Heteroditopic receptor based on crown ether and cyclen units for the recognition of zwitterionic amino acids

Article abstract of DOI:10.1016/j.tet.2012.03.110

The molecular recognition of five targeted amino acids differing in the nature of the side (R)- group and in the size of the aliphatic chain, glycine (Hgly), phenylalanine (Hphe), glutamic acid (Hglu^-), 4-aminobutyric acid (Hgaba), and 6-aminohexanoic acid (Heahx), has been studied with a new heteroditopic receptor based in two distinct macrocycles, a cyclen and a crown ether moiety. The bismacrocycle L was synthesized via the bis-aminal route allowing to obtain the designed compound in gram scale and in good yield. Protonation constants of L and its binding constants with amino acids were determined by potentiometry in H₂O/MeOH (1:1 v/v) solutions at 298.2 K and I=0.10 mol dm^-3 in NMe₄NO₃. Stronger binding ability of the H_nLⁿ⁺ receptor for α-amino acids, Hgly and Hphen, than for the other studied substrates were found. Structural data derived from NMR studies showed that the binding of α-amino acids result from the cooperative participation of hydrogen bonds between the carboxylate group of amino acids and the polyammonium sites of cyclen, and the ion-dipole interactions between the ammonium group of the amino acids and the oxygen atoms of the crown ether.

Full text of DOI:10.1016/j.tet.2012.03.110

Tetrahedron 68 (2012) 4860e4868
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Heteroditopic receptor based on crown ether and cyclen units for the recognition
of zwitterionic amino acids
*
Nicolas Bernier, Catarina V. Esteves, Rita Delgado
ꢀ
ꢀ
Instituto de Tecnologia Química e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The molecular recognition of five targeted amino acids differing in the nature of the side (R)- group and
in the size of the aliphatic chain, glycine (Hgly), phenylalanine (Hphe), glutamic acid (Hgluꢀ), 4-
aminobutyric acid (Hgaba), and 6-aminohexanoic acid (Heahx), has been studied with a new hetero-
ditopic receptor based in two distinct macrocycles, a cyclen and a crown ether moiety. The bismacrocycle
L was synthesized via the bis-aminal route allowing to obtain the designed compound in gram scale and
in good yield. Protonation constants of L and its binding constants with amino acids were determined by
potentiometry in H2O/MeOH (1:1 v/v) solutions at 298.2 K and I¼0.10 mol dmꢀ3 in NMe4NO3. Stronger
Received 23 February 2012
Received in revised form 20 March 2012
Accepted 28 March 2012
Available online 6 April 2012
Keywords:
Bismacrocycle
Heteroditopic receptor
Amino acids
Recognition
binding ability of the HnLnþ receptor for
a
-amino acids, Hgly and Hphen, than for the other studied
substrates were found. Structural data derived from NMR studies showed that the binding of -amino
a
acids result from the cooperative participation of hydrogen bonds between the carboxylate group of
amino acids and the polyammonium sites of cyclen, and the ion-dipole interactions between the am-
monium group of the amino acids and the oxygen atoms of the crown ether.
Ó 2012 Elsevier Ltd. All rights reserved.
Potentiometry
1H NMR
1. Introduction
studies are often restricted to non-aqueous media (e.g., CHCl3,
MeCN, DMSO) and N- or O-protected amino acids.8,9
Nature use amino acids as building blocks for construction of
proteins, and as molecular messengers capable of transmitting in-
formation in living organisms.1 Therefore, molecular recognition of
amino acids providing the chemical information inherent in their
structure and functionality is of great importance.2 Potential fields
of application are the development of efficient methods of amino
acid sensing and analysis,3 the purification and resolution of race-
mates,4 the extension of the acquired knowledge to peptide rec-
ognition,5 the development of carriers for drug delivery,6 etc.
Consequently, is not surprising the interest of supramolecular
chemists in designing receptors for this group of compounds.7
The effective recognition of aminocarboxylic acids at physio-
logical conditions is a difficult task due to its zwitterionic character
and the strong competition that they undergo in polar solvents,
such as water. Moreover, the electronic densities at the carboxylate
and ammonium functions are greatly affected by their mutual vi-
cinity, causing the binding forces of complementary groups of the
receptor less effective for association. Thus, in aqueous solution,
where zwitterionic amino acids enjoy their greatest solubility, hy-
drogen bonds are difficult to establish. Consequently, recognition
The design of receptors capable to confer the required rigidity
and complementarity for the recognition of this type of partner, is
very challenging. However, several receptors9f,10 and metal-
loreceptors11 have been proposed recently. Among them, hetero-
ditopic receptors offer the possible simultaneous bind of cationic
and anionic guests, as well as zwitterions.12 These compounds are
also able to form strong ternary species, especially when various
sites act cooperatively.13,14
Pursuing the work on recognition of anionic substrates already
developed in the group,15,16 the challenge is now the binding of
amino acids in neutral aqueous solution. Thus, herein the prepa-
ration and recognition properties of the bismacrocycle L (Scheme 1)
is carried out. This compound when protonated can act as a syn-
thetic receptor, combining the following structural components: (i)
a tetraazamacrocyclic unit that in its polyammonium form is ca-
pable to interact with carboxylate groups through hydrogen bonds
and coulombic interactions; (ii) a crown ether moiety able to rec-
ognize ammonium groups through ion-dipole interactions; and (iii)
a naphthalene ring that provides an aromatic planar surface for
additional
pep stacking interactions involving side chains of aro-
matic amino acids. The target molecules were the a-amino acids,
glycine (Hgly), phenylalanine (Hphe) and glutamic acid (Hgluꢀ) as
well as 4-aminobutyric acid (Hgaba) and 6-aminocaproic acid or 6-
aminohexanoic acid (Heahx), see Scheme 1. The ability of the
* Corresponding author. Fax: þ351 21 441 12 77; e-mail address: delgado@itq-
b.unl.pt (R. Delgado).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.03.110

N. Bernier et al. / Tetrahedron 68 (2012) 4860e4868
4861
receptor to organize its both macrocycles and aromatic moieties in
multiple and cooperative interactions with the substrates in su-
pramolecular entities is studied. Therefore, potentiometric mea-
surements were performed to obtain thermodynamic data, and
NMR spectroscopy was used in order to obtain structural insights of
the supramolecular entities formed in aqueous solution.
