1H NMR and theoretical studies on the conformational equilibrium of tryptophan methyl ester

Article abstract of DOI:10.1016/j.molstruc.2013.07.024

Selected ³J_HH coupling constants and theoretical calculations were used to explain the conformational equilibrium of L-tryptophan methyl ester (Trp-OMe) in several solvents. The obtained ³J _HαHβ values did not exhibit any significant variability and thus indicate that there are no conformational population variations for the side chain of the Trp-O-Me depending on the solvent. Moreover, the potential energy surfaces obtained at the B3LYP/cc-pVDZ theoretical level produced eight energy minima that were analysed by QTAIM and NBO methods. It was possible to conclude that the Trp-OMe conformational preferences were due to hyperconjugative effects involving the nonbonding electron pairs of the main chain nitrogen atom and certain antibonding orbitals (σC4-C13a?? -, σC1-C4a?? - and σC4-H12a?? -) and also to the steric effects from the nonbonding electron pairs of oxygen atoms and the main and side chain of this system.

Full text of DOI:10.1016/j.molstruc.2013.07.024

Accepted Manuscript
1H NMR and theoretical studies on the conformationalequilibriumoftryptophan
methyl ester
Claudimar J. Duarte, Rodrigo A. Cormanich, Lucas C. Ducati, Roberto Rittner
PII:
S0022-2860(13)00629-7
DOI:
http://dx.doi.org/10.1016/j.molstruc.2013.07.024
MOLSTR 19899
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
13 June 2013
15 July 2013
15 July 2013
Please cite this article as: C.J. Duarte, R.A. Cormanich, L.C. Ducati, R. Rittner, 1H NMR and theoretical studies on
the conformational equilibrium of tryptophan methyl ester, Journal of Molecular Structure (2013), doi: http://
dx.doi.org/10.1016/j.molstruc.2013.07.024
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Manuscript submitted for publication in:
Journal of Molecular Structure
Title: 1H NMR and theoretical studies on the conformational
equilibrium of tryptophan methyl ester
Contents: 15 pages, 3 tables and 3 figures
Supporting Information : 2 tables and 4 figures
Correspondence to:
Professor Roberto Rittner
Instituto de Química – UNICAMP
Caixa Postal 6154
13083-970 – CAMPINAS - S.P.
BRAZIL
E-mail: rittner@iqm.unicamp.br
fax: +55-19-3521-3023
telephone/fax: +55-19-3521-3150
1

_____________________________________________________________________
1H NMR and theoretical studies on the conformational
equilibrium of tryptophan methyl ester
_____________________________________________________________________
Claudimar J. Duarte,a Rodrigo A. Cormanich,a Lucas C. Ducatib and Roberto
Rittnera,*
aChemistry Institute, State University of Campinas, P.O. Box 6154, 13083-970,
Campinas, SP, Brazil
bInstitute of Chemistry, University of São Paulo, P.O. Box 26077, 05513-970
São Paulo, SP, Brazil
* E-mail: rittner@iqm.unicamp.br
_______________________________________________________________
Abstract
3
Selected JHH coupling constants and theoretical calculations were used to explain the
conformational equilibrium of L-tryptophan methyl ester (Trp-OMe) in several solvents.
3
The obtained JHαHβ values did not exhibit any significant variability and thus indicate
that there are no conformational population variations for the side chain of the Trp-O-
Me depending on the solvent. Moreover, the potential energy surfaces obtained at the
B3LYP/cc-pVDZ theoretical level produced eight energy minima that were analysed by
QTAIM and NBO methods. It was possible to conclude that the Trp-OMe
conformational preferences were due to hyperconjugative effects involving the
nonbonding electron pairs of the main chain nitrogen atom and certain antibonding
orbitals (σ*C4-C13, σ*C1-C4and σ*C4-H12) and also to the steric effects from the nonbonding
electron pairs of oxygen atoms and the main and side chain of this system.
Keywords: tryptophan methyl ester, conformational analysis, hydrogen bonding,
stereoeletronic interactions.
_______________________________________________________________
2

