New 1H-Pyrazole-Containing Polyamine Receptors Able to Complex L-Glutamate in Water at Physiological pH Values

Article abstract of DOI:10.1021/ja035671m

The interaction of the pyrazole-containing macrocyclic receptors 3,6,9,12,13,16,19,22,25,26-decaazatricyclo-[22.2.1.1 ^11,14]-octacosa-1(27),11,14(28),24-tetraene 1[L₁], 13,26-dibenzyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-[22.2.1.1 ^11,14]-octacosa-1(27),11,14(28),24-tetraene 2[L₂], 3,9,12,13,16,22,25,26-octaazatricyclo-[22.2.1.1 ^11,14]-octacosa-1(27),11,14(28),24-tetraene 3[L₃], 6,19-dibenzyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-[22.2.1.1 ^11,14]-octacosa-1(27),11,14,(28),24-tetraene 4[L₄], 6,19-diphenethyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-[22.2.1.1 ^11,14]-octacosa-1(27),11,14(28),24-tetraene 5[L₅], and 6,19-dioctyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-[22.2.1.1 ^11,14]-octacosa-1(27),11,14(28),24-tetraene 6[L₆] with L-glutamate in aqueous solution has been studied by potentiometric techniques. The synthesis of receptors 3-6[L₃-L₆] is described for the first time. The potentiometric results show that 4[L₄] containing benzyl groups in the central nitrogens of the polyamine side chains is the receptor displaying the larger interaction at pH 7.4 (K_eff = 2.04 × 10⁴). The presence of phenethyl 5[L₅] or octyl groups 6[L₆] instead of benzyl groups 4[L₄] in the central nitrogens of the chains produces a drastic decrease in the stability [K_eff = 3.51 × 10² (5), K_eff = 3.64 × 10² (6)]. The studies show the relevance of the central polyaminic nitrogen in the interaction with glutamate. 1[L₁] and 2[L₂] with secondary nitrogens in this position present significantly larger interactions than 3[L₃], which lacks an amino group in the center of the chains. The NMR and modeling studies suggest the important contribution of hydrogen bonding and π-cation interaction to adduct formation.

Full text of DOI:10.1021/ja035671m

Published on Web 12/30/2003
New 1H-Pyrazole-Containing Polyamine Receptors Able To
Complex L-Glutamate in Water at Physiological pH Values
Carlos Miranda,† Francisco Escart´ı,‡ Laurent Lamarque,† Mar´ıa J. R. Yunta,§
Pilar Navarro,*,† Enrique Garc´ıa-Espan˜a,*,‡ and M. Luisa Jimeno†
Contribution from the Instituto de Qu´ımica Me´dica, Centro de Qu´ımica Orga´nica Manuel Lora
Tamayo, CSIC, C/ Juan de la CierVa 3, 28006 Madrid, Spain, Departamento de Qu´ımica
Inorga´nica, Facultad de Qu´ımica, UniVersidad de Valencia, c/ Doctor Moliner 50,
46100 Burjassot (Valencia), Spain, and Departamento de Qu´ımica Orga´nica, Facultad de
Qu´ımica, UniVersidad Complutense de Madrid, AVda. Complutense s/n, 28040 Madrid, Spain
Received April 16, 2003; E-mail: enrique.garcia-es@uv.es
Abstract: The interaction of the pyrazole-containing macrocyclic receptors 3,6,9,12,13,16,19,22,25,26-
decaazatricyclo-[22.2.1.111,14]-octacosa-1(27),11,14(28),24-tetraene 1[L1], 13,26-dibenzyl-3,6,9,12,13,16,-
19,22,25,26-decaazatricyclo-[22.2.1.111,14]-octacosa-1(27),11,14(28),24-tetraene 2[L2], 3,9,12,13,16,22,-
25,26-octaazatricyclo-[22.2.1.111,14]-octacosa-1(27),11,14(28),24-tetraene 3[L3], 6,19-dibenzyl-3,6,9,12,13,-
16,19,22,25,26-decaazatricyclo-[22.2.1.111,14]-octacosa-1(27),11,14(28),24-tetraene 4[L4], 6,19-diphenethyl-
3,6,9,12,13,16,19,22,25,26-decaazatricyclo-[22.2.1.111,14]-octacosa-1(27),11,14(28),24-tetraene 5[L5], and
6,19-dioctyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-[22.2.1.111,14]-octacosa-1(27),11,14(28),24-tetra-
ene 6[L6] with L-glutamate in aqueous solution has been studied by potentiometric techniques. The synthesis
of receptors 3-6[L3-L6] is described for the first time. The potentiometric results show that 4[L4] containing
benzyl groups in the central nitrogens of the polyamine side chains is the receptor displaying the larger
interaction at pH 7.4 (Keff ) 2.04 × 104). The presence of phenethyl 5[L5] or octyl groups 6[L6] instead of
benzyl groups 4[L4] in the central nitrogens of the chains produces a drastic decrease in the stability [Keff
) 3.51 × 102 (5), Keff ) 3.64 × 102 (6)]. The studies show the relevance of the central polyaminic nitrogen
in the interaction with glutamate. 1[L1] and 2[L2] with secondary nitrogens in this position present significantly
larger interactions than 3[L3], which lacks an amino group in the center of the chains. The NMR and modeling
studies suggest the important contribution of hydrogen bonding and π-cation interaction to adduct formation.
loss of neurons of specific areas of the brain.5 There is much
evidence that these processes induce, at least in part, neuro-
Introduction
The search for the L-glutamate receptor field has been and
continues to be in a state of almost explosive development.1
L-Glutamate (Glu) is thought to be the predominant excitatory
transmitter in the central nervous system (CNS) acting at a range
of excitatory amino acid receptors. It is well-known that it plays
a vital role mediating a great part of the synaptic transmission.2
However, there is an increasing amount of experimental
evidence that metabolic defects and glutamatergic abnormalities
can exacerbate or induce glutamate-mediated excitotoxic damage
and consequently neurological disorders.3,4 Overactivation of
ionotropic (NMDA, AMPA, and Kainate) receptors (iGluRs)
by Glu yields an excessive Ca2+ influx that produces irreversible
degenerative illnesses such as Parkinson, Alzheimer, Huntington,
AIDS, dementia, and amyotrophic lateral sclerosis (ALS).6 In
particular, ALS is one of the neurodegenerative disorders for
which there is more evidence that excitotoxicity due to an
increase in Glu concentration may contribute to the pathology
of the disease.7 Memantine, a drug able to antagonize the
pathological effects of sustained, but relatively small, increases
in extracellular glutamate concentration, has been recently
received for the treatment of Alzheimer disease.8 However, there
is not an effective treatment for ALS. Therefore, the preparation
of adequately functionalized synthetic receptors for L-glutamate
seems to be an important target in finding new routes for
controlling abnormal excitatory processes. However, effective
recognition in water of aminocarboxylic acids is not an easy
task due to its zwitterionic character at physiological pH values
and to the strong competition that it finds in its own solvent.9
† Centro de Qu´ımica Orga´nica Manuel Lora Tamayo.
‡ Universidad de Valencia.
§ Universidad Complutense de Madrid.
(1) Jane, D. E. In Medicinal Chemistry into the Millenium; Campbell, M. M.,
Blagbrough, I. S., Eds.; Royal Society of Chemistry: Cambridge, 2001;
pp 67-84.
(2) (a) Standaert, D. G.; Young, A. B. In The Pharmacological Basis of
Therapeutics; Hardman, J. G., Goodman Gilman, A., Limbird, L. E., Eds.;
McGraw-Hill: New York, 1996; Chapter 22, p 503. (b) Fletcher, E. J.;
Loge, D. In An Introduction to Neurotransmission in Health and Disease;
Riederer, P., Kopp, N., Pearson, J., Eds.; Oxford University Press: New
York, 1990; Chapter 7, p 79.
(5) Green, J. G.; Greenamyre, J. T. Prog. Neurobiol. 1996, 48, 613-63.
(6) Bra¨un-Osborne, H.; Egebjerg, J.; Nielsen, E. O.; Madsen, U.; Krogsgaard-
Larsen, P. J. Med. Chem. 2000, 43, 2609-2645 and references therein.
(7) (a) Shaw, P. J.; Ince, P. G. J. Neurol. 1997, 244 (Suppl 2), S3-S14. (b)
Plaitakis, A.; Fesdjian, C. O.; Shashidharan, P. CNS Drugs 1996, 5, 437-
456.
(3) Michaelis, E. K. Prog. Neurobiol. 1998, 54, 369-415.
(4) Olney, J. W. Science 1969, 164, 719-721.
(8) Frantz, A.; Smith, A. Nat. ReV. Drug DicoVery 2003, 2, 9.
9
10.1021/ja035671m CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 823-833
823

A R T I C L E S
Miranda et al.
Chart 1. Some Receptors Employed for Dicarboxylic Acid and
Chart 2. New 1H-Pyrazole-Containing Polyamine Receptors Able
N-Acetylglutamate Recognition
To Complex L-Glutamate in Water
There are many types of receptors able to interact with
carboxylic acids and amino acids in organic solvents,10-13
yielding selective complexation in some instances. However,
the number of reported receptors of glutamate in aqueous
solution is very scarce. In this sense, one of the few reports
concerns an optical sensor based on a Zn(II) complex of a 2,2′:
6′,2′′-terpyridine derivative in which L-aspartate and L-glutamate
were efficiently bound as axial ligands (Ks ) 104-105 M-1) in
50/50 water/methanol mixtures.14
Among the receptors employed for carboxylic acid recogni-
tion, the polyamine macrocycles I-IV in Chart 1 are of
particular relevance to this work. In a seminal paper, Lehn et
al.15 showed that saturated polyamines I and II could exert
chain-length discrimination between different R,ω-dicarboxylic
acids as a function of the number of methylene groups between
the two triamine units of the receptor. Such compounds were
also able to interact with a glutamic acid derivative which has
the ammonium group protected with an acyl moiety.15,16
Compounds III and IV reported by Gotor and Lehn interact in
their protonated forms in aqueous solution with protected
N-acetyl-L-glutamate and N-acetyl-D-glutamate, showing a
higher stability for the interaction with the D-isomer.17 In both
reports, the interaction with protected N-acetyl-L-glutamate at
physiological pH yields constants of ca. 3 logarithmic units.
