Calorimetric measurement of the CH/π interaction involved in the molecular recognition of saccharides by aromatic compounds

Article abstract of DOI:10.1021/jo701926r

(Chemical Equation Presented) Can a benzene molecule differentiate between two isomeric carbohydrates? It is generally accepted that two factors govern molecular recognition: complementarity and preorganization. Preorganization requires the presence of cavities for positioning the host's groups of complementary nature to those of the guest. This study shows that, in fact, groups should be complementary to recognize each other (for the case presented here, it is controlled by the CH/π interaction) but preorganization is not essential. Since weak interactions have their origin in dispersion forces, they also have impact on the enthalpic term of the free energy, so it was considered that their participation can be demonstrated by measuring the energy involved. For recognition to happen, two conditions must be satisfied: specificity and associated stabilizing energy. In this study we evaluated the heat of dissolution of different carbohydrates such as methyl 2,3,4,6-tetra-O-methyl- α-D-mannopyranoside and methyl 2,3,4,6-tetra-O-methyl-β-D- galactopyranoside using different aromatic solvents. The solvation enthalpies in benzene were -78.8 ± 3.9 and -88.7 ± 5.5 kJ mol^-1 for each carbohydrate, respectively; and these values yielded a CH/π energy of interaction of 9.9 kJ mol^-1. In addition, NMR studies of the effect of the addition of benzene to chloroform solutions of the two carbohydrates showed that benzene specifically interacts with the hydrogen atoms of the pyranose ring at positions 3, 4, and 5 located on the α face of the methyl-β-galactoside, so it is, in fact, able to recognize it. Thus, the interactions between carbohydrates and the aromatic residues of proteins occur in the absence of the confinement generated by the protein structure. By experimentally measuring the energy associated with this interaction and comparing it to theoretical calculations, it was also possible to unequivocally determine the existence of CH/π interactions between carbohydrates and proteins.

Full text of DOI:10.1021/jo701926r

Calorimetric Measurement of the CH/π Interaction Involved in the
Molecular Recognition of Saccharides by Aromatic Compounds
Lorena Bautista-Iba´n˜ez,† Karla Ram´ırez-Gualito,† Beatriz Quiroz-Garc´ıa,†
Aaro´n Rojas-Aguilar,‡ and Gabriel Cuevas*,†
Instituto de Qu´ımica, UniVersidad Nacional Auto´noma de Me´xico, Apdo. Postal 70213, 04510, Coyoaca´n,
Me´xico, D.F., Me´xico, and Departamento de Qu´ımica, Centro de InVestigacio´n y de Estudios AVanzados,
Instituto Polite´cnico Nacional Apdo. Postal 14-740, 07000 Me´xico, D.F., Me´xico
gecgb@serVidor.unam.mx
ReceiVed September 2, 2007
Can a benzene molecule differentiate between two isomeric carbohydrates? It is generally accepted that
two factors govern molecular recognition: complementarity and preorganization. Preorganization requires
the presence of cavities for positioning the host’s groups of complementary nature to those of the guest.
This study shows that, in fact, groups should be complementary to recognize each other (for the case
presented here, it is controlled by the CH/π interaction) but preorganization is not essential. Since weak
interactions have their origin in dispersion forces, they also have impact on the enthalpic term of the free
energy, so it was considered that their participation can be demonstrated by measuring the energy involved.
For recognition to happen, two conditions must be satisfied: specificity and associated stabilizing energy.
In this study we evaluated the heat of dissolution of different carbohydrates such as methyl 2,3,4,6-
tetra-O-methyl-R-D-mannopyranoside and methyl 2,3,4,6-tetra-O-methyl-â-D-galactopyranoside using
different aromatic solvents. The solvation enthalpies in benzene were -78.8 ( 3.9 and -88.7 ( 5.5 kJ
mol-1 for each carbohydrate, respectively; and these values yielded a CH/π energy of interaction of 9.9
kJ mol-1. In addition, NMR studies of the effect of the addition of benzene to chloroform solutions of
the two carbohydrates showed that benzene specifically interacts with the hydrogen atoms of the pyranose
ring at positions 3, 4, and 5 located on the R face of the methyl-â-galactoside, so it is, in fact, able to
recognize it. Thus, the interactions between carbohydrates and the aromatic residues of proteins occur in
the absence of the confinement generated by the protein structure. By experimentally measuring the
energy associated with this interaction and comparing it to theoretical calculations, it was also possible
to unequivocally determine the existence of CH/π interactions between carbohydrates and proteins.
Introduction
rupole, and quadrupole-quadrupole among others. This is the
origin of some noncovalent interactions that allow a specific
association between a host and its guest.1 This phenomenon is
known as molecular recognition and it is generally accepted
that the existence of host cavities in molecules of biological
The so-called weak interactions such as the hydrogen bonds
or the interactions originating from dispersion forces are the
heart of Supramolecular Chemistry and have their origin in
electrostatic interactions such as dipole-dipole, dipole-quad-
† Universidad Nacional Auto´noma de Me´xico.
(1) Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic Chemistry;
University Science Books: Sausalito, CA, 2006; p 207.
‡ Centro de Investigacio´n y de Estudios Avanzados.
10.1021/jo701926r CCC: $40.75 © 2008 American Chemical Society
Published on Web 01/04/2008
J. Org. Chem. 2008, 73, 849-857
849

Bautista-Ilba´n˜ez et al.
origin such as enzymes and antigens is essential for recognition.2
This phenomenon is known as preorganization. Emil Fischer3-5
was the first scientist to explain the selectivity that is charac-
teristic of enzymatic reactions using the rigid lock and key
model.6 In 1958, Daniel Koshland7,8 proposed that a protein
can show some flexibility that would allow an adaptation to
the substrate. This led to the establishment of the induced fit
model. The two models imply that preorganization plays a
central role. On the other hand, the term complementarity, where
the structure of the host is complementary to that of the guest,
is the other factor that influences molecular recognition.
Apparently, both factors are essential for recognition.
Sugar-protein interactions play an important role in a wide
range of fundamental biological processes that include metabolic
regulation, growth, embriogenesis, and apoptosis among many
others.9-11 The determination of the mechanisms by which
carbohydrates recognize proteins (lectins, antibodies, and en-
zymes among others) remains a fundamental question in
biochemistry. This information will be useful in the future to
control and manipulate the interactions and design structural
mimics of oligosaccharides of pharmaceutical interest.12 For this,
it is important to understand the structural, thermodynamic, and
kinetic phenomena that control the manner in which a carbo-
hydrate is attached to its receptor by the participation of an
aromatic molecule.
