Experimental and theoretical evaluation on the conformational behavior of l-aspartic acid dimethyl ester and its N-acetylated derivative

Article abstract of DOI:10.1039/c4ra14480e

In this work the conformational preferences of l-aspartic acid dimethyl ester (AspOMe) and its N-acetylated derivative (AcAspOMe) were evaluated through spectroscopic data and theoretical calculations. Unlike amino acids, their corresponding amino ester derivatives do not exhibit a zwitterionic structure and are soluble in most organic solvents, enabling their studies in these media. Thus, the conformers of AspOMe and AcAspOMe were theoretically determined both in isolated phase and in solution (IEF-PCM model) at the ωB97X-D/aug-cc-pVTZ level. A joint analysis of the experimental and theoretical ³J_HH coupling constants in several aprotic solvents allowed assigning the most stable conformers, showing excellent agreement between these approaches. Also, IR spectroscopy allowed us to obtain quantitative data on AcAspOMe conformer populations in different solvents. Natural bond orbital (NBO) analysis indicated that both steric and hyperconjugative contributions count in determining the relative conformer stabilities of these compounds. Intramolecular hydrogen bonding, characterized by Quantum Theory of Atoms in Molecules (QTAIM) and Non-Covalent Interactions (NCI) methodologies, represents only a secondary factor to drive the stabilities of AspOMe and AcAspOMe conformers. This journal is

Full text of DOI:10.1039/c4ra14480e

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Experimental and theoretical evaluation on the
conformational behavior of L-aspartic acid
dimethyl ester and its N-acetylated derivative†
Cite this: RSC Adv., 2015, 5, 18013
a
b
a
*
Carolyne B. Braga, Lucas C. Ducati and Roberto Rittner
In this work the conformational preferences of L-aspartic acid dimethyl ester (AspOMe) and its N-acetylated
derivative (AcAspOMe) were evaluated through spectroscopic data and theoretical calculations. Unlike
amino acids, their corresponding amino ester derivatives do not exhibit a zwitterionic structure and are
soluble in most organic solvents, enabling their studies in these media. Thus, the conformers of AspOMe
and AcAspOMe were theoretically determined both in isolated phase and in solution (IEF-PCM model) at
3
the uB97X-D/aug-cc-pVTZ level. A joint analysis of the experimental and theoretical JHH coupling
constants in several aprotic solvents allowed assigning the most stable conformers, showing excellent
agreement between these approaches. Also, IR spectroscopy allowed us to obtain quantitative data on
AcAspOMe conformer populations in different solvents. Natural bond orbital (NBO) analysis indicated
that both steric and hyperconjugative contributions count in determining the relative conformer
stabilities of these compounds. Intramolecular hydrogen bonding, characterized by Quantum Theory of
Atoms in Molecules (QTAIM) and Non-Covalent Interactions (NCI) methodologies, represents only a
secondary factor to drive the stabilities of AspOMe and AcAspOMe conformers.
Received 13th November 2014
Accepted 4th February 2015
DOI: 10.1039/c4ra14480e
www.rsc.org/advances
in experimental studies of isolated amino acids are caused by
their high melting points and associated low vapor pressures.
1. Introduction
They also have low thermal stability, so they tend to decompose
before melting.6
Amino acids and their derivatives have been under investigation
for decades because of the numerous functions that these
compounds can perform. Among these studies, a large number
is addressed to investigate their conformational preferences,1–3
because these are directly related to the interactions that occur
in biological environments and, consequently, with their func-
tions.4,5 However, although signicant advances have occurred
in both computational and spectroscopic methods, it is still
critical to obtain this type of structural information both in
isolated phase and in solution.
In one way, the remarkable conformational exibilities of
amino acids and the amount of possible intramolecular inter-
actions that can be established between their different func-
tional groups give rise to a large number of low-energy
conformers. Thus, gas phase studies are essential, since they
provide a unique opportunity to understand the conformer
structures as well as their intrinsic properties (such as intra-
molecular interactions) without the interference of neighboring
molecules or the solvent. Nevertheless, the obvious difficulties
In contrast, exploring the conformational preferences of
amino acids in solution is essential for a better understanding
of their behavior in biological systems. Indeed, a substantial
increase in the number of theoretical studies about solvation of
amino acids has been recently published.7,8 Notwithstanding,
experimental studies in solution are hindered by the fact that
these compounds have low solubility in organic solvents,
resulting in an additional barrier for experimental studies,
including NMR spectroscopy.
In order to overcome the aforementioned difficulties, an
alternative approach proposed by our group is to analyze their
esteried and N-acetylated derivatives,9–11 which, unlike amino
acids, are soluble in most organic solvents. Moreover, these
compounds properly simulate the amino acid residue in a
polypeptide chain or protein.