Recently, we have shown that 1H-pyrazole-containing mac-
rocycles present desirable properties for the binding of dopam-
ine.18 These polyaza macrocycles, apart from having a high
positive charge at neutral pH values, can form hydrogen bonds
not only through the ammonium or amine groups but also
through the pyrazole nitrogens that can behave as hydrogen bond
donors or acceptors. In fact, Elguero et al.19 have recently shown
the ability of the pyrazole rings to form hydrogen bonds with
carboxylic and carboxylate functions. These features can be used
to recognize the functionalities of glutamic acid, the carboxylic
and/or carboxylate functions and the ammonium group.
Apart from this, the introduction of aromatic donor groups
appropriately arranged within the macrocyclic framework or
appended to it through arms of adequate length may contribute
to the recognition event through π-cation interactions with the
ammonium group of L-glutamate. π-Cation interactions are a
key feature in many enzymatic centers, a classical example being
acetylcholine esterase.20 The role of such an interaction in abiotic
systems was very well illustrated several years ago in a seminal
work carried out by Dougherty and Stauffer.21 Since then, many
other examples have been reported both in biotic and in abiotic
systems.22
(9) Rebek, J., Jr.; Askew, B.; Nemeth, D.; Parris, K. J. Am. Chem. Soc. 1987,
109, 2432-2434.
Taking into account all of these considerations, here we report
on the ability of receptors 1[L1]-6[L6] (Chart 2) to interact
with L-glutamic acid. These receptors display structures which
differ from one another in only one feature, which helps to
obtain clear-cut relations between structure and interaction
(10) Seel, C.; de Mendoza, J. In ComprehensiVe Supramolecular Chemistry;
Vogtle, F., Ed.; Elsevier Science: New York, 1996; Vol. 2, p 519.
(11) (a) Sessler, J. L.; Sanson, P. I.; Andrievesky, A.; Kral, V. In Supramolecular
Chemistry of Anions; Bianchi, A., Bowman-James, K., Garc´ıa-Espan˜a, E.,
Eds.; John Wiley & Sons: New York, 1997; Chapter 10, pp 369-375. (b)
Sessler, J. L.; Andrievsky, A.; Kra´l, V.; Lynch, V. J. Am. Chem. Soc. 1997,
119, 9385-9392.
(12) Fitzmaurice, R. J.; Kyne, G. M.; Douheret, D.; Kilburn, J. D. J. Chem.
Soc., Perkin Trans. 1 2002, 7, 841-864 and references therein.
(13) Rossi, S.; Kyne, G. M.; Turner, D. L.; Wells, N. J.; Kilburn, J. D. Angew.
Chem., Int. Ed. 2002, 41, 4233-4236.
(14) A¨ıt-Haddou, H.; Wiskur, S. L.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem.
Soc. 2001, 123, 11296-11297.
(19) Foces-Foces, C.; Echevarria, A.; Jagerovic, N.; Alkorta, I.; Elguero, J.;
Langer, U.; Klein, O.; Minguet-Bonveh´ı, H.-H. J. Am. Chem. Soc. 2001,
123, 7898-7906.
(20) Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker,
L.; Silman, I. Science 1991, 253, 872-879.
(15) Hosseini, M. W.; Lehn, J.-M. J. Am. Chem. Soc. 1982, 104, 3525-3527.
(16) (a) Hosseini, M. W.; Lehn, J.-M. HelV. Chim. Acta 1986, 69, 587-603.
(b) Heyer, D.; Lehn, J.-M. Tetrahedron Lett. 1986, 27, 5869-5872.
(17) (a) Alfonso, I.; Dietrich, B.; Rebolledo, F.; Gotor, V.; Lehn, J.-M. HelV.
Chim. Acta 2001, 84, 280-295. (b) Alfonso, I.; Rebolledo, F.; Gotor, V.
Chem.-Eur. J. 2000, 6, 3331-3338.
(18) Lamarque, L.; Navarro, P.; Miranda, C.; Ara´n, V. J.; Ochoa, C.; Escart´ı,
F.; Garc´ıa-Espan˜a, E.; Latorre, J.; Luis, S. V.; Miravet, J. F. J. Am. Chem.
Soc. 2001, 123, 10560-10570.
(21) Dougherty, D. A.; Stauffer, D. A. Science 1990, 250, 1558-1560.
(22) (a) Sutcliffe, M. J.; Smeeton, A. H.; Wo, Z. G.; Oswald, R. E. Faraday
Discuss. 1998, 111, 259-272. (b) Kearney, P. C.; Mizoue, L. S.; Kumpf,
R. A.; Forman, J. E.; McCurdy, A.; Dougherty, D. A. J. Am. Chem. Soc.
1993, 115, 9907-9919. (c) Bra¨uner-Osborne, H.; Egebjerg, J.; Nielsen,
E.; Madsen, U.; Krogsgaard-Larsen, P. J. Med. Chem. 2000, 43, 2609-
2645. (d) Zacharias, N.; Dougherty, D. A. Trends Pharmacol. Sci. 2002,
23, 281-287. (e) Hu, J.; Barbour, L. J.; Gokel, G. W. J. Am. Chem. Soc.
2002, 124, 10940-10941.
9
824 J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004

New 1H-Pyrazole-Containing Polyamine Receptors
A R T I C L E S
Scheme 1. Synthesis of the Pyrazole-Containing Macrocyclic
strengths. 1[L1] and 2[L2] differ in the N-benzylation of the
pyrazole moiety, and 1[L1] and 3[L3] differ in the presence in
the center of the polyamine side chains of an amino group or
of a methylene group. The receptors 4[L4] and 5[L5] present
the central nitrogens of the chain N-functionalized with benzyl
or phenethyl groups, and 6[L6] has large hydrophobic octyl
groups.
Receptors
Results and Discussion
Synthesis of 3-6. Macrocycles 3-6 have been obtained
following the procedure previously reported for the preparation
of 1 and 2.23 The method includes a first dipodal (2+2)
condensation of the 1H-pyrazol-3,5-dicarbaldehyde 7 with the
corresponding R,ω-diamine, followed by hydrogenation of the
resulting Schiff base imine bonds. In the case of receptor 3, the
Schiff base formed by condensation with 1,5-pentanediamine
is a stable solid (8, mp 208-210 °C) which precipitated in 68%
yield from the reaction mixture. Further reduction with NaBH4
in absolute ethanol gave the expected tetraazamacrocycle 3,
which after crystallization from toluene was isolated as a pure
compound (mp 184-186 °C). In the cases of receptors 4-6,
the precursor R,ω-diamines (11a-11c) (Scheme 1B) were
obtained, by using a procedure previously described for 11a.24
This procedure is based on the previous protection of the primary
amino groups of 1,5-diamino-3-azapentane by treatment with
phthalic anhydride, followed by alkylation of the secondary
amino group of 1,5-diphthalimido-3-azapentane 9 with benzyl,
phenethyl, or octyl bromide. Finally, the phthalimido groups
of the N-alkyl substituted intermediates 10a-10c were removed
by treatment with hydrazine to afford the desired amines 11a-
11c, which were obtained in moderate yield (54-63%).
In contrast with the behavior previously observed in the
synthesis of 3, in the (2+2) dipodal condensations of 7 with
3-benzyl-, 3-phenethyl-, and 3-octyl-substituted 3-aza-1,5-
pentanediamine 11a, 11b, and 11c, respectively, there was not
precipitation of the expected Schiff bases (Scheme 1A).
Consequently, the reaction mixtures were directly reduced in
situ with NaBH4 to obtain the desired hexaamines 4-6, which
after being carefully purified by chromatography afforded pure
colorless oils in 51%, 63%, and 31% yield, respectively. The
structures of all of these new cyclic polyamines have been
established from the analytical and spectroscopic data (MS(ES+),
1H and 13C NMR) of both the free ligands 3-6 and their
corresponding hydrochloride salts [3‚4HCl, 4‚6HCl, 5‚6HCl,
and 6‚6HCl], which were obtained as stable solids following
the same procedure previously reported18 for 1‚6HCl and 2‚
6HCl.
without an amino group in the center of the side chain, and the
N-substituted ligands 4-6. In 3, the C3,5 signal appears as a
broad singlet. However, in 4-6, it almost disappears within the
baseline of the spectra, and the methylene carbon atoms C6 and
C8 experience a significant broadening. Additionally, a remark-
able line-broadening is also observed in the C1′ carbon signals
belonging to the phenethyl and octyl groups of L5 and L6,
respectively. All of these data suggest that as the N-substituents
located in the middle of the side chains of 4-6 are larger, the
dynamic exchange rate of the pyrazole prototropic equilibrium
is gradually lower, probably due to a relation between proto-
tropic and conformational equilibria.
Acid-Base Behavior. To follow the complexation of L-
glutamate (hereafter abbreviated as Glu2-) and its protonated
forms (HGlu-, H2Glu, and H3Glu+) by the receptors L1-L6,
the acid-base behavior of L-glutamate has to be revisited under
the experimental conditions of this work, 298 K and 0.15 mol
dm-3. The protonation constants obtained, included in the first
column of Table 1, agree with the literature25 and show that
the zwitterionic HGlu- species is the only species present in
aqueous solution at physiological pH values (Scheme 2 and
Figure S1 of Supporting Information). Therefore, receptors for
As usually occurs for 3,5-disubstituted 1H-pyrazole deriva-
tives, either the free ligands 3-6 or their hydrochlorides show
very simple 1H and 13C NMR spectra, in which signals indicate
that, because of the prototropic equilibrium of the pyrazole ring,
all of these compounds present average 4-fold symmetry on
the NMR scale. The quaternary C3 and C5 carbons appear
together, and the pairs of methylene carbons C6, C7, and C8 are
magnetically equivalent (see Experimental Section).