Interestingly it is usual to find aromatic amino acid residues
(tryptophan, tyrosine, and phenylalanine) in the protein active
sites that recognize and bond carbohydrates.13,14 Carbohydrate-
protein complexes that have been studied by X-ray diffraction
show that the carbohydrate is positioned in such a way that at
least three hydrogen atoms of the hydrophobic region (that
includes the C-H bonds of the tetrahydropyran ring) are
oriented toward the amino acid aromatic nucleus (Figure 1).
FIGURE 1. Supramolecular structure of the fucose and benzene
complex determined at MP2/6-31G(d,p) including BSSE during the
optimization process.15
energy, so it was considered that if this could be measured, it
would be a clear sign of their existence and relevance.
Recent studies demonstrated theoretically and experimentally
the existence of stabilizing interactions between fucose (a model
of galactose free of conformational implications due to the
rotation of the CH2-OH segment) and benzene (Figure 1). The
determination of the stabilization energy was performed using
ab initio methods at the MP2/6-31G(d,p) level of theory
considering a counterpoise correction during optimization of
the supramolecule. This energy was on the order of 12.5 kJ
mol-1, approximately 4.16 kJ mol-1 for each C-H bond
involved in the interaction. On the other hand, the structure of
the complex was very sensitive to the inclusion of the correction
for the basis set superposition error (BSSE) during the optimiza-
tion. It was, thus, demonstrated that the use of density
functionals that lack terms that adequately describe the long-
distance interaction is not good for evaluating the energy of
the system since it has its origin in the dispersion forces.15,16 In
addition, the authors established that the interaction produced
by benzene (or phenol) on the hydrogen atoms of the R face of
the methyl-â-galactoside is the most important, because the
For recognition to exist, two conditions should be satisfied:
specificity and stabilizing energy. Since the energy associated
with the recognition process of the system is important, the
evaluation of the energy was considered an extremely important
factor for the molecular recognition process. Thus, the evaluation
of the thermodynamic properties of the carbohydrate-aromatic
compound system is most relevant. Stabilizing weak interactions
play an important role on the enthalpic term of the free Gibbs
1
specific H NMR resonances undergo upfield shifting upon
addition of phenol. This behavior has been taken as direct proof
of the existence of CH/π interaction.15 The fact that this
interaction does not require a well-defined mold or a structured
rigid frame created by the protein where the conditions for
interaction are generated is notable.
A study published recently exposed experimental evidence
of the CH/π interaction through the use of near-IR vibrational
spectra of individual carbohydrate conformers isolated under
molecular beam conditions in the gas phase.17 Since the analysis
of weak interactions is controversial18 and it is important to
establish the mechanisms by which these interactions occur, the
experimental determination of the interaction energy between
an aromatic compound and a carbohydrate where the energy
(2) Smith, M. B.; March, J. March’s AdVanced Organic Chemistry.
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Day, P. J.; Monzingo, A. F.; Ernst, S. R.; Robertus, J. D.; Iglesias, R.;
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piranosa y origen de las interacciones carbohidrato-anillo aroma´tico. Ph.D.
Thesis, Universidad de Vigo-Centro de Investigaciones Biolo´gicas, Vigo,
Spain, 2006.
(16) Jime´nez-Barbero, J.; Can˜ada, F.; Cuevas, G.; Asensio, J. L.; Aboitiz,
N.; Canales, A.; Cha´vez, M. I.; Ferna´ndez-Alonso M. C.; Garc´ıa-Herrero,
A.; Mari, S.; Vidal, P. In Protein-Carbohydrate Interactions: A Combined
Theoretical and NMR Experimental Approach on Carbohydrate-Aromatic
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850 J. Org. Chem., Vol. 73, No. 3, 2008

Molecular Recognition of Saccharides
SCHEME 1. Thermodynamic Determination of the Heat of
Solvation
associated with the CH/π interaction is evaluated is fundamental
and is the topic of the present study. Trying to determine the
contribution of the enthalpic and entropic terms to the free
energy associated with the donor-acceptor-complex equilibria
may prove challenging. This is because the first term includes
the effects of the covalent bonds, the noncovalent interactions
of electrostatic origin, the stereoelectronic effects, and hydrogen
bonds among others, while the second term encompasses the
evaluation of the change of degrees of freedom experienced by
the compounds that participate in the chemical equilibrium under
study. These include the changes in degrees of freedom of the
solvent, the host, and the guest as well as the product of the
interaction, the supramolecule. The contribution of the enthalpic
term to the free conformational energy has allowed the
understanding of conformational processes19,20 as well as
molecular recognition.21 For the latter, it has been recently
demonstrated that the interaction between Amarantus caudatus
Antimicrobial Peptide 2 (AcAMP2)-like peptides and the trimer
of N-acetylglucosamine (GluNAc)3 is enthalpically driven and
that both hydrogen bonds and van del Waals forces contribute
to the stability of the complexes in aqueous solution.22 This is
in sharp contrast to the previous idea that suggested that the
liberation of the molecules from the solvents present at the
binding site would make entropy the dominating term.23 In this
sense, it would be important to establish the role of the enthalpic
term in the interaction and evaluate its magnitude in order to
obtain fundamental information. This information would be quite
useful for the understanding of the selectivity of receptors toward
certain carbohydrates.24-29 This information can be used to
rationally manipulate the carbohydrate-protein interaction to
improve the selectivity of compounds of pharmaceutical im-
portance.30
enthalpy of solvation must be proportional to the magnitude of
the interaction of each isomer with the solvent molecules.
Scheme 1 shows the terms required to determine this thermo-
dynamic property.
The enthalpy of phase change as well as the dissolution
enthalpy can be determined by direct measurement in a
calorimeter.32 However, these numbers can be useful to directly
quantify the interaction energy for solute and solvents through
a second approximation that can be established as:
∆Edis ) Z[E1,2 - 1/2(E1,1 + E2,2)]
(1)
where ∆Edis is the dissolution enthalpy, Z is the number of
molecules in the carbohydrate solvation sphere, E1,2 is associated
with the molecular interaction between the carbohydrate mol-
ecules and the solvent, E1,1 is the energy associated with the
intermolecular interaction of the carbohydrate in the condensed
phase and is directly proportional to the sublimation enthalpy,
and finally E2,2 is the cohesive energy of the liquid molecules
that is proportional to the vaporization enthalpy. The direct
interaction energy between the carbohydrate molecules and the
aromatic solvent can be calculated through eq 1 and can be
easily quantified through the enthalpies of solution and of change
of phases applied to the solvation model.
