A thermodynamic insight into the recognition of hydrophilic and hydrophobic amino acids in pure water by aza-scorpiand type receptors

Article abstract of DOI:10.1039/c4ob02092h

Interactions of different hydrophilic (His, Asp, Glu,) and hydrophobic (Ala, Phe, Tyr, Trp) amino acids in water with a scorpiand aza-macrocycle (L1) containing a pyridine group in the ring and its derivative (L2) bearing a naphthalene group in the tail have been analysed by potentiometric and calorimetric measurements. Theoretical calculations corroborate that major attractive forces that hold the adduct together are hydrogen bonds and salt-bridges, even though other interactions such as π-stacking or NH⁺...π may contribute in the case of hydrophobic amino acids and L2. Calorimetric measurements indicate that the interactions between L1 and the different amino acids are principally driven by entropy, often associated with solvation/desolvation processes.

Full text of DOI:10.1039/c4ob02092h

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A thermodynamic insight into the recognition of
hydrophilic and hydrophobic amino acids in pure
water by aza-scorpiand type receptors†
Cite this: DOI: 10.1039/c4ob02092h
Salvador Blasco,a Begoña Verdejo,*a Carla Bazzicalupi,b Antonio Bianchi,*b
Claudia Giorgi,b Concepción Sorianoa and Enrique García-España*a
Interactions of different hydrophilic (His, Asp, Glu,) and hydrophobic (Ala, Phe, Tyr, Trp) amino acids in
water with a scorpiand aza-macrocycle (L1) containing a pyridine group in the ring and its derivative (L2)
bearing a naphthalene group in the tail have been analysed by potentiometric and calorimetric measure-
ments. Theoretical calculations corroborate that major attractive forces that hold the adduct together are
hydrogen bonds and salt-bridges, even though other interactions such as π-stacking or NH+⋯π may con-
tribute in the case of hydrophobic amino acids and L2. Calorimetric measurements indicate that the inter-
actions between L1 and the different amino acids are principally driven by entropy, often associated with
solvation/desolvation processes.
Received 30th September 2014,
Accepted 30th October 2014
DOI: 10.1039/c4ob02092h
www.rsc.org/obc
amino acids with two scorpiand aza-macrocycles (L1 and L2,
see Chart 1). Amino acid recognition is of great interest in
Introduction
Molecular recognition implies complementarity of electronic different fields like drug delivery,3 protein–nucleic acid recog-
and structural features between the receptor and the target nition4 or amino acid sensing.5 This process is particularly
substrate.1 In the case of biological substrates, the forces challenging, since they have a zwitterionic nature over a wide
involved in the recognition processes are mostly electrostatic pH range and often require host species having functionalities
attractions (charge–charge, dipole–charge, dipole–dipole, and able to simultaneously interact with their positive ammonium
H-bonds). Such electrostatic forces are markedly weakened in and negative carboxylate sites. In this respect, in spite of being
polar solvents, in particular, in water, which, apart from relatively small molecules, L1 and L2 contain different binding
having high polarity, is also an efficient donor and acceptor of sites (amine and/or ammonium groups, hydrophobic moieties)
H-bonds. Hence, molecular recognition in water requires high and have the ability to self-adapt to the guest amino acid due
complementarity between substrates and receptors and the to the flexibility afforded by their hanging arms. Indeed, such
presence of many binding sites on both, as actually occurs in an induced-fit ability is a key point for successful recognition
biological systems. On the other hand, water favours hydro- in many biological systems.
phobic association, which is another important aspect of the
L1 and L2 have been chosen to achieve selectivity with
biological world. Accordingly, the design of synthetic receptors amino acids of different hydrophobicity due to their different
for the recognition of biological substrates in water is very hydrophilic/hydrophobic balance.6 In the case of the more
challenging, but it is also a very attractive objective since water water soluble L1, a complete set of thermodynamic binding
is the solvent of life. To obtain a clear view of the binding data is provided.