different functionalized groups. Among the methodologies de-
veloped in the last three decades,17 the bis-aminal one18 leads
rapidly and selectively to mono-N- and trans-di-N-alkylated tet-
raazamacrocycles.19,20 After the formation of the bis-aminal de-
rivative 2, by condensation of glyoxal on cyclen, the first selective
mono-N-alkylation with 2-bromomethylnaphthalene yielded the
intermediate 3, and the second selective trans-N-alkylation with
dibromo-p-xylene formed the diammonium salt 4. Polyalkylation
reaction was not observed under the conditions used, see
Experimental. Then, the nucleophilic substitution of [18]aneNO5 on
4 yielded the protected bismacrocycle 5, and finally, the bis-aminal
bridge of cyclen in 5 was removed by treatment with hydrazine to
afford the bismacrocycle L. Therefore, the heteroditopic bismacro-
cycle L was obtained in five steps from commercially available
products in good yield (51%).
The structure of the new heteroditopic macrocyclic receptor was
well established from analytical and spectroscopic data (see Figs.
S1eS11 in Supplementary data).
2.2. Binding studies
Scheme 1. Chemical structures of the bismacrocycle L and the amino acids studied
with the corresponding abbreviations used.
The bismacrocyclic compound L was designed with two distinct
binding sites separated by a rigid aromatic spacer. The cyclen
moiety, generally di-protonated under physiological conditions,
should interact with the carboxylate group of amino acids, and the
crown ether unit is appropriate for the recognition of the ammo-
nium group, essentially via electrostatic interactions and hydrogen
bonds.10k,o,12b,f,14b,21,22 Therefore, this receptor when protonated is
a good candidate for associations with amino acids especially when
the recognition process leads to cooperative effects.
2. Results and discussion
2.1. Synthesis and characterization of the bismacrocyclic
compound, L
The design of the heteroditopic bismacrocycle L is based in the
use of cyclen and monoaza crown ether macrocycles as common
building blocks, linked by the p-xylyl spacer. The two building
blocks were assembled via the bis-aminal route depicted in
Scheme 2, which allows easy changes of the structure to modulate
the ability of the receptor to the binding properties of zwitterions.
The synthetic procedure required first the selective protection of
the tetraazamacrocycle 1 allowing two N-trans substitutions with
2.2.1. Acidebase behavior: protonation constants and protonation
sequence of L. Previous to the binding studies, the acid-base be-
havior of each partner (the receptor and substrates) needs to be
known under the experimental conditions of this work, which are
H2O/MeOH (1:1 v/v) solution at 298.2 K and in I¼0.10 mol dmꢀ3
Scheme 2. Preparation of bismacrocycle L.

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N. Bernier et al. / Tetrahedron 68 (2012) 4860e4868
NMe4NO3. The solvent choice took into account the low solubility of
L at pH >7 in aqueous solution, and the interest to study amino
acids at conditions close to the physiological one. The stepwise and
overall protonation constants were compiled in Table 1, and Table
S1 in Supplementary data, respectively. For all the studied amino
acids, the protonation constant of the ammonium group is
0.13e0.63 log units lower while that of the carboxylate group is
0.12e0.89 log units higher than the corresponding values in
aqueous solution taken from the literature (see Table S2 of Sup-
plementary data), testifying the impact of the presence of methanol
in the acidebase behavior.
Table 1
Stepwise protonation constants (KHi ) of L and of the
studied amino acids in H2O/MeOH (1:1 v/v),
T¼298.2ꢁ0.1 K, I¼0.10ꢁ0.01 mol dmꢀ3 in NMe4NO3
a
Equilibrium reaction
logKiH
Compound L
L þ Hþ%HLþ
9.32(1)
7.73(1)
6.38(2)
1.88(4)
HLþ þ Hþ%H2L2þ
H2L2þ þ Hþ%H3L3þ
H3L3þ þ Hþ%H4L4þ
Amino acids
Fig. 1. Species distribution diagram for the bismacrocycle HnLnþ. CL¼2 mmol dmꢀ3
.
The charges were omitted for clarity.
glyꢀ þ Hþ%Hgly
9.33(1)
2.91(1)
8.87(1)
2.84(1)
9.62(1)
4.75(1)
2.78(2)
9.93(1)
4.69(2)
10.10(1)
5.16(2)
Hgly þ Hþ%H2glyþ
pheꢀ þ Hþ%Hphe
Hphe þ Hþ%H2pheþ
glu2ꢀ þ Hþ%Hgluꢀ
Hgluꢀ þ Hþ%H2glu
H2glu þ Hþ%H3gluþ
gabaꢀ þ Hþ%Hgaba
Hgaba þ Hþ%H2gabaþ
eahxꢀ þ Hþ%Heahx
Heahx þ Hþ%H2eahxþ
respectively. On the other hand, at the same pH, all amino acids
substrates present its zwitterionic form. These data indicate that L
can be a good receptor for amino acids only in case of bearing the
two positive charges located at the cyclen moiety at pH about 7.
Based only on potentiometric measurements, it is not possible
to establish the exact scheme of protonation, namely if the second
protonation occurs at a second amine of cyclen or at the azacrown
ether amine. Then, a 1H NMR titration was performed in mixed
D2O/CD3OD solution (1:1 v/v) at 298.2 K using KOD as the titrant,
which is shown in Fig. 2. 2D NMR experiments carried out at pD 3
and 10 allowed to assign all proton resonances, see Fig. S11 in
Supplementary data.
a
Values in parentheses are standard deviations in
the last significant figure.