There are two approaches to determine the interaction energy
of interest. The first one is based on the evaluation of the
solvation energy between the carbohydrate and the aromatic
substrate. The term solvation process refers to the energetic and
structural changes occurring in a system during the process of
transferring molecules from the gas phase into the liquid solvent.
Those changes are not only accompanied by disruption of
intermolecular bonds in the solvent but also involve the
formation of new interactions between the molecules of solute
and solvent.31 So, for both carbohydrates, the difference in the
Results and Discussion
Two carbohydrate derivatives were used for this study. The
first, permethylated galactose,33 is a derivative of fucose that,
according to theoretical calculations and experimental results,
has a CH/π interaction when combined with an aromatic
compound.15 The second, a mannose derivative,34 lacks the
interaction and was used as a reference. Since carbohydrates
have a polar region (hydroxyl groups), they are insoluble in
aromatic substrates that lack the groups that can form strong
hydrogen bonds. The energy associated with the CH/π interac-
tion is less than the energy associated with the hydrogen bonds.
Therefore, the use of aromatic compounds capable of forming
hydrogen bonds to determine the CH/π interaction is inadequate
because it is possible to mask the interaction of interest. Thus,
aromatic solvents such as benzene are useful to determine the
interaction energy of interest. To use benzene and its derivatives
as solvents, it was necessary to modify the carbohydrates
through permethylation using iodomethane (Scheme 2). This
reaction changes the amphiphilic character of the carbohydrate
(19) Torres, L. A.; Rojas, A.; Cuevas, G.; Juaristi, E. J. Phys. Org. Chem.
1994, 7, 561.
(20) Juaristi, E.; Cuevas, G. Tetrahedron, 1999, 55, 359.
(21) Kronis, K. A.; Carver, J. P. Biochemistry 1985, 24, 834.
(22) Chavez, M. I.; Andreu, C.; Vidal, P.; Aboitiz, N.; Freire, F.; Groves,
P.; Asensio, J. L.; Asensio, G.; Muraki, M.; Can˜ada, F. J.; Jime´nez-Barbero,
J. Chem. Eur. J. 2005, 11, 7060.
(23) Quiocho, F. A. Pure Appl. Chem. 1989, 61, 1293.
(24) Davis, A. P.; Wareham, R. S. Angew. Chem., Int. Ed. 1999, 38,
2978. Davies, A. P.; Wareham, R. S. Angew. Chem., Int. Ed. 1998, 37,
2270.
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Am. Chem. Soc. 2004, 126, 16456.
(26) Nativi, C.; Cacciarini, M.; Francesconi, O.; Vacca, A.; Moneti, G.;
Ienco, A.; Roelens, S. J. Am. Chem. Soc. 2007, 129, 4377.
(27) Francesconi, O.; Ienco, A.; Moneti, G.; Nativi, C.; Roelens, S.
Angew. Chem., Int. Ed. 2006, 45, 6693.
(28) Calderone, V.; Fragai, M.; Luchinat, C.; Nativi, C.; Richichi, B.;
Roelens, S. ChemMedChem. 2006, 1, 598.
(29) Cacciarini, M.; Cordiano, E.; Nativi, C.; Roelens, S. J. Org. Chem.
2007, 72, 3933.
(30) Bernardi, A.; Arosio, D.; Potenza, D.; Sa´nchez-Medina, I.; Mari,
S.; Can˜ada, F. J.; Jime´nez-Barbero, J. Chem. Eur. J. 2004, 10, 4395.
(31) Krestov, G. A. Thermodynamics of solVation; Ellis Horwood: New
York, 1991.
(32) Berry, R. S.; Rice, S. A.; Ross, J. Physical Chemistry; John Wiley
and Sons: New York, 1980.
(33) Asres, D. D.; Perreault, H. Can. J. Chem. 1999, 77, 319. Rathbone,
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J. Org. Chem, Vol. 73, No. 3, 2008 851

Bautista-Ilba´n˜ez et al.
SCHEME 2. Synthesis of the Modified Carbohydrates of
Interest
if existent, can be attributed to the CH/π interaction. The second
is that the solvation of compound 2 is not random, even when
there is no cavity or a preorganized site. In fact, the benzene
ring protects the region of positions 3-6 of the carbohydrate
as shown in Figure 2b. The benzene molecule is oriented due
to the presence of the CH bonds at positions 3, 4, and 5 as has
been determined by theoretical calculations,15 as a consequence
of molecular recognition. This supramolecular arrangement does
not occur in compound 1 (Figure 2a), which experiences
solvation but not recognition. This can be observed through the
fact that the most protected proton at position 6 is the pro-R
that tends to orient itself toward the zone where benzene is
located.16 The presence of the equatorial methoxyl group at
position 4 of molecule 1 generates a repulsive environment for
benzene. The equatorial methoxy group at C4 prevents the
arrangement of the three C-H bond pattern that is a condition
for the existence of the CH/π interaction.16
Figure 3 shows the evolution of the chemical shift of the
carbohydrate’s ring protons when benzene is added. These
results provide support to the model where a benzene molecule
approaches selectively to the R face of compound 2. The
negative slopes imply the shielding that is characteristic of
compound 2 while the positive slopes show the unshielding of
compound 1.
by modifying the hydroxyl groups.35,36 The use of the methyl
groups was considered adequate because it does not present
strapping problems due to the steric volume. The modification
was done using standard methods starting with the corresponding
methyl pyranosides. The theoretical calculations and the recently
published NMR experiments15 were useful to find the stabiliza-
tion of methyl-â-galactoside through a CH/π interaction.
However, this interaction was not found for methyl-R-manno-
side. During this study permethylation of the two carbohydrates
rendered methyl 2,3,4,6-tetra-O-methyl-R-D-mannopyranoside33
(1) and methyl 2,3,4,6-tetra-O-methyl-â-D-galactopyranoside
(2)34 with purity greater than 99.8% determined through the use
of gas chromatography.
1
Since the specific H NMR signals of a group of protons
attached to the pyran ring that undergo upfield shifting upon
addition of an aromatic compound has been taken as direct
evidence of the existence of CH/π interactions, the modified
carbohydrates were used for the NMR study.37 Thus, the effect
of benzene on the chemical shift of the hydrogen atoms of the
molecules of interest was evaluated. Tables S1 and S2 (see the
Supporting Information) show the effect that consecutive
additions of the aromatic substrate have on the chemical shifts
of the different hydrogen atoms. Except for the hydrogen atom
at position H2, all other hydrogen atoms of the methyl 2,3,4,6-
tetra-O-methyl-â-D-galactopyranoside (2) show a shielding effect
from the solvent. This effect can be evaluated through the
difference ∆δ ) δ(CDCl3) - δ(C6D6), since the chemical shift
in the initial chloroform solution is slowly modified as the
proportion of benzene increases. A positive value in the shift
of the hydrogen atoms of molecule 2 shows a net shielding.
