Phenanthroline-containing macrocycles as multifunctional receptors for nucleotide anions. a thermodynamic and NMR study

Article abstract of DOI:10.1039/a903059j

The synthesis of the phenanthroline-containing macrocycle 2,6,10,14-tetraaza[15](2.9)cyclo(1,10)phenanthrolinophane (L1) is reported. L1 contains a tetraamine chain connecting the 2,9-positions of a phenanthroline unit. Protonation of L1 has been studied by means of potentiometric and ¹H and ¹³C NMR techniques, allowing the determination of the basicity constants and of the stepwise protonation sites. The protonation features of L1 are compared with those of macrocycle 2,5,8,11-tetraaza[12](2,9)cyclophenanthrolinophane (L2), in which the amine groups are linked by ethylenic chains. Considering the [H₄L1]^4- and the [H₄L2]⁴⁺ species, the acidic protons are located on the aliphatic nitrogens, while phenanthroline is not involved in protonation. Binding of diphosphate, triphosphate, ATP and ADP has been studied by means of potentiometry and ¹H and ³¹P NMR. Both L1 and L2 behave as multifunctional receptors for the nucleotide anions at neutral or slight acidic pHs, giving 1 : 1 complexes. Charge-charge and hydrogen bonding interactions take place between the polyphosphate chain of nucleotides and the polyammonium groups of L1 and L2, while the adenine moiety gives charge-dipole interactions with the ammonium groups and π-stacking with the phenanthroline unit of the receptors. The high upfield displacements in the ¹H NMR spectra exhibited by the adenine protons upon complexation by L1 suggest a partial inclusion of nucleotides inside the macrocyclic cavity.

Full text of DOI:10.1039/a903059j

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Phenanthroline-containing macrocycles as multifunctional receptors
for nucleotide anions. A thermodynamic and NMR study†
Carla Bazzicalupi,a Alessia Beconcini,a Andrea Bencini,*a Vieri Fusi,b Claudia Giorgi,a
Andrea Masottia and Barbara Valtancolia
a Department of Chemistry, University of Florence, Via Maragliano 75/77, 50144 Florence,
Italy
b Institute of Chemical Sciences, University of Urbino, Italy
Received (in Cambridge) 16th April 1999, Accepted 11th June 1999
The synthesis of the phenanthroline-containing macrocycle 2,6,10,14-tetraaza[15](2,9)cyclo(1,10)phenanthrolino-
phane (L1) is reported. L1 contains a tetraamine chain connecting the 2,9-positions of a phenanthroline unit.
Protonation of L1 has been studied by means of potentiometric and 1H and 13C NMR techniques, allowing the
determination of the basicity constants and of the stepwise protonation sites. The protonation features of L1 are
compared with those of macrocycle 2,5,8,11-tetraaza[12](2,9)cyclophenanthrolinophane (L2), in which the amine
groups are linked by ethylenic chains. Considering the [H4L1]4ϩ and the [H4L2]4ϩ species, the acidic protons are
located on the aliphatic nitrogens, while phenanthroline is not involved in protonation. Binding of diphosphate,
triphosphate, ATP and ADP has been studied by means of potentiometry and 1H and 31P NMR. Both L1 and L2
behave as multifunctional receptors for the nucleotide anions at neutral or slight acidic pHs, giving 1:1 complexes.
Charge–charge and hydrogen bonding interactions take place between the polyphosphate chain of nucleotides and
the polyammonium groups of L1 and L2, while the adenine moiety gives charge–dipole interactions with the
ammonium groups and π-stacking with the phenanthroline unit of the receptors. The high upfield displacements
in the 1H NMR spectra exhibited by the adenine protons upon complexation by L1 suggest a partial inclusion of
nucleotides inside the macrocyclic cavity.
The design of host molecules for recognizing anions, such as
carboxylic acids or nucleotides, in aqueous solution is an
important target according to the biological relevance of the
studies in this solvent. This goal is not easily achieved as strong
solvent–guest interactions can efficiently compete with the pro-
cess of selective complexation. To this purpose, polyammonium
macrocycles may behave as efficient receptors for polycharged
anions in aqueous solution.1–14 Protonated macrocyclic poly-
amines strongly bind to nucleotides via electrostatic inter-
actions between the cationic binding sites (ammonium groups)
of the receptor and the negatively charged polyphosphate
chain. It is accepted, however, that selective coordination
requires the incorporation of sites for multiple interactions with
the substrates. To achieve a better recognition of nucleotides,
receptors need to contain other binding sites capable of inter-
actions with the sugar moiety15–17 or the nucleic base,2c,18–20 in
addition to the anion binding sites. In particular, base-selective
recognition is attainable either by hydrogen bonds to suitably
constructed receptors or by stacking interactions with π-
systems incorporated into the host molecule.
the nucleobase. Recently, polyazacyclophanes, i.e. macrocycles
containing phenylene subunits as an integral part of the
cyclic framework, have been used as selective recognizers of
nucleotides. In the case of ligands 23 and 3,5c as well as stacking
interactions, the inclusion of nucleobases into the macro-
cyclic cavity is a peculiar structural motif of their adducts with
nucleotides.
