Water encapsulation in a polyoxapolyaza macrobicyclic compound

Article abstract of DOI:10.1021/jo300799s

A new heteroditopic macrobicyclic compound (t₂pN ₅O₃) containing two separate polyoxa and polyaza compartments was synthesized in good yield through a [1 + 1] tripod-tripod coupling strategy. The X-ray crystal structure of H₃t ₂pN₅O₃³⁺ revealed the presence of one encapsulated water molecule accepting two hydrogen bonds from two protonated secondary amines and donating a hydrogen bond to one amino group. The acid-base behavior of the compound was studied by potentiometry at 298.2 K in aqueous solution and at ionic strength 0.10 M in KCl. The results revealed unusual protonation behavior, namely a surprisingly low fourth protonation constant contrary to what was expected for the compound. ¹H NMR and DOSY experiments, as well as molecular modeling studies, showed that the water encapsulation and the conformation observed in the solid state are retained in solution. The strong binding of the encapsulated water molecule, reinforced by the cooperative occurrence of a trifurcated hydrogen bond at the polyether compartment of the macrobicycle, account for the very low log K₄ ^H value obtained.

Full text of DOI:10.1021/jo300799s

Article
pubs.acs.org/joc
Water Encapsulation in a Polyoxapolyaza Macrobicyclic Compound
Pedro Mateus,† Rita Delgado,*,† Patrick Groves,† Sara R. R. Campos,† Antonio M. Baptista,†
́
Paula Brandao,‡ and Vítor Felix§
†Instituto de Tecnologia Química e Biolog
́
̃
́
ica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras, Portugal
‡Departamento de Química, CICECO, and Secca
̧ o Autonoma de Ciencias da Saude, Universidade de Aveiro, 3810-193 Aveiro,
́ ̂
́
̃
́
§
Portugal
S
* Supporting Information
ABSTRACT: A new heteroditopic macrobicyclic compound
(t2pN5O3) containing two separate polyoxa and polyaza
compartments was synthesized in good yield through a [1 +
1] “tripod−tripod coupling” strategy. The X-ray crystal
3+
structure of H3t2pN5O3 revealed the presence of one
encapsulated water molecule accepting two hydrogen bonds
from two protonated secondary amines and donating a
hydrogen bond to one amino group. The acid−base behavior
of the compound was studied by potentiometry at 298.2 K in
aqueous solution and at ionic strength 0.10 M in KCl. The
results revealed unusual protonation behavior, namely a
surprisingly low fourth protonation constant contrary to
1
what was expected for the compound. H NMR and DOSY
experiments, as well as molecular modeling studies, showed that the water encapsulation and the conformation observed in the
solid state are retained in solution. The strong binding of the encapsulated water molecule, reinforced by the cooperative
H
occurrence of a trifurcated hydrogen bond at the polyether compartment of the macrobicycle, account for the very low log K4
value obtained.
Chart 1. Compounds Used Previously in Water Binding
Studies
INTRODUCTION
■
In the biological sciences it is widely recognized that the
water−protein interactions are not only fundamental in folding,
conformational stability and internal dynamics of proteins, but
are also important as modulators of recognition, assembling
and catalysis.1 In fact, the role of buried water molecules in
protein stability and function is difficult to estimate
experimentally,2 consequently it is useful to resort to model
systems to investigate the modulation of physicochemical
properties of host molecules by included water.
However the role played by the water in molecular
recognition by synthetic receptors is often overlooked and it
is rarely seen as an active player. In the very few examples
where water binding was investigated, it was observed that
encapsulation of water by nitrogen based receptors leads to
abnormal protonation behavior.3 Lehn et al. reported that in a
varied ring sizes (18 to 24-membered rings). In the case of the
18-membered macrocycle (L2, Chart 1) the protonation
constant of the pyridine nitrogen (log K1 = 4.95) is 1.59 log
H
units to higher than that of 2,6-bis(methoxymethyl)pyridine, its
noncyclic analogue, which was attributed to a stabilization of
the protonated species by the presence of a water molecule
accepting a hydrogen bond from the pyridinium group and
donating two hydrogen bonds to ether oxygen atoms, as
evidenced by X-ray crystal structures.3b Bharadwaj et al.
reported that a small polyoxapolyaza macrobicyclic compound
(L3, Chart 1) behaves in the same way as Lehn’s macrotricyclic
one: the first two protonation constants are of the same
tetraoxatetraaza spheroidal macrotricyclic compound (L1, Chart
H
1) the second protonation is as easy as the first one (log K1
≈
log K2H = 10.5) whereas the third one is only possible at much
lower pH values (log K3 = 5.3). This result led to the
H
formulation of the diprotonated species as a water cryptate, the
water molecule being held in an ideal tetrahedral array of
hydrogen bonds, accepting two hydrogen bonds from the
ammonium sites and donating two hydrogen bonds to the
amine sites.3a Reinhoudt et al. reported a study on the acidity
and water binding properties of 2,6-pyrido crown ethers of
Received: April 19, 2012
© XXXX American Chemical Society
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Scheme 1. Synthetic Procedure of t2pN5O3
H
H
magnitude (log K1 ≈ log K2 = 10.45) and the third one is
much lower (log K3H = 5.56). This result was also attributed to
the presence of an encapsulated water molecule. A crystal
structure also showed the water molecule held in a tetrahedral
array of hydrogen bonds, accepting two hydrogen bonds from
the ammonium sites and donating two hydrogen bonds to the
amine sites.3d
phenyl alcohols used by those authors. Alternatively, 2-(4-
bromomethyl-phenyl)-5,5-dimethyl-[1,3]dioxane (b) was suc-
cessfully reacted with triethanolamine (a) to prepare the
tripodal intermediate (Scheme 1). After deprotection of the
aldehyde groups, the trialdehyde tripodal intermediate (c) was
obtained for subsequent reaction with tren in a Schiff-base
condensation cyclization step, which afforded t2pN5O3 in 60%
yield after imine reduction with sodium borohydride.