event in water, enthalpic and entropic contributions give fun-
damental information. Nonetheless, these types of data related
to pure water are very scarce in the literature.2
Here we report on the interactions of a selection of hydro-
philic (His, Asp, Glu) and hydrophobic (Ala, Phe, Tyr, Trp)
aInstituto de Ciencia Molecular, C/Catedrático José Beltrán 2, 46980 Paterna,
Valencia, Spain. E-mail: enrique.garcia-es@uv.es
bDipartimento di Chimica, Via Della Lastruccia 3, 50019 Sesto Fiorentino, Italy.
E-mail: antonio.bianchi@unifi.it
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob02092h
Chart 1
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Table 1 Thermodynamic data for the protonation of L1 and L2 in 0.15
M NaClO4, at 298.1 K
Results and discussion
Protonation of L1 and L2
ΔG°
ΔH°
TΔS°
(kcal
The equilibrium constants for the protonation of L1 and L2
were reported in a previous paper where it was anticipated that
L2 undergoes important conformational changes upon proto-
nation.6 In the mono- and diprotonated forms, the molecule
assumes a folded conformation stabilized by intramolecular
hydrogen bonds, involving the amino group of the pendant
arm and the ammonium group(s) of the macrocyclic moiety,
and π-stacking interaction between the two aromatic units.
Further protonation of L2, occurring on the amino group of
the pendant arm, causes the opening of such a conformation
with loss of all intramolecular interactions to form a more
expanded structure where the three positive charges of
[H3L2]3+ are brought as far apart as possible to minimize the
electrostatic repulsion between them.7
This was clearly evidenced by NMR spectra recorded for
ligand solutions at different pH values and by the crystal
structures obtained for L2, [H(L2)]ClO4·H2O and [H3(L2)]-
(H2PO4)3·H2O.6 Furthermore, stacking of pyridine and
naphthalene moieties for L2 was also evidenced by changes in
the UV-Vis spectra as a function of pH (see Fig. 1).
(kcal
(kcal
Reaction
Log K
mol−1
)
mol−1
)
mol−1
)
L1 + H+ ⇄ [HL1]+
10.19(6)a −13.90(8) −10.19(7)
3.7(1)
0.5(1)
[HL1]+ + H+ ⇄ [H2L1]2+
[H2L1]2+ + H+ ⇄ [H3L1]3+
9.19(7)
7.94(5)
−12.54(9) −12.05(9)
−10.83(7) −11.46(5) −0.6(1)
L2 + H+ ⇄ [HL2]+
10.01(1)
8.71(1)
7.27(1)
−13.65(1)
−11.88(2) −13.2(6)
−9.92(2) 0.1(7)
−8.6(4)
5.1(4)
−1.3(6)
10.0(5)
[HL2]+ + H+ ⇄ [H2L2]2+
[H2L2]2+ + H+ ⇄ [H3L2]3+
a Numbers in parentheses are standard deviations in the last
significant figure.
appears to be almost insensitive to these structural modifi-
cations (Table 1). As shown in Table 1, successive protonation
of L1 is accompanied by invariably favourable enthalpy
changes, while the entropic contributions become less and
less favourable with increasing ligand protonation and conse-
quent increasing solvation, according to a general trend
observed for polyamines.8 A similar behaviour is found for the
first two protonation stages of L2, while the third one is an
athermic process moved by a largely favourable entropy contri-
bution (Table 1): the breaking of intramolecular bonds, occur-
ring at this stage, compensates the favourable enthalpy
contribution coming from protonation of the amine group but
gives the molecules a greater conformation freedom.
The calorimetric study performed in this work showed that
such conformational changes are clearly manifested by the
enthalpic and entropic contributions to ligand protonation, in
contrast to the free energy change (stability constants) which
Therefore, it appears that to achieve a closed conformation,
both intramolecular hydrogen bonding and π-stacking inter-
action need to occur simultaneously in this kind of receptors.