1. Introduction
Amino acids play an important role in nature as the building blocks of proteins [1].
Indeed, the conformational analysis of amino acids has gained special attention with an
increasing number of experimental and theoretical studies reported in the literature [2-
16]. Amino acids in neutral form (NH2-CHR-COOH; R = amino acid side chain)
predominate in the gas phase, while ionic or zwitterionic forms (+NH3-CHR-COO-) are
observed in aqueous solution and crystal structures. However, amino acids are
thermally unstable solids with high melting points and low vapour pressures [7].
Despite these characteristics of amino acids, which generate many gas-phase
experimental-based approach difficulties, these studies have gained increasing
popularity in the literature, while the conformational analysis of these compounds in
water and organic solvents is rare, due to either the zwitterionic form or low solubility.
Moreover, amino acid zwitterionic structures are distinct from the neutral ones present
in polypeptide chains. Therefore, most studies are performed in the gas phase using
both experimental [4,7,17-20] and theoretical [1, 9,21-26] approaches. An alternative
method to avoid these difficulties, as recently reported for the valine methyl ester [27],
is to use amino acid derivatives obtained by esterification or acetylation [28,29].
Tryptophan is an important serotonin [30] precursor neurotransmitter, whose
conformational analysis has been extensively studied in the literature [2, 17, 18, 23, 26,
31-36]. In general, the conformational preferences of tryptophan and other amino acids
have been attributed to intramolecular hydrogen bonding (HB) between the carboxylic
acid (COOH) and the amino (NH2) functional groups, while classic and quantum effects
have been ignored. However, in recent publications, it was shown that the ubiquitous
steric and hyperconjugative interactions, and not HB, are the forces responsible for
glycine [37], alanine [38], sarcosine and N,N-dimethylglycine [39] and valine methyl
ester [27] conformational preferences.
3

In the present study, we performed the conformational analysis of L-tryptophan
methyl ester (Trp-OMe) (Fig.1), a compound that is soluble in certain organic solvents
1
and that does not present the zwitterionic structure in this media, using H NMR spin-
spin coupling constants (3JHH) together with DFT theoretical calculations, the quantum
theory atoms in molecules (QTAIM) [40-44] and natural bond orbital (NBO) [45]
methods.
2 Experimental
2.1 Synthesis
Trp-OMe was prepared by the deprotonation of L-tryptophan methyl ester
hydrochloride (from Sigma-Aldrich Company) in THF (tetrahydrofuran) using zinc
powder according to a described procedure [46]. To a suspension of the Trp-OMe.HCl
(4 mmol; 1.015 g) in 40 mL of THF, commercial zinc powder (400 mg) was added in
one portion. The mixture was stirred for ca. 10 minutes. Subsequently, the mixture was
filtered, and the solvent was evaporated. The Trp-OMe was obtained as a free ester
crystalline solid (0.687 g; 79.0 % yield). Experimental monoisotopic molecular ion M+ =
218.1055; calcd. for C12H14N2O2 M+ = 218.10553.
2.2 NMR Experiments
The solvents (acetonitrile-d3, methanol-d4, pyridine-d5 and DMSO-d6) were
1
commercially available and used without further purification. H NMR spectra were
recorded on a Bruker Avance III spectrometer equipped with a 5-mm probe operating
at 600 MHz for 1H (Fig. 2). The internal reference used was TMS. Typical conditions for
1H spectra were as follows: eight transients, spectral width 5102Hz with 48076 data
4