The 1H NMR spectra of L at the entire pD range (see Fig. S12) is
rather complicated due to the low symmetry of the molecule. For
instance at pD¼5, in the aromatic region of the spectrum, six dif-
ferent sets of signals, corresponding to nine magnetically non-
equivalent protons, are observed, while at upper field the number
of signals, or sets of signals, increases to eight. Among the latter
signals, those assigned to Hken protons of the crown ether unit (see
Fig. 2 for labeling) appear as a broad signal that does not move
along the pD. On the other hand, large shifts of Hh and Hi proton
resonances, followed by those of Hj and Hb,c, were observed. The
Ha,d, He and Ho resonances undergo small shifts.
By decreasing the pD from a solution of L completely deproto-
nated, the Hb,c proton resonances shift downfield at 10.5e9.5 pD
region, but He and Ho also slightly move, indicating that the first
protonation occurs at the cyclen ring, mainly on the secondary
amines although a small percentage of the protons also spread at
the tertiary amines. Otherwise, the small He and Ho shift can be
a result of hydrogen bond formation þNeH/N inside the cyclen
ring moiety. At the 8.7e6.0 pD range, corresponding to the second
and the third protonation constants, all the resonances close to
amine groups shift downfield, except Hken. However, Hj shifts only
at 8.7e7.5 and Hb,c shifts more at 7.5e6.0 pD region. Additionally,
Hh and Hi have significant shifts in this entire pD range (8.7e6.0).
These data indicate that in this pD region several amine centers are
simultaneously protonated although in different percentage at each
pD value. The resonances close to the tertiary amines of cyclen,
Haed, He, and Ho, only slightly shift indicating a very low percentage
of protonation of these centers at this pD region. Therefore, it is
possible to conclude that the second and third protonations mainly
occur on the amine of the crown ether moiety and the secondary
The bismacrocycle L contains three amino groups behaving as
strong to moderate bases, and the fourth one as a very weak base.
The fifth and last protonation constant could not be detected in the
pH range (2e11) investigated. This behavior is in part explained by
electrostatic repulsions minimization between positive charges in
polyprotonated species. Cyclen derivatives generally exhibit two
high protonation constants for which protons are located in trans
nitrogen atoms and are involved in strong hydrogen bonds with
the two adjacent amino groups leading to two high and two
very low protonation constants in the ranges 9.5e11 and <2.5,
respectively.23 The cyclen moiety of L behaves similarly, conse-
quently two of the three highest protonation constants must cor-
respond to protonations of the cyclen ring, and the third one must
be ascribed to the tertiary amino group of the crown ether unit (for
the exact assignement see below). The fourth proton has to be lo-
cated in one amine contiguous to protonated ones of the cyclen ring
exhibiting the very low protonation constant. However, the three
highest protonation constants of L are lower than those found for
other cyclen derivatives in aqueous solution.23 As for amino acids,
the presence of methanol in the solvent mixture affects the values,
but the main effect arise from the presence of nearby electron-
withdrawing groups, such as the aromatic rings (naphthalene and
the p-xylene spacer) and the oxygen donor atoms of the crown
ether moiety. Additionally, the steric effect induced by the
presence of bulky groups can lead to decrease the stability of hy-
drogen bonds either with adjacent nonprotonated amino groups
or with solvent molecules.10m
The species distribution diagram, presented in Fig.1, reveals that
at physiological pH value (pH¼7.4) the main species in solution is
H2L2þ (63%), while HLþ and H3L3þ exist only in 31% and 6%,

N. Bernier et al. / Tetrahedron 68 (2012) 4860e4868
4863
Fig. 2. pD values in function of experimental 1H NMR chemical shifts of some resonances of the bismacrocycle L.
amines of the cyclen ring, although the percentage of protonation
in the former center is larger for the highest pD values and the
protonation of the second secondary amine of cyclen occurs mainly
at lower pD (7.5e6.0 pD region). Besides, the symmetry of the
compound is kept at acidic pD values (between 5 and 2.5) in-
dicating that, in the NMR time scale, the positive charges in the
triprotonated species have an average homogeneous distribution
overall nitrogen atoms. Therefore, these data revealed that, at pH
values about 7, the first proton is located in the cyclen moiety in the
monoprotonated form of the receptor (existing in 31%), whereas
the second proton coexists in amine centers of the two macro-
cycles, with larger percentage at the crown ether moiety of the
receptor (existing in 63%).
Table 2
Stepwise association constants (K
) of L in H2O/MeOH (1:1 v/v), T¼298.2ꢁ0.1 K,
HhLlAa
I¼0.10ꢁ0.01 mol dmꢀ3 in NMe4NO3
Equilibrium reactiona
b
logKH
hLlAa
H3L3þ þ H2glyþ%H5Lgly4þ
2.99(5)
2.77(5)
H3L3þ þ Hgly%H4Lgly3þ
H2L2þ þ Hgly%H3Lgly2þ
HLþ þ Hgly%H2Lglyþ
2.66(5)
2.39(5)
2.76(5)
2.91(4)
2.56(4)
2.47(5)
2.37(5)
2.57(4)
3.11(1)
2.65(2)
2.38(2)
2.16(3)
1.77(4)
2.55(3)
2.36(3)
2.19(4)
1.93(5)
1.97(6)
2.32(4)
2.24(5)
2.00(6)
1.75(8)
1.70(10)
L þ Hgly%HLgly
H3L3þ þ H2pheþ%H5Lphe4þ
H3L3þ þ Hphe%H4Lphe3þ
H2L2þ þ Hphe%H3Lphe2þ
HLþ þ Hphe%H2Lpheþ
HLþ þ pheꢀ%HLphe
H3L3þ þ H3gluþ%H6Lglu4þ
H3L3þ þ H2glu%H5Lglu3þ
H3L3þ þ Hgluꢀ%H4Lglu2þ
H2L2þ þ Hgluꢀ%H3Lgluþ
HLþ þ Hgluꢀ%H2Lglu
2.2.2. Binding studies of the bismacrocycle HnLnþ receptor with
amino acids
. 2.2.2.1. Association constants. The association constants of the
protonated receptor HnLnþ with five targeted amino acids dif-
fering in the nature of the side (R)-group and in the size of the
aliphatic chain (see Scheme 1) were determined by potentiom-
H3L3þ þ H2gabaþ%H5Lgaba4þ
H3L3þ þ Hgaba%H4Lgaba3þ
H2L2þ þ Hgaba%H3Lgaba2þ
HLþ þ Hgaba%H2Lgabaþ
L þ Hgaba%HLgaba
etry in H2O/MeOH (1:1 v/v) solution, at 298.2
K and
I¼0.10 mol dmꢀ3 in NMe4NO3. Only species of 1:1 (receptor/
substrate) stoichiometry were found, in the entire pH scale, al-
though titration data of different ratios were used. The stepwise
binding constants were collected in Table 2 and a selected spe-
ciation diagram was shown in Fig. 3. They were derived from the
overall association constants bH L A values (corresponding to the
H3L3þþH2eahxþ%H5Leahx4þ
H3L3þ þ Heahx%H4Leahx3þ
H2L2þ þ Heahx%H3Leahx2þ
HLþ þ Heahx%H2Leahxþ
L þ Heahx%HLeahx
a
a
h
l
L¼free receptor in its completely deprotonated form.
