;
(c) K. Bowman-James, Acc. Chem. Res., 2005, 38, 671.
3 A. Metzger, V. A. Lynch and E. V. Anslyn, Angew. Chem., Int. Ed.
Engl., 1997, 36, 862.
The 31P NMR spectra were recorded on a Bruker Avance PX
300 MHz operating at 121.495 MHz. Chemical shifts are relative
to an external reference of 85% H3PO4. Adjustments to the desired
pH were made using drops of DCl or NaOD solutions. The pD
was calculated from the measured pH values using the correlation,
pH = pD − 0.4.24
4 (a) M. Demeunynck, C. Bailly and W. D. Wilson, Small Molecule
DNA and RNA Binders, From Synthesis to Nucleic Acid Complexes,
Wiley-VCH, New York, 2003; (b) H.-J. Schneider and A. Yatsimirsky,
Principles and Methods in Supramolecular Chemistry, Wiley, Chichester,
UK, 2000; (c) D. Kumar Chand, H.-J. Schneider, J. A. Aguilar, F. Escart´ı
and E. Garc´ıa-Espan˜a, Inorg. Chim. Acta, 2001, 316, 71; (d) H.-J.
Schneider and T. Blatter, Angew. Chem., Int. Ed. Engl., 1992, 31, 1207.
5 For a preliminary report on this behaviour, see: N. Lomadze, E.
Gogritchiani, H.-J. Schneider, M. T. Albelda, J. Aguilar, E. Garc´ıa-
Espan˜a and S. V. Luis, Tetrahedron Lett., 2002, 43, 7801.
6 For a recent review on tripodal amines, see: A. G. Blackman,
Polyhedron, 2005, 24, 1.
7 A report on the interaction of the tripodal receptors with nucleic acid
models can be seen in: N. Lomadze, H.-J. Schneider, M. T. Albelda, E.
Garc´ıa-Espan˜a and B. Verdejo, Org. Biomol. Chem., 2006, 4, 1755.
8 M. T. Albelda, E. Garc´ıa-Espan˜a, L. Gil, J. C. Lima, C. Lodeiro, J. S.
De Melo, M. J. Melo, A. J. Parola, F. Pina and C. Soriano, J. Phys.
Chem. B, 2003, 107, 6573.
9 (a) A. Bencini, A. Bianchi, E. Garc´ıa-Espan˜a, M. Micheloni and
J. A. Ram´ırez, Coord. Chem. Rev., 1999, 188, 97; (b) C. Frassinetti, L.
Alderighi, P. Gans, A. Sabatini, A. Vacca and S. Ghelli, Anal. Bioanal.
Chem., 2003, 376, 1041; (c) J. E. Sarnesky, H. L. Surprenant, F. K.
Molen and C. N. Reilley, Anal. Chem., 1975, 47, 2116; (d) D. N.
Hague and A. D. Moreton, J. Chem. Soc., Perkin Trans. 2, 1994,
265.
10 R. M. Smith and A. E. Martell, NIST Stability Constants Database,
version 4.0 National Institute of Standards and Technology, Washington,
DC, 1997.
1H paramagnetic NMR measurements were acquired on a
Bruker Avance 400 spectrometer operating at 399.91 MHz. One-
dimensional spectra were recorded in D2O solvent with presat-
uration of the H2O signal during part of the relaxation delay to
eliminate the H2O signal. Relaxation delay times of 50–200 ms, 30–
80 kHz spectral widths ranging and acquisition times of 60–200 ms
were used. 1D spectra were processed using exponential line-
broadening weighting functions as apodization with values of 20–
40 Hz. Chemical shifts were referenced to residual solvent protons
of D2O resonating at 4.76 ppm (298 K) relative to TMS. Sample
1
concentrations for paramagnetic H NMR were 2.5 mmol dm−3
Cu2TAL complexes. The longitudinal relaxation times of the
hyperfine shifted resonances were determined using the inversion
recovery pulse sequence d1 − 180◦ − s − 90◦ − acq,25 where d1 is the
relaxation delay and acq the acquisition time), 14 values of s were
selected between 0.4 ms and 300 ms, (d1 + acq) values were at least
five times the longest expected T1 ranging from 100 to 400 ms, and
the number of scans was 8000. The T1 values were calculated from
the inversion–recovery equation. Transversal relaxation times were
obtained measuring the line broadening of the isotropically shifted
11 (a) F. Thaler, C. D. Hubbard, F. W. Heinemman, R. Van Eldik, S.