In the 13C NMR spectra registered in CDCl3 solution,
significant differences can be observed between ligand 3,
(23) Ara´n, V. J.; Kumar, M.; Molina, J.; Lamarque, L.; Navarro, P.; Garc´ıa-
Espan˜a, E.; Ram´ırez, J. A.; Luis, S. V.; Escuder, B. J. Org. Chem. 1999,
64, 6137-6146.
(24) (a) Yuen Ng, C.; Motekaitis, R. J.; Martell, A. E. Inorg. Chem. 1979, 18,
2982-2986. (b) Anelli, P. L.; Lunazzi, L.; Montanari, F.; Quici, S. J. Org.
Chem. 1984, 49, 4197-4203.
9
J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004 825

A R T I C L E S
Miranda et al.
Table 1. Protonation Constants of Glutamic Acid and Receptors L1-L6 Determined in NaCl 0.15 mol dm-3 at 298.1 K
reaction
Glu
L1a
L2a
L3b
L4
L5
L6
L + H ) LHc
9.574 (2)d
4.165 (3)
2.18 (2)
9.74 (2)
8.86 (2)
7.96 (2)
6.83 (2)
4.57 (3)
3.18 (3)
8.90 (3)
8.27 (2)
6.62 (3)
5.85 (4)
3.37 (4)
2.27 (6)
9.56 (1)
8.939 (7)
8.02 (1)
7.63 (1)
9.25 (3)
8.38 (3)
6.89 (5)
6.32 (5)
2.72 (8)
9.49 (4)
8.11 (5)
7.17 (6)
6.35 (6)
2.84 (9)
9.34 (5)
8.13 (5)
7.46 (7)
5.97 (8)
3.23 (9)
LH + H ) LH2
LH2 + H ) LH3
LH3 + H ) LH4
LH4 + H ) LH5
LH5 + H ) LH6
∑ log KH L
41.1
35.3
34.2
33.6
34.0
34.1
n
a Taken from ref 23. b These data were previously cited in a short communication (ref 26). c Charges omitted for clarity. d Values in parentheses are the
standard deviations in the last significant figure.
Scheme 2. L-Glutamate Acid-Base Behavior
Table 2. Percentages of the Different Protonated Species at pH
7.4
H La
H L
2
H L
3
H L
4
1
L1
L2
L3
L4
L5
L6
1
10
0
0
11
8
18
77
8
8
54
42
64
13
34
34
32
48
17
0
58
58
3
2
a Charges omitted for clarity.
the different protonated species existing in solution at pH 7.4
for receptors L1-L6. As can be seen, except for the receptor
with the pentamethylenic chains L3 in which the tetraprotonated
species prevails, all of the other systems show that the di- and
triprotonated species prevail, although to different extents.
glutamate recognition able to address both the negative charges
of the carboxylate groups and the positive charge of ammonium
are highly relevant.
Interaction with Glutamate. The stepwise constants for the
interaction of the receptors L1-L6 with glutamate are shown
in Table 3, and selected distribution diagrams are plotted in
Figure 1A-C.
The protonation constants of L3-L6 are included in Table 1,
together with those we have previously reported for receptors
L1 and L2.23 A comparison of the constants of L4-L6 with those
of the nonfunctionalized receptor L1 shows a reduced basicity
of the receptors L4-L6 with tertiary nitrogens at the middle of
the polyamine bridges. Such a reduction in basicity prevented
the potentiometric detection of the last protonation for these
ligands in aqueous solution. A similar reduction in basicity was
previously reported for the macrocycle with the N-benzylated
pyrazole spacers (L2).23 These diminished basicities are related
to the lower probability of the tertiary nitrogens for stabilizing
the positive charges through hydrogen bond formation either
with adjacent nonprotonated amino groups of the molecule or
with water molecules. Also, the increase in the hydrophobicity
of these molecules will contribute to their lower basicity. The
stepwise basicity constants are relatively high for the first four
protonation steps, which is attributable to the fact that these
protons can bind to the nitrogen atoms adjacent to the pyrazole
groups leaving the central nitrogen free, the electrostatic
repulsions between them being therefore of little significance.
The remaining protonation steps will occur in the central
nitrogen atom, which will produce an important increase in the
electrostatic repulsion in the molecule and therefore a reduction
in basicity. As stated above, the tertiary nitrogen atoms present
in L4-L6 will also contribute to this diminished basicity.
To analyze the interaction with glutamic acid, it is important
to know the protonation degree of the ligands at physiological
pH values. In Table 2, we have calculated the percentages of
All of the studied receptors interact with glutamate forming
adduct species with protonation degrees (j) which vary between
8 and 0 depending on the system (see Table 3). The stepwise
constants have been derived from the overall association
constants (L + Glu2- + jH+ ) HjLGlu(j-2)+, log âj) provided
by the fitting of the pH-metric titration curves. This takes into
account the basicities of the receptors and glutamate (vide supra)
and the pH range in which a given species prevails in solution.
In this respect, except below pH ca. 4 and above pH 9, HGlu-
can be chosen as the protonated form of glutamate involved in
the formation of the different adducts. Below pH 4, the
participation of H2Glu in the equilibria has also to be considered
(entries 9 and 10 in Table 3). For instance, the formation of the
H6LGlu4+ species can proceed through the equilibria HGlu- +
H5L5+ ) H6LGlu4+ (entry 8, Table 3), and H2Glu + H4L4+
)
H6LGlu4 (entry 9 Table 3), with percentages of participation
that depend on pH. One of the effects of the interaction is to
render somewhat more basic the receptor, and somewhat more
acidic glutamic acid, facilitating the attraction between op-
positely charged partners.
A first inspection of Table 3 and of the diagrams A, B, and
C in Figure 1 shows that the interaction strengths differ markedly
from one system to another depending on the structural features
of the receptors involved. L4 is the receptor that presents the
highest capacity for interacting with glutamate throughout all
of the pH range explored. It must also be remarked that there
are not clear-cut trends in the values of the stepwise constants
as a function of the protonation degree of the receptors. This
suggests that charge-charge attractions do not play the most
(25) (a) Martell, E.; Smith, R. M. Critical Stability Constants; Plenum: New
York, 1975. (b) Motekaitis, R. J. NIST Critically Selected Stability
Constants of Metal Complexes Database; NIST Standard Reference
Database, version 4, 1997.
9
826 J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004

New 1H-Pyrazole-Containing Polyamine Receptors
A R T I C L E S
Table 3. Stability Constants for the Interaction of L1-L6 with the Different Protonated Forms of Glutamate (Glu)
entry
reactiona
L1
L2
L3
L4
L5
L6
1
2
3
4
5
6
7
8
9
Glu + L ) GluL
3.30 (2)b
3.65 (2)
3.89 (2)
3.49 (2)
3.44 (2)
3.33 (2)
3.02 (2)
3.11 (3)
2.54 (3)
2.61 (6)
4.11 (1)
4.11 (1)
4.48 (1)
3.89 (1)
3.73 (1)
3.56 (2)
3.26 (2)
3.54 (2)
3.05 (2)
2.73 (4)
HGlu + L ) HGluL
3.68 (2)
3.96 (2)
3.71 (2)
4.14 (2)
4.65 (2)
4.77 (2)
6.76 (3)
5.35 (3)
5.51 (3)
3.38 (4)
3.57 (4)
Glu + HL ) HGluL
HGlu + HL ) H2GluL
HGlu + H2L ) H3GluL
HGlu + H3L ) H4GluL
HGlu + H4L ) H5GluL
HGlu + H5L ) H6GluL
H2Glu + H4L ) H6GluL
H2Glu + H5L ) H7GluL
H3Glu + H4L ) H7GluL
2.37 (4)
2.34 (3)
2.66 (3)
2.58 (3)
2.46 (4)
2.74 (3)
2.87 (3)
4.96 (3)
3.66 (4)
3.57 (4)
2.61 (7)
2.55 (7)
2.91 (5)
4.47 (3)
3.56 (3)
3.22 (8)
4.12 (9)
3.88 (2)
4.82 (2)
10
11
a Charges omitted for clarity. b Values in parentheses are standard deviations in the last significant figure.
Figure 2. Representation of the variation of Kcond (M-1) for the interaction
of glutamic acid with (A) L1 and L3, (B) L2, L4, L5, and L6. Initial
concentrations of glutamate and receptors are 10-3 mol dm-3
.
amounts of complexed species and those of free receptor and
substrate at a given pH [eq 1].
Kcond
)
[(H L)‚(H Glu)]/{ [H L] [H Glu]} (1)
i j i j
∑
∑ ∑
Figure 1. Distribution diagrams for the systems (A) L1-glutamic acid,
(B) L4-glutamic acid, and (C) L5-glutamic acid.
In Figure 2 are presented the logarithms of the effective
constants versus pH for all of the studied systems. Receptors
L1 and L2 with a nonfunctionalized secondary amino group in
the side chains display opposite trend from all other receptors.
While the stability of the L1 and L2 adducts tends to increase
with pH, the other ligands show a decreasing interaction.
Additionally, L1 and L2 present a close interaction over the entire
pH range under study. The tetraaminic macrocycle L3 is a better
outstanding role and that other forces contribute very importantly
to these processes.26
However, in systems such as these, which present overlapping
equilibria, it is convenient to use conditional constants because
they provide a clearer picture of the selectivity trends.27 These
constants are defined as the quotient between the overall
(27) (a) Bianchi, A.; Garc´ıa-Espan˜a, A. J. Chem. Educ. 1999, 12, 1725-1732.
(b) Aguilar, J. A.; Celda, B.; Garc´ıa-Espan˜a, E.; Luis, S. V.; Mart´ınez, M.;
Ram´ırez, J. A.; Soriano, C.; Tejero, B. J. Chem. Soc., Perkin Trans. 2 2000,
7, 1323-1328.