Clearly, macrocyclic and macropolycyclic compounds can be
useful as model systems to investigate the interaction of water
molecules with host compounds and how it affects their
properties. Macrobicyclic compounds in particular possess well-
defined and preorganized three-dimensional cavities, able to
encapsulate a wide range of cationic,4 anionic5 and neutral
substrates.6 Many reported crystal structures of macrobicyclic
compounds show individual water molecules and even water
clusters occupying molecular cavities,3d,7 as well as mediating
hydrogen bonding interactions between receptors and sub-
strates,8 however little is known on how water affects the
conformation and chemical properties of this class of
compounds in solution.
Crystallographic Studies. Single-crystal X-ray diffraction
analysis reveals that t2pN5O3 crystallizes from an asymmetric
unit composed of one [(H3t2pN5O3)(H2O)]3+ cation, seven
water molecules, one of them disordered over two positions
(see below), and three chloride counterions, which is consistent
with the molecular formula [(H3t2pN5O3)(H2O)]Cl3·7H2O.
Furthermore, the positions of N−H hydrogen atoms were
unequivocally located from the last calculated difference Fourier
maps showing that two secondary amines of the tren moiety
and the tertiary amine of the tripodal polyether subunit of
3+
H3t2pN5O3 are protonated. One water molecule is encapsu-
lated into the cryptand cavity establishing, as shown in Figure 1,
two N−H···O hydrogen bonds with both protonated secondary
amines at N···O distances of 2.751(2) and 2.773(2) Å and
corresponding angles of 174(3) and 171(2)°, respectively. This
water molecule is also tightly O−H···N hydrogen bonded to
the third secondary amine from the tren moiety at an N···O
distance of 2.597(2) Å and an angle O−H···N of 173(2)°. The
proton of the tripodal nitrogen is sited from the three oxygen
atoms of the polyether subunit at H···O short distances of 2.34,
2.34, and 2.38 Å, which are consistent with the existence of a
trifurcated hydrogen bond with the proton encapsulated in a
tetrahedral environment, as reported for TEA.11 Three N−H
binding sites from the tren moiety, pointing to the outside of
the cryptand cavity, establish independent hydrogen bonds with
two chloride counterions [N···Cl = 3.161(2) and 3.380(2) Å
and N−H···Cl angles of 171(2) and 173(2)°, respectively] and
with one crystallization water [N···O of 2.871(3) Å and O−
H···N of 153(2)°]. In fact, the crystallization water molecules
and chloride counterions are involved in the formation of two-
dimensional network of O−H···Cl and O−H···O bonding
interactions with hydrogen bond parameters listed in Table S1
(Supporting Information).
Herein is described a new heteroditopic polyoxapolyaza
macrobicyclic compound whose conformation and protonation
properties are shown to be dramatically influenced by an
encapsulated water molecule.
RESULTS AND DISCUSSION
■
Synthesis of the cryptand. The synthesis of heteroditopic
macrobicyclic compounds requires two critical steps: formation
of a tripodal intermediate and macrobicyclization through a [1
+ 1] “tripod−tripod coupling” strategy.9 This synthetic strategy
requires a bifunctional building block in which one of the
functionalities is used to react in the tripodal formation reaction
and the other in the macrobicyclization step.
Bharadwaj et al. have used 2-hydroxybenzaldehyde or 3-
hydroxybenzaldehyde as the bifunctional building blocks in the
preparation of polyoxapolyaza heteroditopic macrobicyclic
compounds, which after reaction with tris(2-chloroethyl)amine
in alcoholic solvents yielded the tripodal intermediate.10
We were interested in enlarging the cavity of such
compounds by using a p-xylyl spacer. However an attempt to
use 4-(hydroxymethyl)benzaldehyde as the bifunctional reagent
and reaction with tris(2-chloroethyl)amine using the same
conditions described by Bharadwaj et al., failed due to the lower
nucleophilicity of the benzyl alcohol when compared with the
Potentiometric Studies. The protonation constants of
t2pN5O3 were determined by potentiometry in aqueous
solution at 298.2 K and ionic strength 0.10 M in KCl. Two
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Figure 1. Perspective views of [H3t2pN5O33+⊂H2O] showing different
structural features of the inclusion compound: (a) view showing the
encapsulated water molecule and the proton at the tripodal nitrogen
involved in three hydrogen-bond interactions with H3t2pN5O33+; (b)
space-filling model showing the water molecule encapsulated into the
cryptand cage. Carbon, nitrogen, hydrogen, oxygen, and chlorine
atoms are shown in gray, blue, white, red, and green, respectively. The
hydrogen-bonding interactions to the tripodal proton are drawn as
light blue dashed lines and the remaining ones as yellow dashed lines.
Figure 2. Species distribution diagram of the protonation of t2pN5O3
(b). Ct2pN5O3 = 1.0 × 10−3 M. Charges were omitted for clarity.
secondary amines and a tertiary one.12 The tertiary amine of
the tren subunit can only be protonated at very low pH values
due to electrostatic repulsions.5c Interesting to note is the fact
that the protonation constants of t2pN5O3 do not correspond
to a combination of those of tbN4 and TEA (see Table 1). The
first two protonation constants of t2pN5O3 can be unambig-
uously attributed to the protonation of two secondary amines
(see the confirmation of the sequence of protonation in the
NMR studies, below), and the values are in excellent agreement
with the first two protonation constants of tbN4. The third and
fourth protonation constants, however, are quite different from
the expected values, taking into account the protonation
constant of TEA and the third protonation constant of tbN4.