Taking into account that these types of intramolecular inter-
actions are precluded to L1, it can be assumed that this recep-
tor is not able to adopt a “closed” conformation.
Binding of amino acids by L1 and L2
The equilibrium constants for the interaction of protonated
forms of L1 and L2 with Ala, Phe, Tyr, Trp, His, Asp and Glu
were determined by means of pH-metric titrations in 0.15 M
NaClO4 solutions at 298.1 K. Analyses of the titrations curves
by means of the HYPERQUAD9 program afforded the equili-
brium constants for the general reaction (1):
L þ Anꢀ þ mHþ ⇄ HmLAðmꢀnÞþ
ð1Þ
where An− is the amino acid in its completely deprotonated
form (n = 1 for Ala, Phe, His and Trp; n = 2 for Tyr, Asp and
Glu). It has been shown for many similar assemblies that the
supramolecular interaction does not alter significantly the pro-
tonation patterns and the distribution of the charged groups
in the interacting species; that is, the interacting partners
maintain their identity, and no significant redistribution of
protons occurs.10 Accordingly, the m protons of the HmLA(m−n)+
complexes were assumed to be localized on the ligand or on
the amino acid in accordance with their basicity, and the rele-
vant binding equilibria were derived as reported in Table 2 for
Fig. 1 (A) 1H NMR spectra in D2O of L2 recorded from the top to the
bottom at pD = 4.1, pD = 8.2 and pD = 9.4. (B) pH dependence of the
absorption spectra of L2.
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Table 2 Thermodynamic data for the formation of selected amino acid
complexes with L1 in 0.15 M NaClO4 at 298.1 K
ΔH°
TΔS°
(kcal
(kcal
Reaction
Log K
mol−1
)
mol−1
)
HL+ + Tyr2− ⇄ [HL(Tyr)]−
3.27(1)a
3.61(1)
3.79(1)
3.31(1)
3.39(1)
9.0(4)
5.0(3)
7.2(4)
6.2(3)
7.0(4)
13.5(4)
9.9(3)
12.3(4)
10.7(3)
11.6(4)
HL+ + HTyr− ⇄ [HL(HTyr)]
H2L2+ + HTyr− ⇄ [H2L(HTyr)]+
H2L2+ + H2Tyr ⇄ [H2L(H2Tyr)]2+
H3L3+ + H2Tyr ⇄ [H3L(H2Tyr)]3+
HL+ + Asp2− ⇄ [HL(Asp)]−
3.2(1)
5.1(2)
3.4(2)
4.1(2)
4.6(2)
4.3(2)
9.5(2)
8.2(2)
8.9(2)
8.3(2)
10.2(2)
HL+ + HAsp− ⇄ [HL(HAsp)]
H2L2+ + HAsp− ⇄ [H2L(HAsp)]+
H3L3+ + HAsp− ⇄ [H3L(HAsp)]2+
H3L3+ + H2Asp ⇄ [H3L(H2Asp)]3+
3.48(6)
3.47(6)
3.72(6)
4.26(6)
a Numbers in parentheses are standard deviations in the last
significant figure.
Tyr and Asp with L1 and in Tables S1 and S2† for all the
amino acids with both ligands.
A comparison of the equilibrium information collected in
these tables evidence significant differences in the binding
properties of the two ligands towards amino acids. Very
impressive is the ability of the neutral (not protonated and not
charged) L2 ligand to form stable complexes with anionic
forms of amino acids. These are the most stable complexes
formed by L2, the stability of the other complex species
decreasing with increasing ligand protonation. Conversely, L1
requires at least one positive charge, [HL1]+, to interact with
the amino acids and the stability of its complexes generally
increases with ligand protonation. Accordingly, L2 is a better
receptor for amino acids in neutral-to-alkaline media while L1
extends its binding properties to the acidic region. To better
visualize and quantify such differences, the stability constants
Fig. 2 Log Keff vs. pH for complexes with L1 (A) and L2 (B).