(26) Escart´ı, F.; Miranda, C.; Lamarque, L.; Latorre, J.; Garc´ıa-Espan˜a, E.;
Kumar, M.; Ara´n, V. J.; Navarro, P. Chem. Commun. 2002, 9, 936-937.
9
J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004 827

A R T I C L E S
Miranda et al.
Table 4. Percentages of the Different Protonated Adducts
[HGlu‚HjL](j-1)+, Overall Percentages of Complexation, and
Conditional Constants (KCond) at pH 7.4 for the Interaction of
Glutamate (HGlu-) with Receptors L1-L6 at Physiological pH
The quality of a computer simulation depends on two
factors: accuracy of the force field that describes intra- and
intermolecular interactions, and an adequate sampling of the
conformational and configuration space of the system.28 The
additive AMBER force field is appropriate for describing the
complexation processes of our compounds, as it is one of the
best methods29 in reproducing H-bonding and stacking stabiliza-
tion energies.
The experimental data show that at pH 7.4, L1-L6 exist in
different protonation states. So, a theoretical study of the
protonation of these ligands was done, including all of the
species shown in 5% or more abundance in the potentiometric
measurements (Table 4). In each case, the more favored
positions of protons were calculated for mono-, di-, tri-, and
tetraprotonated species. Molecular dynamics studies were
performed to find the minimum energy conformations with
simulated solvent effects.
[H L‚HGlu]a
n
n ) 1
n ) 2
n ) 3
n ) 4
∑{[H L‚HGlu]}
K )
cond (M-1
n
L1
L2
L3
L4
L5
L6
3
9
27
47
1
37
10
12
23
7
10
37
10
12
53
63
24
81
22
24
2.44 × 103
4.12 × 103
3.99 × 102
2.04 × 104
3.51 × 102
3.64 × 102
13
5
2
2
a Charges omitted for clarity.
receptor at acidic pH, but its interaction markedly decreases on
raising the pH. These results strongly suggest the implication
of the central nitrogens of the lateral polyamine chains in the
stabilization of the adducts.
Molecular modeling studies were carried out using the
AMBER30 method implemented in the Hyperchem 6.0 pack-
age,31 modified by the inclusion of appropriate parameters.
Where available, the parameters came from analogous ones used
in the literature.32 All others were developed following Koll-
man33 and Hopfinger34 procedures. The equilibrium bond length
and angle values came from experimental values of reasonable
reference compounds. All of the compounds were constructed
using standard geometry and standard bond lengths. To develop
suitable parameters for NH‚‚‚N hydrogen bonding, ab initio
calculations at the STO-3G level35 were used to calculate atomic
charges compatible with the AMBER force field charges, as
they gave excellent results, and, at the same time, this method
allows the study of aryl-amine interactions. In all cases, full
geometry optimizations with the Polak-Ribiere algorithm were
carried out, with no restraints.
Ions are separated far away and well solvated in water due
to the fact that water has a high dielectric constant and hydrogen
bond network. Consequently, there is no need to use counteri-
ons36 in the modelization studies. In the absence of explicit
solvent molecules, a distance-dependent dielectric factor quali-
tatively simulates the presence of water, as it takes into account
the fact that the intermolecular electrostatic interactions should
vanish more rapidly with distance than in the gas phase. The
same results can be obtained using a constant dielectric factor
greater than 1. We have chosen to use a distance-dependent
dielectric constant (ꢀ ) 4Rij) as this was the method used by
Weiner et al.37 to develop the AMBER force field. Table 8
shows the theoretical differences in protonation energy (∆Ep)
of mono-, bi-, and triprotonated hexaamine ligands, for the
Among the N-functionalized receptors, L4 presents the largest
interaction with glutamate. Interestingly enough, L5, which
differs from L4 only in having a phenethyl group instead of a
benzyl one, presents much lower stability of its adducts. Since
the basicity and thereby the protonation states that L4 and L5
present with pH are very close, the reason for the larger stability
of the L4 adducts could reside on a better spatial disposition
for forming π-cation interactions with the ammonium group of
the amino acid. In addition, as already pointed out, L4 presents
the highest affinity for glutamic acid in a wide pH range, being
overcome only by L1 and L2 at pH values over 9. This
observation again supports the contribution of π-cation inter-
actions in the system L4-glutamic because at these pH values
the ammonium functionality will start to deprotonate (see
Scheme 2 and Figure 1B).
Table 4 gathers the percentages of the species existing in
equilibria at pH 7.4 together with the values of the conditional
constant at this pH. In correspondence with Figure 1A, 1C and
Figure S2 (Supporting Information), it can be seen that for L1,
L2, L5, and L6 the prevailing species are [H2L‚HGlu]+ and [H3L‚
HGlu]2+ (protonation degrees 3 and 4, respectively), while for
L3 the main species are [H3L‚HGlu]+ and [H4L‚HGlu]2+
(protonation degrees 4 and 5, respectively). The most effective
receptor at this pH would be L4 which joins hydrogen bonding,
charge-charge, and π-cation contributions for the stabilization
of the adducts. To check the selectivity of this receptor, we
have also studied its interaction with L-aspartate, which is a
competitor of L-glutamate in the biologic receptors. The
conditional constant at pH 7.4 has a value of 3.1 logarithmic
units for the system Asp-L4. Therefore, the selectivity of L4
for glutamate over aspartate (Kcond(L4-glu)/Kcond(L4-asp)) will
be of ca. 15. It is interesting to remark that the affinity of L4
for zwiterionic L-glutamate at pH 7.4 is even larger than that
displayed by receptors III and IV (Chart 1) with the protected
dianion N-acetyl-L-glutamate lacking the zwitterionic charac-
teristics. Applying eq 1 and the stability constants reported in
ref 17, conditional constants at pH 7.4 of 3.24 and 2.96
logarithmic units can be derived for the systems III-L-Glu and
IV-L-Glu, respectively.
(28) Urban, J. J.; Cronin, C. W.; Roberts, R. R.; Famini, G. R. J. Am. Chem.
Soc. 1997, 119, 12292-12299.
(29) Hobza, P.; Kabelac, M.; Sponer, J.; Mejzlik, P.; Vondrasek, J. J. Comput.
Chem. 1997, 18, 1136-1150.
(30) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M., Jr.;
Ferguson, D. M.; Spelmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P.
A. J. Am. Chem. Soc. 1995, 117, 5179-5197.
(31) Hyperchem 6.0 (Hypercube Inc.).
(32) (a) Fox, T.; Scanlan, T. S.; Kollman, P. A. J. Am. Chem. Soc. 1997, 119,
11571-11577. (b) Grootenhuis, P. D.; Kollman, P. A. J. Am. Chem. Soc.
1989, 111, 2152-2158. (c) Moyna, G.; Hernandez, G.; Williams, H. J.;
Nachman, R. J.; Scott, A. I. J. Chem. Inf. Comput. Sci. 1997, 37, 951-
956. (d) Boden, C. D. J.; Patenden, G. J. Comput.-Aided Mol. Des. 1999,
13, 153-166.
Molecular Modeling Studies. Molecular mechanics-based
methods involving docking studies have been used to study the
binding orientations and affinities for the complexation of
glutamate by L1-L6 receptors.
(33) http://www.amber.ucsf.edu/amber.
(34) Hopfinger, A. J.; Pearlstein, R. A. J. Comput. Chem. 1984, 5, 486-499.
(35) Glennon, T. M.; Zheng, Y.-J.; Le Grand, S. M.; Shutzberg, B. A.; Merz,
K. M., Jr. J. Comput. Chem. 1994, 15, 1019-1040.
(36) Wang, J.; Kollman, P. A. J. Am. Chem. Soc. 1998, 120, 11106-11114.
9
828 J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004

New 1H-Pyrazole-Containing Polyamine Receptors
A R T I C L E S
Table 5. Differences in Theoretical Protonation Energies (∆Ep, kJ
mol-1) as a Function of Macrocyclic Nitrogen Positions and the
Number of Protonated Nitrogens of Hexaamine Receptors L1, L2,
L4-L6
energies of the glutamate and ligand.
Ecomplexation ) Ecomplex - (Eglutamate + Eligand
)
(2)
Table 6 shows the calculated values of complexation energies
for each species (∆ECn, kJ mol-1, where n is the protonation
state), the contribution to the total energy as a function of their
relative abundance (%∆ECn, kJ mol-1), and total energy for
complexation of each receptor with glutamate (∆ETC) as the
sum of all of these partial contributions ∑[(%∆ECn], together
with experimental values of effective association stability
constants (Keff, M-1). Relative values of ∆ETC show that our
modeling calculations are in excellent agreement with experi-
mental Keff data obtained for L1-L6 as they exhibit the same
trend: L4 . L2 > L1 . L3 > L6 > L5.
Molecular docking and dynamics were performed on each
ligand with the glutamate in zwitterionic form (HGlu). The
orientation for each compound discussed here represents the
best orientation and is representative of all possible interactions
within the ligand cavity.
Taking into account that L4 is the ligand forming the more
stable complexes, Figure 3 shows the minimum energy con-
formers for the adduct species [H2L4]HGlu (HpHp)(%∆EC2
)
)
[HL]∆E (2 − 1)
[H L]∆E (4 − 3)
[H L]∆E (6 − 5)
p
2
p
3
p
-101.67 kJ mol-1) and [H3L4]HGlu (HpHpHc)(%∆EC2
L1
L2
L4
L5
L6
-5.81
8.44
18.40
11.39
20.23
-4.35
1.38
-2.89
-8.40
-4.05
-126.18 kJ mol-1), both of them present in 37% at pH 7.4
(Table 4). In [H2L4]HGlu, the carboxylate groups interact
through hydrogen bonds with the ammonium and amine groups
closer to the pyrazole moieties, as well as with the NH protons
of the pyrazole rings. In [H3L4]HGlu species, in addition to the
above-mentioned interactions, a COO-‚‚‚HN+ hydrogen bond
with the central ammonium group of L4 is observed, together
with a π-cation interaction between the ammonium glutamate
group and one of the benzene rings.