The third protonation constant of t2pN5O3 could, in
principle, correspond to the protonation of either the tertiary
amine of the polyether subunit or the third secondary amine of
the tren subunit, as the protonation constant of the third
secondary amine of tbN4 is 7.03 (in log units) and that of the
tertiary amine of TEA 7.80 log units (see Table 1). The value of
the fourth protonation constant is 4.41 (in log units), which is
surprisingly lower than expected. This low value cannot be only
explained by electrostatic repulsions within the cryptand cavity,
since many polyaza cryptands of comparable size are known to
be hexaprotonated at pH values of about 6,5c as is the case of
t2pN8, Chart 1. The protonation constants of t2pN8, also
determined at 298.2 K and ionic strength 0.10 M in KCl (see
Table 1), have similar magnitude in pairs, as they correspond to
protonation of amine centers at alternating positions in the
macrobicyclic backbone, far from each other, and therefore the
difference in values for each pair are mainly due to statistical
tripodal compounds, representing the polyamine and polyether
compartments of t2pN5O3, tbN4, and TEA, respectively (Chart
2), were also studied under the same experimental conditions
Chart 2. Compounds Studied in This Work
for comparison purposes. The results are collected in Table 1,
the corresponding species distribution diagram are represented
in Figure 2, and the titration curves are presented in the
Supporting Information (Figures S1−S4).
Four protonated species of t2pN5O3 were found in the
working pH range, corresponding to the protonation of three
a
Table 1. Stepwise Protonation (KiH) Constants of t2pN5O3, tbN4, TEA, and t2pN8 in Aqueous Solution
b,c
log KiH
equilibrium reaction
t2pN5O3
tbN4
TEA
t2pN8
L + H+ ⇌ HL+
9.25(1); [9.21(2)]
8.50(1); [8.62(2)]
8.18(1); [8.07(2)]
4.41(1); [4.74(2)]
9.22(1)
8.43(1)
7.03(1)
1.56(2)
7.80(1)
9.18(1)
9.03(1)
7.76(1)
7.35(1)
6.37(1)
5.73(1)
HL+ + H+ ⇌ H2L2+
H2L2+ + H+ ⇌ H3L3+
H3L3+ + H+ ⇌ H4L4+
H4L4+ + H+ ⇌ H5L5+
H5L5+ + H+ ⇌ H6L6+
a
b
c
T = (298.2 0.1) K; I = (0.10 0.01) M in KCl. Values in parentheses are standard deviations in the last significant figures. Values in brackets
are log KiH values obtained after conversion of the log KiD determined in D2O from H NMR titration; see NMR titration below.
1
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Figure 3. Titration of H4t2pN5O34+ in D2O with KOD followed by 1H NMR at 298.2 K. (a) Experimental chemical shifts as a function of pD: The
solid lines are drawn by joining calculated values, obtained using the refined equilibrium constants and the individual chemical shifts. (b) First
derivative of the calculated chemical shifts. Both these diagrams were drawn over the corresponding speciation diagram.
factors. Therefore, no abnormal protonation behavior was
found in t2pN8.
NMR and Theoretical Studies. In order to confirm the
protonation sequence in solution, the titration of t2pN5O3 was
The crystal structure of [(H3t2pN5O3)(H2O)]3+ (Figure 1)
suggests that the fourth protonation occurs at a secondary
amine and that its unusual low log K4H value is the result of the
enclosed water molecule, which is so tightly bound that the
protonation of the amine involved in the O−H···N hydrogen
bond is highly disfavored. In addition, the trifurcated hydrogen
bond taking place at the polyether compartment should
contribute to stabilize a conformation of the macrobicycle,
which reinforces the tight binding of the encapsulated water
molecule. Indeed, it is very likely that the protonation of the
tertiary amine of the polyether compartment and consequent
trifurcated hydrogen bond formation preorganizes the macro-
bicycle in such way that binding of water is easier than a
binding of another proton. Comparison of the protonation
constants of t2pN5O3 with those of L1 and L3 (Chart 1)
confirms this point of view. In L1 and L3, the second
protonation constant is unusually high and of the same value
of the first, whereas the third is abnormally low, which means
that the diprotonated species is highly stabilized by the
presence of a water molecule.3a,c In both cases, this is due to
the small size of the cavities and the ideal tetrahedral array of
hydrogen bonds, in which the water molecule accepts two
hydrogen bonds from the ammonium sites and donates two
hydrogen bonds to the amine sites. The case of t2pN5O3 is
different: the first two protonation constants have normal
values for tren-derived secondary amines (for instance, they are
identical to those of model compound tbN4, see Table 1),
which means that in this case the diprotonated species is not
particularly stabilized and no water molecule is bound at this
stage. It is only when the tertiary amine of the polyether
compartment is protonated that a water molecule is bound and
has the effect of lowering the log K4H of t2pN5O3. It should also
followed by H NMR in D2O, at 298.2 K (see Figure 3a and
1
Figure S5 in the Supporting Information).
As pD decreases from 12.0, resonances 1, 2, 3, and 4,
corresponding to protons on the polyamine compartment, shift
downfield, as expected for resonances nearby secondary amines
being protonated. In the 9.5−8.0 pD region, resonances 5, 6, 7,
and 8, corresponding to protons in the polyether subunit, shift
downfield due to the protonation of the tertiary amine.
Consequently these results confirm that the third protonation
occurs at the tertiary amine, as anticipated by the determined
crystal structure. As the pD drops from 6.0 to 2.5, the
protonation of the third secondary amine takes place, as
evidenced by the downfield shift of resonances 1, 3, and 4. After
conversion of the log KiD determined from this titration to log
KiH,13 it was found that both sets of values are in very good
agreement (see Table 1).