In this paper we report the synthesis of the new macrocyclic
receptor L1, which contains a large heteroaromatic moiety,
phenanthroline, as a non-pendant integral part of a polyamine
macrocyclic structure. The insertion of phenanthroline may
provide a further binding site for nucleotide anions. In fact,
this large heteroaromatic moiety may offer an optimal binding
site for the coordination of nucleotides, through stacking
and hydrophobic interactions. Since anion coordination by
polyamine ligands is strictly related to their basicity properties,
we have analyzed the protonation features of L1. Protonation
of L1 gives [HnL1]nϩ cations, potential multifunctional recep-
tors for nucleotides: they contain a polyammonium chain which
may interact with the anionic polyphosphate chain of nucleo-
tides and a large heteroaromatic unit as binding site for the
nucleic bases. Furthermore, charge–dipole interactions between
the polyammonium groups and the adenine ring may take
place, as actually found for the adducts of 3 with nucleotides.5a,c
Earlier, Lehn and Hosseini reported one of the first syn-
thetic ligands able to behave as a multifunctional receptor
for nucleotides (1 in Scheme 1).2c 1 contains two recognition
sites, a macrocyclic polyammonium moiety, as anion binding
site, and an acridine pendant arm for stacking interactions with
1
Therefore, we have carried out a thermodynamic and H and
31P NMR study on the interaction of this receptor with ATP
and ADP. For comparison, the binding properties of L1
towards inorganic diphosphate and triphosphate have been also
investigated. In order to analyze the effect of the dimensions
of the macrocyclic cavity on nucleotide coordination, we
have also studied the binding features of macrocycle L2.21
Similarly to L1, L2 contains a tetraamine unit linking the
2,9-phenanthroline positions; on the other hand this ligand
possesses a smaller cavity than L1, due to the ethylenic chains
connecting the aliphatic nitrogens.
1
† Supplementary data available: plots of the H chemical shifts of the
signals of selected aromatic protons of ligands and nucleotides against
the nucleotide/ligand ratio (Fig. S1–S4) and plots of the H chemical
1
shifts of L1 in the presence and in the absence of ATP and of ATP in
the presence and in the absence of L1 as a function of pH (Fig. S5). For
direct electronic access see http://www.rsc.org/suppdata/p2/1999/1675,
otherwise available from BLDSC (SUPPL. NO. 57587, pp. 5) or the
RSC Library. See Instructions for Authors available via the RSC web
page (http://www.rsc.org/authors).
J. Chem. Soc., Perkin Trans. 2, 1999, 1675–1682
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Table 1 Protonation constants (log K) of L1 and L2 determined by
means of potentiometric measurements in 0.1 mol dmϪ3 NMe4Cl aque-
ous solution at 298.1 K
Log K
Reaction
L1
L2
L ϩ Hϩ = HLϩ
10.48(1)a
9.06(2)
7.39(3)
6.08(3)
9.72b
8.71
6.18
2.18
HLϩ ϩ Hϩ = H2L2ϩ
H2L2ϩ ϩ Hϩ = H3L3ϩ
H3L3ϩ ϩ Hϩ = H4L4ϩ
a Values in parentheses are standard deviations on the last significant
figure. b From reference 21b.
Fig. 1 Distribution diagram of the protonated species formed by L1
as a function of pH (0.1 mol dmϪ3 NMe4Cl, 298.1 K, [L1] = 1 × 10Ϫ3
mol dmϪ3).
solution (Fig. 1). On the other hand, a decrease of absorbance
is observed at strongly acidic pHs (λmax = 282 nm, ε = 25950
molϪ1 dm3 cmϪ1 at pH 1.5), suggesting that protonation of
phenanthroline takes place only at very acidic pH values. These
data suggest that the first four protonation steps of L1 and L2
take place on the four aliphatic amine groups. It is also to be
noted that the basicity constants of L1 are remarkably higher
than those of L2 in each step of protonation. This behavior
can be ascribed to the larger ϩI inductive effect of the longer
propylenic chains, which increases the basicity of each amine
group, as well as to the longer distance between amine nitro-
gens, which allows a better minimization of the electrostatic
repulsion between protonated ammonium groups in the
[HnL1]nϩ polycharged cations with respect to the [HnL2]nϩ ones.
This effect has been also observed in aliphatic diamines, such
as N,NЈ-dimethyl-1,2-diaminoethane (log K1 = 10.05 and log
K2 = 7.12)24 and N,NЈ-dimethyl-1,3-diaminopropane (log K1 =
10.80 and log K2 = 9.1),25 which contain two secondary amine
groups linked respectively by an ethylenic and a propylenic
chain.
Scheme 1
Results and discussion
Protonation of L1
The protonation equilibria of L1 have been studied in 0.1 mol
dmϪ3 NMe4Cl solution at 298.1 0.1 K by means of potentio-
metric measurements. The protonation constants are reported
in Table 1, together with those previously reported for L2.21b
The distribution diagram for the species present in solution
as a function of pH for the system L1/Hϩ is shown in Fig. 1.