NMR Studies. To gain insight into these interactions and
check the speciation results with complementary techniques, we
have performed a 1H and 13C NMR study on the interaction of
the receptor 4[L4] with L-glutamate. The experiments were
carried out in D2O at pH 7.4 (pH ) pD - 0.4.38) with a 3:1
excess of either the receptor (Table 7) or the L-glutamate (Table
8).
3.18
different possible positions of the protons. As can be seen,
theoretical values agree with the experimental results previously
obtained for L1 and L2 from a NMR study in D2O at variable
pH.23 In L1, the pattern for the protonation sequence is as
follows: two protons are preferably located on the central
nitrogen atoms of the macrocyclic cavity, HcHc (4), while the
more stable triprotonated species correspond to a HpHpHc (6)
disposition, with two protons located on nitrogen atoms adjacent
to the pyrazole ring of one chain and the third one in the middle
of the opposite chain (see scheme of Table 5).
On the other hand, in L2 the theoretical protonation energies
favor the following dispositions: Hp for monoprotonated
compounds (1), HpHp for the diprotonated ones (3), and
HpHpHp for the triprotonated species (5), with all successive
protons on adjacent positions to the pyrazole nitrogen. Finally,
in L4-L6, the protonation sequence is as follows: two protons
prefer to locate on adjacent positions to the pyrazole nitrogen
HpHp (3), while the more stable triprotonated species correspond
to a HpHpHc (6). Nevertheless, theoretical energy differences
for HpHpHp (5) and HpHpHc (6) species are small enough [∆Ep
(6 - 5) ) 8.5 kJ mol-1] to consider both possibilities in the
interaction with glutamate. Lowest energy conformers for all
protonation states showing more than 5% abundance in poten-
tiometric assays were located by performing simulated annealing
at 400 K. These conformers were used for docking.
The 1H-induced shifts experienced by L-glutamate upon
interaction with L4 are very small (<0.1 ppm). However, the
13C-induced chemical shifts (∆δC) are much more significant
and provide valuable information. All of the carbon atoms
experience strong ∆δC values which are larger for both
carboxylate groups -OOC-R and -OOC-γ (ca. -1.2 ppm) than
for the methine carbon (C-R) and methylene carbon atoms (C-â
and C-γ) (ca. -1.0 ppm) (see Figure 4A).
With respect to the chemical shifts induced on the receptor
by the addition of an excess of L-glutamate at pH 7.4 (Table
8), one can notice that complexation produces a general upfield
shift of all the protons of L4 in the 1H spectrum. This effect is
maximum for the pyrazole ring (H-4, ∆δH ) -0.31 ppm), and
it gradually diminishes for the aliphatic protons as the distance
from the heteroaromatic ring increases [Table 8A and Figure
4B (values of ∆δ in parentheses)]. Such a behavior suggests
that the carboxylate groups should preferentially interact with
The energy of complexation was calculated for each com-
pound after minimization of the selected trajectory frames, as
the difference between the energy of the complex and individual
+
the NH2 groups of the receptor placed close to the pyrazole
(37) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.; Alagona,
(38) Covington, A. K.; Paabo, M.; Robison, R. A.; Bates, R. G. Anal. Chem.
1968, 40, 700-706.
G.; Profeta, S., Jr.; Weiner, P. J. Am. Chem. Soc. 1984, 106, 765-784.
9
J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004 829

A R T I C L E S
Miranda et al.
Table 6. Theoretical L-Glutamate Complexation Energies for Mono-, Di-, Tri-, and Tetraprotonated Species (∆ECn, kJ mol-1), Energetic
Contribution of Each Species as a Function of Its Relative Abundance (%∆ECn, kJ mol-1), and Total Complexation Energies (∆ETC, kJ
mol-1) in Comparison with Experimental Association Stability Constants (Keff, M-1
)
L1
L2
L3
L4
L5
L6
[HL]HGlu (%)
∆EC1
Hp (9)
-176.86
-15.93
HpHp (47)
-259.33
-121.89
HpHpHp (7)
-274.08
-19.18
%∆EC1
[H2L]HGlu (%)
∆EC2
HcHc (27)
-267.85
-72.31
HpHp (37)
-274.79
HpHp (10)
-208.79
-20.87
HpHpHp (10)
-296.07
-29.61
HpHp (12)
-288.29
-34.59
HpHpHp (12)
-198.55
-23.83
%∆EC2
-101.67
[H3L]HGlu (%)
∆EC3
HpHpHc (23)
-337.91
-77.72
HpHpHp (10)
-279.47
HpHpHc (37)
-341.04
-126.18
%∆EC3
-27.95
[H4L]HGlu (%)
∆EC4
HpHpHpHp (13)
-349.49
%∆EC4
-45.43
∆ETC
-150.03
-157.00
-73.38
-227.85
-50.49
-58.52
K
eff (M-1
)
2.44 × 103
4.12 × 103
3.99 × 102
2.04 × 104
3.51 × 102
3.64 × 102
Table 7. Chemical Shifts (∆δ, ppm) Induced in L-Glutamate 1H
and 13C NMR Spectra (D2O) at pH 7.4 by Complexation with 4[L4]
[L:Glu (3:1)]
G1u
L4−Glu
Glu
L4−Glu
HR
Hâ
Hγ
3.57
dd, 1H
2.17
m, 2H
1.90
m, 2H
3.56
2dd, 1H
2.18
m, 2H
1.97
m, 2H
OOC-R
CR
175.90
174.65
55.98
28.28
54.83
27.11
Câ
Cγ
OOC-γ
34.73
182.55
33.69
181.35
Table 8. Chemical Shifts (∆δ, ppm) Induced in 4[L4] 1H and 13C
NMR Spectra (D2O) at pH 7.4 by Complexation with L-Glutamate
[L:Glu (1:3)]
L4
L4−Glu
L4
L4−Glu
HC4
H2C6
H2C7
H2C8
H2C1′
Ho
6.44
s, 2H
4.11
s, 8H
3.04
t, 8H
2.71
t, 8H
3.59
s, 4H
7.26
m, 4H
7.26
m, 4H
7.26
m, 2H
6.13
s, 2H
3.85
s, 8H
2.86
t, 8H
2.65
t, 8H
3.55
s, 4H
7.18
m, 4H
7.18
m, 4H
7.18
m, 2H
C3,5
C4
141.58
143.81
108.65
44.00
46.28
51.24
58.51
137.86
131.08
107.01
44.15
46.60
51.46
59.57
138.42
131.05
C6
C7
C8
C1′
Cipso
Co
Hm
Hp
Cm
Cp
130.13
129.31
129.99
129.17
With respect to the ∆δC, produced on the receptor L4 by
addition of an excess of L-glutamate at pH 7.4 (Table 8, Figure
4B), in general, they are again of sign opposite to those
experienced by these and related molecules when protonation
occurs.23,39 Upon protonation of the ligands, the carbon nuclei
bear shielding effects that are particularly large for the atoms
C3,5 of the pyrazole ring placed in the â-position with respect
to the nitrogen that protonates. This effect is of the same sign
(39) (a) Andre´s, A.; Burguete, M. I.; Garc´ıa-Espan˜a, E.; Luis, S. V.; Miravet,
J. F.; Soriano, C. J. Chem. Soc., Perkin Trans. 2 1993, 4, 749-755. (b)
Bianchi, A.; Escuder, B.; Garc´ıa Espan˜a, E.; Luis, S. V.; Marcelino, V.;
Miravet, J. F.; Ram´ırez, J. A. J. Chem. Soc., Perkin Trans. 2 1994, 6, 1253-
1259. (c) Andre´s, A.; Bazzicaluppi, C.; Bianchi, A.; Garc´ıa Espan˜a, E.;
Luis, S. V.; Miravet, J. F.; Ram´ırez, J. A. J. Chem. Soc., Dalton Trans.
1994, 20, 2995-3004. (d) Aguilar, J. A.; Garc´ıa-Espan˜a, E.; Guerrero, J.
A.; Llinares, J. M.; Ram´ırez, J. A.; Soriano, C.; Luis, S. V.; Bianchi, A.;
Ferrini, L.; Fusi, V. J. Chem. Soc., Dalton Trans. 1996, 2, 239-246.
Figure 3. Molecular models and interactions for L-glutamate and L4
complexes.
moiety, producing a neutralizing effect of their positive charge,
because the protonation of other pyrazole-based polyaminic
receptors produces a downfield shift opposite effect.23
9
830 J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004

New 1H-Pyrazole-Containing Polyamine Receptors
A R T I C L E S
benzyl groups (L4) yields a sharp increase in stability (Keff
)
2.0 × 104 at pH 7.4), while the introduction of phenethyl groups
originates the opposite effect (Keff ) 3.51 × 102 at pH 7.4).
Molecular modeling and NMR analysis suggest that in
addition to COO-‚‚‚+H2N, COO-‚‚‚+HNBn, COO-‚‚‚HN, and
COO-‚‚‚HPz hydrogen bonds, π-cation interactions may be
contributing to the extra stabilization of the glutamic adducts
with L4. In the case of this receptor, we have also studied its
association constants with L-aspartate. The data show a 15-fold
selectivity of L-glutamate over L-aspartate at pH 7.4.
Experimental Section
The starting materials were purchased from commercial sources and
used without further purification. The solvents were dried using standard
techniques. All reactions were monitored by thin-layer chromatography
using DC-Alufolien silica gel 60PF254 (Merck; layer thickness, 0.2 mm).
Compounds were detected UV light (254 nm), with iodine, or with
phosphomolybdic acid reagent. Melting points were determined in a
1
Reichert-Jung hot-stage microscope and are uncorrected. H and 13C
Figure 4. (A) ∆δC induced upon addition of an excess of L4 to a
L-glutamate D2O solution at pH 7.4 [L4:Glu (3:1)]. (B) ∆δC and ∆δH (in
parentheses) induced upon addition of an excess of L-glutamate to a L4
D2O solution at pH 7.4 [L4:Glu (1:3)].