points, giving an acquisition time of 4.71 s. The resolution of spectra obtained by these
parameters is 0.1 Hz.
2.3 Theoretical calculations
Potential energy surfaces (PESs) for the Trp-OMe compound were built at the
B3LYP/cc-pVDZ theoretical level by scanning the [N-C-C-Ctorsion angle from
0º to 360º in steps of 10º, while the  [LP-N-C-C(O)] (LP represents the nonbonding
electron pair of the nitrogen atom) and [N-C-C=O] dihedral angles were kept static
(Fig. 1). The  and  dihedral angles have previously been optimised for the alanine
methyl ester [27], which provided 6 stable conformers. Thus, the 6 alanine methyl ester
main chain geometries were used as starting points to build the Trp-OMe dihedral
angle PES by exchanging an alanine methyl ester hydrogen atom for an indolyl group.
In addition, the dihedral angle [O=C-O-CH3] was maintained in the cis form because
this form is the most stable O=C-O-CH3 dihedral angle geometry, as demonstrated for
several methyl esters [47]. Each minimum obtained from the Trp-OMe PES was fully
optimised at the B3LYP/aug-cc-pVDZ theoretical level, which already showed good
agreement to CCSD/CBS and experimental data for both glycine and alanine
conformers [48,49]. Some of the  [LP-N-C-C(O)] and  [N-C-C=O] dihedral angles
were similar to the ones from the starting geometries of alanine methyl esters, but this
procedure did not interfere with the results, since a final full optimization was performed
for the geometries of the methyl ester of tryptophan. To these calculated energies, the
zero point energy (ZPE) corrections were added. Subsequently, each Trp-OMe energy
minimum was optimised by considering solvent effects through the IEF-PCM implicit
solvent model [50] in solvents with different () dielectric constants (methanol, pyridine
and DMSO). The NBO analysis was performed over the wave functions obtained from
the B3LYP/aug-cc-pVDZ optimisations in the same level of theory. The spin-spin
5

coupling constant calculations were performed at the B3LYP functional and with the
EPR-III (for C and H) and the aug-cc-pVDZ (for O and N) basis sets. All calculations
were performed using the Gaussian 09 package of programmes [51].
Moreover, topological analyses and the evaluation of integral properties over the
atomic basins () were conducted with the AIMALL programme [52] over the electron
density obtained from the B3LYP/aug-cc-pVDZ optimisation calculations. As usual, the
accuracy of these calculations was checked by the integral values of the Laplacian of
the charge density in each atom, which did not show absolute values larger than 10-3
atomic units (au) [27, 37-39].
3 Results and Discussion
The side chain rotational isomerism of amino acids and their derivatives is usually
represented in terms of a mixture of three staggered rotamers (“a”, “b” and “c”) as
shown in Fig. 3 for Trp-OMe. In this context, the 3JHH vicinal coupling constant between
the and β hydrogen atoms is one important parameter, which is dependent on the
dihedral angle of rotation around the Cα-Cβ single bond and is useful to evaluate the
relationship between the side and main chain of these compounds. Thus, to
1
understand the conformational changes induced by each solvent, H NMR spectra
were recorded in solvents of varying dielectric constant values: acetonitrile, methanol,
3
pyridine and DMSO (Table 1). The JHαHβ values did not show significant changes
(values between 6.39 and 7.14 Hz for 3JHαHβ1and between 5.57 and 5.86 Hz for 3JHαHβ2
)
and thus indicate that there are no conformational population variations concerning the
side chain of Trp-O-Me caused by changing the solvent. Once internal rotation is
sufficiently rapid, the observed spin-spin coupling constant values are weighted
averages obtained for the three rotamers, and thus experimental data from NMR alone
does not explain the Trp-OMe rotational isomerism.
6

To interpret the experimental results, theoretical calculations were performed. PESs
were built for the Trp-OMe compound (ggg. S1, Supporting Information) by using each
of the 6 stable conformers of alanine. Although 18 energy minima have been identified
(3 from each PES), only 8 conformers (named according to the increasing order of
energy, Trp1 to Trp8) converged to stable structures at the B3LYP/aug-cc-pVDZ level
(Fig. S2, Supporting Information). Subsequently, each energy minimum was optimised
by including solvent effects using the IEF-PCM implicit solvation model for the solvents
methanol, pyridine and DMSO. The contribution of each “i” conformer in a given solvent
for the observed 3JHαHβ value may be obtained by the equation:
where ni/nT and Ji are the fractional population and the intrinsic coupling constant of
conformer “i”, respectively. The population for each conformer may be easily obtained
by the Boltzmann distribution:
where ΔEi is the relative energy of the conformer “i”, R is the Boltzmann constant, and
3
T is the temperature. The values of ni/nT x JHH1 for the Trp-OMe conformers
calculated in DMSO, pyridine and methanol are shown in Fig. S3 (Supporting
3
Information). The values of JHH1 do not vary considerably with increased dielectric
constant, which is in good agreement with the experimental data (Table 1).
Hydrogen bond (HB) formation is commonly assumed to govern amino acid relative
energy values. In fact, several rotamers of Trp-OMe exhibit suitable geometry for
possible HB formation. However, other interactions such as steric hindrance and
hyperconjugative effects may operate in these systems. Thus, to investigate the
7