Both L1 and L2 behave as tetraprotic bases in the pH range
3–11. Phenanthroline nitrogens are characterized by far lower
basicity than aliphatic amine nitrogens: the first protonation
constant of phenanthroline is 4.93 log units,22 while the first
protonation constant of secondary amines usually ranges
between 9.5–11 log units.23 Therefore, it is expected that at least
the first protonation steps take place on the amine groups of
the aliphatic chains. Indeed, UV spectra recorded on solutions
containing L2 at various pH values do not show significant
variations in the pH range 2.5–11 (λmax = 275 nm, ε = 29500
molϪ1 dm3 cmϪ1 at pH 5) indicating that the aromatic nitrogens
are not involved in protonation. In the case of L1, the UV
spectra of the ligand do not change significantly in the pH
range 11–3.5 (λmax = 273 nm, ε = 31200 molϪ1 dm3 cmϪ1 at pH
5), where the macrocycle binds up to four protons in aqueous
To get further information on the protonation pattern of
1
L1 we have also analyzed the variations with pH of its H and
13C NMR spectra. All the assignments have been made on
the basis of 1H–1H homonuclear and 1H–13C heteronuclear
correlation experiments at the different pH values studied.
The 13C spectrum of L1 at pH 12.0, where the unprotonated
amine predominates in solution, exhibits twelve peaks, six
for the aliphatic carbons CF1–CF6 and six for the aromatic
1
carbons CF7–CF12. The H spectrum of L1 at this pH shows
two multiplets at 1.46 and 1.61 ppm (each integrating four
protons and attributed to the hydrogen atoms HF1 and HF4
respectively), a multiplet at 2.42 ppm (eight protons, the hydro-
gens of HF2 and HF3), a triplet at 2.61 ppm (4 protons, HF5),
a singlet at 3.95 ppm (4 protons, HF6), and a singlet and
two doublets for the aromatic protons HF12, HF8 and HF9.
These spectral features indicate a C2v time averaged symmetry.
This symmetry is preserved throughout all the pH range
investigated.
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ation of these species dramatically affects the pattern of the
1H and 13C NMR spectra. Fig. 2a shows that the signals of HF5
and HF6 undergo a remarkable downfield shift. Considering
the 13C NMR spectra recorded in the same pH range, the
resonances of CF4 and CF7, in the β-position with respect
to N2, exhibit a marked upfield shift. These spectral features
indicate that the second and third protonation steps take place
on the benzylic nitrogens N2 and N2Ј. As can be seen in the dis-
tribution diagram (Fig. 1), L1 is mainly in its tetraprotonated
form at pH < 6. In the 1H NMR spectrum recorded below pH
6, the signals of the hydrogens HF2 and HF3 shift downfield,
suggesting that the nitrogens N1 and N1Ј are involved in the
fourth protonation step.
It is worth noting that the first four protonation steps
1
involve only the nitrogens of the aliphatic chain. The H and
13C signals of the aromatic moiety (with the exception of
CF7) do not vary significantly in the pH range 12–3. Only
at strongly acidic pH values do the protons of the phenan-
throline moiety show a downfield shift, probably due to the
involvement of the aromatic nitrogens in the binding of the
fifth proton.
1
Considering the ligand L2, the analysis of the H and 13C
NMR spectra recorded at different pH values shows a similar
protonation mechanism; in the [H4L2]4ϩ species the four
protons are located on the aliphatic amine groups, while
phenanthroline is not protonated even at strongly acidic pHs
(pH 2).
In conclusion, these protonation features make L1 and L2
promising receptors for nucleotides. They give polyprotonated
forms at neutral pH. These polycharged species display a
polyammonium chain, a potential binding site for the anionic
phosphate moiety of nucleotides, and a phenanthroline unit,
which may give π-stacking interactions with the nucleobase.
Phosphate anion binding
Binding of ATP, ADP, diphosphate and triphosphate by L1
and L2 has been studied by means of potentiometric measure-
1
ments and, in the case of ATP and ADP, by H and 31P NMR
measurements.
Protonation of the receptors gives charged species which
enable L1 and L2 to form stable complexes with anionic forms
of these nucleotides in aqueous solution. The formation of such
species is strictly pH-dependent and, therefore, the relevant
equilibria can be studied by pH-metric titrations. Table 2
collects the cumulative and stepwise equilibrium constants
Fig. 2 Experimental 1H chemical shifts (a) and 13C NMR chemical
shifts (b) of L1 as a function of pH (the signals of the aromatic carbons
CF8–CF12 have been omitted for clarity. Their chemical shifts do not
significantly change in the pH range investigated).
4Ϫ
for the species formed by L1 and L2 with ATP, ADP, P2O7
and P3O10 .
5Ϫ 27 The stability constants of the L2 complexes with
triphosphate cannot be determined, due to the low solubility of
the adducts. Although the formation in some cases of both 1:1
and 2:1 anion–macrocycle complexes has been observed,28
the data analysis with the program HYPERQUAD29 under
our experimental conditions revealed predominantly 1:1
stoichiometries for all the species detected. This is confirmed by
1
Fig. 2a shows the H NMR spectra recorded at various pH
values and Fig. 2b reports the 13C NMR chemical shifts of L1
as a function of pH.