Although several studies have been dealt with the confor-
mational preferences of glycine and alanine, amino acids with
longer side chains have been less explored, since they present a
more complex behavior, such as a larger number of low energy
conformers. Specically, few studies have been performed with
L-aspartic acid and their derivatives,1,7 despite their biological
importance. There is no doubt that the conformation of resi-
dues of this amino acid contributes signicantly to the three
dimensional shape of polypeptides and proteins, and can
aPhysical Organic Chemistry Laboratory, Chemistry Institute, University of Campinas,
P.O. Box 6154, 13083-970, Campinas, SP, Brazil. E-mail: rittner@iqm.unicamp.br
bChemistry Institute, University of Sao Paulo, 05508-900, Sa
̃o Paulo, SP, Brazil
† Electronic supplementary information (ESI) available: Experimental and
theoretical evaluation on the conformational behavior of L-aspartic acid
dimethyl ester and its N-acetylated derivative. See DOI: 10.1039/c4ra14480e
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change their structures and functions in an active biological
system.
2.4. Theoretical calculations
The conformational investigation to nd the possible energy
minima of AspOMe was performed starting from the six
conformers (I, III, IV1, IV2, V1 and V2) previously obtained for
alanine methyl ester (AlaOMe),9 which have the less energetic
arrangement of the backbone [CH3–O–C(O)–CH(NH2)–]. To this
end, a methyl hydrogen atom (side chain) of AlaOMe was
replaced by the C(O)–O–CH3 group, giving rise to the side chain
of aspartic acid. Thus, six potential energy surfaces (PES, Fig. S1
in the ESI†) were built from these six new geometries
by simultaneously scanning the c1 [C(O)–C–C–C(O)] and c2
(O]C–C–C) dihedral angles (Fig. 1a) in 36 steps from 0ꢀ to 360ꢀ,
at B3LYP/cc-pVDZ level. In this stage, the earlier optimized f
[nN–N–C–C(O)] and j [N–C–C]O] dihedral angles were kept
xed to preserve the optimized geometry of the main chain.
The 34 minima resulting from the PES were subsequently
fully reoptimized using DFT different functionals (B3LYP,16
CAM-B3LYP,17 M05-2X,18 M06-2X,19 B97-D20 and uB97X-D21)
with the aug-cc-pVTZ basis set, and their harmonic frequencies
were calculated and also zero-point energy (ZPE) correction. The
obtained data were referenced to the single point calculations at
the MP2/aug-cc-pVTZ level of theory. Some conformers were
discarded, since they: (i) have imaginary harmonic frequencies
(are not true energy minima) or (ii) do not present signicant
contribution to the conformational equilibrium of the
compound in isolated and including the solvent effect, result-
ing in 8 stable conformers for AspOMe.
Another aspect that is worth mentioning is the lack of
detailed evaluation about the driving effects responsible for the
conformational preferences of this kind of compounds, since
they are limited to identify the number of conformers and their
relative energies. Moreover, it is surprising that only intra-
molecular hydrogen bonding (IHB) is taken into account, while
recent studies indicated that the balance between steric and
hyperconjugative effects, and not just IHB, are responsible for
the conformer stabilities of the amino acids.12–14
Therefore, this study is aimed to investigate the conforma-
tional preferences of L-aspartic acid dimethyl ester (AspOMe)
and its N-acetylated derivative (AcAspOMe) in the isolated phase
as well as in several aprotic solvents and to evaluate the intra-
molecular interactions responsible for the stabilities of the
most stable conformers. For this purpose, theoretical and
experimental 3JHH coupling constants were used in the analysis
of their conformational equilibra. Also, infrared data were
employed as a complement to determine AcAspOMe pop-
ulations. Quantum Theory of Atoms in Molecules (QTAIM),
Non-Covalent Interactions (NCI) and Natural Bond Orbital
(NBO) analysis have also been carried out for the interpretation
of the obtained results.
2. Experimental section
2.1. Synthesis of the compounds
Aer, these lowest energy geometries found for the amino
AspOMe was commercially available as a hydrochloride and was ester were used as starting points to determine the AcAspOMe
deprotonated using activated zinc powder.15 The synthesis of conformers. For each previously optimized AspOMe geometry,
AcAspOMe consisted in the esterication of N-acetyl-L-aspartic the N-acetyl group was added by replacing one of the hydrogen
atoms of the amine group, giving rise to an amide linkage, and
thus eight potential energy curves (PEC) were obtained by
rotating the q [C–C(]O)–N–C] dihedral angle (Fig. 1b), at the
B3LYP/cc-pVDZ level. Each PEC presented two stereoisomers
(cis and trans); hence the sixteen minima geometries found were
fully reoptimized at the uB97X-D/aug-cc-pVTZ level, which
showed, for AspOMe, appreciable correlation with the MP2 one,
and their frequencies were calculated with ZPE correction.
Thereaer, the resulting conformers of both compounds
were fully optimized by using the IEF-PCM (Integral Equation
Formalism Polarizable Continuum Model)22 in aprotic solvents
of different dielectric constants, at the uB97X-D/aug-cc-pVTZ
acid. The detailed procedures are described in the ESI.†
2.2. NMR spectra
1H NMR spectra were recorded on a Bruker Avance III spec-
trometer operating at 600.17 MHz for 1H. Spectra were
obtained using solutions of ca. 15 mg in 0.7 mL of deuterated
solvents (C6D6, CDCl3, CD2Cl2, acetone-d6, CD3CN and
DMSO-d6), referenced to internal TMS. The typical conditions
used were: probe temperature of 25 ꢀC, 16 transients,
spectral width around 6.0 kHz and 64k data points with an
acquisition time of ca. 6 s. The free induction decays (FID)
were zero-lled to 128k, providing a digital resolution of 0.09
Hz per point.