Thus, the H3 hydrogen atom goes from 3.15 to 2.97 ppm, H4
goes from 3.65 to 3.45 ppm, and H5 goes from 3.57 to 3.33
ppm. This behavior is in sharp contrast to the effect that the
same solvent has over compound 1, where an opposed effect is
observed on the same hydrogen atoms, i.e., H3 goes from 3.49
to 3.68 ppm, H4 goes from 3.42 to 3.75 ppm, and H5 goes
from 3.55 to 3.82 ppm.
It is interesting to point out that a protective effect of benzene
over the protons at position 6 of carbohydrate 2 is observed.
However, the effect is not protective for carbohydrate 1. All
methyl groups are exposed to the solvent and in all cases show
a protective effect that is increased as the benzene concentration
is increased in the solution. These results support previously
reported observations15 and unequivocally confirm the presence
of a CH/π interaction between benzene and the carbohydrate
only for compound 2 that substitutes chloroform from the first
solvation sphere.
With these results, a calorimetric study of the dissolution
process of carbohydrates in aromatic solvents was designed to
determine the energy associated with the interaction. Table 1
shows the solvation enthalpy data of compounds 1 and 2
obtained through the solvation model (Scheme 1). The solvents
used for this study were 1-methoxy-4-methylbenzene (3),
o-xylene (4), m-xylene (5), p-xylene (6), 1,2-dimethoxybenzene
(7), 1,3-dimethoxybenzene (8), and benzene (9). In all cases,
the substituents make the aromatic compound a better donor in
comparison to benzene. It would be expected that if the
electronic richness of benzene is increased, in the Hammett
sense,38 the interaction would be stronger if it had a hydrogen
bond character. The solvents used to modify the term ∆disHo
m
can be observed in Table 1. The data used to estimate the
enthalpies of dissolution for every solvent are described in the
Supporting Information.
The term ∆disHo is a measure of the amount of heat needed
m
to break the molecular interactions of compound 1 that has a
liquid nature and the crystalline network of compound 2. This
explains the magnitude of these values that is considerably larger
for the solid than for the liquid. A positive value of this quantity
is useful to establish that the compound is more stable in pure
form than in solution because breaking the crystalline structure
requires energy. An exothermic dissolution can be observed for
only two of the cases and both belong to solutions of
1,3-dimethoxybenzene and benzene.
On the xylene series (4-6) the dissolution is markedly
endothermic while the compounds that have oxygen (3, 7, 8)
make it less endothermic and even exothermic. This trend is
also observed in the dissolution heat of compound 2 even when,
for these results, all observed cases are endothermic.
Two important conclusions can be drawn from these results.
The first one is the fact that even when the solvent-methyl
group interactions are approximately similar for both com-
pounds, the difference in the solvent-solute interaction energies,
The solvation process is global, as described in the introduc-
tion of this paper. In this case, once the crystalline network is
broken, the solvent interacts with the solute and it is possible
to evaluate the energy exchanged in the process. Since both
compounds under study vary in the configuration of the
(35) Quiocho, F. A. Biochem. Soc. Trans. 1993, 21, 442.
(36) Elgavish, S.; Sanan, B. J. Mol. Biol. 1998, 277, 917.
(37) Terraneo, G.; Potenza, D.; Canales, A.; Jime´nez-Barbero, J.;
Baldridge, K. K.; Bernardi, A. J. Am. Chem. Soc. 2007, 129, 2890.
(38) Johnson, C. D. The Hammett Equation; Cambridge University
Press: Cambridge, England, 1973.
852 J. Org. Chem., Vol. 73, No. 3, 2008

Molecular Recognition of Saccharides
FIGURE 2. Effect of the CH/π interaction in the chemical shift in ppm of carbohydrates studied here measured by ∆δ ) δ(CDCl3) - δ(C6D6).
(a) benzene-mannoside solution and (b) benzene-galactoside supramolecule.
FIGURE 3. Dependence of the ring protons chemical shift of compounds 1 (a) and 2 (b) with respect to the addition of benzene in solutions of
CDCl3.
TABLE 1. Enthalpies of Solvation (in kJ mol-1) of Methyl 2,3,4,6-Tetra-O-methyl-r-D-mannopyranoside (1) and Methyl
2,3,4,6-Tetra-O-methyl-â-D-galactopyranoside (2) in Different Aromatic Solvents (1:10, mol:mol) Determined by Calvet Microcalorimetry at
303.15 Ka
1
2
disHom
solv
Ho σtot (()b
∆
disHom
Ho σtot (()b
∆
δsolv
solvent
∆
m
m
3
4
5
6
7
8
9
0.938
2.623
2.996
2.625
1.055
-1.069
-3.649
-74.239
-72.554
-72.181
-72.552
-74.122
-76.246
-78.826
3.898
3.899
3.897
3.897
3.897
3.899
3.906
17.391
18.914
19.386
19.249
17.970
16.175
16.537
-87.854
-86.331
-85.859
-85.996
-87.275
-89.070
-88.708
5.655
5.536
5.547
5.564
5.582
5.545
5.536
a The value of ∆lgHom for 1 is constant: 75.177 kJ mol-1. ∆sgHmo ) 105.245 for 2. b σtot is the overall uncertainty, calculated from the uncertainties on the
enthalpies of dissolution and phase change (see the Supporting Information).
stereogenic center at position 4, the region that shows the most
changes in the NMR study, compound 1 cannot have the CH/π
interaction, while compound 2 does have the interaction (Figure
2).
The solvation energy ∆solvHom of compound 1 establishes a
more stable system for benzene with a value of -78.8 ( 3.9
kJ mol-1 in relation to the other aromatic solvents. This is
followed in terms of stability by solvation processes where
oxygenated solvents are present with the least stable systems
being those formed with xylene isomers. Of the xylene isomers,
the more stable interactions are those where o- and p-xylene
are present. The energy difference between the least and the
most stable systems is of 6.6 kJ mol-1. As can be expected the
∆
solvHom values are considerably higher for compound 2
(-88.7 ( 5.5 kJ mol-1). For this solute, benzene does not
generate the most stable system. In fact, the most stable system
is generated by 1,3-dimethoxybenzene (Table 1). Xylenes
contribute to a lesser extent to stability, but m-xylene contributes
the least stabilization energy. The energetic difference between
the extremes is 3.2 kJ mol-1
.