In the pH range 12–10, where the first proton binds to the
ligand, the signals of the hydrogens HF2 and HF3, in the α-
position with respect to N1, shift downfield; a slight downfield
shift is also observed for the protons HF4 and HF1 of the
central methylene of the propylenic chains, while the other
signals do not appreciably shift. This suggests that the first
protonation step involves the nitrogens N1 and N1Ј. This
hypothesis is confirmed by the 13C NMR spectra recorded in
the same pH range, which show that the resonances of the
carbon atoms CF1 and CF4, in the β-position with respect to
N1, shift upfield (Fig. 2b), in good agreement with the β-shift
reported for the protonation of polyamines.26 The higher pro-
ton affinity of the nitrogen atoms N1 and N1Ј in comparison
with benzylic nitrogens N2 and N2Ј can be ascribed to the
inductive effect of the heteroaromatic moiety on the adjacent
N2 and N2Ј amine groups.
1
Job plots of the H NMR chemical shifts of the signals of
selected aromatic protons of L1 or L2 and ATP or ADP against
the nucleotide/ligand ratio, which show sharp breaks at ligand/
nucleotide = 1.
By examining the different values of stability constants,
several main features can be readily noticed. For all the studied
anions, the interactions with the macrocycles start to be detec-
table for a minimum of protonation of two, except for the
4Ϫ
5Ϫ
systems L1–ADP3Ϫ, L2–P2O7 and L1–P3O10 for which at
least three protons are required on the macrocycle to make the
interaction detectable.
For a given macrocycle the strength of the interaction
generally increases with its degree of protonation. For instance,
the stability constants for the interaction of ATP4Ϫ with L1
and L2 vary respectively from log K = 2.57 and 2.93 for the
diprotonated macrocycles to log K = 4.05 and 5.26 for the
In the pH range 10–6 the macrocycle binds two further
protons, giving the [H2L1]2ϩ and [H3L1]3ϩ species. The form-
J. Chem. Soc., Perkin Trans. 2, 1999, 1675–1682
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Table 2 Stability constants (log K) of the ADP, ATP, diphosphate and triphosphate adducts with L1 and L2, determined by means of potentio-
metric measurements in 0.1 mol dmϪ3 NMe4Cl at 298.1 K
Reaction
L1
L2
Reaction
L1
L2
A = ADP3Ϫ
A = ATP4Ϫ
L ϩ 2Hϩ ϩ A3Ϫ = H2LAϪ
L ϩ 3Hϩ ϩ A3Ϫ = H3LA
L ϩ 4Hϩ ϩ A3Ϫ = H4LAϩ
L ϩ 5Hϩ ϩ A3Ϫ = H5LA2ϩ
L ϩ 6Hϩ ϩ A3Ϫ = H6LA3ϩ
—
20.8(1)a
28.62(5)
33.49(6)
38.32(8)
40.73(8)
L ϩ 2Hϩ ϩ A4Ϫ = H2LA2Ϫ
L ϩ 3Hϩ ϩ A4Ϫ = H3LAϪ
L ϩ 4Hϩ ϩ A4Ϫ = H4LA
L ϩ 5Hϩ ϩ A4Ϫ = H5LAϩ
L ϩ 6Hϩ ϩ A4Ϫ = H6LA2ϩ
22.11(7)
30.98(3)
39.31(3)
45.34(4)
47.76(6)
21.36(6)
29.87(4)
34.31(5)
39.33(5)
—
30.12(3)
38.43(2)
43.65(2)
—
H2L2ϩ ϩ A3Ϫ = H2LAϪ
H3L3ϩ ϩ A3Ϫ = H3LA
—
2.4(1)
H2L2ϩ ϩ A4Ϫ = H2LA2Ϫ
H3L3ϩ ϩ A4Ϫ = H3LAϪ
H3L3ϩ ϩ HA3Ϫ = H4LA
H4L4ϩ ϩ HA3Ϫ = H5LAϩ
H4L4ϩ ϩ H2A2Ϫ = H6LA2ϩ
2.57(7)
4.05(3)
5.20(3)
5.15(4)
3.24(4)
2.93(6)
5.26(6)
2.52(6)
6.36(4)
—
3.19(3)
3.78(3)
4.05(5)
—
4.01(5)
2.29(5)
4.94(6)
3.38(6)
H3L3ϩ ϩ HA2Ϫ = H4LAϩ
H4L4ϩ ϩ HA2Ϫ = H5LA2ϩ
H4L4ϩ ϩ H2AϪ = H6LA3ϩ
Reaction
L1
L2
Reaction
L1
4Ϫ
5Ϫ
A = P2O7
A = P3O10
L ϩ 2Hϩ ϩ A4Ϫ = H2LA2Ϫ
L ϩ 3Hϩ ϩ A4Ϫ = H3LAϪ
L ϩ 4Hϩ ϩ A4Ϫ = H4LA
22.4(1)
30.7(1)
38.7(1)
—
30.2(1)
36.8(1)
L ϩ 3Hϩ ϩ A5Ϫ = H3LA2Ϫ
L ϩ 4Hϩ ϩ A5Ϫ = H4LAϪ
L ϩ 5Hϩ ϩ A5Ϫ = H5LA
30.22(3)
38.45(3)
45.12(4)
H2L2ϩ ϩ A4Ϫ = H2LA2Ϫ
H2L2ϩ ϩ HA3Ϫ = H3LAϪ
H3L3ϩ ϩ HA3Ϫ = H4LA
2.8
2.4
2.9
—
3.0
3.4
H2L2ϩ ϩ HA4Ϫ = H3LA2Ϫ
H3L3ϩ ϩ HA4Ϫ = H4LAϪ
H4L4ϩ ϩ HA4Ϫ = H5LA
2.18(3)
3.02(3)
3.61(4)
a Values in parentheses are standard deviations on the last significant figure.