HTyr− and H2Tyr (log K = 3.79 and 3.31, respectively, Table 2)
are considered. Other examples of this type can be found in
Tables S1 and S2 (see ESI†). Evidently, other forces than
charge–charge interactions contribute to determine the stabi-
lity of these amino acid complexes.
However, for L2 a clearly different behaviour is observed
depending on the hydrophilic/hydrophobic character of the
amino acids studied. As can be seen in Fig. 2B, in comparison
with Ala, amino acids like Glu or Asp show an increasing inter-
action from acidic to basic pH values that can be attributed to
the additional carboxylate group present in the side chain.
Nevertheless, interaction with amino acids containing aro-
matic moieties (Phe, Tyr, Trp, His) is observed only for pH
values above 5. This behaviour can be principally ascribed to
the π-stacking interactions between the aromatic rings present
in the amino acid side chain and the naphthalene moiety
of L2.
reported in Tables S1 and S2† can be used to calculate
11
effective stability constants (Keff
,
eqn (2)) for complexation
equilibria involving total amounts of reactants and products in
the form:
Keff ¼ ½HxþyLAꢁ=½ðHxLÞꢁ½ðHyAÞꢁ
ð2Þ
Plots of log Keff as a function of pH for all the complex
systems here studied are shown in Fig. 2. As can be seen, for
L1 all complexes show the same general trend with a slight
increase of log Keff at around pH 3, where deprotonation of the
carboxylic group present in the amino acid takes place.
This interaction remains constant in a wide pH range until
total deprotonation of L1 occurs at around pH 9, a value from
which a marked decline in log Keff is observed. If the overall
charge of the amino acids is considered, we observe that
complex stability is not determined by merely electrostatic
attractions. For instance, the equilibrium constant for the
interaction of [H3L1]3+ with H2Asp (log K = 4.26, Table 2) is
higher than the constant for the interaction of the same ligand
form with the more charged HAsp species (log K = 3.72,
Table 2), in contrast to electrostatic expectations, while the
opposite trend, obeying Coulomb’s law, is observed when
1H NMR spectra recorded with solutions containing L2 and
Glu showed that opening of the ligand structure upon proto-
nation ([H2L2]2+ → [H3L2]3+) occurs also in the presence of
amino acids (Fig. S1†). As already discussed for the free
ligand, such a conformational change has a considerable
enthalpic cost which is overcompensated by the entropic gain
originating from the increased freedom achieved by the ligand
when passing from closed to open conformation. Nevertheless,
if the open ligand conformation is constrained by interaction
with an amino acid, this favourable entropic gain vanishes and
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the corresponding complex loses stability. Most likely, this is
the reason why the amino acid complexes with L2 in open con-
formation ([H3L2]3+), when formed, are the less stable com-
plexes produced by this ligand. In contrast, L1, maintaining an
open conformation in all its forms, does not experience a
similar depression of complex stability in acidic solution and,
instead, the greater positive charge of its highly protonated
forms enhances the complex stability.
In agreement with the 1H NMR information, UV-vis spectro-
scopic data obtained for L2 and its Glu and Ala complexes
confirm that the presence of the amino acid, in [H3L(GluH)]2+
species, forces the ligand to remain longer in its open confor-
mation, while in the presence of Ala the effect is more
reduced.12,13
Theoretical calculations
To rationalize these behaviours, we performed a modeling
study on the complexes formed by L1 and L2. The modeling
was performed using simulated annealing procedures based
on a molecular mechanics method. The empirical force field
was used as implemented in the HyperChem program.14 In all
cases, the solvent effects were implicitly simulated. The
minimum energy structures of the different complexes formed
by L1 with Tyr and by L2 with Glu, which are used here as an
example, are shown in Fig. 3 and 4, respectively, while those
for the other complex systems are shown in Fig. S2–S7.†
Fig. 4 Minimum energy structures calculated for the complexes [L2-
(Glu)]2− (a), [HL2(Glu)]− (b), [L2(HGlu)]− (c), [HL2(HGlu)] (d), [H2L2(HGlu)]+
(e), and [H3L2(HGlu)]2+ (f).