NMR spectra were recorded on Varian Inova-300, Varian Inova-400,
and Varian Unity-41500 spectrometers. The chemical shifts are reported
in ppm using dioxane (δ ) 67.4 ppm) as an internal reference in D2O.
All assignments have been performed on the basis of 1H-13C
heteronuclear multiple quantum coherence experiments (gHSQC and
gHMBC). In the NMR study of the complexation of ligands with
glutamic acid in D2O, the pH was calculated from the measured pD
values using the correlation pH ) pD - 0.4. IR spectra were recorded
with a Perkin-Elmer Spectrum One spectrophotometer. The mass spectra
(MS) were registered in a Hewlett-Packard 1100 MSD spectrometer
by electrospray positive mode (ES+). Elemental analyses were provided
by the Departamento de Ana´lisis, Centro de Qu´ımica Orga´nica “Manuel
Lora Tamayo”, CSIC, Madrid, Spain.
although of lower magnitude for the carbon atoms placed in
the R-position, while for the γ-carbon C4 is of opposite sign.
Interestingly, the interaction of L4 with glutamate at pH 7.4
also affects the chemical shifts of the benzyl groups, particularly
those of the methylenic carbon C1′ (∆δ ) 1.06 ppm) and the
quaternary carbon atom Cipso (∆δ ) 0.56 ppm). The aromatic
carbon atoms Co and Cp also experience some shift (-0.14 ppm).
All of these facts suggest that the interaction of L-glutamate
with L4 brings about significant conformational changes of the
benzylic groups and that in addition to the secondary amino
groups closer to the pyrazole rings, the central tertiary N-Bn
substituted amino groups are also participating in the interaction
with L-glutamate.12
Finally, we have measured NOE effects (from ROESY and
NOESY NMR spectra) of L4 in the presence of an excess of
L-glutamate in H2O/D2O at pH 7.4. The spectra did not show
evidence of intermolecular cross-peaks. The only NOE effects
observed were intramolecular cross-peaks. Taking into account
that molecular modeling studies indicate that the closest
distances between the protons of L-glutamate and those of L4
are above 4 Å in both conformers (Figure 3a and 3b) and that
beyond the 3.0 Å range the NOE is very weak, these results
indicate that in agreement with the theoretical geometries it is
not possible to detect intermolecular distances in the L4-L-
glutamate complex.
Preparation of the 3,5-Pyrazoledicarbaldehyde (7a). It was
prepared from 3,5-bis(hydroximethyl)pyrazole by oxidation with MnO2
in DME as previously reported.23
Preparation of Precursor Amines. 1,5-Diphthalimido-3-azapen-
tane (9). A mixture of 1,5-diamino-3-azapentane (10.3 g, 0.10 mol)
and phthalic anhydride (33.2 g, 0.20 mol) in 160 mL of glacial acetic
acid was refluxed for 4 h. The solvent was removed under reduced
pressure, and then 160 mL of hot ethanol was added with stirring until
a solid appeared. The product was collected and washed with cold
ethanol. Yield 23.9 g (81%), mp 181-183 °C, lit.24a 182-183 °C. 1H
NMR (DMSO-d6, 300 MHz): 7.79 (m, 4H, H3′,6′), 7.74 (m, 4H, H4′,5′),
3.59 (t, J ) 6.3 Hz, 4H, H1,5), 2.76 (t, 4H, H2,4). 13C NMR (75 MHz,
DMSO-d6): 167.85 (CO), 134.10 (C4′,5′), 131.63 (C1′,2′), 122.74 (C3′,6′),
46.16 (C2,4), 37.18 (C1,5). MS (ES+, MeOH): m/z 364 (MH+). Anal.
Calcd (%) for C20H17N3O4 (363.37): C, 66.11; H, 4.72; N, 11.56.
Found: C, 66.30; H, 4.80; N, 11.88.
General Procedure for Alkylation. A solution of 9 (7.260 g, 20
mmol) and a variable amount of benzyl, phenethyl, or octyl bromide
in 300 mL of MeCN was refluxed with stirring in the presence of
K2CO3. The solvent was removed on a rotatory evaporator, and the
dry residue was treated with H2O (100 mL) and extracted with
dichloromethane (3 × 50 mL). This extract was dried with anhydrous
MgSO4 and evaporated to give a solid, which was purified by flash
column chromatography on silicagel (hexane-EtOAc, 95:5 to 40:60).
3-Benzyl-1,5-diphthalimido-3-azapentane (10a). This was prepared
by reaction of 9, benzyl bromide (4.27 g, 25 mmol), and potassium
carbonate (3.45 g, 25 mmol) for 12 h to give a white solid. Yield 8.23
Conclusions
Hexaaza 2+2 dipodal macrocycles containing 1H-pyrazole
rings as spacers show a good ability for interacting with
L-glutamic acid in water. Controlled structural modifications
introduced in the receptors allow for the derivation of the
following general considerations regarding the structure-
interaction strength relationship: (i) the central nitrogens of the
chain are implicated in the interaction with pyrazole as shown
by the decrease in stability observed when exchanging these
nitrogens by carbon atoms; (ii) the receptors with secondary
nitrogens in the middle of the side chains show a reverse
dependence of the effective constants with pH; and (iii) the
functionalization of the central nitrogens of the side chains with
1
g (91%), mp 130-131 °C, lit.24b 130-132 °C. H NMR (400 MHz,
CDCl3): 7.71 (m, 4H, H3′,6′), 7.67 (m, 4H, H4′,5′), 7.05 (d, J ) 7.2 Hz,
2H, Ho), 7.02 (t, J ) 7.3 Hz,1H, Hp), 6.92 (d, 2H, Hm), 3.76 (t, J ) 6.3
Hz, 4H, H1,5), 3.66 (s, 2H, H1′′), 2.80 (t, 4H, H2,4). 13C NMR (100 MHz,
CDCl3): 168.08 (CO), 138.67 (Cipso), 133.52 (C4′,5′), 132.33 (C1′,2′),
128.86 (Co), 127.94 (Cm), 126.73 (Cp), 122.94 (C3′,6′), 58.16 (C1′′), 51.62
9
J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004 831

A R T I C L E S
Miranda et al.
(C2,4), 35.70 (C1,5). MS (ES+, MeCN/MeOH): m/z 454 (MH+). Anal.
Calcd (%) for C27H23N3O4 (453.59): C, 71.51; H, 5.11; N, 9.27.
Found: C, 71.80; H, 5.43; N, 9.52.
3,9,12,13,16,22,25,26-Octaazatricyclo-[22.2.1.111,14]-octacosa-
1(27),2,9,11,14(28),15,22,24-octaene (8). Working under an argon
atmosphere, a solution of 3,5-pyrazoledicarbaldehyde (496 mg, 4 mmol)
in methanol (200 mL) was added dropwise to a stirred solution of 1,5-
pentanediamine (448 mg, 4.4 mmol) in methanol (120 mL). After the
mixture was stirred for 12 h, a white solid was formed, and then it was
filtered off, successively washed with methanol and Et2O, and dried in
3-Phenethyl-1,5-diphthalimido-3-azapentane (10b). This was pre-
pared by reaction of 9, phenethyl bromide (11.10 g, 60 mmol), and
potassium carbonate (8.28 g, 60 mmol) for 4 days to give a white solid.
Yield 4.48 g (48%). 1H NMR (400 MHz, CDCl3): 7.74 (m, 4H, H3′,6′),
7.67 (m, 4H, H4′,5′), 7.18 (m, 2H, Hm), 7.15 (m, 2H, Ho), 7.08 (m, 1H,
Hp), 3.74 (t, J ) 6.6 Hz, 4H, H1,5), 2.86 (t, 4H, H2,4), 2.81 (s, 2H, H1′′),
2.62 (s, 2H, H2′′). 13C NMR (100 MHz, CDCl3): 168.24 (CO), 140.07
(Cipso), 133.69 (C4′,5′), 132.23 (C1′,2′), 128.75 (Cm), 128.20 (Co), 125.81
(Cp), 123.08 (C3′,6′), 55.64 (C1′′), 51.58 (C2,4), 35.81 (C1,5), 33.78 (C2′′).
IR (KBr, cm-1): 1771, 1701. MS (ES+, MeCN/MeOH): m/z 468
(MH+). Anal. Calcd (%) for C28H25N3O4 (467.52): C, 71.93; H, 5.39;
N, 8.99. Found: C, 72.21; H, 5.60; N, 9.04.
1
vacuo. Yield 529 mg (68%), mp 208-210 °C. H NMR (300 MHz,
DMSO-d6): δ 8.13 (s, 4H, H6), 6.58 (s, 2H, H4), 3.52 (m, 8H, H7),
1.60 (m, 8H, H8), 1.07 (m, 4H, H9). IR (KBr, cm-1): 1645. MS (ES+,
MeOH): m/z 381 (MH+). Anal. Calcd (%) for C20H36N8‚0.5H2O
(389.50): C, 61.67; H, 7.50; N, 28.77. Found: C, 61.91; H, 7.81; N,
28.34.