3
level. Also, from these IEF-PCM calculations the JHH coupling
2.3. Infrared spectra
Infrared spectra were recorded on a Shimadzu FT-IR Prestige-
21 spectrometer using samples with a concentration of 0.03
mol Lꢁ1 and a NaCl cell with an optical path of 0.5 mm. The
solutions were prepared with solvents of different polarities:
CCl4, CHCl3, CH2Cl2 and CH3CN. The spectra were acquired
with 64 scans and resolution of 1 cmꢁ1. The overlapped
carbonyl bands were deconvoluted by means of the GRAMS
curve tting program. The equipment was purged with dry
nitrogen gas.
Fig. 1 Analyzed dihedral angles: (a) c1 [C(O)–C–C–C(O)] and c2
(O]C–C–C) for AspOMe and (b) q [C–C(]O)–N–C] for AcAspOMe.
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Fig. 2 Spacial representation of the most stable conformers of AspOMe, defined by the dihedral angles c1 and c2 and obtained at the uB97X-D/
aug-cc-pVTZ level of theory. The conformers are ordered according to their relative energies in isolated phase.
which was used as the reference. However, the energies varied
considerably with the theoretical level used. On the one hand,
the popular B3LYP, as well as CAM-B3LYP, showed the most
discrepant values related to the MP2, but a good correlation was
obtained for the uB97X-D functional compared with the refer-
ence, which has also been the method used in a previous work
of the corresponding derivatives of the amino acid L-proline.10
Fig. 3 Newman projections showing the dispositions a, b and c of the
side chain, resulting from rotation around the Ca–Cb bond.
Therefore, uB97X-D, which includes empirical atom–atom
dispersion corrections important to describe non-covalent
interactions (such as hydrogen bonding), was used as the
main computational method for the subsequent calculations.
The geometries of the eight most stable conformers involved
in the conformational equilibrium of the AspOMe (at uB97X-D/
aug-cc-pVTZ level of theory) are shown in Fig. 2. Each conformer
was named with a roman numeral followed by the letters a, b or
c. The numbers indicate the order of stability in isolated phase
(considering the relative energies with ZPE at uB97X-D) and the
letters represent the relationship between side and main
chains, which are depicted on the Newman projections of Fig. 3.
The relatively large number of conformers is due to the
presence of a polar side chain (R ¼ –CH2COOCH3), which can
act as a proton acceptor, multiplying the possible combinations
of intramolecular interactions that can be established between
the different functional groups. Of these, the conformer Ia is the
most stable for the isolated compound and accounts for 30.4%
constants were obtained for each conformer using the uB97X-D
functional and the EPR-III23 basis set for the hydrogen and
carbon atoms, whereas oxygen and nitrogen were represented
by aug-cc-pVTZ basis set, since this level reproduces JHH with
good accuracy.24
Lastly, natural bond orbital (NBO)25 analysis was performed
on the same uB97X-D/aug-cc-pVTZ level, as well as QTAIM and
NCI calculations. All calculations employed the Gaussian09
program package, Revision D.01,26 excepting the QTAIM and
NCI, which were carried out with AIMALL27 and NCIPLOT 3.0
(ref. 28) programs, respectively.
3
3. Results and discussion
3.1. AspOMe
The eight most stable geometries found in the PES (Fig. S1 in of the conformational population. However, the conformer IIa
the ESI†) were optimized using different DFT methods and ab has also a relatively large stability (26.8%) compared to the
initio MP2 with aug-cc-pVTZ basis set. A complete comparison remaining conformers, since together with Ia represents more
between the energies, relative energies and investigated dihe- than half of the conformational equilibrium of AspOMe in
dral angles obtained using these different methods is shown in isolated phase. The others conformers have similar energies
Table S1 (ESI†). It was observed that all methods gave values of and they contribute much less to the overall conformational
the dihedral angles very close to the ones from the MP2 method, population.
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Fig. 4 QTAIM molecular graphs for the AspOMe conformers, obtained at the uB97X-D/aug-cc-pVTZ level of theory. Dotted lines represent the
bond paths (BPs), the green dots represent the bond critical points (BCPs) and the ring critical points (RCPs) are indicated by red dots.
Inspection of conformers Ia and IIa shows a great similarity Nevertheless, it can be stated that this interaction has a small
in their spatial arrangements, with values of about 60ꢀ and contribution to the stability of VIb and VIIIa, given their high
0ꢀ for the dihedrals c1 and c2, respectively. The only structural relative energies.
difference between these two most stable conformers is the
Although the QTAIM is a widely used method for charac-
dihedral j [N–C–C]O], which is syn for the former and anti for terizing IHB, some studies have shown that it fails in detecting
the latter. Assuming that these conformers are almost iso- some weak interactions.31,32 Moreover, unlike Bader, other
energetics, it can be concluded, therefore, that this dihedral reports in the literature also indicate that BP not necessarily
does not affect their stabilities. Then, which effects are related
with the observed conformational preferences?