It is generally accepted that the energy difference between
compounds 1 and 2 is due mainly to the CH/π energy. This
can be estimated through eq 2.
CH/π energy ) ∆solVHom(1) - ∆solvHmo (2)
(2)
J. Org. Chem, Vol. 73, No. 3, 2008 853

Bautista-Ilba´n˜ez et al.
It is possible to take into account that the ∆solvHom term
as is demonstrated here, and this has been described already.22
Nevertheless, entropic contributions evaluated by NMR tech-
niques must be carefully interpreted, for example, in small
molecules their contribution to the conformational equilibrium
is very high and this cannot be easily explained.20 However,
the elevated values have not been questioned in other processes
associated with proteins where the same techniques have been
used. Thus, the elevated entropies may be due to issues directly
related to the determination and would not really represent the
physical phenomenon.
The model proposed by Berry, Rice, and Ross was used to
compare the interaction energy values obtained through the
solvation energy.32 The results for both compounds are shown
in Table 2. The determination of the ∆gl Hmo values is described
on the Experimental Section. The vaporization enthalpy values
for aromatic solvents were taken from the literature.43,44 Since
this energy has not been determined for 1-methoxy-4-methyl-
benzene and 1,3-dimethoxybenzene, values of the isomers
1-methoxy-3-methylbenzene and 1,2-dimethoxybenzene were
used, so the values derived from either one of these compounds
are only approximations.
(1)
includes only the interactions between the methyl groups and
the solvent, while the ∆solvHom term also includes the CH/π
(2)
interaction (due to recognition); thus, the interaction between
the carbohydrates and benzene gives a CH/π energy of 9.9 kJ
mol-1. This is in agreement with the value calculated at the
MP2/6-31G(d,p) level when the base superposition correction
is included (12.5 kJ mol-1),15 and with previous results showing
that enthalpy is a relevant contribution for the binding process.22
Recently, the protein-carbohydrate interaction was evaluated
using a simple model that was useful to establish that the energy
of interaction is approximately 20.9 kJ mol-1 37
. This was done
using the B3LYP functional and the DZV(2d,p) base for the
optimization of the molecular geometry followed by a single
point calculation using the MP2 method with the same basis.
This was done taking advantage of an intramolecular interaction
that is free of BSSE. The calculation of the energy was done
using an isodesmic reaction where the energy difference between
the elements of the reaction is attributed only to the CH/π
interaction. Since the nature of the exo-anomeric effect present
in both molecular systems is different, it is possible that the
final value of the energy obtained at the MP2/DZV(2d,p)//
B3LYP/DZV(2d,p) level has some of this contribution. In
addition, the original geometries were calculated at the B3LYP/
DZV(2d,p) level. However, it is well-known that this level does
not describe long-distance interactions adequately, thus the
geometry reported using this erroneous calculation is far from
the best. The carbohydrate-aromatic compound interactions
have been widely studied using computational methods and the
energy values oscillate between 10.5 and 20.9 kJ mol-1 when
the MP2 method is used. Calculations using the Hartree-Fock
method and hybrid functionals describe small and even repulsive
interactions.39-41 Therefore, the different levels of theory can
be used to somewhat justify the repulsive interactions as well
as different magnitudes of attractive interactions. So, it is very
important to have the experimental reference value of 9.9 kJ
mol-1 to estimate the quality of the theoretical values available.
The presence of the substituents increases the interaction
energy because it increases the ring’s donating capacity.
However, xylenes produce similar stabilizing interactions (13.8
kJ mol-1 for 4, 13.4 for 6, and 13.7 for 5) with respect to ethers
(13.2 kJ mol-1 for 7 and 12.8 kJ mol-1 for 8). Ether 3 generates
a CH/π energy of 13.6 kJ mol-1 similar to that of m-xylene 5.
This shows that benzene derivatives with strong donating groups
do not necessarily produce the more stabilizing interactions,
because the stronger methoxy group produces a weaker enthalpic
effect with respect to the methyl group, in contrast with the
previous suggestion.22
The values described are useful to confirm the participation
of the CH/π interaction in the stabilization of compound 2 since
E1,2 is always higher for compound 2 in relation to compound
1. In this case, the dispersion of values is small, and different
from the previous case, the benzene yields the most stabilized
system. This fact can be justified if the steric size of the aromatic
compounds is considered.
The use of the 1 to 10 ratio of the carbohydrate in relation to
the aromatic solvent guaranteed the saturation of the solvation
sphere. It can be accepted that, in accordance with Figure 1
and Tables S1 and S2 (Supporting Information), this sphere is
saturated with three benzene molecules for each carbohydrate
molecule. At this point, most protons attached to the pyrane
ring stop suffering an important effect on the chemical shift
(Figure 3). This implies that the effect remains approximately
constant. Nevertheless, the methyl groups exposed to the solvent
suffer continuous shielding. The calculation of the interaction
energy considering, in an extreme case, only three molecules
of solvent for each one of the solutes is increased to practically
4.0 kJ mol-1 when compared to 10 molecules for the case of
molecule 2. However, it remains practically constant for
molecule 1 (Table 2). This can be attributed to the fact that the
interaction energy is distributed between a smaller number of
neighboring molecules. In consequence, the difference in the
interaction energy between compounds 1 and 2 is also increased
to 4 kJ mol-1 making it more favorable by 20.5 kJ mol-1 for
compound 2. This is in comparison with the 16.6 kJ mol-1
obtained using 10 molecules in the solvation sphere. This for
benzene, for example, makes the energy go from 17.1 to 21.7
kJ mol-1. These results are in line with the fact that compound
2 experiences a stabilizing interaction in accordance with
molecular recognition. This is followed by solvation while
molecule 1 only experiences solvation. This implies the interac-
tion of a benzene molecule with one carbohydrate molecule15
because both the energy observed experimentally and the
calculated energy where the carbohydrate interacts with only
one molecule of benzene are comparable. It is important to
These results show that the enthalpic term is a relevant
contribution in the carbohydrate-aromatic compound recogni-
tion process. At 303 K with benzene being the molecule of
interaction, the entropic term would be the same as the enthalpic
term when it reaches 32.67 J K mol-1, which is possible because
high ∆So values have been reported,42 over 32.67 J K mol-1
.
The final interaction value would be the balance between both
terms and where the enthalpic term is present and is relevant
(39) Spiwok, V.; Lipovova, P.; Skalova, T.; Vondrackova, E.-; Dohnalek,
J.; Hasek, J.; Kralova, B. J. Comput-Aided Mol. Des. 2005, 19, 887.
(40) Sujatha, M. S.; Sasidhar, Y. U.; Balaji, P. V. Biochemistry 2005,
44, 8554.