3,9,12,13,16,22,25,26-Octaazatricyclo-[22.2.1.111,14]-octacosa-1(27),-
11,14(28),24-tetraene (3). To a stirred suspension of the Schiff base 8
(389 mg, 1 mmol) in methanol (150 mL) was added sodium borohydride
(456 mg, 12 mmol) portionwise. The mixture was stirred for 2 h, the
solvent was then removed, and the dry residue was purified by
extracting with toluene in a Soxhlet apparatus to give a solid which
was recrystallized from toluene. Yield 432 mg (54%), mp (toluene)
3-Octyl-1,5-diphthalimido-3-azapentane (10c). This was prepared
by reaction of 9, octyl bromide (11.58 g, 60 mmol), and potassium
carbonate (8.28 g, 60 mmol) for 4 days to give a yellow oil. Yield
8.26 g (87%). 1H NMR (400 MHz, CDCl3): 7.75 (m, 4H, H3′,6′), 7.68
(m, 4H, H4′,5′), 3.73 (t, J ) 6.7 Hz, 4H, H1,5), 2.78 (t, 4H, H2,4), 2.50
(t, J ) 7.1 Hz, 2H, H1′′), 1.15 (m, 12H, H2′′, H3′′, H4′′, H5′′, H6′′, H7′′),
0.84 (t, J ) 7.1 Hz, 3H, H8′′). 13C NMR (100 MHz, CDCl3): 168.25
(CO), 133.66 (C4′,5′), 132.17 (C1′,2′), 123.01 (C3′,6′), 53.92 (C1′′), 51.49
(C2,4), 35.84 (C1,5), 31.75 (C6′′), 29.51, 29.21, 27.22 (C2′′, C3′′, C4′′, C5′′),
22.59 (C7′′), 14.08 (C8′′). IR (KBr, cm-1): 1755, 1695. MS (ES+, MeCN/
MeOH): m/z 476 (MH+). Anal. Calcd (%) for C28H33N3O4 (475.58):
C, 70.71; H, 6.99; N, 8.84. Found: C, 70.89; H, 7.21; N, 8.91.
1
184-186 °C. H NMR (300 MHz, CDCl3): δ 6.00 (s, 2H, H4), 3.79
(s, 8H, H6), 2.62 (t, J 5.8 Hz, 8H, H7), 1.48 (m, 8H, H8), 1.48 (m, 4H,
H9). 13C NMR (75 MHz, CDCl3): 147.01 (broad singlet, C3,5), 102.51
(C4), 48.23 (C7), 45.68 (C6), 28.51 (C8), 23.85 (C9). IR (KBr, cm-1):
3380. MS (ES+, MeOH): m/z 389 (MH+). Anal. Calcd (%) for
C20H36N8 (388.55): C, 61.82; H, 9.34; N, 28.84. Found: C, 61.70; H,
9.48; N, 28.59.
6,19-Dibenzyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-
[22.2.1.111,14]-octacosa-1(27),11,14(28),24-tetraene (4). 3,5-Pyrazoledi-
carbaldehyde (248 mg, 2 mmol) was dissolved in hot methanol (120
mL). This solution was then cooled to room temperature and added
dropwise under an argon atmosphere to a stirred solution of the diamine
11a (387 mg, 2 mmol) in methanol (200 mL). The reaction was
monitored by TLC (Cl3CH/MeOH 10:1), and when it was complete
(ca. 12 h), sodium borohydride (456 mg, 24 mmol) was added
portionwise. After 2 h of reaction, the solvent was evaporated to dryness
under reduced pressure. The residual syrup was purified by flash column
chromatography on silica gel (MeOH/30% aqueous NH3 48:2). The
fractions containing the product of Rf 0.37 (TLC, MeOH/ 30% aqueous
NH3 10:1) were evaporated to dryness to give a pure colorless syrup.
Yield 295 mg (51%). 1H NMR (CDCl3, 400 MHz): 7.29 (m, 4H, Ho),
7.24 (m, 4H, Hm), 7.18 (m, 2H, Hp), 5.94 (s, 2H, H4), 3.75 (s, 8H, H6),
3.61 (s, 4H, H1′), 2.80 (t, J ) 5.3 Hz, 8H, H7), 2.69 (t, 8H, H8). 13C
NMR (100 MHz, CDCl3): 146.21 (very broad signal, C3,5), 138.92
(Cipso), 128.74 (Co), 128.26 (Cm), 126.93 (Cp), 101.09 (broad singlet,
C4), 59.00 (C1′), 53.05 (broad singlet, C8), 46.98 (C7), 46.25 (broad
singlet, C6). IR (KBr, cm-1): 3435. MS (ES+, MeOH): m/z 572 (MH+).
Anal. Calcd (%) for C32H46N10‚0.25OHNH4 (578.75): C, 66.34; H, 8.16;
N, 24.79. Found: C, 66.22; H, 8.40; N, 24.82.
General Procedure of Deprotection. Working under argon atmo-
sphere, a solution of 10a, 10b, or 10c (12 mmol) and hydrazine
monohydrate (12.0 g, 240 mmol) in 500 mL of EtOH was refluxed
with vigorous stirring for 36 h. The mixture was filtered and washed
with EtOH. The solvent was removed, and then 300 mL of CHCl3 was
added, and after the mixture was stirred overnight, the insoluble
phthalhydrazide was filtered off. Evaporation of CHCl3 gave a yellow
oil which was purified by distillation under reduced pressure to give
the corresponding diamine.
1,5-Diamino-3-benzyl-3-azapentane (11a). Colorless oil. Yield 1.48
1
g (63%), bp 51-54 °C (0.1 mmHg). H NMR (300 MHz, CDCl3):
7.26 (m, 5H, Ho, Hm, Hp), 3.57 (s, 2H, H1′), 2.74 (t, J ) 6.0 Hz, 4H,
H
1,5), 2.50 (t, 4H, H2,4), 1.29 (bs, 4H, NH). 13C NMR (75 MHz,
CDCl3): 139.44 (Cipso), 128.65 (Co), 128.11 (Cm), 126.81 (Cp), 59.01
(C1′), 57.20 (C2,4), 39.63 (C1,5). IR (KBr, cm-1): 3350-3100. MS (ES+,
MeOH): m/z 194 (MH+). Anal. Calcd (%) for C11H19N3 (193.29): C,
68.35; H, 9.91; N, 21.74. Found: C, 67.98; H, 10.21; N, 22.04.
1,5-Diamino-3-phenethyl-3-azapentane (11b). Colorless oil. Yield
1.42 g (57%), bp 89-94 °C (0.5 mmHg). 1H NMR (300 MHz,
CDCl3): 7.24 (m, 2H, Hm), 7.14 (m, 3H, Ho, Hp), 2.81 (m, 2H, H1′),
2.66 (t, J ) 5.9 Hz, 4H, H1,5), 2.62 (m, 2H, H2′), 2.49 (t, 4H, H2,4),
1.42 (bs, 4H, NH). 13C NMR (75 MHz, CDCl3): 140.49 (Cipso), 128.58
(Co), 128.18 (Cm), 125.81 (Cp), 56.96 (C2,4), 55.98 (C1′), 39.64 (C1,5),
33.56 (C2′). IR (KBr, cm-1): 3400-3200. MS (ES+, MeOH): m/z 208
(MH+).
6,19-Diphenethyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-
[22.2.1.111,14]-octacosa-1(27),11,14(28),24-tetraene (5). This compound
was prepared as described for 4 from the diamine 11b (414 mg) to
give a colorless syrup of Rf 0.56 (TLC, MeOH/30% aqueous NH4OH
1
1,5-Diamino-3-n-octyl-3-azapentane (11c). Colorless oil. Yield 1.39
10:1). Yield 380 mg (63%). H NMR (CDCl3, 400 MHz): 7.06 (d, J
1
g (54%), bp 66-68 °C (0.1 mmHg). H NMR (300 MHz, CDCl3):
) 7.2 Hz, 4H, Ho), 6.98 (t, 4H, Hm), 6.81 (t, 2H, Hp), 5.91 (s, 2H, H4),
3.64 (s, 8H, H6), 2.68 (m, 8H, H7), 2.63 (m, 8H, H8, H1′, H2′). 13C
NMR (100 MHz, CDCl3): 145.73 (very broad signal, C3,5), 140.81
(Cipso), 128.63 (Co), 128.01 (Cm), 125.72 (Cp), 100.91 (broad singlet,
C4), 55.06 (very broad singlet, C1′), 52.13 (broad singlet, C8), 46.71
(C7), 46.42 (broad singlet, C6), 33.11 (C2′). IR (KBr, cm-1): 3430. MS
(ES+, MeOH): m/z 600 (MH+). Anal. Calcd (%) for C34H50N10‚1.5H2O
(625.0): C, 65.28; H, 8.48; N, 22.40. Found: C, 65.55; H, 8.33; N,
22.56.
2.71 (t, J ) 6.0 Hz, 4H, H1,5), 2.45 (t, 4H, H2,4), 2.38 (m, 2H, H1′),
1.32 (m, 12H, H2′, H3′, H4′, H5′, H6′, H7′), 0.85 (t, J ) 6.4 Hz, 3H, H8′).
13C NMR (75 MHz, CDCl3): 57.26 (C2,4), 54.51 (C1′), 39.56 (C1,5),
31.84 (C6′), 29.55, 29.32, 27.49, 27.19 (C2′, C3′, C4′, C5′), 22.64 (C7′),
14.09 (C8′). IR (KBr, cm-1): 3400-3100. MS (ES+, MeOH): m/z 216
(MH+). Anal. Calcd (%) for C12H29N3 (215.38): C, 66.92; H, 13.57;
N, 19.51. Found: C, 66.67; H, 13.85; N, 19.70.
Preparation of Macrocyclic Ligands. Ligands 1 and 2 were
prepared by reaction of the 3,5-pyrazoledicarbaldehyde 1H- or 1-benzyl
substituted, respectively, with 1,5-diamino-3-azapentane as previously
reported.23
6,19-Dioctyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-[22.2.1.111,14]-
octacosa-1(27),11,14(28),24-tetraene (6). This compound was prepared
as described for 4 from the diamine 11c (430 mg) to give a colorless
9
832 J. AM. CHEM. SOC. VOL. 126, NO. 3, 2004

New 1H-Pyrazole-Containing Polyamine Receptors
A R T I C L E S
syrup of Rf 0.54 (TLC, MeOH/30% aqueous NH4OH 10:1). Yield 204
mg (31%). H NMR (CDCl3, 400 MHz): 6.11 (s, 2H, H4), 3.81 (s,
concentration probe by titration of well-known amounts of HCl with
1
CO2
-free NaOH solutions and determination of the equivalent point
by Gran’s method,42 which gives the standard potential E°′ and the
ionic product of water [pKw ) 13.73(1)]. The computer program
HYPERQUAD43 was used to calculate the protonation and stability
constants, and the HYSS44 program was used to obtain the distribution
diagrams. The titration curves for each system (ca. 200 experimental
points corresponding to at least three measurements, pH 2-11,
concentration of ligands 1 × 10-3 to 5 × 10-3 M) were treated either
as a single set or as separated curves without significant variations in
the values of the stability constants. Finally, the sets of data were merged
together and treated simultaneously to give the final stability constants.