When the conformational equilibra of amino acids and their
Table
1 QTAIM topological parameters obtained for AspOMe
derivatives are described in literature, rares are the examples
that do not explain the observed conformational stabilities in
terms of intramolecular hydrogen bonding (IHB). However,
recent works published by our research group9,10 have shown
that, in many cases, other interactions are responsible for the
conformational preferences, as hyperconjugative and steric
effects. Therefore, to identify the intramolecular effects that
occur in this isolated system, NBO calculations and QTAIM and
NCI topological analyses were performed.
The molecular graphs obtained by the QTAIM (Fig. 4) char-
acterize an IHB in conformers Ia, IIa, VIb and VIIIa, which show
a bond critical point (BCP) and a bond path (BP). According to
Bader, the presence of BCP and BP are necessary and sufficient
conditions for two atoms to be considered chemically bonded.29
In addition, the topological criteria30 were also evaluated (Table
1), conrming that the IHB exhibited by QTAIM are stable.
conformers [electron density (rHBCP) and Laplacian of the electron
density (V2rHBCP) at the hydrogen bond BCP and integrated atomic
properties of the atom H7a] calculated at the uB97X-D/aug-cc-pVTZ
level of theory
rHBCP V2r HBCP q(H7)
E(H7)
M1(H7) V(H7) 3HBCP
b
Ia
0.0111 +0.044
0.0127 +0.051
+0.374 ꢁ0.4804 0.173
+0.379 ꢁ0.4784 0.172
+0.355 ꢁ0.4879 0.187
+0.379 ꢁ0.4775 0.173
+0.366 ꢁ0.4850 0.176
29.02 0.6507
28.32 0.3716
IIa
Vcc
VIb
—
—
32.43
—
0.0116 +0.045
28.59 0.6575
29.35 0.5565
VIIIa 0.0114 +0.047
a
The numbering of atoms is in Fig. 2; q (U) is the atomic charge, E (U) is
the total energy, M (U) is the dipolar polarization and V (U) is the atomic
volume of given atom. b rHBCP, V2rHBCP, q(H7), E(H7), M1(H7) and V(H7)
c
in atomic units. The conformer Vc does not form an IHB and,
therefore, the QTAIM properties of H7 of this conformer can be used
as reference to evaluate IHB in the others.
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Table 2 Relative total energy of the system (Erel,Tot)a, relative energy of
the steric (Erel,Lewis) and hyperconjugative (Erel,Hyper) interactions for the
AspOMe conformers, calculated at uB97X-D/aug-cc-pVTZ level of
theory
IIa. In turn, the conformers b are those with the lowest values of
Erel,Lewis (the carboxyl groups are on opposite sides, in order to
reduce their steric hindrances), but also the lowest Erel,Hyper
.
Therefore, these results show that an interplay between steric
and hyperconjugative effects determines the conformational
preferences of this compound, and the conformers Ia and IIa
present the most favorable energy balances, contributing to
their stabilities in isolated phase.
Ia
IIa
IIIb
IVb
Vc
VIb
VIIb
VIIIa
Erel,Tot
Erel,Lewis
Erel,Hyper
0.00
5.67
7.12
0.15
6.94
8.25
0.63
0.27
1.10
1.10
0.00
0.36
0.93
4.60
5.13
1.37
3.59
3.67
1.50
0.05
0.00
1.42
7.24
7.27
The energies of the hyperconjugative interactions which
most contribute to Erel,Hyper were also analyzed from the NBO
calculations (Table S2 in the ESI†). Although there are small
energy variations, no major differences are observed among the
a
Relative energies in kcal molꢁ1
.
represent a bonding situation.33 Thus, as complement to the conformers. So, a specic interaction cannot be pointed as the
results obtained by Bader's theory (QTAIM), the recently devel- only responsible for stabilizing a given geometry.
oped NCI analysis was also used, which is based on data of
It was also veried the presence of charge transfer interac-
electron density gradient and not only in punctual density tions between LP2(O17) / s*N5–H7 in Ia and IIa, as well as
values, as QTAIM. The plots of the reduced density gradients LP2(O2) / s*N5–H7 in IIIb, characteristics of intramolecular
[s(r)] versus sign (l2)r(r) (Fig. S1 in the ESI†), as well as the hydrogen bonding (IHB). Their energies are 0.50, 0.72 and 0.85
gradient isosurfaces (Fig. S2 in the ESI†), were obtained for all kcal molꢁ1 in the isolated phase, respectively. However, no
conformers of AspOMe. The negative values of sign (l2)r(r) hyperconjugation with energy above 0.50 kcal molꢁ1 was
shown in the Fig. S1† are an indication of attractive interac- observed in conformers VIb and VIIIa.