b
equilibria l L þ a A þ h H%HhLlAa, where the charges of the
species were not considered) provided by fitting the titration
curves, and the values obtained were compiled in Table S3 of
Supplementary data. As amino acids are also pH dependent, the
plot of the effective association constants Keff as a function of the
pH, defined as Keff¼S[HiþjLA]/S[HiA]ꢂS[HjL], was used for the
correct assessment of the stepwise equilibria, see Fig. 4.24
The binding constant for the association of the receptor
with Hgly has a reasonable value, which decrease for the other
amino acids, following the sequence at pH 7: Hgly>Hphe
>HgluꢀzHgaba>Heahx. Although these values seem small when
compared with those of the binding of some dianions with ap-
propriate receptors, they are among the best ones for the binding of
similar partners from the literature in aqueous solution.9f,10neq
On the other hand, the decrease of the receptor and/or substrate
charges did not lead to clear-cut trends of the association constants,
except when the receptor and the amino acids are completely
Values in parentheses are standard deviations in the last significant figures.
deprotonated, which occurat about pH 9. In fact, the largest constant
values would be expected to result from the interaction of H3L3þ or
H2L2þ receptor species with the zwitterionic form of the substrates,
when carboxylate group of amino acids interact with the protonated
amines of cyclen (ꢀO/þNeH), and the ammonium group of amino
acids interact with oxygen atoms of the crown ether moiety
(þNeH/O). This is not the case here, as shown in Fig. 4, the Keff
values only slightly decrease along the 2e9 pH range. This suggests
that electrostatic interactions play the main role, but hydrogen
bonds are also very important to achieve the recognition process.
As can be observed by the plot of Keff as a function of the pH
depicted in Fig. 4, the HnLnþ receptor clearly prefer
a-amino acids in
acidic medium, and also at neutral pH although for slightly lower
Keff values (except glutamic acid). This preference trend must result

4864
N. Bernier et al. / Tetrahedron 68 (2012) 4860e4868
Fig. 3. Species distribution diagram of HnLnþ with Hgly as a function of pH. Percent-
Fig. 5. Distribution diagram of the overall amount of the receptor in the competitive
ꢀ3
ages are calculated with respect to the total amount of receptor. CL¼2 mmol dmꢀ3
,
system HnLnþ/Hgly/Hgluꢀ as a function of pH. CL¼CA¼CA ¼2 mmol dm
.
0
CA¼2 mmol dmꢀ3
.
amino acids by HnLnþ. In this regard, the 1H NMR spectra of 1:1 and
1:2 L/A molar ratios of the receptor and the amino acid substrates
were recorded in D2O/CD3OD (1:1 v/v) solvent at pD¼7.0. The shifts
of resonances are similar at both L/A molar ratios. The proton res-
onance shifts of the associated entity in relation to the free receptor
(Ddmax) of the most representative signals were collected in Table 3.
In all cases, only one set of signals was observed for the free re-
ceptor and for the associated entities, indicating fast equilibrium on
the NMR time scale.
In general the signals Haed assigned to the cyclen ring present
the larger shifts indicating that the strongest interactions mainly
affect the protonated cyclen moiety. Indeed, the protonation
studies showed that the cyclen ring can form mono- and di-
protonated species at physiological pH, leading to the formation of
hydrogen bonds. The Hb and Hc signals shifts are larger than the ones
of Ha and Hd, indicating that the secondary nitrogen atoms are more
involved in the binding event. The very small shifts of He and Ho
signals of the benzylic protons are consistent with this hypothesis.
However, the shifts are downfield, which is unexpected for forma-
tion of an ion-pair interaction, which can result from a possible
structural modification of the receptor around the cyclen moiety
induced by the binding of the carboxylate group. Indeed, for cyclic
polyamines, the inversion of the configuration of the nitrogen atoms
need to be considered.25 Protons are generally stabilized by the
formation of intracyclic hydrogen bonds involving the adjacent
secondary amino groups23 directed towards the center of the cavity
(in). However, by interaction with the negatively charged substrate,
new hydrogen bonds are formed and the nitrogen atoms may un-
dergo an inversion by rotation of the bonds of the cycle (out), leading
a strong structural modification and different chemical shifts.