macrocycles in their triprotonated forms. A similar increase of
stability with the increasing protonation degree of the receptors
is generally observed for the other systems. An increasing
number of protonated polyammonium functions increases the
receptor’s ability to give charge–charge and hydrogen bonding
interactions with the anionic substrates. High protonation
degrees imply protonation of nucleotides and, therefore, a
low negative charge of the substrates. As a consequence, hexa-
protonated adducts with ATP and ADP are not detected by
potentiometry or display low formation constants. Finally,
no interaction between protonated macrocycles and the
H3ADP and H4ATP uncharged substrates was found in our
experimental conditions. As a consequence, the formation of
the substrate–polyammonium receptor adducts takes place
mainly from weakly alkaline to slightly acidic pHs, as shown
in Fig. 3, which displays the distribution diagrams for the
ADP–L1 and ATP–L1 systems. For the system ATP–L1 the
percentage of overall complexed species is more than 70% in
the pH range 4–7, while in the case of the system ADP–L1 the
percentage is more than 50% in the pH range 4.5–7.5. In both
cases, the overall percentages of substrate–receptor complexes
decrease at more strongly alkaline or acidic pH. For instance
the percentage of ATP–L1 adduct is less than 25% at pH 2 and
no interaction between ATP and L1 is detected above pH 10.
The comparison of Fig. 3a and 3b also shows a lower
interaction of ADP with L1 with respect to ATP. As can be
seen from Table 2, for the same protonation degree, the
ADP complexes with L1 and L2 show a lower stability in
comparison with the ATP adducts. This behaviour can be
reasonably ascribed to weaker charge–charge interactions in the
ADP complexes, due to a lower negative charge on this anion.
Finally, for a determined protonation degree of both
reagents, anion and macrocycle, a decrease in stability occurs
from L2 to L1. This may be ascribed to a decrease in charge
density as the macrocyclic size increases.
Fig. 3 Distribution diagrams for the systems L1–ADP (a) (0.1 mol
dmϪ3 NMe4Cl, 298.1 K, [L1] = [ADP] = 1 × 10Ϫ3 mol dmϪ3) and L1–
ATP (b) (0.1 mol dmϪ3 NMe4Cl, 298.1 K, [L1] = [ATP] = 1 × 10Ϫ3 mol
dmϪ3).
induced 31P chemical shifts (CIS) for the systems nucleotides–
L1 or –L2.
Considering the data in Fig. 4 and Table 3, some general
features can be outlined: (a) complexation of substrates pro-
duces significant variations in the 31P chemical shifts, as already
observed for analogous complexes with other polyammonium
macrocyclic receptors.2,3 Considering ADP complexation, the
resonances of both the phosphate groups shift downfield in
the presence of the macrocycles. In the case of the ATP com-
plexes with L1 and L2, only the signals of two phosphate
Anion complexation has also been followed by recording 31
P
NMR spectra on solutions containing receptors and substrates
in 1:1 molar ratio at different pH values. Fig. 4 shows the 31P
chemical shifts of the phosphate groups of ATP in the presence
of L1 (1:1 molar ratio) at different pH values, together with
those of free ATP, and Table 3 reports the complexation
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Table 3 31P NMR shifts (δ, ppm) for the L1 and L2 adducts with ATP
and ADP and complexation-induced 1H NMR chemical shifts (CIS,
ppm) for selected protons, measured in D2O solution at pH 4.5 (systems
ATP–L1 and ADP–L1) and 4.8 (systems ATP–L2 and ADP–L2),
298 K
L1
L2
ADP
δ(ppm)
CIS
Pα
Pβ
Ϫ4.21
3.33
Pα
Ϫ7.97
0.21
Pβ
Ϫ6.09
1.45
Ϫ7.69a
0.49b
L1
L2
ATP
δ(ppm) Ϫ8.21
CIS 0.05
Pα
Pβ
Pγ
Pα
Ϫ8.22
0.0
Pβ
Pγ
Ϫ19.59 Ϫ6.84
0.52 0.82
Ϫ19.35 Ϫ6.95
0.67 0.73
a From measurements in D2O solution at pH 4.5 (systems ATP–L1 and
ADP–L1) and 4.8 (systems ATP–L2 and ADP–L2), 298 K, with a
receptor–substrate 1:1 molar ratio. In these conditions the complex-
ation degrees are 40% (system ADP–L1), 72% (ATP–L1), 49% (ADP–
L2) and 53% (ATP–L2). b CIS (for 100% complexation) calculated
based on equilibrium constants from Table 2.
Fig. 5 Overall percentages of L1 complexed species as a function of
pH in competing systems containing ATP and triphosphate ([L1] =
Fig. 4 Experimental 31P chemical shifts of free ATP (-----) and of ATP
in the presence of L1 (——) as a function of pH ([L1] = [ATP] =
1 × 10Ϫ2 mol dmϪ3, 298 K).
[ATP] = [triphosphate] = 1 × 10Ϫ3 mol dmϪ3
) (a) and ADP and
diphosphate ([L1] = [ADP] = [diphosphate] = 1 × 10Ϫ3 mol dmϪ3) (b).