interactions that rule the conformational equilibrium of Trp-OMe, QTAIM [41-45] and
(NBO) methods [46] were employed.
Popelier criteria [45], which use QTAIM parameters, are useful for HB
characterisation. According to Popelier’s first criterion, if a HB is formed, atomic
interaction lines [called bond paths (BP) when the conformer is in the equilibrium
geometry] must be formed connecting the hydrogen atom and the hydrogen acceptor
atom, along with a bond critical point (BCP). The molecular graphs for all conformers
studied are shown in Fig. S4 (Supporting Information). Only conformers Trp2, Trp4,
Trp6, Trp7 and Trp8 exhibit BPs and BCPs relative to an unusual HB [53] involving the
Trp-OMe main and side chain. Because the presence of a BCP and a BP is the
necessary condition for atoms to be bonded from the QTAIM standpoint, only these
rotamers could show stable HBs.
To evaluate the remaining criteria, the electronic density (BCP) and its Laplacian
(∇2BCP) values in the HB and BCP were obtained as well as the hydrogen atomic
energy E(H), hydrogen atomic charge q(H), hydrogen dipole moment M1(H) and
hydrogen atomic volume V(H) for the hydrogen atoms possibly involved in a HB (Table
2). In addition, the conformer Trp1, which does not show any HBs, was used as
reference. In line with Popelier's criteria, the analysis of Table 2 shows that only the
conformation Trp8 exhibits hydrogen bonding. The Trp2, Trp4, Trp6 and Trp7
conformers show ∇2outside of the 0.0024-0.139 au range. According to Popelier’s
criteria, a hydrogen atom involved in a HB should lose q(H) while the M1(H) and V(H)
decreases and the E(H) increase in magnitude, when compared with an isolated, HB-
uninvolved hydrogen atom. Therefore, only Trp8 fully meets all criteria. However, this
conformer was found to be more unstable than the remaining conformers, and thus
hydrogen bonding is not the interaction responsible for the stability of Trp-OMe.
NBO analysis was performed to evaluate the steric and hyperconjugative
contributions for Trp-OMe conformational preferences. In this way, all interactions
8