The 1H NMR titration also provided very important
structural information. For instance, it was found that
resonance of protons 2 shift slightly upfield in the 6.0 to 2.5
pD region, instead of the expected downfield shift, which in
addition should be of the same magnitude as resonance of
protons 3 (Figure 3b). This suggests that a conformational
rearrangement takes place in which protons 2 are positioned
within the shielding region created by the p-xylyl ring, as further
evidenced by the appearance of a cross peak correlating
resonances of protons 2 and 4 in a NOESY spectrum recorded
at pD 2.4, absent in the NOESY spectrum recorded at pD 6.8
(Figures S6 and S7 in the Supporting Information). Resonances
of protons 6−8 are also shifted upfield in the 6.0−2.5 pD
region (Figure 3b). As no protonation takes place near protons
6−8, the upfield shift of the corresponding resonances must
also be due to the conformational rearrangement that should
cause the benzene rings to change orientation in such a way
that protons 6−8 become shielded. This is also in agreement
with the fact that resonance 6, the closest proton to the
benzene ring, is the most shifted.
H
be noted that the log K3 of t2pN5O3 (which corresponds to
the protonation of the tertiary amine of the polyether
compartment) is higher than that of model compound TEA,
which suggests that water binding and the formation of the
trifurcated hydrogen bond occurs in a cooperative manner.
Assuming that in the absence of an encapsulated water
molecule the log K4H of t2pN5O3 would have the same value as
the log K3H of tbN4 (see Table 1), it is possible to estimate that
the association constant of the included H2O molecule is
around 2.60 log units, which is a high value for the binding of a
neutral molecule in highly polar medium.
The change in the value of the vicinal H−1H coupling
1
constants of the protons of the ethylenic groups in the
compound at the 2.5−6.0 pD region (Figure 4) gave further
evidence of the conformational rearrangement. As shown in
Figure 4, at pD 2.4, the resonances of protons 1 and 2 look
3
3
essentially like perfect triplets, because JAB ≈ JAB′ = 6.8 Hz.
This happens when the populations of the anti and gauche
D
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much smaller than expected from the increased molecular mass
of the TsO− complex compared to a Cl− complex (791 Da
versus 637 Da). Therefore, we conclude that t2pN5O3 forms a
compact structure at pH 6.8 that is consistent with an organized
cavity and tightly bound water molecule. This is supported by
molecular dynamics simulations of the tri- and tetraprotonated
species of t2pN5O3, whose distributions of radius of gyration
(Figure 5) correspond to an average increase of 5% with pH
Figure 4. AA′BB′ splitting patterns of the ethylenic protons of the
compound recorded at pD 6.8 and 2.4 at 298.2 K in a 400 MHz
spectrometer.
conformations are close to statistical values (33% anti; 67%
gauche) as a consequence of unconstrained rotation of the
3
3
XCH2−CH2Y bond. At pD 6.8, JAB and JAB′ are no longer
equal as seen by the different AA′BB′ splitting pattern (3JAB
=
3
6.7 Hz and JAB′ = 3.2 Hz), which is in agreement with an
increase of the population of the gauche conformations and a
more restricted rotation of the XCH2−CH2Y bond, consistent
with the encapsulation of a water molecule, as seen in the
crystal structure. Spectra recorded in the 278.2−328.2 K
temperature range and at pD = 6.8 (see Figure S8 in the
Supporting Information) showed that, although the rotation of
the XCH2−CH2Y bond have increased with the temperature,
the free rotation is not achieved even at the highest temperature
recorded.
The JAB and JAB′ of the protons of the ethylenic groups of
the polyether compartment are different (3JAB = 6.1 Hz and
3JAB′ = 3.6 Hz), which indicates a preference for the gauche
conformers. Interestingly, the AA′BB′ splitting pattern of
resonances of protons 7 and 8 is the same at both pD values
which means that the conformation rearrangement of the
compound does not affect the population distribution of the
rotamers of the polyether subunit. This is in agreement with the
trifurcated hydrogen bonding taking place at the polyether
compartment which should increase the preference for the
gauche rotamers at both pD values.
In order to provide further evidence of the conformational
rearrangement, diffusion ordered spectroscopy (DOSY) NMR
experiments were performed with samples of t2pN5O3 at pD =
2.4 and 6.8. DOSY experiments allow the measurement of the
diffusion coefficient of a given molecular species that relates
directly to its hydrodynamic radius according to the Stokes−
Einstein equation.14 It has been shown that this technique
provides a way of determining small conformational or shape
changes in molecular and supramolecular systems.15
Figure 5. Distributions of the radius of gyration for H3t2pN5O33+ (red)
and H4t2pN5O3 (blue), obtained from molecular dynamics
simulations.
4+
3
3
decrease, in agreement with the 6% increase of hydrodynamic
radius indicated by the diffusion coefficients reported above;
the radius of gyration is the mass-weighted root-mean-square
atomic distance from the center of mass and is usually
proportional to the hydrodynamic radius,17 so that their relative
changes should be similar.
These data suggest that at low pH the XCH2−CH2Y bonds
of the polyamine compartment rotate unconstrained while at
higher pH such rotation is hindered because of the presence of
an encapsulated water molecule, as depicted in Figure 6.
CONCLUSION
■
A new heteroditopic polyoxapolyaza macrobicyclic compound
was synthesized in good yield through a [1 + 1] “tripod−tripod
coupling” strategy.
3+
The X-ray crystal structure of H3t2pN5O3 revealed an
encapsulated water molecule strongly bound by accepting two
hydrogen bonds from two protonated secondary amines and
donating a hydrogen bond to an amino group. On the other
hand, the study of the acid−base properties of t2pN5O3 showed
an unusual protonation behavior, namely a value of 4.41 log
units for the forth protonation constant, which is surprisingly
lower than the expected one, and that cannot be explained by
electrostatic repulsions within the cryptand cavity alone.
Structural information in aqueous solution provided by NMR
experiments showed that rotation of the XCH2−CH2Y bonds
A recent study using the same NMR instrument16 illustrated
that log Dt measurements could be measured with high
precision for concentrated samples (greater than 1 mM). A
change from log Dt = −9.394 at pD 2.4 to log Dt = −9.368 at
pD 6.8 (+0.026 0.003 log units) was determined for samples
with chloride as the counterion.