Percentages are calculated with respect to L1.
groups, namely Pβ and Pγ, show a clear downfield shift upon
complexation, while the chemical shift of Pα is not influenced
by the interaction with the receptors. These observations seem
to indicate that in both ATP and ADP binding the poly-
ammonium functions of the macrocycles interact with two
contiguous phosphate groups of nucleotides (Pβ and Pγ in the
case of ATP). (b) As shown in Fig. 4 for the system ATP–L1,
the variations of chemical shifts are strongly pH dependent,
being greater in the pH range 5–7 and almost negligible above
pH 8 and below pH 3.5. A similar behavior is also observed
for the other systems under investigation. This result is in good
accord with the potentiometric study of these systems, which
has shown that large amounts of the 1:1 receptor–substrate
adducts are formed from slight alkaline to acidic pHs, i.e., in
the pH region where highly protonated species of the receptors
and anionic species of ADP or ATP are simultaneously present
in solution.
with L1 is lower than the ATP one. On the other hand, such
comparisons may sometimes be misleading in the analysis of
selectivity in anion coordination, due to the different proton-
ation degrees of different substrates at the same pH values. An
appropriate way to visualize selectivity in anion coordination is
to consider a ternary system containing the ligands and the two
anions in equimolar concentrations and calculate the overall
percentages of the two complexed anions over a wide pH
range.11b Plots of the percentages vs. pH produce species distri-
bution diagrams from which the binding ability of the receptor
can be interpreted in terms of selectivity. In Fig. 5 are
reported similar diagrams calculated for the ATP–P3O105Ϫ–L1
and ADP–P2O74Ϫ–L1 systems. As can be seen, the formation of
nucleotide adducts with L1 prevails over a wide pH range,
i.e., nucleotide anions are selectively bound with respect to their
inorganic counterparts (all over the pH range investigated in
the case of the ATP–P3O105Ϫ–L1 system, from pH 8 to acidic
pH in the case of the ADP–P2O74Ϫ–L1 system).
Both potentiometric and 31P NMR data confirm the impor-
tant roles played by electrostatic forces and hydrogen bonding
in this kind of interaction. The formation of the adducts takes
place mainly via the charged polyphosphate chains of ADP
or ATP, which allows the formation of multiple electrostatic
and hydrogen bond interactions with the polyammonium
macrocycles. In the case of ATP, however, one of the phosphate
groups (Pα) does not interact with the polyammonium sites.
Comparing nucleotide and inorganic phosphate anion
(P2O74Ϫ and P3O105Ϫ) binding, the data in Table 2 seem to show
that pyrophosphate gives weaker interactions with L1 and L2
than ADP, which contains a diphosphate chain similar to that
of P2O74Ϫ. Similarly, the stability of the triphosphate adducts
These experimental observations suggest that, besides
electrostatic interactions between the phosphate chain and
the polyammonium macrocycles, other effects (hydrogen bond
interactions between adenine nitrogens and/or hydroxy groups
of nucleotides and polyammonium functions, hydrophobic
and/or π-stacking interactions between the heteroaromatic
moieties and cation–π system interactions between the charged
ammonium group and adenine) may contribute to the complex
stability.
Indeed, 1H NMR spectra of these systems provide
unambiguous evidence for the participation of π-stacking
J. Chem. Soc., Perkin Trans. 2, 1999, 1675–1682
1679

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interactions in the stabilization of the adduct species with L1
and L2 (for the labeling, see Scheme 1). For both ligands,
throughout the pH ranges in which interaction occurs, signifi-
cant upfield displacements are observed for the resonances of
the adenine protons H2, H8 and for the anomeric proton H1Ј
of the nucleotides as well as for the signals of phenanthroline
(HF8, HF9 and HF12). Minor shifts are observed for the
benzylic protons F6 (less than 0.3 ppm) and for the other pro-
tons of the aliphatic chains (less than 0.2 ppm). Fig. 6 shows the
1H chemical shifts for the phenanthroline hydrogens of L1 in
the absence and in the presence of ADP (Fig. 6a) and for the
hydrogens H2, H8 and H1Ј of ADP (Fig. 6c) in the absence and
in the presence of L1 (1:1 molar ratio). A distribution diagram
displaying the overall percentages of complexed ADP is also
reported (Fig. 6b). As previously observed for the 31P reson-
ances of the phosphate chains, the 1H NMR displacements are
strongly pH dependent, being larger at neutral or slight acidic
pHs, where the largest extent of complexation occurs (Fig. 6). It
is interesting to remark that, once more, these differences are
remarkably reduced at strongly acidic or alkaline pHs, where
the interaction vanishes. Similar considerations can be made
also for ATP complexation by L1 (Fig. S5, supplementary
material).† Table 4 reports the complexation induced chemical
shift (CIS) for the interaction of L1 and L2 with ADP and ATP.