On the other hand, small shifts are observed for the Hm,n signals
corresponding to the crown ether, which undergo a downfield shift
only when Hgly or Hphe are added. This suggests the presence of
ion-dipole interactions between the ammonium groups of these
substrates and the oxygens of the crown ether moiety. In opposi-
tion, protons Hi resonated at similar chemical shifts upon addition
of the substrate, suggesting that the amine/ammonium function of
the crown ether do not participate in the recognition event. Simi-
larly, no evident change was observed for the aromatic resonances
of both receptor and phenylalanine (naphthyl moiety Hpev or
p-xylene spacer Hf,g of the receptor and benzene ring of phenylal-
Fig. 4. Plot of the effective association constant Keff versus pH for the supramolecular
species formed between the bismacrocycle HnLnþ and the amino acids studied.
CL¼CA¼2 mmol dmꢀ3
.
from geometrical requirements between the three-dimensional
location of the binding groups of the receptor involved in the rec-
ognition process and the size of the aliphatic chain of different
substrates. In this sense, both macrocyclic binding units might or-
ganize multiple and cooperative interactions with zwitterionic
a
-amino acids, in particular. On the other hand, was not observed
any special preference by the receptor for Hphen, suggesting that
no interactions with the aromatic arms of the cyclen moiety
pep
and the side chain aromatic substituent of Hphen is involved in the
recognition event.
The dicarboxylate nature of Hgluꢀ induces a higher charge con-
centration at neutral-basic pH when other amino acids are negatively
charged or in zwitterionic form, causing a stronger competition be-
tween the desolvation process and the possible hydrogen bonds for-
mation, leading to a decrease of the association constant compared to
the other a-amino acids. This can be observed by the competition be-
tween Hgly and Hgluꢀ for the receptor shown in Fig. 5.
anine). As suggested by the potentiometric data, no
teraction with the side chain of the substrate is involved in the
recognition event.
pep in-
2.2.2.2. NMR spectroscopic studies. NMR spectroscopy was used
to provide structural data of the supramolecular entities formed in
solution and to propose a binding model for the recognition of

N. Bernier et al. / Tetrahedron 68 (2012) 4860e4868
4865
Table 3
Induced chemical shifts (Ddmax, ppm) of selected receptor signals in the presence of 2 equiv of amino acids at pD¼7.0 (5 mmol dmꢀ3 in D2O/CD3OD (1:1 v/v), T¼298.2ꢁ0.1 K,
400 MHz)a
Substrate
Hn
Hm
Hh
He,o
Hb,c
Ha,d
Hgly
ꢀ0.012
d
ꢀ0.017
ꢀ0.010
d
þ0.013
d
ꢀ0.012
d
ꢀ0.010
d
ꢀ0.060
ꢀ0.032
ꢀ0.011
ꢀ0.034
d
ꢀ0.055
ꢀ0.027
ꢀ0.008
ꢀ0.030
þ0.015
ꢀ0.022
ꢀ0.012
d
ꢀ0.027
ꢀ0.015
d
Hphe
Hgluꢀ
Hgaba
Heahx
d
ꢀ0.010
d
d
d
d
ꢀ0.010
d
d
d
ꢀ0.014
d
ꢀ0.014
d
d
d
d
d
a
Induced chemical shifts (Ddmax, ppm) lower than 0.010 ppm were not shown, which include ICS of aromatic Hpev, Hf,g, some of the crown ether resonances Hiel of the
receptor and amino acids resonances.
Moreover, the shifts of proton resonances by the supramolecular
entity formation are a function of the nature of the associated
amino acid. Thus, the relative variation of chemical shift of a given
signal can be considered proportional to the amount of supramo-
lecular species formed in solution, the comparison of these varia-
tions of the 1:1 L/A molar ratio solutions lead to the following
trend: Hgly>Hphe>HgluꢀzHgabazHeahx, which is in agreement
with the Keff values at neutral pH, see Fig. 4. Besides, stronger shifts
of Haed and Hm,n with Hgly and Hphe than the others amino acids
were obtained. These results support the involvement of both the
crown ether and the protonated cyclen moieties in the binding of
these two amino acids yielding to a likely cooperative effect in the
neutral pH. In spite of the low association constants obtained due to
the strong competition of the water used as solvent, a preference
trend for the a-amino acids, Hgly and Hphen, was observed. This
preference is probably caused by multiple and cooperative in-
teractions between both macrocycles of the receptor and the
zwitterionic substrates. Consequently the bismacrocycle L acts as
a ditopic receptor. Unfortunately, the additional aromatic groups of
L (naphthalene and p-xylene) do not participate to the recognition
event,
pep stacking interaction being too low to be detected in our
conditions.
Finally, this study contributed to obtain further informations on
the understanding of factors involved in the selective binding of
zwitterionic amino acids by polyammonium artificial receptors.
Besides, this knowledge will contribute to improve the design of
receptors based on related structures, an area of the supramolec-
ular chemistry that must be developed.
best cases, which means with short aliphatic a-amino acids sub-
strates (except glutamic acid, as discussed before).
Finally, in agreement with our experimental findings suggested
by NMR experiments, a schematic representation of the recognition
event can be suggested for [H3Lgly]2þ (mainly present at physio-
logical pH), see Fig. 6. A 1:1 (receptor/substrate) stoichiometric
ratio of the associated species is depicted, showing a total of five
4. Experimental
4.1. General
Microanalyses were carried out by the ITQB Microanalytical
Service. Mass spectra have been acquired in the positive and neg-
ative polarity mode in an API-ION TRAPmass spectrometer equip-
ped with ESI source, at the ITQB Mass Spectrometry Laboratory. 1H
and 13C NMR spectra were recorded on a Bruker Avance III 400 MHz
and a Bruker Amx 300 MHz spectrometers at 298.2 K, respectively.
The internal reference used for the 1H NMR measurements in CDCl3
was TMS, and in D2O was 3-(trimethylsilyl) propionic acid-d4. The
NMR samples were prepared using Rainin EDP2 automatic micro-
pipets of 25, 100 or 1000 mL, and the pH* was measured with an
Orion 420A measuring instrument fitted with a combined Hamil-
ton Spintrode microelectrode. Resonance assignments are based on
chemical shift, peak integration and multiplicity for 1H, 13C NMR
spectra and for COSY, NOESY and HMQC experiments.