(41) Sujatha, M. S.; Sasidhar, Y. U.; Balaji, P. V. Protein Sci. 2004, 13,
2502.
(43) Majer, J.; Svoboda, V.; Lencka, M. J. Chem. Thermodyn. 1985,
17, 365.
(44) Cox, J. D.; Pilcher, G. Thermochemistry of Organic and Organo-
metallic Compounds; Academic Press: New York, 1970.
(42) Kronis, K. A.; Carver, J. P. Biochemistry 1985, 24, 834.
854 J. Org. Chem., Vol. 73, No. 3, 2008

Molecular Recognition of Saccharides
TABLE 2. Enthalpies of Interaction of Methyl 2,3,4,6-Tetra-O-methyl-r-D-mannopyranoside (1) and Methyl
2,3,4,6-Tetra-O-methyl-â-D-galactopyranoside (2) in Different Aromatic Solvents (1:3 and 1:10, mol:mol). Determined from Calvet
Microcalorimetry at 303.15 K (in kJ mol-1
)
1
2
∆gl Hom
E12
E12
σ
tot (()
∆gl Hom
E12
E12
σ
tot (()
∆E1,2
∆E1,2
a
b
a
b
a
b
solvent
3
4
5
6
7
8
9
51.500c
43.450
42.680
40.000
66.900d
66.900d
33.920
63.432
59.576
59.228
57.851
71.144
70.931
54.183
63.651
60.188
59.927
58.463
71.390
70.682
53.332
2.121
1.949
1.959
1.959
2.212
2.212
1.959
51.500c
43.450
42.680
40.000
66.900d
66.900d
33.920
80.112
76.239
75.901
74.547
87.869
87.690
71.236
84.170
80.652
80.425
79.039
92.063
91.464
75.095
2.893
2.767
2.774
2.775
2.959
2.958
2.774
16.680
16.663
16.673
16.696
16.725
16.759
17.053
20.518
20.464
20.497
20.575
20.672
20.782
21.763
a Value for 10 molecules. b Value for 3 molecules. c The value is the value reported for the isomer 1-methoxy-3-methylbenzene.43-44 d The value is from
1,2-dimethoxybenzene.43-44
TABLE 3. Enthalpies of Dissolution and Solvation (in kJ mol-1) of
concluded that the energy associated with the pyranoside 1
corresponds to five times this value, so the additivity principle
is preserved and undoubtedly, the value associated with
Methyl Acetate in Different Aromatic Solvents (1:10, mol:mol),
Determined by Calvet Microcalorimetry at 303.15 Ka
solvent
Q
dis (J)
∆
disHom
∆
solvHom
compound 2 has its origin on the stabilization that goes with
the CH/π interaction.45
3
4
5
6
7
9
1.72
4.68
4.8
4.42
3.44
4.21
0.642
1.75
1.83
1.725
1.35
1.073
-28.700 ( 0.026
-27.570 ( 0.018
-27.490 ( 0.049
-27.600 ( 0.009
-27.970 ( 0.035
-28.250 ( 0.007
Since saccharides may be considered as amphipathic sub-
stances,46 the substitution of the carbohydrate’s hydroxyl groups
with methyl groups increases the hydrophobic properties and
facilitates the access of the aromatic compound to the proximity
of the pyrane ring. Solvation in the absence of specific
interactions would be random. However, a pattern originated
by the specific and stabilizing CH/π interaction would make
the system orderly. NOE experiments were performed to detect
the possibility of close proximity between the carbohydrate and
benzene. For both carbohydrates the signal of a mixture of
benzene-d6 and benzene (1:1) was inverted with a 180 selective
pulse, and the corresponding NOEs were monitored after mixing
periods of 1300 ms. The different behavior for both compounds
is notorious. For 2 small but detectable NOEs (Figure 4b) were
observed for all the annular protons. The larger NOEs (integrated
in relation to the solvent) are H1 (0.28), H2 (0.15), H3 (0.56),
H4 (0.16), H5 (0.32), H6a (0.15), and H6b (0.14), which confirm
a structured complex. For compound 1 the determined NOEs
of the ring protons were very small, close to zero or negative:
H3 (0.14), H4 (0.08), H5 (0.06), H6a (0.14), and H6b (0.07),
but 0.67 for H1 (Figure 4a). Interestingly, the H1 proton of
compound 1 is the only one that suffers shielding by adding
benzene (Figure 3a), a fact that is relevant to build the first
solvation sphere. As is expected, all the methyl groups showed
significant NOEs because of the direct interaction with benzene.
a The value of ∆lgHmo is constant: ∆Hvap (303.15) ) 29.32 kJ mol-1
.
differentiate between the recognition and the solvation processes.
The former implies only one benzene molecule associated with
a specific region of the carbohydrate through weak interactions.
The latter includes several solvent molecules randomly associ-
ated with the carbohydrate.
In summary, the results are not affected importantly when
three or ten molecules are considered within the solvation sphere
because, in fact, regardless of the excess of solvent, the number
of molecules involved in the first solvation sphere of the
carbohydrate is three. Only one molecule is responsable for the
recognition process and, as a consequence, its main energetic
contribution.
During the NMR experiment, when benzene is added, the
methyl groups of both carbohydrates show a shift to the lower
field as was stated before. This led us to several questions: What
is the effect in energetic terms of the contribution of methyl
groups once it is solvated? Could this mask the CH/π interaction
of interest? In order to answer these questions, a study on the
effect of benzene over a methyl acetate in chloroform solution
was conducted (Table S18, Supporting Information) and the heat
of solvation of methyl acetate in different aromatic compounds
was also determined. The results are shown in Table 3.
The NMR study was useful to establish that both methyl
groups, of different chemical nature due to the acidic nature of
the hydrogens of the acetyl group, suffer equally the protective
effect of benzene. So, the methyl acetate is solvated by the
benzene and it orients itself toward the hydrogen atoms. This
behavior must be similar to that expected experimentally on
the five methoxy groups of the carbohydrates.
Conclusions
When the calorimetric and NMR results are combined with
the previously reported computational calculations,15 it can be
demonstrated that the CH/π interaction is stabilizing in a
magnitude that ranges from 9 to 13 kJ mol-1, depending on the
aromatic substrate in use, and is of enthalpic nature. These
values were determined using the solvation energy method that
is useful to obtain an energy value that can be compared with
that calculated using the MP2/6-31G(d,p) level of theory
including the BSSE during optimization. The calculation of the
interaction energy confirmed the existence of a carbohydrate-
It can be observed that the ∆solvHom value presented in Table
3 is slightly exothermic and has negligible variation with the
nature of the aromatic compound in this study. This is also
usually the case for the carbohydrates used in the study. Since
both methyl groups can be solvated in the same manner, for
the case of benzene, it can be considered that each methyl group
contributes with approximately 14.1 kJ mol-1, so it can be
(45) For example, see: Kopp, H. as reported in: Glasstone, S. Textbook
of Physical Chemistry, 2nd ed.; Van Nostrand: New York, 1946; p 525.