The DOSY data clearly indicate that the diffusion properties
of t2pN5O3 are pH-dependent. The faster diffusion at higher
pH indicates a smaller hydrodynamic radius, which is best
explained by a more compact shape. We measured a sample of
t2pN5O3 with the much bulkier TsO− counterion at pD 2.4 to
discriminate between shape changes related to the tightly
bound water molecule observed by other methods, and the
possibility of counterion association at low pD. The diffusion
coefficient for the TsO− sample at pD 1.96 is log Dt = −9.402.
This is slightly slower than the Cl− sample, but the difference of
3+
of the polyamine compartment of H3t2pN5O3 is hindered,
suggesting also the presence of one encapsulated water
molecule. DOSY experiments indicated a decrease in hydro-
dynamic radius at higher pD, which was also supported by
molecular dynamics simulations, which is consistent with a
more compact molecular shape.
All these results suggest that the fourth protonation occurs at
−0.008
0.003 log units is equivalent to about 30 Da and
a secondary amine and that its unusual low log K4H value is the
E
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a saturated solution of NaHCO3, dried over anhydrous sodium sulfate,
filtered, and evaporated to dryness, yielding 4-(bromomethyl)-
benzaldehyde (2.190 g, 95%) which was used without purification in
1
the next step: H NMR (400 MHz; CDCl3) δ 4.45 (s, 2H, BrCH2p-
xylyl), 7.49 (d, 2H, J = 8.0 Hz, H2/H6 of p-xylyl), 7.80 (2H, d, J = 8.0
Hz, H3/H5 of p-xylyl) and 9.95 (1H, s, HCO) ppm; 13C NMR
(100 MHz; CDCl3) δ 32.1 (BrCH2p-xylyl), 129.8 (C2/C6 of p-xylyl),
130.3 (C3/C5 of p-xylyl), 136.3 (C4 of p-xylyl), 144.4 (C1 of p-xylyl),
191.6 (CO) ppm.
2-(4-Bromomethylphenyl)-5,5-dimethyl[1,3]dioxane (b, Scheme
1). A mixture containing 4-(bromomethyl)benzaldehyde (2.190 g, 11
mmol), 2,2-dimethyl-1,3-propanediol (2.293 g, 22 mmol), and
toluenesulfonic acid (7 mg) in toluene (13 mL) was heated to reflux
for 4 h, and the azeotrope of water was received in a Dean−Stark trap.
The solution was allowed to cool to rt, and the solvent was evaporated
to dryness. The compound was purified by dry column vacuum
chromatography using n-hexane/ethyl acetate (9:1) as eluent to yield
1
the desired compound (2.767 g, 88%): H NMR (400 MHz; CDCl3)
δ 0.73 (s, 3H, CH3), 1.21 (s, 3H, CH3), 3.58 (d, 2H, J = 12.0 Hz,
CH2), 3.70 (d, 2H, J = 12.0 Hz, CH2), 4.41 (s, 2H, BrCH2p-xylyl),
5.31 (s, 1H, CH), 7.32 (d, 2H, J = 8.0 Hz, H2/H6 of p-xylyl), 7.41 (d,
2H, J = 8.0 Hz, H3/H5 of p-xylyl) ppm; 13C NMR (100 MHz; CDCl3)
δ 22.0 (CH3), 23.2 (CH3), 30.4 (C(CH3)2), 33.3 (BrCH2p-xylyl), 77.8
(OCH2), 101.4 (CH), 126.8 (C3/C5 of p-xylyl), 129.2 (C2/C6 of p-
xylyl), 138.5 (C1 of p-xylyl), 138.9 (C4 of p-xylyl).
Figure 6. Proposed conformation rearrangements of t2pN5O3 shown
in schematic representation (top) and with two representative
conformers from the molecular dynamics simulations (bottom).
Tripodal Trialdehyde (c, Scheme 1). To a solution of triethanol-
amine (388 mg, 2.6 mmol) in dry THF (8 mL) was added NaH (60%
dispersion in oil) (312 mg, 7.8 mmol) under nitrogen. When bubbling
ceased, 2-(4-bromomethylphenyl)-5,5-dimethyl[1,3]dioxane (2.280 g,
8.0 mmol) dissolved in dry THF/DMF 3:1 mixture (32 mL) was
added during 30 min. The solution was allowed to stir for 24 h and
then poured into water (100 mL) and extracted with CH2Cl2 (3 × 100
mL). The organic portions were washed with water (3 × 100 mL) and
brine (100 mL), collected in an Erlenmeyer flask, dried over
anhydrous sodium sulfate, filtered, and evaporated to dryness. The
solid was dissolved in CH2Cl2 (100 mL) to which water (100 mL) and
CF3COOH 99% (100 mL) were added. The mixture was allowed to
stir for three days at 40 °C. The mixture was transferred to a separating
funnel, and the aqueous phase was rejected. The organic portion was
then washed with NaHCO3 saturated solution (3 × 100 mL), dried
over anhydrous sodium sulfate, filtered, and evaporated to dryness to
give the tripodal trialdehyde (c) (1.178 g, 90%) which was used
result of the enclosed water molecule, which is so tightly bound
that the protonation of the amine involved in the O−H···N
hydrogen bond is highly disfavored. In addition, the trifurcated
hydrogen bond taking place at the polyether compartment
should contribute to stabilize a conformation of the macrobi-
cycle, which reinforces the tight binding of the encapsulated
water molecule. Indeed, the trifurcated hydrogen bond
formation preorganizes the macrobicycle in such way that the
binding of water is easier than a binding of another proton. The
estimated association constant of the included H2O molecule is
around 2.60 log units, which is a high value for the binding of a
neutral molecule in highly polar medium.