involving antibonding and Rydberg orbitals were deleted, i.e., conformer energies were
computed as a Lewis structure, which does not account for hyperconjugative
interactions. In this hypothetical situation, it is possible to obtain the contributions of
steric and hyperconjugation effects because the conformer energies before and after
hyperconjugation deletion are known (Table 3). In the real system (EFull), Trp1 is the
most stable conformer. However, when the hyperconjugation is removed, Trp5 is the
most stable conformation by an energy difference greater than 14 kJ mol-1 (ELewis).
These results indicate that Trp5 is the conformer that experiences the least steric
effects, but they also indicate that hyperconjugation has marked importance for the
stabilisation of Trp1. The contribution of hyperconjugative effects is 66.13 kJ mol-1
greater for conformer Trp1 in comparison to conformer Trp5 (Table 3), which is the
least stabilised conformer by hyperconjugative effects, followed by Trp3 and Trp7.
The importance of hyperconjugation is highlighted by the values shown in Table S1
(Supporting Information), which shows the main orbital interactions involving the
nitrogen and oxygen lone pairs for the Trp-OMe conformers. The Trp1, Trp3 and Trp6
conformers present LP(N5) directed to the σ*C4-C13 antibonding orbital (ϕ≈300º), resulting
in the interaction energies (LP(N5)→σ*C4-C13) contributing to the effective stabilisation of
these conformers. Indeed, for the first conformer, one additional interaction involving
LP(N5) and σ*C4-H12 is also predominant (12.46 kJ mol-1) when compared with Trp3 and
Trp6, although this interaction shows larger values for Trp4 and Trp8 (35.20 and 26.75
kJ mol-1, respectively), due to a favourable dihedral angle (ϕ≈90º). Therefore, the
hyperconjugative interactions that reinforce the stability of Trp1, in relation to the
remaining conformers, are the sum of LP(N5)→σ*C4-C13 and LP(N5)→σ*C4-H12. Moreover,
the Trp2, Trp5 and Trp7 conformers (ϕ≈180º) are stabilised by the interaction
LP(N5)→σ*C1-C4 with a larger influence for Trp2 because it has a better spatial
arrangement between these two orbitals. Additionally, for the Trp2 conformer, the
LP(N5) is distant from LP(O2), minimising the steric repulsion between these lone pairs.
Therefore, the LP(N5) orientation plays an important role in Trp-OMe orbitals’ interaction
9

energies and contributes effectively to the conformational equilibrium of this compound.
For the hyperconjugative interactions involving non-bonding electrons of oxygen atoms,
the values observed are practically identical for all conformers.
In addition, to evaluate the importance of steric effects for this system, the AIM
electronic population N(Ω) and atomic electronic energy E(Ω) parameters were
obtained. As noted previously [37,38], the loss of N(Ω) of an atom Ω from one
conformer (which experiences less steric effects) to another, may be understood as an
atomic electronic density rearrangement involved in steric effects. Consequently, this
rearrangement affects the atomic energies E(Ω) of the atoms, which lose and receive
N(Ω). Table S2 (Supporting Information) shows the main N(Ω) and E(Ω) variations for
the conformers of Trp-OMe relative to Trp5, the conformer that shows the smaller
Lewis energy (Table 3). The Trp1 conformer shows a decrease in N(Ω) to C1, C4 and
H6 atoms, while the inverse may be observed to N5. These N(Ω) variations can be
explained by high steric hindrance resulting from the interactions between H6 and
LP(O2), C4 and LP(O3) and C1 and LP(N5), which are closer in space relative to the Trp5
conformer. For these interactions, N5 acts as an electronic density acceptor from H6,
C1 and C4 atoms, and Trp1 therefore shows high N(N5) and low E(N5) when
compared to Trp5. Because Trp1 shows higher ΔELewis (Table 3), these interactions
should be the main sources of instability for this conformer. However, these steric
effects are compensated by the larger hyperconjugation energy when compared with
the remaining conformers, and thus Trp1 represents the most stable form of Trp-OMe.
For Trp2 and Trp6, the loss of N(H25), together with an increase of N(O2), can be
explained by transferring electron density involving these atoms, due the proximity
between the main and side chains. Additionally, this last conformer seems to
experience steric effects for the interaction between C1 and LP(N5). A similar effect
present in Trp2 and Trp6, involving the side and main chain, is observed for the Trp8
conformer, though it is mediated by H22 and LP(N5). Indeed, a steric repulsion occurs
between H7 and C1 atoms, and thus the electron density from these atoms is
10