Schlinder, I. Fa´bian, A. M. Dittler-Klingemann, F. E. Hahn and C.
Orvig, Inorg. Chem., 1998, 37, 4022; (b) J. R. Hartam, R. W. Bachet, W.
Pearson, R. J. Wheat and J. H. Callahan, Inorg. Chim. Acta, 2003, 343,
119.
−1
signals at half-height through the equation T2 = pDm1/2
.
Electrospray mass spectrometry
12 G. Anderregg and V. Gramlich, Helv. Chim. Acta, 1994, 77, 685.
The electrospray mass spectrum was recorded on a Bruker
Esquire 3000plus, Bruker Daltonics mass spectrometer (Agilent
Headquarters, Palo Alto, USA). ES-MS was carried out in the
positive ion mode. Scanning was performed from m/z = 300 to
1400. For electrospray ionization, the drying gas was set at a flow
rate of 2.5 lL min−1, with capillary voltage of 166 V. Non-buffered
solutions containing AMP : TAL ([TAL] = 1.00 × 10−3 mol dm−3,
molar ratio 6 : 1) in water were injected into the mass spectrometer
source with a syringe pump (Cole-Parmer Instruments Company,
Illinois, USA) at a flow rate of 2 L min−1. The sampling cone
voltage (VC) was set at 40 V.
13 The logarithms of the protonation constants in 0.15 mol dm−3 NaClO4
at 298.1 K are: log KHL/H·L = 10.39(3), log KH
= 10.33(3),
L·H = 8.02(2),
2 L/HL·H
log KH
L·H = 9.64(2), log KH
L·H = 8.89(2), log KH
3 L/H2
4 L/H3
5 L/H4
log K
= 7.39(2).
H6 L/H5 L·H
14 Protonation constants for AMP determined at 298.1 K in 0.15 mol dm−3
NaCl: log KHL/H·L = 6.32(1); log KH
= 10.26(1).
2 L/HL·H
15 For a recent review on anion coordination chemistry, see for instance:
S. Kubik, C. Reyheller and S. Stu¨we, J. Inclusion Phenom. Macrocycl.
Chem., 2005, 52, 137.
16 C. Miranda, F. Escart´ı, L. Lamarque, E. Garc´ıa-Espan˜a, P. Navarro, J.
Latorre, F. Lloret, H. R. Jime´nez and J. R. Yunta, Eur. J. Inorg. Chem.,
2005, 189.
17 B. Verdejo, J. Aguilar, A. Dome´nech, C. Miranda, P. Navarro, H. R.
Jime´nez, C. Soriano and E. Garc´ıa-Espan˜a, Chem. Commun., 2005,
3086.
18 M. Bera, W. T. Wong, G. Aromi and D. Ray, Eur. J. Inorg. Chem., 2005,
Acknowledgements
2526.
19 E. Garc´ıa-Espan˜a, M.-J. Ballester, F. Lloret, J.-M. Moratal, J. Faus and
A. Bianchi, J. Chem. Soc., Dalton Trans., 1988, 101.
20 M. Fontanelli and M. Micheloni, Proceedings of the Spanish-Italian
Congress on Thermodynamic of Metal Complexes, Diputacio´n de
Castello´n, Castello´n, Spain, 1990.
We would like to thank Prof. H.-J. Schneider for helpful dis-
cussion. Financial support from Grupos S03/196 and DGICYT
project BQU2003-09215-CO3-01 (Spain) is gratefully acknowl-
edged. J. M. Ll. thanks MCYT of Spain for a Ramo´n y Cajal
contract.
21 G. Gran, Analyst, 1952, 77, 661; F. J. C. Rossotti and H. Rossotti,
J. Chem. Educ., 1965, 42, 375.
22 P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43, 1739.
23 E. Martell, R. M. Smith and R. J. Motekaitis, NIST Critically Selected
Stability Constants of Metal Complexes Database, NIST Standard
Reference Database, version 4, 1997.
24 A. K. Convington, M. Paabo, R. A. Robinson and R. G. Bates, Anal.
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This journal is
The Royal Society of Chemistry 2006
Dalton Trans., 2006, 4474–4481 | 4481
©