In conclusion, a large set of experiments performed in
aqueous solution, using methods so different as potentiometric
measurements, NMR titration, NOESY and DOSY, and
molecular dynamics, point for the presence of a water molecule
1
without purification in the next step. H NMR (400 MHz; CDCl3): δ
2.83 (t, 6H, J = 5.9 Hz, NCH2CH2O), 3.54 (t, 6H, J = 5.9 Hz,
NCH2CH2O), 4.51 (s, 6H, CH2p-xylyl), 7.40 (d, 6H, J = 7.9 Hz, H2/
H6 of p-xylyl), 7.76 (d, 6H, J = 7.9 Hz, H3/H5 of p-xylyl) and 9.92 (s,
3H, HCO) ppm; 13C NMR (100 MHz; CDCl3) δ 54.9
(NCH2CH2O), 69.6 (NCH2CH2O), 77.6 (OCH2p-xylyl), 127.7
(C2/C6 of p-xylyl), 130.0 (C3/C5 of p-xylyl), 135.9 (C4 of p-xylyl),
145.7 (C1 of p-xylyl), 192.0 (CO) ppm.
3+
encapsulated into the cavity of H3t2pN5O3 forming the
[H3t2pN5O33+⊂H2O] entity in agreement with the single-
crystal X-ray structure determination.
EXPERIMENTAL SECTION
t2pN5O3. A solution of tren (220 mg, 1.5 mmol) in MeCN (60 mL)
was added dropwise over 20 min to a magnetically stirred solution of
the tripodal trialdehyde (c) (758 mg, 1.5 mmol) in MeCN (200 mL).
The mixture was allowed to stir overnight. The solvent was evaporated
to dryness, the product was redissolved in MeOH (80 mL), and solid
NaBH4 (1.7 g, 45 mmol) was added in small portions. After the
addition was completed, the mixture was allowed to stir at rt for 2 h
and under reflux overnight. The solution was allowed to cool to rt,
filtered, and evaporated under vacuum almost to dryness, water (20
mL) was added, and the entire methanol was evaporated. The solution
was made strongly basic with 6 M KOH and extracted with CHCl3 (3
× 50 mL). The organic portions were collected in an Erlenmeyer flask,
dried over anhydrous sodium sulfate, filtered, and evaporated to
dryness. The crude product was recrystallized from n-hexane to give
■
General Considerations. All solvents and chemicals were
commercially purchased reagent grade quality and used as supplied
without further purification, except for triethanolamine hydrochloride,
which was recrystallized from ethanol. The tbN4, t2pN8 (4-
diethoxymethylphenyl)methanol, and 1,3,5-tris(bromomethyl)-2,4,6-
triethylbenzene compounds were prepared according to literature
methods.18−21 NMR spectra used for characterization of products
were recorded on a 400 MHz instrument. NMR spectra used in the 1H
NMR titration were recorded on a 300 MHz instrument. The DOSY
experiments were carried out in a 500 MHz instrument. TMS was used
as reference for the 1H NMR measurements in CDCl3 and in D2O the
3-(trimethylsilyl)propanoic acid-d4-sodium salt. Peak assignments are
1
based on peak integration and multiplicity for 1D H spectra and on
1
pure t2pN5O3 (535 mg, 60%) as a white solid: mp 116−117 °C; H
2D COSY, NOESY and HMQC experiments (Figures S9−S19 in the
Supporting Information).
Syntheses. 4-(Bromomethyl)benzaldehyde. A mixture of (4-
diethoxymethylphenyl)methanol (2.447 g, 11.6 mmol) and HBr 65%
(65 mL) was refluxed for 1 h. The solution was cooled to rt and
extracted with CH2Cl2 (100 mL). The organic phase was washed with
NMR (400 MHz; CDCl3): δ 1.58 (br s, 3H, N−H), 2.56 (t, 6H, J =
5.0 Hz, NCH2CH2NH), 2.69 (t, 6H, J = 5.0 Hz, NCH2CH2NH), 2.71
(t, 6H, J = 5.0 Hz, NCH2CH2O), 3.53 (s, 6H, NHCH2p-xylyl), 3.59 (t,
6H, J = 5.0 Hz, NCH2CH2O), 4.50 (s, 6H, p-xylylCH2O), 6.78 (d, 6H,
J = 7.6 Hz, H3/H5 of p-xylyl), 7.02 (d, 6H, J = 7.6 Hz, H2/H6 of p-
F
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The Journal of Organic Chemistry
Article
xylyl); 13C NMR (100 MHz; CDCl3) δ 48.1 (NCH2CH2O), 54.2
(NCH2CH2NH), 54.6 (NHCH2p-xylyl), 55.7 (NCH2CH2NH), 68.7
(NCH2CH2O), 73.1 (p-xylylCH2O), 127.6 (C2/C6 of p-xylyl), 127.8
(C3/C5 of p-xylyl) 137.4 (C4 of p-xylyl), 139.1 (C1 of p-xylyl); ESI−
MS (MeOH) m/z 602.3 [M + H]+. Anal. Calcd for C36H51N5O3: C,
71.85; H, 8.58; N, 11.64. Found: C, 71.80; H, 8.60; N, 11.73.
Crystals of [H3t2pN5O3(H2O)]Cl3·7H2O. The cryptand t2pN5O3 (5.0
mg, 8.3 μmol) was dissolved in acetone (200 μL), and 37% HCl (2
μL) was added. A white precipitate was formed immediately. Water
(20 μL) was added and the solution heated until it was clear, and then
the mixture was allowed to slowly cool to rt. Single colorless crystals
suitable for X-ray crystallographic determination were obtained
overnight.