It is to be noted that the CIS values for ATP and ADP com-
plexation are remarkably high (more than 1 ppm for adenosine
protons of ADP complexed by L1 or L2), if compared with the
CIS value found for ATP or ADP complexation by poly-
ammonium macrocycles containing phenylene spacers, such
as 2 in Scheme 1.3 This can be attributed to the insertion into
the macrocyclic framework of a large heteroaromatic system,
phenanthroline, which can give strong π-stacking interactions
with the adenine moiety of nucleotides. Furthermore, high CIS
values may be indicative of a partial inclusion of the substrate
inside the macrocyclic cavity, as already observed in the case of
the receptors 23 and 3.5c A partial inclusion may allow for a
simultaneous involvement of electrostatic and π-stacking inter-
actions in the stabilization of the adducts and, additionally,
adenine nitrogens and/or hydroxy groups of the ribose sub-
units could be properly disposed to give hydrogen bonds with
the polyammonium groups of the receptor. A proposed mode
of interaction between ATP and H4L14ϩ is reported in Fig. 7.
Table 4 also shows that the CIS values of the adenine protons
of both ATP and ADP are larger for the interaction with macro-
cycle L1, which presents a larger cavity than L2. Furthermore,
for the same macrocycle, the CIS values of adenine protons of
1
Fig. 6 (a) Experimental H chemical shifts for the aromatic protons
......
of free L1 (——) and of L1 in the presence of ADP ( ); (b) overall
percentages of free ADP, L1 and ADP–L1 complexes; (c) experimental
1H chemical shifts for the H2, H8 and H1Ј protons of free ADP (——)
Ϫ2
......
and of ADP in the presence of L1 ( ) ([L1] = [ADP] = 1 × 10 mol
dmϪ3, 298 K).
Table 4 1H NMR shifts (δ, ppm) for the L1 and L2 adducts with ATP and ADP and complexation-induced 1H NMR chemical shifts (CIS, ppm)
for selected protons, measured in D2O solution at pH 4.5 (systems ATP–L1 and ADP–L1) and 4.8 (systems ATP–L2 and ADP–L2), 298 K
HF8
HF9
HF12
7.47
HF6
H8
H2
H1
L1
ATP
δ
δ
7.69a
Ϫ0.19b
7.71
8.21
Ϫ0.36
8.27
4.64
Ϫ0.12
4.579
Ϫ0.03
4.44
7.61
Ϫ0.91
7.31
Ϫ0.95
5.02
Ϫ1.12
CIS
Ϫ0.52
L2
ATP
δ
δ
7.55
Ϫ0.46
7.05
7.98
Ϫ0.52
7.77
Ϫ0.46
5.46
Ϫ0.66
CIS
Ϫ0.13
7.48
Ϫ0.31
7.91
L1
ADP
δ
δ
7.2
6.58
4.04
CIS
Ϫ0.44
7.6
Ϫ0.74
8.23
Ϫ1.01
7.49
Ϫ0.25
4.61
Ϫ1.36
Ϫ1.73
Ϫ2.15
L2
ADP
δa
7.34
4.7
δ
7.50
CIS
Ϫ0.14
Ϫ0.35
Ϫ0.52
Ϫ0.10
Ϫ1.06
Ϫ0.97
Ϫ1.49
a From measurements in D2O solution at pH 4.5 (systems ATP-L1 and ADP–L1) and 4.8 (systems ATP–L2 and ADP–L2), 298 K, with a receptor–
substrate 1:1 molar ratio. In these conditions the complexation degrees are 40% (system ADP–L1), 72% (ATP–L1), 49% (ADP–L2) and 53% (ATP–
L2). b CIS (for 100% complexation) calculated based on equilibrium constants from Table 2.
1680
J. Chem. Soc., Perkin Trans. 2, 1999, 1675–1682

View Article Online
2,6,10,14-Tetratosyl-2,6,10,14-tetraaza[15](2,9)cyclo(1,10)-
phenanthrolinophane (6). A solution of 5 (2.3 g, 6.2 mmol) in
dry CH3CN (200 cm3) was added over a period of 6 h to a
refluxing and vigorously stirred suspension of 4 (5 g, 6.2 mmol)
and K2CO3 (8.6 g, 0.062 mol) in CH3CN (400 cm3). After the
addition was completed, the solution was refluxed for an
additional two hours. The resulting suspension was filtered out
and the solution was vacuum evaporated to give a crude oil.
The product was purified by chromatography on neutral
alumina using CH2Cl2–ethyl acetate (100:4) as eluting
solvent, affording product 6 as a white solid. Yield: 1.6 g (27%).
Elemental anal., found: C, 62.3; H, 5.6; N, 5.6. Calc. for
C51H56N6S4O8: C, 62.43; H, 5.75; N, 5.71%.
2,6,10,14-Tetraaza[15](2,9)cyclo(1,10)phenanthrolinophane
tetrahydrobromide (L1ؒ4HBr). 6 (2.2 g, 2.19 mmol) and phenol
(29 g, 0.308 mol) were dissolved in 33% HBr–CH3COOH (240
cm3). The reaction mixture was kept under stirring at 90 ЊC for
22 hours until a precipitate was formed. The solid was filtered
out and washed several times with CH2Cl2. The tetrahydro-
bromide salt was recrystallized from an EtOH–water 2:1
mixture, yield 1.5 g (96%). Elemental anal., found: C, 38.5; H,
5.0; N, 11.6. Calc. for C23H32N6ؒ4HBr: C, 38.57; H, 5.07; N,
Fig. 7 Schematic representation for the interaction of tetraprotonated
receptor L1 with HATP in the [H5L1ATP]ϩ adduct.