tions, in this case the N–H/O IHB, which are spotted in blue
Then, the evaluation of the rotational isomerism taking into
color on gradient isosurfaces. There are also positive values of account the solvent effects was also performed using a joint
sign (l2)r(r), which characterize repulsive interactions and are analysis of NMR spectroscopy and theoretical calculations. It is
marked in red color on isosurfaces, and lastly, van der Waals well known that the vicinal spin–spin coupling constant (3JHH
)
interactions (green) are related to values of sign (l2)r(r) near exhibits a dihedral angle dependence and, therefore, allows us
zero. Therefore, the NCI analysis conrmed the occurrence of to examine the conformational preferences of different
IHB in conformers Ia, IIa, VIb and VIIIa and, also, it revealed a compounds.33 For amino acid residues, specically, such
weak interaction in IIIb, which was not identied by QTAIM, constant can help in the identication of the most stable
providing more reliable results.
conformers by relating the experimental 3JHH with the dihedral
It is worth mentioning that for the conformations with IHBs, angles of the backbone and side chains. However, due to the
BSSE values could also be considered. However, although fast interconversion rate between the conformers, the experi-
3
signicant intramolecular BSSE effects can affect the single mentally observed JHH,obs in solution is a weighted average of
molecules, where there can be noncovalent interactions the contribution of each conformer i (hix3JHH,i), which can be
between different parts of the molecule, studies have shown estimated through the following equation:
that larger basis sets are used as an approximation to correct
intramolecular BSSE.34 Therefore, the basis set applied in this
work, the aug-cc-pVTZ, can be considered large enough to
3
Table 3 Experimental 1H NMR chemical shift (in ppm) and JHaHb
coupling constant (in Hz)a values for the AspOMe compound obtained
in different solvents
include these BSSE effects. Other attempt to correct the intra-
molecular BSSE is made through using the counterpoise (CP)
correction (by dividing the molecule into fragments). The main
problem with this simplication of the system is that there is no
unique way to dene the fragments.35–37 Thus, based on arbi-
trariness regarding the appropriate correction procedure for the
intramolecular BSSE, we nd more suitable to use a larger basis
set instead using CP correction.
Next, NBO analysis was performed in order to nd the
contributions of steric and hyperconjugative effects for the
energy of each conformer. According to the results of the Table
2, the order of destabilization in relation to the steric effects is:
IVb < VIIb < IIIb < VIb < Vc < Ia < IIa < VIIIa. It is evident from
these relations that the conformers a are those exhibiting the
greatest steric effects, which is consistent, because they have
three bulky groups targeted to the same region of space (Fig. 3).
Although the conformers a are the most destabilized forms by
steric effects, they are also the most stabilized due to hyper-
conjugations, as follows: VIIb < IVb < IIIb < VIb < Vc < Ia < VIIIa <
3
3
Solvent
3b
dNH dHa dHb1 dHb2 dHc dHd JHaHb1 JHaHb2
C6D6
CDCl3
CD2Cl2
Acetone-d6 20.7
CD3CN
DMSO-d6 46.7
2.3
—
3.89 2.88 2.76 3.25 3.28 4.2
4.8 4.54 4.26 3.21 3.12 3.73 3.80 4.2
4.13 3.13 3.08 3.72 3.79 4.1
4.17 3.23 3.11 3.68 3.76 4.8
37.5 3.65 4.05 4.03 3.98 3.67 3.73 4.8
6.0
5.6
5.8
5.2
5.7
6.3
9.1
—
—
—
3.80 3.73 2.68 3.60 3.64 6.3
a
b
Error in the measurements of J ¼ ꢂ0.05 Hz. Dielectric constant.
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P
3JHH,obs
¼
hix3JHH,i
where hi represents the molar fraction of conformer i in the
3
conformational equilibrium and JHH,i the intrinsic coupling
constant of conformer i. Moreover, as 3JHH,obs is directly related
to the conformer populations, which, in turn, can vary
depending on the medium, it is possible to evaluate the solvent
effect on the conformational preferences of the compounds
through JHH,obs. In this sense, 1H NMR spectra for AspOMe
3
were acquired in aprotic solvents of nonpolar (C6D6, CDCl3 and
CD2Cl2) and polar (acetone-d6, CD3CN and DMSO-d6) nature
3
with different dielectric constants (3), and the JHaHb coupling
constants measured are shown in Table 3.
3
The values listed show that the experimental JHaHb1 and
3JHaHb2 values vary with the change of solvent indicating,
therefore, changes in the conformer populations. In nonpolar
solvents, the diastereotopic hydrogens (Hb1 and Hb2) are at very
different chemical environments and couple with the alpha-
3
hydrogen (Ha) with different values of JHaHb. By increasing
the solvent polarity, on the other hand, such constants of Hb1
and Hb2 with Ha exhibit closer values, until in DMSO-d6, the
solvent used of higher dielectric constant, they become equal
(3JHaHb1
¼
3JHaHb2 ¼ 6.3 Hz). Considering that the rotational
isomerism of amino acids is usually composed by forms a, b
and c, shown in the Newman projections of Fig. 3, the analysis
of these three possible side chain arrangements in relation to
the main chain reveals that the geometry a shows only gauche
dispositions between hydrogens Ha and Hb, while one
arrangement anti and one gauche are observed for conforma-
tions b and c. For this reason, it is expected that the 3JHaHb1 and
3JHaHb2 values are greater in c and b, respectively, than in a,
since anti hydrogens present spin–spin coupling constants
larger than gauche hydrogens. Thus, it can be suggested that in
solvents with higher dielectric constants there would be a
stabilization of arrangements b and c in relation to the forms a,
explaining the fact that the experimental vicinal coupling
constants have shown larger values.