Potentiometric Measurements. Reagents and Solutions. All
the solutions were prepared using dematerialized water. Carbonate-
free solutions of the KOH titrant were prepared from a commercial
ampule diluted with 1000 mL of water (freshly boiled for about 2 h
and allowed to cool under nitrogen). These solutions were discarded
every time carbonate concentration was about 0.5% of the total
amount of base. The titrant solutions were standardized (tested by
Gran’s method).22
constants, βiD, of the compound were calculated by fitting the data
obtained with the HYPNMR program.26 The chemical shifts of all the
1
resonances present in the H NMR spectra of t2pN5O3 were used in
the refinement calculations. A total of 29 data sets were used (each
consisting in a measured pD value and eight recorded chemical shifts)
corresponding to a total of 261 experimental values. The initial
computations were obtained in the form of overall (deuteron)
protonation constants, βiD = [DiL]/[D]i[L]. Conversion of the log KiD
determined from this titration to log KiH was performed using the
equation log KiD − log KiH = 0.076 log KiH − 0.05.13
Determination of Vicinal 1H−1H Coupling Constants. Solutions of
the (H4t2pN5O3)(Cl)4 were prepared in D2O (2.0 × 10−3 M) and the
pD was adjusted to 2.40 and 6.80 by addition of DCl or KOD with pH
meter instrument fitted with a combined microelectrode. The 1H
NMR spectra were recorded on a 400 MHz instrument. The AA′BB′
spin systems involving were simulated with the spectral analysis
routine NUMMRIT27 (including iteration) within the software
SpinWorks.28
1
DOSY. Selected samples prepared in D2O were analyzed by H
DOSY NMR spectra, recorded on a 500 MHz instrument fitted with a
triple resonance probe and Great 50/10 gradient synthesizer (53.5 G/
cm maximum output). The protocol followed that published according
to Groves et al.29 The ledbpgs2s pulse sequence was used with values
of 200 ms for the diffusion delay (D20, big delta, Δ) and 1 ms for the
bipolar diffusion gradient (p30, little delta, δ = 2 ms). A series of 32
spectra were stepped over the 2−95% gradient range and processed
with Topspin 3.1 software. After processing into an 8k × 1k matrix,
columns containing the projected diffusion parameters were summed
to create the diffusion profiles from which the experimental diffusion
coefficients were taken.
Crystallography. The single-crystal X-ray data of [H3t2pN5O3(H2O)]-
Cl3·7H2O were collected at 150(2) K with graphite-monochromatized
Mo Kα radiation (λ = 0.71073 Å). The selected crystal was positioned
35 mm from the CCD and the spots were measured with a counting
time of 100 s. Data reduction including a multiscan absorption
correction was carried out using the SAINT-NT software package. The
structure was solved by a combination of direct methods with
subsequent difference Fourier syntheses and refined by full matrix
least-squares on F2 using the SHELX-97 suite.30 Anisotropic thermal
displacements were used for all non-hydrogen atoms. The hydrogen
atoms of the C−H bonds were placed at geometrical positions and
refined with Uiso = 1.2Ueq of the atom to which they are attached. The
atomic positions of the hydrogen atoms of the amine groups and of
seven crystallization water molecules were discernible from difference
Fourier maps, and they were included in the structure refinement with
individual isotropic thermal parameters. The eighth water molecule
was found to be disordered over two positions, which were inserted in
the structure refinement with refined occupancies of 1 − x and x, x
being 0.55(1). Therefore, the hydrogen atoms of this single molecule
were not considered in the last refinements of the crystal structure.
Molecular diagrams were drawn with PyMOL software.31 The crystal
data and refinement details are summarized in Table S2 in the
Supporting Information.
Equipment and Working Conditions. The equipment used was
described before.8f The ionic strength of the experimental solutions
was kept at 0.10
0.01 M with KCl, and the temperature was
maintained at 298.2 0.1 K. Atmospheric CO2 was excluded from the
titration cell during experiments by passing purified nitrogen across
the top of the experimental solution.
Measurements. The [H+] of the solutions was determined by the
measurement of the electromotive force of the cell, E = E′° + Q log
[H+] + Ej. The term pH is defined as −log [H+]. E′°, Q, Ej, and Kw
were determined by titration of 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 Kw
was determined from data obtained in the alkaline range of the
titration, considering E′° and Q valid for the entire pH range and
found to be equal to 10−13.76 under our experimental conditions.
Before and after each set of titrations, the glass electrode was calibrated
as a [H+] probe by titration of 1.000 × 10−3 M standard HCl solution
with standard KOH. Every measurement was carried out with 0.040
mmol of ligand in a total volume of 30 mL. The exact amount of
ligand was obtained by determination of the excess of acid present in a
mixture of the ligand and standard 1.0 × 10−2 M HCl by titration with
standard KOH solution. Each titration curve consisted typically of one
hundred points in the 3.0−11.0 pH range, with three replicates
undertaken.
Calculation of Equilibrium Constants. Overall protonation
constants, βiH, of the ligand were calculated by fitting the
potentiometric data obtained for all the performed titrations in the
same experimental conditions with the HYPERQUAD program.23 The
initial computations were obtained in the form of overall protonation
constants, βiH = [HiL]/[H]i[L]. Differences, in log units, between the
values of two consecutive constants provide the stepwise (log K)
reaction constants (being KIH = [HiL]/[Hi−1L][H]). The errors
quoted are the standard deviations of the overall protonation constants
given directly by the program for the input data, which include all the
experimental points of all titration curves. The HYSS program24 was
used to calculate the concentration of equilibrium species from the
calculated constants from which distribution diagrams were plotted.