A general observation coming from the analysis of the
minimum energy structures of the complexes formed with all
amino acids is that when the amino acid contains an
ammonium group, the interacting partners approach each
other in such a way as to locate the positively charged
ammonium groups as far apart as possible. Hence, although
at a macroscopic level (stability constants) electrostatic forces
appear not to play a fundamental role, at the molecular level
they can be fundamental in regulating the mutual orientation
of the interacting partners within the complex.
As a specific example, the minimum energy structures for
the complexes with tyrosine are shown in Fig. 3. According to
the calculated structure, the [HL1(Tyr)]− complex, formed by
[HL1]+ with the completely deprotonated, 2− charged form of
tyrosine, is held together by a bifurcated hydrogen bond invol-
ving the two tyrosine carboxylate oxygen atoms and a single
proton of the ligand ammonium group, and by a NH+–π inter-
action between this ammonium group and the aromatic ring
of the amino acid (Fig. 3a). Upon the protonation of the
phenolate tyrosine oxygen to give [HL1(HTyr)], strengthening
of the complex is observed thanks to the formation of two salt-
bridges between the oxygen atoms of the carboxylate groups of
tyrosine and two hydrogen atoms of the ligand ammonium
groups accompanied by π-stacking interaction between the aro-
matic residues of the interacting partners (Fig. 3b). Such struc-
tural features are consistent with the fair increase of complex
stability observed in solution (from log K = 3.27 to log K = 3.61,
Table 2). Protonation of the ligand to form the [H2L1(HTyr)]+
Fig. 3 Minimum energy structures calculated for the complexes [HL1-
(Tyr)]− (a), [HL1(HTyr)] (b), [H2L1(HTyr)]+ (c), [H2L1(H2Tyr)]2+ (d), and
[H3L1(H2Tyr)]3+ (e).
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species gives rise to a modest reorganization of the complex
structure (Fig. 3c), mostly consisting in a modification of
the binding mode of the carboxylate tyrosine group, while the
π-stacking interaction between the aromatic residues of the
two partners is retained. In [H2L1(HTyr)]+, the carboxylate
groups still form a couple of salt-bridges, as in [HL1(HTyr)],
but this time only one oxygen atom is used, the other one
being involved in a hydrogen bond with the unprotonated sec-
ondary amine group of the ligand. On the other hand, only
one ammonium group was used by [HL1]+ to form salt-bridges
with HTyr−. All in all, only a modest stabilization can be
expected for the formation of [H2L1(HTyr)]+, relative to [HL1-
(HTyr)], as actually observed in solution (log K = 3.79, Table 2).
Upon protonation of the amine group of tyrosine to form
[H2L1(H2Tyr)]2+, the resulting ammonium group moves away
from the protonated ligand, likely due to electrostatic repul-
sion, as shown in Fig. 3d. This causes breaking of the π-stack-
ing interaction observed in the previous adduct and loss of
one hydrogen bond, while a NH+⋯π interaction is formed.
Such a weakening of the substrate–receptor interaction corre-
sponds to a lower stability of the complex in solution (log K =
3.31, Table 2). Successive protonation of the ligand, to form
[H3L1(H2Tyr)]3+, causes the ammonium group on the ligand
pendant to move away from the macrocycle, far from the sub-
strate molecule, most likely due to the electrostatic repulsion
with the two ammonium groups on the ring. As a conse-
quence, the NH+⋯π interaction formed by these groups is
broken, while the aromatic groups of the interacting partners
restore the π-stacking interaction and the number of salt-
bridges (2) does not change (Fig. 3e). Despite the considerable
modification of the mutual arrangements of the interacting
partners in the last two complexes, the strength of the inter-
action is not expected to change significantly, in agreement
with the stability shown by these adducts in solution (log K =
3.31(1) and 3.39(1), respectively, Table 2).