Rossini, F. D. J. Res. Natl. Bur. Stand. 1931, 6, 37:7, 329. Rossini, F. D.
J. Res. Natl. Bur. Stand. 1934, 13, 21. Prosen, E. J.; Johnson, W. H.; Rossini,
F. D. J. Res. Natl. Bur. Stand. 1946, 37, 51.
(46) Quiocho, F. A. Curr. Top. Microbiol. Inmunol. 1988, 139, 135.
J. Org. Chem, Vol. 73, No. 3, 2008 855

Bautista-Ilba´n˜ez et al.
FIGURE 4. Comparative NOE effect of benzene-permethylated mannoside (a) and benzene-permethylated galactoside (b) at 298 K.
was dissolved in dimethyl sulfoxide (18 mL). NaOH aq disolution
(50%, w/w) (1.8 mL, 8.75 mmol, 1.7 equiv) was added slowly.
The mixture was stirred to form a gel-suspension and CH3I (1.93
mL, 30.99 mmol, 6.00 equiv) was added dropwise. The reaction
mixture was stirred for 2 h. Water (100 mL) was added. The phases
were separated and the aqueous phase was extracted with CH2Cl2
(3 × 100 mL).48 The combined organic phases were washed with
sat. aq Na2S2O3 disolution (200 mL), dried (Na2SO4), filtered, and
concentrated in vacuo. The reaction mixture was dissolved in EtOAc
(30 mL), activated charcoal was added (20%, w/w), then the black
suspension was stirred into a water-bath for 20 min. The mixture
was filtered over celite after 8 h. Finally the product methyl 2,3,4,6-
tetra-O-methyl-â-D-galactopyranoside was recrystallized from hex-
ane (g90%) and the methyl 2,3,4,6-tetra-O-methyl-R-D-mannopy-
ranoside was treated with activated charcoal three more times, to
obtain 99.8% purity for each compound. NMR signal assignments
were done using HSQC, HMBC, NOESY, and COSY. 2D experi-
ments are included in the Supporting Information.
aromatic compound interaction that is stabilizing in energy and
allowed to establish that at least three molecules of benzene
are associated with the solute. These values can be used to
establish that the HF and B3LYP theoretical methods are not
useful to approach this problem because the dispersion terms
are not included and should not be used for this purpose in the
future. The value of the magnitude of the CH/π interaction does
not vary with the electronic nature of the benzene substituent
in the Hammett sense. Interestingly, the carbohydrate-aromatic
compound interaction does not require the existence of a specific
cavity or an environment generated by protein residues (con-
finement, preorganization) to occur and has a relevant enthalpic
contribution. By experimentally measuring the energy associated
with this interaction and comparing it to theoretical calculations
it was possible to unequivocally determine the existence of CH/π
interactions between carbohydrates and proteins.
It is well-known that solvents in general have properties that
make them a macroscopic continuum characterized by physical
properties such as density, the dielectric constant, the refraction
index, etc.;47 but as a discontinuum, it has individual molecules
that interact among themselves through specific interactions.
We show that these specific interactions are the origin of
molecular recognition and these interactions in turn have their
origin, at least partially, in electrostatic interactions.
Methyl 2,3,4,6-Tetra-O-methyl-r-D-mannopyranoside (1).34
Yield: 3.475 g (90%). Yellow oil. IR (film): 2980, 2910, 2829,
1451, 1377, 1325, 1291, 1191, 1114, 1065, 997, 972, 925, 883,
1
844, 796, 663, 633 cm-1. H NMR (500 MHz, C6D6): δ 3.14 (s,
3H), 3.24 (s, 3H,), 3.24 (s, 3H), 3.24(s, 3H), 3.47 (m, 1H), 3.48 (s,
3H), 3.62 (dd, J ) 2.0, 10.5, 1H), 3.67 (dd, J ) 5.5, 10.5, 1H),
3.68 (dd, J ) 3.0, 9.0, 1H), 3.75 (t, J ) 9.5, 1H), 3.82 (ddd, J )
2.0, 5.5, 9.5, 1H), 4.68 (d, J ) 2.0, 1H). 13C NMR (125 MHz,
C6D6): δ 54.4, 57.2, 58.9, 59.0, 60.5, 72.5, 72.6, 77.1, 77.4, 82.6,
99.0. CI-MS: 251 ([M + H]+, [C11H22O6]H+).
Experimental Section
General Procedure To Carry Out the Permethylation of
Carbohydrates. Methyl-D-pyranoside (1 g, 5.15 mmol, 1.0 equiv)
(48) Wang, H.; Sun, L.; Glazebnik, S.; Zhao, K. Tetrahedron Lett. 1995,
36, 2953. Majer, J.; Svoboda, V.; Lencka, M. J. Chem. Thermodyn. 1985,
17, 365.
(47) Reichardt, C. SolVents and SolVent Effects in Organic Chemistry,
2nd ed.; VCH: Weinheim, Germany, 1990.
856 J. Org. Chem., Vol. 73, No. 3, 2008

Molecular Recognition of Saccharides
Methyl 2,3,4,6-Tetra-O-methyl-â-D-galactopyranoside (2).33
Yield: 735 mg (73%). White solid. mp 43.34 °C. IR (KBr): 2932,
2824, 1551, 1388, 1256, 1185, 1124, 1079, 1050, 1000, 965, 754,
In each experiment of sublimation, samples of around 10.0 mg
of the solid carbohydrates were placed inside the sublimation pan
and were weighed on a Sartorius 4503 microbalance sensitive to
1.0 µg.
1
692 cm-1. H NMR (500 MHz, C6D6): δ 2.97 (dd, J ) 3.0, 9.5
1H), 3.10 (s, 3H), 3.27 (s, 3H), 3.33 (ddd, J ) 1.5, 6.5, 13.0, 1H),
3.37 (s, 3H), 3.45 (dd, J ) 1.0, 3.0, 1H), 3.46 (s, 3H), 3.50 (dd, J
) 5.5, 9.0, 1H), 3.56 (s, 3H), 3.64 (dd, J ) 7.5, 9.5, 1H), 3.68,
(dd, J ) 7.5, 10.0, 1H), 4.14 (d, J ) 7.5, 1H). 13C NMR (75 MHz,
CDCl3): δ 56.6, 58.2, 59.1, 60.6, 61.1, 70.8, 73.1, 74.9, 80.6, 84.1,
104.5. CI-MS: 251 ([M + H]+, [C11H22O6]H+).
Once the prepared sample pans are loaded in the calorimetric
sensor, temperature and heat flux are stabilized and data acquisition
begins. Five minutes is enough to get a good initial baseline, then
a valve connected to the vacuum pump is opened, and the pressure
inside the vacuum chamber is downloaded quickly to promote the
sublimation process.