All commercial reagents were analytical grade and were used
without further purification, unless noted otherwise. The cyclen
and the 1-aza-18-crown-6 compounds were obtained from
Chematech and Aldrich, respectively. The intermediate bis-aminal
2 starting from cyclen was prepared as previously published.19
Triethylamine was previously distilled before use. All amino acids
were analytical grade and were used without further purification.
Fig. 6. Schematic representation of the proposed structure with the hydrogen bond
network (dashed bonds in blue) for the [H3Lgly]2þ entity mainly present at physio-
logical pH.
hydrogen bonds formed between individual molecules led to an
associated species where cyclen and crown ether moieties face
each other. This structure provide a three-dimensional cavity of
suitable shape and size for amino acids recognition.
Glycine (>99.7%) was purchased from Merck,
(>99.0%), was purchased from Fluka, -Glutamic acid (>99%) is
Carlo Erba product, 4-Aminobutyric acid (>97%) and 6-
L-Phenylalanine
L
a
3. Conclusions
Aminohexanoic acid (>98%) were purchased from Aldrich.
The synthesized heteroditopic bismacrocycle L showed in-
teresting features in its interaction with biologically relevant amino
acids. Both potentiometric and NMR results showed that hydrogen
bonds between the protonated cyclen moiety and the carboxylate
group of the substrate together with ion-dipole interactions be-
tween the crown ether and the ammonium group of the amino
acids contribute unambiguously to the recognition process at
4.2. Synthesis (see Scheme 1 for labeling)
4.2.1. Preparation of 3. To a stirred solution of the bis-aminal 2
(676 mg, 3.48 mmol) dissolved in dry THF (5 cm3), a solution of 2-
bromomethylnaphthalene (770 mg, 3.48 mmol) in dry THF (5 cm3)
was slowly added at rt. A white precipitate is immediately formed.

4866
N. Bernier et al. / Tetrahedron 68 (2012) 4860e4868
The mixture was stirred overnight at rt. The precipitate was col-
lected by filtration, washed with THF and diethyl ether, then was
dried in vacuo, giving 3 as a white powder (1.31 g, 91%). Found: C,
60.31; H, 6.40; N, 13.54. Calcd for C21H27BrN4$0.1H2O: C, 60.46; H,
6.57; N, 13.43%. dH (400.13 MHz; D2O; Me3Si(CH2)3SO3Na)
2.40e3.60 (16H, m, CH2N), 3.86 (2H, d, 4J¼2.2 Hz, HBn), 4.78 (1H, d,
3J¼13.2 Hz, Haminal), 4.93 (2H, d, 3J¼13.2 Hz, Haminal), 7.55 (1H, d,
3J¼8.7 Hz), 7.60 (2H, m), 7.95 (2H, d, 3J¼7.5 Hz), 8.01 (1H, d,
3J¼8.5 Hz), 8.06 (1H, s) (the last five correspond to Har). dC
(100.61 MHz; D2O; Me3Si(CH2)3SO3Na) 43.8, 47.4, 47.5, 48.1, 48.2,
51.2 (2C), 56.8 (CH2N), 61.1 (CBn), 71.6, 82.7 (Caminal), 124.1, 127.4,
127.8, 128.2, 128.3, 128.4, 129.3, 132.6, 133.0, 133.5 (Car).
3J¼7.7 Hz). dC (100.61 MHz; CDCl3; Me4Si) 45.3 (2C), 51.6, 51.8, 53.7
(CH2Ncyclenþether), 57.7, 59.7, 60.2 (CBn), 69.9, 70.3, 70.7 (2C), 70.8
(CH2O), 125.6, 126.0, 127.1, 127.6, 127.7, 127.8, 128.1, 128.8 (2C), 132.8,
133.3, 136.5, 137.5, 138.5 (Car). m/z (ESI-MS; CHCl3; positive ion
mode) 678.6 [MþH]þ.
4.2.5. Preparation of the nitrate salt of the bismacrocycle L. The
bismacrocycle L (373 mg, 0.401 mmol) was dissolved in a minimum
amount of absolute ethanol. Then, w4 equiv of w2 M nitric acid
was added (94ꢂ10ꢀ3 cm3) and the compound was precipitated by
adding a large amount of diethyl ether (30 cm3). The product was
filtered and washed several times with diethyl ether, dried in vacuo,
giving L$3.34HNO3 as a yellow powder (94.5 mg, 26%). Found: C,
51.40; H, 7.12; N, 12.82. Calcd for C39H59N5O5$H2O$3.34HNO3: C,
51.68; H, 6.78; N, 12.89%.
4.2.2. Preparation of 4. To a stirred solution of a,a
0-dibromo-p-xy-
lene (3.32 g, 12.6 mmol) dissolved in dry DMF (30 cm3), the com-
pound 3 (1.31 g, 3.16 mmol) in dry acetonitrile (30 cm3) was slowly
added at rt. The mixture was stirred during 4 days at rt. The white
precipitate was filtered off, washed three times with DMF and
diethyl ether, and dried in vacuo giving 4 as a white powder (1.94 g,
90%). Found: C, 51.21; H, 5.12; N, 8.22. Calcd for C29H35N4Br3: C,
51.27; H, 5.19; N, 8.25%. dH (400.13 MHz; CD3OD; Me4Si) 3.24e3.82
(16H, m, CH2N), 4.35e4.55 (4H, m, HBn), 5.11 (2H, d, 3J¼13.2 Hz,
Haminal), 5.16 (2H, d, 3J¼13.2 Hz, Haminal), 5.32 (2H, d, 4J¼3.0 Hz,
CH2Br), 7.47 (2H, d, 3J¼8.0 Hz), 7.55 (2H, m), 7.60 (2H, d, 3J¼8.0 Hz),
7.65 (1H, d, 3J¼8.3 Hz), 7.86 (1H, d, 3J¼7.7 Hz), 7.93 (2H, d,
3J¼8.1 Hz), 8.21 (1H, s), Har. dC (100.61 MHz; CD3OD; Me4Si) 32.58
(CH2Br), 43.9, 44.0, 47.6 (2C), 55.9, 56.1, 61.0, 62.2 (CH2N), 62.3 (2C)
(CBn), 79.2, 79.3 (Caminal), 125.3, 128.0, 128.4, 128.9, 129.2, 129.3,
129.6, 130.8,131.5,134.1,134.5,134.6,135.6,143.1 (Car). m/z (ESI-MS;
CH3OH; positive ion mode) 599.0 [MꢀBr]þ, (negative ion mode)
757.3, [MþBr]ꢀ.