ADP are remarkably higher than those of ATP. These data
suggest that the extent of the inclusion of nucleotides inside the
macrocyclic cavity depends on the dimensions of both the
receptor cavity and the nucleotide, i.e., the smaller nucleotide,
ADP, is better accommodated into the larger cavity of L1.
1
11.73%. H NMR (D2O, pH = 4): δ(ppm): 2.24 (m, 4H), 2.37
(m, 4H), 3.36 (t, 4H), 3.42 (t, 4H), 3.61 (t, 4H), 4.76 (s, 4H), 7.88
(d, 2H), 8.06 (s, 2H), 8.59 (d, 2H); 13C NMR (D2O, pH = 4):
δ(ppm): 22.1, 23.6, 44.4, 44.6, 46.1, 53.3, 124.5, 128.3, 130.1,
140.2, 145.3, 152.3.
Concluding remarks
EMF Measurements
Protonation of L1 and L2 takes place on the amine groups of
the aliphatic chain, while the heteroaromatic moieties are not
involved in protonation. In the case of L1, the aromatic
nitrogens protonate only at strongly acidic pHs. Therefore,
L1 and L2 present in their protonated species a molecular
organization which enables their multipoint binding with
anionic forms of nucleotides. The protonated macrocycles con-
tain two main binding sites: i) the polyammonium groups,
which can form salt bridges with the phosphate chains and,
at the same time, charge–dipole interactions with the π-system
of nucleotides, ii) the phenanthroline unit, which can give π-
stacking and hydrophobic interactions with the adenine moiety
of substrates.
Electrostatic and hydrogen bonding interactions between the
polyammonium binding sites and the anionic phosphate chain
give the most important contribution to the stability of the
complexes. On the other hand, both ADP and ATP give a
stronger interaction with the protonated receptors than their
inorganic counterparts, namely di- and triphosphate, indicating
that the adenosine moiety also contributes to complex stabiliz-
ation. In particular, the presence of a large heteroaromatic unit
as an integral part of the macrocyclic frameworks leads to a
marked π-stacking interaction with the adenine moiety of
nucleotides. The extent of this mode of interaction seems to be
modulated by the dimensions of the macrocyclic cavity and the
length of the phosphate chain of nucleotides. Larger staking
effects are observed in the case of the interaction of macrocycle
L1 with ADP, suggesting a better “fitting” of the smaller
ADP anion inside the larger macrocyclic cavity of this poly-
ammonium receptor.
The protonation constants of L1 and the formation constants
of the anion complexes with L1 and L2 were determined by
means of potentiometric measurements (pH = Ϫlog [Hϩ]),
carried out in 0.1 mol dmϪ3 NMe4Cl at 298.1 0.1 K, in the
pH range 2.5–11, using the equipment that has already been
described.31 The reference electrode was an Ag/AgCl electrode
in saturated KCl solution. The glass electrode was calibrated
as a hydrogen concentration probe by titrating known amounts
of HCl with CO2-free NaOH solutions and determining the
equivalent point by Gran’s method32 which allows us to
determine the standard potential Eo, and the ionic product of
water (pKw = 13.83 0.01). Ligand concentration was about
1 × 10Ϫ3 M, while substrate concentration was in the range
5 × 10Ϫ3–5 × 10Ϫ4 M. At least three measurements (about 100
experimental points each one) were performed for each system.
The computer program HYPERQUAD29 was used to calculate
the protonation constants and the stability constants of the
ATP and ADP complexes from EMF data. The titration curves
for each system were treated either as a single set or as separate
entities without significant variations in the values of the
basicity constants.
NMR Spectroscopy
200.0 MHz 1H and 50.32 MHz 13C NMR spectra in D2O
solutions at different pH values were recorded at 298 K in a
Bruker AC-200 spectrometer. In 1H NMR spectra peak
positions are reported relative to HOD at 4.75 ppm. Dioxane
was used as reference standard in 13C NMR spectra (δ = 67.4
ppm). 1H–1H and 1H–13C 2D correlation experiments were
performed to assign the signals. Small amounts of 0.01 mol
dmϪ3 NaOD or DCl solutions were added to a solution of
L1ؒ4HBr or L2ؒ4HBr to adjust the pD. The pH was calcu-
lated from the measured pD values using the following
relationship:33 pH = pD Ϫ 0.40. The 31P NMR spectra were
recorded at 81.01 MHz in a Bruker AC-200 spectrometer.
Chemical shifts are relative to an external reference of 85%
H3PO4.
Experimental
Synthesis
Macrocycle L2,21a 1,5,9,13-tetratosyl-1,5,9,13-tetraazatridecane
(4) and 2,9-bis(bromomethyl)-1,10-phenanthroline (5) were
prepared as previously described.30
J. Chem. Soc., Perkin Trans. 2, 1999, 1675–1682
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V. Fusi, C. Giorgi, A. Granchi, P. Paoletti and B. Valtancoli,
J. Chem. Soc., Perkin Trans. 2, 1997, 775.
Acknowledgements
Financial support by Italian Ministero dell’Università e della
Ricerca Scientifica e Tecnologica (quota 40%) and by Italian
Research Council (CNR) is gratefully acknowledged.
12 M. P. Mertes and K. B. Mertes, Acc. Chem. Res., 1990, 23, 413 and
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Paper 9/03059J
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J. Chem. Soc., Perkin Trans. 2, 1999, 1675–1682