8H, H6), 2.84 (m, 8H, H7), 2.65 (m, 8H, H8), 2.48 (m, 4H, H1′), 1.41
(m, 4H, H2′), 1.21 (m, 20H, H3′, H4′, H5′, H6′, H7′), 0.83 (t, J ) 6.7 Hz,
6H, H8′). 13C NMR (100 MHz, CDCl3): 145.67 (very broad signal,
C3,5), 102.47 (broad singlet, C4), 54.18 (broad singlet, C1′), 51.29 (broad
singlet, C8), 46.98 (C7), 45.30 (broad singlet, C6), 31.79 (C6′), 29.49,
29.25, 27.51, 25.88 (C2′, C3′, C4′, C5′), 22.6 (C7′), 14.1 (C8′). IR (KBr,
cm-1): 3422. MS (ES+, MeOH): m/z 616 (MH+). Anal. Calcd (%)
for C34H66N10‚2H2O (833.72): C, 62.73; H, 10.84; N, 21.52. Found:
C, 63.07; H, 10.95; N, 21.78.
General Procedure for Preparation of the Hydrochlorides.
Ligands 3, 4, 5, and 6 descompose partially after some time, and it is
preferable to store them as the corresponding hydrochlorides, prepared
as follows: A mixture of the corresponding ligand (0.5 mmol) and 1
M aqueous hydrochloric acid (3 mL) was stirred for 12 h. After addition
of ethanol (50 mL), the solvent was evaporated to dryness. Next, 30
mL of ethanol was added, and the resulting solution was stirred for 1
h. A white solid was formed, filtered off, and dried over phosphorus
pentoxide at 60 °C for 48 h.
Molecular Modeling. L-Glutamic acid was modeled as zwitterion,
as this was the principal structure in the pH range of experimental
studies, and ligands were modeled in all posible protonation states
showed to exist in the pH range of experimental data in 5% or more
abundance. Starting structures for ligands were built by using Hyper-
chem capabilities. Its geometry was minimized to a maximum energy
gradient of 0.4 kJ/(A mol) with the AMBER force field, using the Polak-
Ribiere (conjugate gradient) algorithm, and the “simulated annealing”
procedure was used to cover all conformational space runnning
molecular dynamics at 400 K. This geometry was always used in all
calculations of host/guest complexes. To optimize host/guest inter-
actions, the L-glutamic acid was moved and/or rotated around the C2-
C3 bond and the structure of the acid was docked into the “cavity” of
the ligand in all different possible orientations, and then the energy of
the complex was minimized with no restraints, following the same
procedure as above.
1
[3]‚4HCl. Yield 219 mg (82%), mp 225-227 °C. H NMR (D2O,
300 MHz): δ 6.68 (s, 2 H, H4), 4.35 (s, 8 H, H6), 2.96 (t, J ) 6.3 Hz,
8 H, H7), 1.63 (m, 8 H, H8), 1.37 (m, 4 H, H9). 13C NMR (D2O, 75
MHz): 139.00 (C3,5), 108.55 (C4), 45.99 (C7), 41.99 (C6), 24.83 (C8),
22.84 (C9). IR (KBr, cm-1): 3395. MS (ES+, H2O): m/z 389 ([MH -
4HCl]+). Anal. Calcd (%) for C20H36N8‚4HCl (534.40): C, 44.95; H,
7.54; N, 20.97. Found: C, 44.61; H, 7.17; N, 20.82.
1
[4]‚6HCl. Yield 210 mg (52%), mp 238-240 °C. H NMR (D2O,
300 MHz): δ 7.35 (s, 10 H, Ho, Hm, Hp), 6.58 (s, 2 H, H4), 4.16 (s, 8
H, H6), 3.89 (s, 4 H, H1′), 3.22 (t, J ) 6.3 Hz, 8 H, H7), 3.01 (t, 8 H,
H8). 13C NMR (D2O, 75 MHz): 139.95 (C3,5), 132.53 (Cipso), 131.86,
130.64 (Co, Cm), 131.00 (Cp), 109.91 (C4), 59.64 (C1′), 50.51 (C8), 43.87
(C6), 43.59 (C7). IR (KBr, cm-1): 3440. MS (ES+, H2O): m/z 572
([MH - 6HCl - H2O]+). Anal. Calcd (%) for C32H46N10‚6HCl‚H2O
(807.55): C, 47.59; H, 6.74; N, 17.34. Found: C, 47.24; H, 7.11; N,
16.84.
Acknowledgment. This work is devoted to the memory of
Prof. Manuel Lora-Tamayo. Financial support by the Spanish
“Comisio´n Interministerial de Ciencia y Tecnolog´ıa” (CICYT,
Proyects SAF-99-0063, and BQU2003-09215-CO3-01) is grate-
fully acknowledged.
1
Supporting Information Available: Potentiometric data:
Figure S1 (distribution diagrams for the systems Ln-H+ and
Glu-H+) and Figure S2 (distribution diagrams for the systems
L2-glutamic acid, L3-glutamic acid, and L6-glutamic acid).
Molecular modelization data: Figures S3 and S4 (charges and
AMBER atom type for 3,5-bis[(N-methylamino)methyl]-1H-
pyrazol and for 1-benzyl-3,5-bis[(N-methylamino)-methyl]-
pyrazol), Table S1 (nonstandard force field parameters used in
this work), Table S2 (energy variations for ligands L1-L6 with
protonation pattern), Tables S3-S8 (calculation of complexation
energy for L1-L6), Table S9 (stability constants for the system
Asp-L4), Figure S5 (representation of the variation of Kcond
(M-1) for the interaction of glutamic and aspartic acids with
L4; initial concentrations of acids and receptors are 10-3 M),
and Figures S6-S10 (molecular models and interactions for the
L-glutamate adducts formed from ligands L1-L3 and L5,L6)
(PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
[5]‚6HCl. Yield 318 mg (78%), mp 221-223 °C. H NMR (D2O,
500 MHz): δ 7.24 (t, 4 H, Ho, Hm), 7.17 (m, 6 H, Hp), 6.58 (s, 2 H,
H4), 4.16 (s, 8 H, H6), 3.89 (s, 4 H, H1′), 3.22 (t, J ) 6.3 Hz, 8 H, H7),
3.01 (t, 8 H, H8). 13C NMR (D2O, 125 MHz): 139.79 (C3,5), 137.41
(Cipso), 130.30 (Cm), 130.01 (Co), 128.58 (Cp), 110.10 (C4), 55.98 (C1′),
50.00 (C8), 43.87 (C6), 42.44 (C7), 30.54 (C2′). IR (KBr, cm-1): 3435.
MS (ES+, H2O): m/z 600 ([MH - 6HCl]+). Anal. Calcd (%) for
C34H50N10‚6HCl (817.59): C, 49.95; H, 6.90; N, 17.13. Found: C,
50.16; H, 7.00; N, 17.18.
1
[6]‚6HCl. Yield 312 mg (75%), mp 228-231 °C. H NMR (D2O,
400 MHz): 6.57 (s, 2H, H4), 4.22 (s, 8H, H6), 3.36 (m, 8H, H8), 3.30
(m, 8H, H7), 3.04 (t, J ) 8.0 Hz, 4H, H1′), 3.22 (m, 4H, H2′), 1.06 (m,
10H, H3′, H4′, H5′, H6′, H7′), 0.61 (t, J ) 6.6 Hz, 6H, H8′). 13C NMR
(D2O, 100 MHz): 139.68 (C3,5), 110.18 (C4), 55.52 (C1′), 49.87 (C8),
43.89 (C6), 41.81 (C7), 32.11 (C6′), 29.33 (C4′, C5′), 26.73 (C3′), 23.97
(C2′), 23.13 (C7′), 14.55 (C8′). IR (KBr, cm-1): 3434. MS (ES+, H2O):
m/z 616 ([MH - 6HCl]+). Anal. Calcd (%) for C34H66N10‚6HCl
(833.72): C, 48.98; H, 8.70; N, 16.80. Found: C, 48.69; H, 8.76; N,
16.72.
Electromotive Force (emf) Measurements. The potentiometric
titrations were carried out in 0.15 M NaCl at 298.1 ( 0.1 K by using
the experimental procedure (buret, potentiometer, cell, stirrer, micro-
computer, etc.) that has been fully described elsewhere.40 The acquisi-
tion of the emf data was performed with the computer program
PASAT.41 The reference electrode was a Ag/AgCl electrode in saturated
KCl solution. The glass electrode was calibrated as a hydrogen ion
JA035671M
(41) Fontanelli, M.; Micheloni, M. Proceedings of the 1st Spanish-Italian
Congress on Thermodynamics of Metal Complexes; Diputacio´n de Cas-
tello´n: Pen˜´ıscola (Castello´n), Espan˜a, 3-6 Junio 1990.
(42) (a) Gran, G. Analyst (London) 1952, 77, 661. (b) Rossoti, F. J. C.; Rossoti,
H. J. Chem. Educ. 1965, 42, 375-378.
(43) Gans, P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739-1753.
(44) Gans, P. Program to determine the distribution of species in multiequilibria
systems from the stability constants and mass balance equations.
(40) Garc´ıa-Espan˜a, E.; Ballester, M. J.; Lloret, F.; Moratal, J.-M.; Faus, J.;
Bianchi, A. J. Chem. Soc., Dalton Trans. 1988, 2, 101-104.
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