In order to understand and explain in more detail these
assumptions, theoretical calculations with solvent inclusion
using the IEF-PCM model were also performed (Table 4) and
show that the minimum Ia remains as the most stable, and the
increase of dielectric constant of the solvent causes a subtle
stabilization in this geometry. At the same time, there is a
destabilization of IIa (from 26.8% for the isolated phase to
14.3% in DMSO), which becomes almost isoenergetic with IVb
in polar solvents, being the third more stable in solution. Thus,
since these three geometries represent about 60% of these
3
conformational equilibria of AspOMe, the experimental JHaHb
values are mainly due to these three conformers. Small varia-
tions in populations are exhibited by the other conformers,
verifying that their energies become even closer to each other
with the increase of solvent polarities.
3
The JHaHb,i coupling constants for the individual
conformers were calculated (Fig. 5a), as well as the estimated
3
contributions of each conformer (hix3JHaHb,i) to JHaHb1 and
3JHaHb2 (Fig. 5b) experimentally obtained (see also these values
in Table S3 and S4 in the ESI†, respectively). Fig. 5a shows that
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Fig. 5 (a) Individual coupling constants 3JHaHb,i for the AspOMe conformers and (b) conformational contributions (hix3JHaHb,i) for the observed
3JHaHb,obs coupling constants, calculated at the uB97X-D/EPR-III level of theory, by using the IEF-PCM in several solvents. The values are
presented in Hz.
3
variations in JHaHb,i of each conformer are insignicant, but the weakening or even breaking of an IHB due the solvation
the results of Fig. 5b support the changes in experimental carried by the solvent. The same is true for the dipole repulsion.
3
3JHaHb. In nonpolar solvents, the experimental JHaHb1 is about So, the conformer interactions in solution were also checked.
4.2 Hz, in agreement with the individual values of this constant Table S5 (in the ESI†) shows that the conformer Ia undergoes a
for conformers Ia and IIa (approximately 3.3 and 3.7 Hz,
respectively), which are among the main responsible for the
observed values. In more polar solvents, although these
conformers again exhibit the largest contributions to the
experimental values, there is a secondary factor that causes the
increase of the experimental 3JHaHb. In this case, the increase in
the polarity of the medium decreases the difference among the
conformer energies, especially those whose forms are b and c.
3
Therefore, it was found that the experimental JHaHb values are
in agreement with the theoretical results, and the combined use
of theoretical calculations and NMR data allowed showing the
inuence of the solvent on the conformational equilibrium of
AspOMe.
One fact that called our attention was the inuence of the
solvent on the relative order of stabilities, mainly for IIa and
IIIb, since the latter is the third most stable in isolated phase
and becomes the most energetic in DMSO. Although IHB and a
balance between hyperconjugative and steric effects are the
factors governing the conformational preferences of this
compound in isolated phase, it is known that intramolecular
AcAspOMe, defined by the q [C–C(]O)–N–C] dihedral angle and
effects can be disturbed in solution. A well known example is obtained at the uB97X-D/aug-cc-pVTZ level of theory.
Fig. 6 Spacial representations of the most stable conformers of
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Table
Paper
5
Conformer energiesa, relative energies, populations and
as well as in isolated phase, the joint contribution of hyper-
conjugations and steric effects accounts for its greater stability
in solution. This is also valid for other conformers, whose
relative energies resulting of stabilizing and destabilizing
contributions, namely Erel,Tot, exhibit exactly the same trend
observed in Table 4.
Thus, based on the data presented for the AspOMe, one can
say that both in isolated phase and solution the conformational
preferences are governed by a balance between steric and
hyperconjugative interactions and, as a side effect, the IHB.
investigated dihedral anglesb in isolated phase for the most stable
conformers of AcAspOMe, optimized using uB97X-D/aug-cc-pVTZ
level of theoryd
Parameters
trans-Ia
trans-IIIb
trans-Vc
E (hartrees)
ꢁ743.42574
0.00
ꢁ743.42060
3.23
ꢁ743.42052
3.28
Erel (kcal molꢁ1 c
P (%)
)
100.0
3.8
0.0
0.6
0.0
4.9
u [O]C1–O–C]
j [N–C–C]O]
c1 [C(O)–C–C–C(O)]
c2 [O]C16–C–C]
q [C–C(]O)–N–C]
10.3
56.7
17.8
22.3
173.6
55.7
142.9
70.7
32.0
3.2. AcAspOMe
174.9
169.0
172.8
A similar study to that performed for AspOMe was conducted
for the AcAspOMe, its N-acetylated derivative. The three more
stable conformers of sixteen minima found from PEC are shown
in Fig. 6. Their energies, populations and geometric parameters
are presented in Table 5. The other conformers will be not
mentioned in this discussion, since they have negligible pop-
ulations (less than 5%) both in isolated phase and solution.