4.3. Potentiometric measurements
4.3.1. Instrumentation. The potentiometric setup for conventional
titrations consisted of a 50 cm3 glass-jacketed titration cell sealed
from the atmosphere and connected to a separate glass-jacketed
reference electrode cell by a Wilhelm-type salt bridge containing
0.10 mol dmꢀ3 KCl solution. The measuring instrument fitted with
a glass electrode and a Ag/AgCl reference electrode. The glass
electrode was pre-treated by soaking it in the H2O/MeOH 1:1 v/v
solution over a period of two days, in order to prevent erratic re-
sponses. The ionic strength of the experimental solutions was kept
at 0.10ꢁ0.01 mol dmꢀ3 NMe4NO3. The temperature was controlled
at 298.2ꢁ0.1 K using a thermostat and atmospheric CO2 was ex-
cluded from the titration cell during experiments by passing puri-
fied nitrogen across the top of the experimental solution. Titrant
solutions were added through capillary tips at the surface of the
experimental solution with a very precise automatic burette. Ti-
tration procedure is automatically controlled by software after se-
lection of suitable parameters, allowing for long unattended
experimental runs.
4.2.3. Preparation of 5. Compound 4 (1.94 g, 2.86 mmol), 1-aza-18-
crown-6 (752 mg, 2.86 mmol) and triethylamine (0.398 cm3,
2.86 mmol) were dissolved in dry DMF (500 cm3). The solution was
stirred overnight at 50 ꢃC. The solvent was removed under vacuo,
then CH3CN (30 cm3) was added to the residue and the mixture
stirred for more 30 min. After cooling the mixture to 0 ꢃC, a white
precipitate was collected by filtration, washed three times with
diethyl ether and dried in vacuo. This operation was repeated twice
giving 5 as a white powder (1.90 g, 77%). Found: C, 56.92; H, 6.35; N,
7.98. Calcd for C41H59N5O5Br2: C, 57.14; H, 6.90; N, 8.13%. dH
(400.13 MHz; D2O; Me3Si(CH2)3SO3Na) 3.12 (4H, m, CH2Nether),
3.30e3.90 (38H, m, CH2NcyclenþCH2OetherþCH2Nether), 4.36 (2H, m,
CH2Ncyclen), 4.80e5.10 (4H, m, HaminalþCH2Ncyclen), 7.58e7.68 (7H,
m), 7.97 (2H, d, 3J¼8.6 Hz), 8.04 (1H, d, 3J¼7.5 Hz), 8.13 (1H, s). dC
(100.61 MHz; D2O; Me3Si(CH2)3SO3Na) 42.7, 42.8, 46.1, 52.5, 54.0,
54.7, 54.8, 55.0 (CH2N), 60.2, 61.1, 63.5 (CBn), 69.4, 69.5 (2C), 69.6,
69.8 (CH2O), 77.7, 77.9 (Caminal), 123.5, 127.5, 127.8 (2C), 128.1, 128.2,
128.4, 129.6 (2C), 132.6, 132.7, 133.1, 133.2, 133.8 (Car). m/z (ESI-MS;
CHCl3; positive ion mode) 861.9 [MBr2þH]þ, 782.2 [MBr]þ, m/z
700.1 [MꢀH]þ.
4.3.2. Preparation of the solutions. All solutions for potentiometric
titrations were prepared in water/methanol 1:1 v/v solvent mix-
ture, except the amino acids stock solutions that were prepared in
aqueous solution. A special care was taken during the preparation
of all solutions in water/methanol 1:1 v/v for which the tempera-
ture was constantly controlled and maintained at 293 K. The
demineralized water was freshly obtained from a Millipore Milli-Q
system, boiled for about 2 h and allowed to cool to rt under
nitrogen.
Stock solutions of the compound
L were prepared at
w2 mmol dmꢀ3 with addition of w4 equiv of standard
0.1 mol dmꢀ3 HNO3 solution. Stock solutions of amino acids were
prepared at 80 mmol dmꢀ3 from the analytical grade compounds,
and standardized by titration with standard HNO3 solutions.
Carbonate-free solutions of the titrant 0.10 mol dmꢀ3 NMe4OH
were obtained by reacting freshly prepared silver oxide with a so-
lution of NMe4I under nitrogen. These solutions were discarded
every time carbonate concentration was about 0.5% of the total
amount of base. For the back titrations, standard 0.100 mol dmꢀ3
HNO3 solutions were used. The titrant solutions were standardized
(using Gran’s method).26 The salt NMe4NO3 was prepared by neu-
tralization of a commercially available aqueous solution of 25%
NMe4OH with concentrated HNO3 under nitrogen. The salt was
evaporated to dryness under vacuum line, then the 0.75 mol dmꢀ3
NMe4NO3 solution was prepared.
4.2.4. Preparation of bismacrocycle L. Hydrazine monohydrate
(10 cm3) was added to compound 5 (1.726 g, 2.00 mmol) and the
solution was refluxed during 2 h. After cooling the mixture to rt, the
insoluble residue was collected by filtration and washed three
times with 1 cm3 of hydrazine monohydrate. Then, the residue was
dissolved in absolute ethanol and the solvent removed in vacuo.