Table 3 Stepwise stability constants for the formation of selected
amino acid complexes with L2 in 0.15 M NaClO4 at 298.1 K
Reaction
Ala
Tyr
Glu
L + A2− ⇄ [LA]2−
—
3.11(3)
—
3.25(5)
3.17(5)
—
—
3.30(2)
—
4.68(2)
—
4.98(2)
4.44(2)
—
—
4.38(2)
—
L + A− ⇄ [LA]−
3.11(5)a
—
L + HA− ⇄ [L(HA)]−
HL+ + A2− ⇄ [HLA]−
L + HA ⇄ [L(HA)]
—
3.33(8)
3.05(8)
—
3.01(6)
—
HL+ + A− ⇄ [HLA]
HL+ + HA− ⇄ [HL(HA)]
HL+ + HA ⇄ [HL(HA)]+
H2L2+ + HA− ⇄ [H2L(HA)]+
H2L2+ + HA ⇄ [H2L(HA)]2+
H3L3+ + HA− ⇄ [H3L(HA)]2+
3.29(5)
—
—
3.83(2)
—
2.98(2)
2.5(1)
—
a Numbers in parentheses are standard deviations in the last
significant figure.
complex stability observed in solution (log K = 4.44(2) and
log K = 4.98(2) respectively, Table 3). The next protonation to
form the [HL2(HGlu)] species gives rise to a reorganization of
the complex structure (Fig. 4d), mostly consisting in a modifi-
cation of the binding mode of the carboxylate groups, main-
taining constant the NH+⋯π interaction between the
naphthalene moiety and the ammonium group of Glu. In [HL2-
(HGlu)], one of the carboxylate groups forms a couple of salt-
bridges, as in [L2(HGlu)]−, but this time only one oxygen atom
is used; the other one is involved in a salt-bridge with the pro-
tonated amino group of the macrocyclic core. This fact can
explain certain destabilization observed for the formation of
[HL2(HGlu)] relative to [L2(HGlu)]− (log K = 4.38(2), Table 3).
Upon protonation of the secondary amino group in the
macrocyclic core to form [H2L2(HGlu)]+, the ammonium group
of the amino acid moves away from the protonated ligand,
likely due to electrostatic repulsion, as shown in Fig. 4e. Such
a weakening of the substrate–receptor interaction corresponds
to the lower stability of the complex in solution (log K =
3.83(2), Table 3). The third protonation of L2, to form the
[H3L2(HGlu)]2+ species, promotes a conformational change to
a more expanded structure of the receptor to minimize the
electrostatic repulsions between the three ammonium groups
of [H3L2]3+ (Fig. 4f). In this sense, only the formation of salt-
bridges between the two carboxylate groups of the amino acid
and the ammonium groups of the receptor stabilizes the
[H3L2(HGlu)]2+ species (log K = 2.98(2), Table 3). Through
similar considerations based on modeling studies and taking
into account that salt-bridges contribute to the stability of the
L1 complexes more than simple hydrogen bonds and that salt-
bridges involving shared atoms contribute less than salt-
bridges involving non-shared ones,15 we can draw reasonable
justifications for the stability trends observed for the other
amino acid complexes (see ESI, Fig. S2–S7†).
On the other hand, and in accordance with the calculated
structure, the [L2(Glu)]2− complex, formed by the completely
deprotonated L2 and the 2− charged form of glutamic acid, is
held together by a single hydrogen bond involving one of the
glutamic carboxylate oxygen atoms and a single proton of the
ligand amine group (Fig. 4a). Furthermore, L2 presents a
closed conformation associated with the π–π interaction
between the aromatic moieties present in the receptor.