During loading, thermal stabilization and pressure change due
to initial evacuation and a small fraction of the substance sublimes
or vaporizes, therefore, the calculation of mass lost is necessary
and was performed by independent experiments as has been
previously described.49,50
Data acquisition and integration of the area under the change of
phase curve were performed using the Pyris software of the DSC7
calorimeter. Knowing the area under the sublimation curve, the
initial mass, the loss of mass during loading, and thermal stabiliza-
tion, the enthalpy of sublimation was obtained using the relation:
General Procedure To Measure the Enthalpies of Disolution
by Heat Flux Calorimetry. Dissolution experiments were per-
formed by heat flux calorimetry, using a differential Setaram C80
Calvet calorimeter working in isothermal mode. The sensors of the
calorimeter are two fluxmeters with a calorimetric resolution of
0.12 µW and a detection limit in power of 2 to 5 µW, which are
assembled inside of a calorimetric block with a temperature control
of at least (0.001 K. An adequate temperature control in the
environmental conditions of our laboratory is attained maintaining
the calorimetric block at 303.15 K, so all the dissolution experiments
were performed at this temperature. For this set of experiments,
stainless steel mixing with membrane vessels was employed,
utilizing masses around 50 mg for each pyranoside and an amount
of 350 to 55 mg of aromatic solvent, which were the largest masses
able to be located in each of the containers of the vessel, in order
to generate a maximal thermal signal. The resulting molar relation
of the carbohydrate:solvent, after the dissolution process, was 1:10.
The masses of the substances involved in a dissolution experiment
with this technique were measured in a MC210 P Sartorius balance
sensitive to 10 µg.
∆Hsub or vap ) change phase area (W‚s)/{initial mass (g) -
lost mass (g)} (3)
The tables providing detailed experimental data of the change
of phase experiments are supplied in the Supporting Information.
There the uncertainty associated to the average value of enthalpy
of sublimation or vaporization of the pyranosides represents the
standard deviation.
Currently the calorimetric holder assembly associated with the
vacuum system and that of the commercial DCS7 are calibrated
for energy and temperature using high-purity samples of indium
and zinc.
NMR Experiments. Modified methyl pyranoside (108 mg, 0.43
mmol) was dissolved in CDCl3 (0.5 mL) and the NMR spectrum
was measured in a 500 MHz spectrometer. After that C6D6 (19.4
µL, 0.42 mmol) was added several times starting from 1:0.5
(carbohydrate/benzene) to get a carbohydrate saturated dissolution
with benzene (see Tables S1 and S2, Supporting Information) and
in each case the spectrum was recorded. NOE difference were done
using the cycling technique51 (cyclenOe) with mixing times of 1300
ms and internal subtraction of data acquired by on-resonance and
off-resonance selective excitation on alternate scans.
Once the disolution vessels are loaded in the fluxmeters of the
Calvet Calorimeter, temperature and heat flux are stabilized by
around 60 min then data acquisition begins. Five minutes is enough
to get a good initial baseline, then the Teflon membrane of the
mixing vessel is broken and reversing of the C80 is performed
several times to promote the disolution process.
Analysis of the amplified dissolution curves, generated by the
data treatment software of the C80 calorimeter, showed that for
the quantity of energy of 7.0 J involved in some of the dissolution
experiments, the transfer of heat finished before 120 min. At the
end of the experiment of dissolution, the difference between the
initial and final baseline is not more than 0.01 mW, which
introduces a maximal uncertainty in the measurement of the area
under the measured curve of (0.069 J. The C80 calorimeter works
at constant pressure, consequently the integration of the curve of
heat flux as a function of time releases directly the enthalpy of
dissolution of each carbohydrate in the respective aromatic solvent.
In Table 1 the uncertainty associated with the average value of
enthalpy of dissolution represents the standard deviation. The tables
including all the experimental data involved in the dissolution
experiments are provided in the Supporting Information.
Enthalpies of Sublimation and Vaporization by Differential
Scanning Calorimetry. The calorimetric measurements of enthal-
pies of sublimation or vaporization of the carbohydrates were
performed using the isothermal mode operation of a modified DSC7
calorimeter.49,50
The sensitive element of this device is a Perkin-Elmer DSC7
calorimetric holder assembly within a vacuum chamber connected
to the DSC7 control device by an electrical feed. The vaporization
system is evacuated with a rotary vacuum pump and residual
pressure inside of the chamber is monitored by a pressure gauge,
which is relayed to a Pirani gauge control. The Perkin-Elmer
commercial aluminum open standard pans were utilized as sublima-
tion cells. A working temperature of 333.15 K was established as
the most recommendable from preliminary experiments.
Acknowledgment. L.B.I. acknowledges Conacyt for finan-
cial support. We also thank Francisco Ginez Carbajal for his
technical assistance for the disolution experiments. We are
grateful to the reviewers for their useful comments that led to
the improvement of this paper. This work was supported by
Consejo Nacional de Ciencia y Tecnolog´ıa (CONACYT) for
financial support via grants 49921-Q, P47679-Q, and DGAPA
grant IN-209606. We are grateful to Rebeca Lo´pez-Garc´ıa for
the revision of the English version of this manuscript.
Supporting Information Available: 1H NMR chemical shifts
(ppm) of compounds 1 and 2 (Tables S1-S2, pp S4 and S5),
enthalpies of solution of compounds 1 and 2 in solvents 3-9 (Tables
S3-S15, pp S6-S18), and enthalpies of Vaporization of com-
pounds 1 and 2 (Tables S16-S17, pp S19-S20), 1H NMR chemical
shifts (ppm) of methyl acetate and mixtures of benzene and
chloroform (Table S18, p S21), 1H NMR chemical shifts (ppm) of
compounds 1 and 2 of benzene-d6 and chloroform-d (Table S18, p
S22), Sidgwick’s temperature correction (p S23), and 1H, 13C,
NOESY, COSY, HMBC, and HSQC 500 MHz NMR spectra of
compounds 1 and 2. This material is available free of charge via
the Internet at http://pubs.acs.org.
JO701926R
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