This operation was repeated three times giving L as a colorless oil
(1.10 g, 81%). Found: C, 69.20; H, 8.39; N, 10.45. Calcd for
C39H59N5O5: C, 69.10; H, 8.77; N, 10.33%. dH (400.13 MHz; CDCl3;
Me4Si) 2.61 (8H, m, CH2Ncyclen), 2.65 (8H, m, CH2Ncyclen), 2.80 (4H, t,
3J¼5.8 Hz, CH2Nether), 3.61 (2H, s, CH2Ar), 3.65 (20H, m, CH2Oether),
3.66 (2H, s, CH2Ar), 3.76 (2H, s, CH2Ar), 7.30 (4H, m), 7.44 (2H, m),
7.53 (1H, d, 3J¼8.5 Hz), 7.69 (1H, s), 7.79e7.81 (2H, m), 7.86 (1H, d,
4.3.3. Measurements. The [Hþ] of the solutions was determined
by the measuþrement of the electromotive force of the cell,
E¼Eꢃ0þQ log [H ]þEj. The term pH is defined as ꢀlog [Hþ]. Eꢃ0 and Q

N. Bernier et al. / Tetrahedron 68 (2012) 4860e4868
4867
were determined by titrating a solution of known hydrogen-ion
concentration at the same ionic strength, using the acid pH range
of the titration. The liquid-junction potential Ej was found to be
negligible under the experimental conditions used. The value of
Kw¼[Hþ][OHꢀ] was determined from data obtained in the alkaline
range of the titration, considering Eꢃ0 and Q valid for the entire pH
range and found to be equal to 10ꢀ13.88 in our experimental con-
ditions. Before and after each set of titrations a calibration of the
system was performed by titration of a 1.0 mmol dmꢀ3 standard
HNO3 solution. Measurements during conventional titrations were
carried out with 0.04 mmol of compound L (20.00 cm3 of the stock
solution) in a total volume of ca. 30 cm3 (care was taken to maintain
unaltered the water/methanol 1:1 volumetric ratio), in the absence
of substrate and in the presence of each substrate for which the
CL:CA ratio was 1:1 and 1:2. Each titration curve consisted typically
of 50e60 points in the 2.5e11.0 pH range.
Spectrometry Service. N.B. thanks the financial support from ITQB
(055/BI/2007).
Supplementary data
These data include: 1H and 13C NMR spectra for compounds
3e5; 13C, 1H, COSY, HMQC, HMBC, ROESY NMR spectra of L in CDCl3;
COSY NMR spectra of L at pD¼3 and 10 in D2O/CD3OD (1:1 v/v),
298 K; Overall protonation (bH L) constants of L and amino acids;
Overall association (bH L A ) cohnstants of L with amino acids; 1H
NMR spectra at differenta pD values of the bismacrocycle L.
Supplementary data related to this article can be found online at
doi:10.1016/j.tet.2012.03.110.
h
l
References and notes
1. (a) Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; Wiley: New York, 1995; (b)
Excitatory Amino Acid Receptors: Design of Agonist and Antagonist; Krogsgaard-
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4.3.4. Calculation and equilibrium constants. Overall equilibrium
constants bHi and bH L A (being bH L A ¼[HhLlAa]/[H]h[L]l[A]a) were
a
a
calculated by fitting the potentiomel tric data from performed ti-
trations with the HYPERQUAD program.27 Overall protonation
constants obtained were used as fixed values in order to obtain the
equilibrium constants of the associated species. The errors quoted
are the standard deviations of the overall association constants
given directly by the program for the input data, which include all
the experimental points of all titration curves. Species distribution
diagrams were plotted from the calculated constants with the HYSS
program.28 The species considered in the model were justified by
NMR studies and the principles of supramolecular chemistry.
h
l
h
2. (a) Lehn, J.-M. Supramolecular Chemistry; VCH: New York, NY, 1995; (b) Su-
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4.4.1. 1H NMR titration of L. A solution of L at 9.82 mmol dmꢀ3 in
D2O/CD3OD 1:1 v/v solvent mixture was prepared and the titration
was carried out using freshly prepared CO2-free 0.10 mol dmꢀ3 KOD
or 0.10 mol dmꢀ3 DCl in the same mixture of solvents (D2O/CD3OD
1:1 v/v). Care was taken to keep the volumetric ratio of the solvent
mixture. The measurements were undertaken with a pH meter
coupled with a combined microelectrode, which was previously
calibrated with commercial buffer solutions of standard pH 4.00,
8.00, and 12.00. The electrode was also soaked in the H2O/MeOH
1:1 v/v solution over a period of two days. The titration curve
consisted of 19 points in the 4e12 pH* range. The pH* was then
converted in pD values using the equation pD¼pH*þ0.40ꢁ0.02.29
4.4.2. 1H NMR spectra of L and amino acids. Stock solutions of
compound L (5.15 mmol dmꢀ3) in D2O/CD3OD 1:1 v/v solvent
mixture and of amino acids (100 mmol dmꢀ3, except for glutamic
acid for which 58.7 mmol dmꢀ3 was used due to solubility reasons)
in D2O where prepared and the pD was adjusted to 7.00ꢁ0.05
by addition of 0.1 mmol dmꢀ3 NMe4OD/D2O solution or
0.1 mmol dmꢀ3 DCl/D2O solution freshly prepared. Then, solutions
of amino acids and L in 1:1 and 1:2 molar ratios were prepared.
Care was taken to keep the D2O/MeOD 1:1 volumetric ratio. The
pH* was checked and adjusted again if necessary. 1H NMR spectra
were finally acquired.
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Acknowledgements
The NMR spectrometers used in the work are part of the Na-
tional NMR Network and were purchased in the framework of the
National Program for Scientific Re-equipment, contract REDE/1517/
RMN/2005, with funds from POCI 2010 (FEDER) and FCT. The au-
thors also thank M. C. Almeida for providing elemental analysis
and ESI-MS data from the ITQB Elemental Analysis and Mass
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