Depending on the amino group where protonation occurs,
two different closed conformations are possible. If protonation
occurs over one of the amino groups of the macrocyclic core
to give [HL2(Glu)]−,
a rearrangement can be observed
accompanied by the formation of two new salt-bridges
between the oxygen atoms of the carboxylate groups of Glu
and the hydrogen atoms of the receptor ammonium group
(Fig. 4b). However, if protonation occurs over the Glu’s amino
group, a most favourable [L2(HGlu)]− complex is formed due
to the presence of an additional NH+⋯π interaction between
Enthalpic and entropic contributions to complexation
the ammonium group of amino acids and the naphthalene The formation of amino acid complexes with L1 was also
moiety, thereby strengthening the complex (Fig. 4c). Such studied by means of isothermal titration calorimetry in 0.15 M
structural features are consistent with the fair increase of NaClO4, at 298.1 K, to obtain the enthalpy changes and
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derived entropic terms reported in Tables 2 and S1.† Unfortu- L1 compares well with the values (0.89–0.92) previously
nately, an analogous study was not possible with L2, since its obtained for the binding of phosphate and pyrophosphate
complexes are not enough soluble to analyse with the appar- anions by acyclic and macrocyclic polyammonium receptors,17
atus described in the Experimental section. As can be seen in suggesting, by analogy with the latter, a good adaptability of
Tables 2 and S1,† most of the complexation reactions are L1 in all its protonated forms to the amino acidic substrates,
endothermic and promoted by favourable entropy changes, despite the ligand stiffening occurring upon accumulation of
although there are a number of complexation equilibria, positive charge on the ligand.
mostly related to the formation of histidine and alanine com-
plexes, that are weakly exothermic or nearly athermic. The
enthalpy changes associated with similar interactions result
from a subtle combination of favourable and unfavourable
Experimental
contributions deriving from the formation of weak bonds EMF measurements
(favourable) and desolvation effects (unfavourable) the latter
being strictly related to the local neutralization of charge
occurring upon substrate–receptor interaction. Desolvation
The potentiometric titrations were carried out at 298.1 0.1 K
using 0.15 M NaClO4 as the supporting electrolyte. The experi-
mental procedure (burette, potentiometer, cell, stirrer, micro-
effects are normally dominating when oppositely charged
computer, etc.) has been fully described elsewhere.18 The
species are involved, leading to positive (unfavourable)
acquisition of the emf data was performed with the computer
enthalpy changes and positive (favourable) entropic gains.2
program PASAT.19 The reference electrode was a Ag/AgCl elec-
The favourable entropic contribution caused by the release of
trode in saturated KCl solution. The glass electrode was cali-
solvent molecules occurring upon charge neutralization is nor-
brated as a hydrogen-ion concentration probe by titration of
mally large enough, as in the present case, to overcome the
previously standardized amounts of HCl with CO2-free NaOH
loss of entropy due to substrate–receptors association. As
solutions and the equivalent point was determined by Gran’s
shown by the thermodynamic parameters in Tables 2 and S1,†
method,20 which gives the standard potential, E°′, and the
these effects give rise to some compensation between the
ionic product of water (pKw = 13.73(1)).
enthalpy and entropy changes for the relevant complexation
The computer program HYPERQUAD was used to calculate
reactions. A good linear ΔH° − TΔS° correlation (correlation
coefficient (R) of 0.98) is obtained for the complete set of data
reported in Table S1† (Fig. 5).
the protonation and stability constants.8 The pH range investi-
gated was 2.5–11.0 and the concentration of the amino acids
and of the ligands ranged from 1 × 10−3 to 5 × 10−3 mol dm−3
with the A : L molar ratio 1 : 1. The different titration curves for
each system (at least two) 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.