The name of each conformer was derived from the respective
AspOMe starting geometry, including the cis–trans
a
b
c
ZPE correction was included. Dihedral angles in degrees. 1 au ¼
627.5095 kcal molꢁ1
.
The numbering of atoms is in Fig. 6.
d
small stabilization in more polar solvents, contrary to expecta-
tions, which would be increase its energy because weakening (or
even the rupture) of the IHB. As much as its energy difference of
steric repulsion has increase according the solvent polarity, the
same was observed for the stabilizing energy (Erel,Hyper). That is,
Fig. 7 (a) QTAIM molecular graphs, (b) NCI isosurfaces generated with s ¼ 0.5 au and blue-green-red scaling from ꢁ2 au < (l2)r(r) < 2 au, and (c)
NCI plots of the reduced density gradients [s(r)] versus sign (l2)r(r) for the AcAspOMe conformers, obtained at the uB97X-D/aug-cc-pVTZ level
of theory.
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Table 6 Relative total energy of the system (Erel,Tot)a, relative energy of
the steric (Erel,Lewis) and hyperconjugative (Erel,Hyper) interactions for the
AcAspOMe conformers, calculated at uB97X-D/aug-cc-pVTZ level of
theory
(Fig. 7b), obtained by NCI method, a blue volume between the
carbonyl oxygen (side chain) and hydrogen attached to
nitrogen, representing an attractive non-covalent interaction,
which corresponds to an IHB. The red volume near to this blue
volume appears as consequence of the formation of the ve-
membered ring, and can be related to the RCP of QTAIM.
These two types of interactions encompassing a single volume
in isosurface suggest a weak IHB in this conformer, explaining
the fact of not having been detected by QTAIM. Moreover, the
peak at negative sign (l2)r(r) (ꢁ0.018 au) in Fig. 7c indicates also
the presence of an IHB. In accordance with the QTAIM, the NCI
analysis shows no IHB in conformers trans-IIIb and trans-Vc.
The contribution of the steric repulsion and hyper-
conjugative effects in the conformational preferences of the
studied compound were evaluated through the NBO analysis.
According to the data shown in Table 6, it appears that, as
occurred for the AspOMe, a balance between these effects is
crucial to the order of stability. Although the conformer trans-
IIIb is the one that suffers minor steric repulsion (Erel,Lewis), it is
also the geometry less stabilized by hiperconjugations
(Erel,Hyper). The opposite is valid for trans-Ia and trans-Vc.
Therefore, it was veried that the conformer trans-Ia has the
resulting more favorable energetic balance in isolated phase,
giving its greater stability. Furthermore, the severe increase of
steric repulsion in conformers of AcAspOMe compared to the
AspOMe was remarkable from NBO analysis.
trans-Ia
trans-IIIb
trans-Vc
Erel,Tot
Erel,Lewis
Erel,Hyper
0.00
16.18
20.00
3.81
0.00
0.00
3.59
16.25
16.47
a
Relative energies in kcal molꢁ1
.
denomination, related to the rotation of q [C–C(]O)–N–C]
dihedral angle (Fig. 1b), which indicates the position of the
amide methyl group with respect to the C(O)–OCH3. Therefore,
the most stable conformers of AcAspOMe are trans, of which the
trans-Ia is more stable than the second less energetic (trans-
IIIb) by 3.23 kcal molꢁ1, corresponding to a population of about
100% of the conformational equilibrium, in isolated phase. It is
noteworthy that this conformer presents a spatial arrangement
very similar to the most stable conformer of AspOMe (Ia, Fig. 2),
whose structural difference occurs only in small deviations of
the angles due the additional presence of the acetyl group. So,
this additional group in the N-acetylated derivative increases
the steric repulsion interactions, disadvantaging almost all
conformers, except the trans-Ia.
Although steric disturbance in AcAspOMe is signicant
compared to the AspOMe, other effects, such as IHB and
hyperconjugation, are also likely to be important contributions
to its observed conformational preferences. In this sense, these
interactions were analyzed by QTAIM, NCI and NBO analysis.