Molecular Modeling. Molecular dynamics simulations of
3+
4+
H3t2pN5O3 and H4t2pN5O3 in water were performed with
GROMACS 4.0.4.32 Parameters compatible with the GROMOS96
53A6 force field33 were obtained using the Automated Topology
Builder34 for both species of t2pN5O3, and the SPC model was used for
water.35 The nonbonded interactions were treated using a twin-range
cutoff of 8/14 Å, a neighbor lists update every 10 fs, and the reaction
field method to treat long-range electrostatics with a dielectric
constant of 54.36,37 Berendsen coupling38 was used to maintain the
temperature at 300 K (temperature coupling of 0.1 ps) and pressure at
1 atm (isothermal compressibility of 4.5 × 10−5 bar−1 and pressure
coupling of 0.5 ps). The time step was 2 fs, and all bonds were
constrained with the LINCS algorithm.39 A standard protocol was
used to minimize and equilibrate the system before running the 50 ns
long simulations.
1
NMR Studies. H NMR Titration. A solution of the (H4t2pN5O3)-
(Cl)4 was prepared in D2O (2.0 × 10−3 M) and during titration
procedure the pD was adjusted by addition of CO2-free KOD
solutions with a microsiringe and measured with a pH meter
instrument fitted with a combined microelectrode. The pH* was
measured directly in the NMR tube after calibration of the
microelectrode with commercial buffer aqueous solutions of standard
pH 4.01 and 7.96. The final pD was calculated from the equation pD =
pH* + (0.40 0.02).25 The 1H NMR spectra were recorded on a 300
MHz instrument using 3-(trimethylsilyl)propanoic acid-d4 sodium salt
(DSC) as internal reference. Overall (deuteron) protonation
G
dx.doi.org/10.1021/jo300799s | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
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ASSOCIATED CONTENT
* Supporting Information
Titration curves of all studied compounds, H NMR spectra
■
S
1
4+
recorded in the course of the titration of H4t2pN5O3 with
KOD in D2O; NOESY spectra of t2pN5O3 in D2O at pD = 2.4
1
and 6.8; variable-temperature H NMR spectra t2pN5O3 in
D2O at pD = 6.8; NMR (1H, 13C, COSY, NOESY, and
HMQC) and ESI-mass spectra of the reported compounds;
table of hydrogen bonding parameters of the crystal structure;
table with the crystal data and selected refinement details. This
material is available free of charge via the Internet http://pubs.
acs.org.
R.; Brandao, P.; Fel
(f) Mateus, P.; Delgado, R.; Brandao, P.; Carvalho, S.; Felix, V. Org.
́
ix, V. J. Org. Chem. 2009, 74, 8638−8646.
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Biomol. Chem. 2009, 7, 4661−4637.
(9) Dietrich, B.; Hosseini, M. W.; Lehn, J.-M.; Sessions, R. B. Helv.
Chim. Acta 1985, 68, 289−299.
AUTHOR INFORMATION
Corresponding Author
*Tel: (+351) 21 446 9737. Fax: (+351) 21 441 1277. E-mail:
delgado@itqb.unl.pt.
■
(10) (a) Chand, D. K.; Bharadwaj, P. K. Inorg. Chem. 1996, 35,
3380−3387. (b) Ghosh, P.; Gupta, S. S.; Bharadwaj, P. K. J. Chem. Soc.,
Dalton Trans. 1997, 935−938.
(11) Naiini, A. A.; Pinkas, J.; Plass, W.; Young, V. G., Jr.; Verkade, J.
G. Inorg. Chem. 1994, 33, 2137−2141.
Notes
(12) Bencini, A.; Bianchi, A.; Garcia-Espana, E.; Micheloni, M.;
̃
The authors declare no competing financial interest.
Ramirez, J. A. Coord. Chem. Rev. 1999, 188, 97−156.
(13) Kręzel, A.; Bal, W. J. Inorg. Biochem. 2004, 98, 161−166.
̇
ACKNOWLEDGMENTS
■
(14) Cohen, Y.; Avram, L.; Evan-Salem, T.; Frish, L. In Analytical
Methods in Supramolecular Chemistry; Schalley, C., Ed.; Wiley-VCH:
Weinheim, 2007; pp 163−219.
We acknowledge Pedro Lamosa for fruitful discussions and M.
C. Almeida for providing elemental analysis and ESI-MS data
from the Elemental Analysis and Mass Spectrometry Service at
(15) Giuseppone, N.; Schmitt, J.-L.; Allouche, L.; Lehn, J.-M. Angew.
Chem., Int. Ed. 2008, 47, 2235−2239.
ITQB. P.M. thanks the Fundaca̧ o para a Ciencia e a Tecnologia
̂
̃
(16) Reinstein, O.; Neves, M. A. D.; Saad, M.; Boodram, S. N.;
Lombardo, S.; Beckham, S. A.; Brouwer, J.; Audette, G. F.; Groves, P.;
Wilce, M. C. J.; Johnson, P. E. Biochemistry 2011, 50, 9368−9376.
(17) Teraoka, I. Polymer Solutions; Schalley, C., Ed.; Wiley: New
York, 2002; pp 184−188.
(FCT) for the grant (SFRH/BPD/79518/2011). We acknowl-
edge FCT and POCI, with coparticipation of the European
Regional Development Fund (FEDER), for financial support
under project PTDC/QUI/67175/2006. The NMR spectrom-
eters are part of The National NMR Network (REDE/1517/
(18) Collman, J. P.; Fu, L.; Herrmann, P. C.; Wang, Z.; Rapta, M.;
RMN/2005), supported by “Programa Operacional Cien
̂
cia e
Broring, M.; Schwenninger, R.; Boitrel, B. Angew. Chem., Int. Ed. 1998,
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Inovacao (POCTI) 2010” and FCT. This work was also
̧
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37, 3397−3400.
supported by FCT through grant no. PEst-OE/EQB/LA0004/
2011.
(19) Lakshminarayanan, P. S.; Ravikumar, I.; Suresh, E.; Ghosh, P.
Cryst. Growth Des. 2008, 8, 2842−2852.
(20) Sarri, P.; Venturi, F.; Cuda, F.; Roelens, S. J. Org. Chem. 2004,
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