Similar ΔH° − TΔS° compensatory relationships hold in
general for complexation reactions involving weak interactions,
i.e., van der Waals, hydrogen bonding, dipole–dipole, and ion–
dipole interactions, and have been used to obtain quantitative
estimations of ligand conformational changes resulting from
complex formation, according to the general observation that
the slope α of the ΔH° − TΔS° plots (TΔS° = αΔH° + I)
increases with the adaptability of the ligand binding sites
to the specific substrates.16 From this point of view, the high
Calorimetric studies
α value (1.02(4)) obtained for the amino acid complexes with The enthalpies of ligand protonation and anion binding were
determined in the same ionic media as those of potentio-
metric measurements by means of an automated system com-
posed of a Thermometric AB thermal activity monitor (model
2277) equipped with a perfusion–titration device and a Hamil-
ton Pump (model Microlab M) coupled with a 0.250 cm3 gas-
tight Hamilton syringe (model 1750 LT). The microcalorimeter
was checked by determining the enthalpy of reaction of strong
base (KOH) with strong acid (HCl) solutions. The value
obtained (−56.7(2) kJ mol−1) was in agreement with the litera-
ture values.21 In a typical experiment, a NaOH solution
(0.10 M, addition volumes 15 μL) was added to acidic solutions
of the ligand (5 × 10−3 M, 1.2 cm3), containing equimolar
quantities of the amino acid in the binding experiments.
Corrections for heats of dilution were applied. The corres-
ponding enthalpies of reaction were determined from the
calorimetric data by means of the AAAL program.22 ΔH° and
Fig. 5 Enthalpy–entropy compensation plot for the complexation of
TΔS° values for the protonation of anions were redetermined
different amino acids with L1 (see Table S1† for the original data). Corre-
lation: TΔS° = (1.02 0.04)(−ΔH°) + (4.69 0.15), r = 0.98.
under our experimental conditions (Table S3, ESI†).
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Theoretical calculations
Spanish Ministerio de Economía y Competitividad for a Juan
de la Cierva Postdoctoral contract.
Molecular modelling investigations on the adducts formed by
different protonated forms of L1 and L2 with some amino
acids were performed according to the location of acidic
protons on the interacting partners reported in Tables 2, 3 and
S1† (tables of stability constants). The potential energy surface Notes and references
of all systems was explored by means of simulated annealing
(empirical force field method AMBER3,23 T = 600 K, equili-
bration time = 5 ps, run time = 10 ps and cooling time = 15 ps,
time step = 1.0 fs, atomic partial charges evaluated at the
PM324 semi-empirical level of theory). For each system, 80 con-
formations were sampled.
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Å
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Conclusions
Interaction of several hydrophilic (His, Asp, Glu) and hydro-
phobic (Ala, Phe, Tyr, Trp) amino acids in water with two scor-
piand aza-macrocycles (L1 and L2) has been analysed and two
clearly differentiated behaviours have been observed. The
stability of L1 complexes with all amino acids remains almost
constant in a wide pH range until total deprotonation of the
receptor forces it to decrease. Conversely, L2 complexes show
an increasing stability at high pH values that can be attributed
to the action of the naphthalene moiety attached to the
pendant arm of the receptor.
In all cases, theoretical calculations show that the main
interactions take place through the carboxylate groups of the
amino acids. In this sense, the major attractive forces that
hold the adduct together are hydrogen bonds and salt-bridges,
even though other interactions such as π-stacking or NH+–π
may give a significant contribution, particularly for hydro-
phobic amino acids and L2.
Calorimetric measurements indicate that the interaction
between L1 and the different amino acids is principally driven
by entropy changes, often associated with solvation/desolva-
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Acknowledgements
Financial support by the Spanish Ministerio de Economía y
Competitividad (projects CONSOLIDER INGENIO CSD-2010- 15 G. Gilli and P. Gilli, The Nature of the Hydrogen Bond:
00065) and Generalitat Valenciana (project PROMETEO 2011/
008) is gratefully acknowledged. B.V. wishes to thank the
Outline of a Comprehensive Hydrogen Bond Theory, Oxford
University Press, New York, 2009.
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