The molecular graphs (Fig. 7a) show no BP or BCP referring
to an IHB, indicating the absence of this interaction by QTAIM
methodology. However, it is observed in trans-Ia isosurface
In order to assess the conformational variations induced by
1
solvent effects, the AcAspOMe H NMR parameters were recor-
ded in several aprotic solvents (Table 7). There are meaningful
3JHaHb value variations with the change of solvent, suggesting
that the conformational populations are also varied. Indeed,
when solvents were included in the calculations (Table 8),
changes in the populations were noted. These variations are in
accordance with the dipole moment of each conformer, since
Table 7 Experimental 1H NMR chemical shifts (in ppm) and 3JHaHb coupling constants (in Hz)a values for the AcAspOMe compound obtained in
different solvents
Solvent
3b
2.3
4.8
9.1
20.7
37.5
46.7
dNH
dHa
dHb1
dHb2
dHc
dHd
dHe
3JHaHb1
3JHaHb2
3JHaH(N)
C6D6
CDCl3
CD2Cl2
Acetone-d6
CD3CN
DMSO-d6
6.28
6.51
6.45
7.43
6.84
8.37
4.90
4.86
4.81
4.79
4.71
4.61
2.80
3.04
2.98
2.84
2.80
2.78
2.72
2.86
2.82
2.81
2.77
2.69
3.28
3.77
3.73
3.67
3.67
3.62
3.21
3.70
3.67
3.63
3.64
3.61
1.49
2.04
1.98
1.92
1.89
1.83
4.5b
4.3
4.6
5.9
6.0
6.0
4.7
4.5
4.6
5.9
5.6
7.3
8.1
7.9
8.1
8.1
8.1
7.8
a
b
Error in the measurements of J ¼ ꢂ0.05 Hz. Dielectric constant.
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those with the highest values (trans-IIIb and trans-Vc) are the
most stabilized in more polar solvents. At the same time, the
trans-Ia, with the lowest dipole moment, was destabilized as the
solvent becomes more polar. Although such destabilization, the
conformer trans-Ia remains as the most stable, even in DMSO
(about 72%), resulting from a more favorable energy balance
due to the joint effect of stabilizing (hyperconjugation) and
destabilizing (steric effects) energies (Table S6†).
Besides 3JHaHb, the coupling constant 3JHH H–N–Ca–H can be
observed in spectra, which provides important information on
the value of the dihedral angle between these atoms. However,
3
the experimental value of JHaH(N) (Table 7) is practically
constant with the change of the medium, which was expected,
since the three conformers have similar H–N–Ca–H dihedral
angles. Although these results are not able to indicate the
predominance of one conformer, they conrm the agreement
between experimental and theoretical data.
The carbonyl region of the infrared spectra for AcAspOMe is
shown in Fig. 8. The experimental curve was deconvoluted in
two bands, verifying the larger predominance of a component
in all solvents, which was attributed to the conformer trans-Ia.
The minor component, correspondent to trans-IIIb and trans-
Vc, shows a progressive stabilization as the solvent polarity
increases (from 7.0% in CCl4 to 20.9% in CH3CN). These
experimental results presented an excellent agreement with the
predicted ones by theoretical calculations, conrming the
higher stability of trans-Ia.
3
Analogously to the AspOMe, the calculated JHaHb for each
conformer of the AcAspOMe (Fig. S4a in the ESI†) are invariable
with the solvent exchange. However, as shown in Fig. S4b (in the
3
ESI†), the experimental variations of JHaHb result from the
contribution values (hix3JHaHb,i) of each conformer. In nonpolar
solvents, the conformer trans-Ia presents a conformational
contribution much larger than the other two geometries. Since
3
its calculated JHaHb values are about 4.0 Hz (Fig. S4a†), the
corresponding experimental values listed in Table 7 (about 4.5
Hz) can be essentially attributed to this geometry. In turn, the
3
increase of the experimental JHaHb in more polar solvents (i.e.
3JHaHb1 ¼ 6.0 and 3JHaHb2 ¼ 7.3 Hz, in DMSO) can be explained
by the stabilization of forms b and c. It is worth noting that the
higher variations in the 3JHaHb for the AcAspOMe, compared to
the AspOMe, corroborate with the fact that their conformers,
mainly the trans-Ia, present more signicant changes in their
1
populations in solution. Thus, the experimental H NMR data
showed excellent concordance with the theoretical calculations,
enabling the examination of the conformational equilibrium of
the AcAspOMe including the solvent effects.
4. Conclusions
A joint application of experimental and theoretical data allowed
us to make predictions about the conformational preferences of
AspOMe and AcAspOMe. The experimental and calculated 3JHH
couplings constants were found to be in good agreement and
provided insights into the conformational behavior of the
compounds in solution. The same is valid with respect to the IR
data for AcAspOMe. In turn, the theoretical results can explain
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Fig. 8 IR spectra of AcAspOMe showing the analytically resolved carbonyl stretching band in: (a) carbon tetrachloride, (b) chloroform, (c)
dichloromethane and (d) acetonitrile.
the experimental ones, since they were used to determine the
most stable conformers and their relative energies, both in
isolated phase and solution. They also indicated that inclusion
of implicit solvent model (IEF-PCM) leads to small changes in
conformer populations related to the isolated phase, but occurs
the prevalence of the Ia and IIa conformers for AspOMe and
trans-Ia for AcAspOMe.
NBO, QTAIM and NCI analysis showed that although the
presence of an IHB has been observed in some conformers of
these compounds, both steric and hyperconjugative effects have
remarkable importance in their conformational preferences, as
in isolated phase as in solution.
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Acknowledgements
˜
The authors thank a grant #2012/03933-5 from Sao Paulo
Research Foundation (FAPESP) for nancial support of this
work and for a scholarship (to C.B.B. #2012/18567-4 and L.C.D).
Thanks also to Conselho Nacional de Pesquisa (CNPQ) for a
fellowship (to R.R.).
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