transferred to the N5 acceptor atom. Therefore, although Trp8 forms an intramolecular
HB that contributes to its stabilisation, the repulsions between LP(N5) and H22 and
between H7 and C1 increase its energy, making it more unstable than the other
conformers. In the Trp4 conformer, the main N(Ω) and E(Ω) variations are observed for
C1 and H7 and N5 and H25, which interact repulsively with each other.
Moreover, from the QTAIM point view, Trp3 does not show significant changes of
N(Ω) and E(Ω), except for N(C1) and N(N5), when compared to Trp5. The Trp3
conformer exhibits low ΔELewis (Table 3) due to the perpendicular relationship between
LP(N5) and COOCH3. This spatial arrangement minimises the repulsion between LP(N5)
and the nonbonding electron pairs of the oxygen atoms, and this result corroborates
the NBO analysis. Trp7 shows decreased N(H7) and N(H25) values and a slight
increase in the N(N5) value. Thus, the repulsion between H7 and O2 and between
LP(N5) and H25 atoms results in a transfer of electron density to N5, which in turn
decreases its E(N) value.
These results indicate that the Trp-OMe spatial arrangement cannot be attributed to
the formation of an intramolecular hydrogen bond, but due to hyperconjugation and
steric effects together, which have remarkable importance for the Trp-OMe
conformational isomerism.
4. Conclusions
In summary, using experimental NMR parameters together with theoretical
calculations and NBO and QTAIM analysis, it was demonstrated that both
hyperconjugation and steric effects are responsible for the conformational isomerism of
Trp-OMe. It was also demonstrated that an intramolecular HB is not an important
interaction for the Trp-OMe conformational preferences because this interaction is
observed only in one relatively unstable conformer.
11

Acknowledgments
The authors thank a grant # 2012/03933-5, São Paulo Research Foundation
(FAPESP) for providing financial support for this research, for a scholarship [to C.J.D. #
2011/11098-6 and to R.A.C. #2011/01170-1, FAPESP) and for a fellowship (to L.C.D.
#2010/15764-4, FAPESP), and the authors thank CNPq for a fellowship (to R.R.
#300379/2009-9).
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15

Figure Captions
Figure 1: Rotation of dihedral angle [N-C-C-C]provided the conformers of Trp-
OMe.
1
Figure 2: 600 MHz H NMR spectra of tryptophan methyl ester in DMSO-d6 (a);
expanded spectral region of 3-4 ppm in: DMSO-d6 (b), MeOD (c), pyridine-d5 (d) and
acetonitrile-d3 (e).
Figure 3: Representation of staggered rotamers relative to the side chain of Trp-O-Me.
16

*Highlights (for review)
Highlights
The conformational equilibrium of tryptophan methyl ester was analysed.
Experimental 3JHH and theoretical calculations were employed.
Conformer populations were not dependent on the solvent.
Hyperconjugation and steric effects govern the ester’s conformational
isomerism.
Graphical Abstracts
1H NMR and theoretical studies on the conformational equilibrium of
Tryptophan methyl ester
Claudimar J. Duarte, Rodrigo A. Cormanich, Lucas C. Ducati and Roberto Rittner

Figure(s)
Figure 1

Figure(s)
Figure 2

Figure(s)
Figure 3

Table(s)
Table 1
Coupling constants (J, in Hz) and Chemical shifts (, in ppm) to the Trp-O-Me rotamers
in solvents of different dielectric constants ().
Solvent
3J
3J






Pyridine-d5 12.3
3.89
4.13
4.12
3.89
3.12
3.28
3.34
3.11
3.18
3.37
3.34
3.17
6.46
7.14
------
6.39
5.78
5.57
------
5.86
MeOD
32.7
37.5
46.7
CD3CN
DMSO-d6

Table 2
Electronic density (BCP) and electronic density Laplacian
(
∇2BCP
at the H-bond BCP
)
and integrated atomic properties in au units.
Parameter
Conformer
Trp1
H
q(H)
E(H)
M1(H)
V(H)

BCP (H) ∇2BCP (H)
H(9)
---
---
---
---
---
---
---
---
+0.020
+0.025
+0.032
-0.003
-0.605
-0.603
-0.596
-0.612
0.135
0.134
0.130
0.129
47.84
47.47
49.40
51.40
H(10)
H(22)
H(25)
H(10)
H(25)
0.002
0.006
+0.007
+0.020
+0.030
+0.022
-0.603
-0.606
0.132
0.118
49.06
47.59
Trp2
Trp4
Trp6
H(10)
H(25)
0.002
0.006
+0.007
+0.020
+0.030
+0.022
-0.603
-0.606
0.132
0.118
49.06
47.59
H(9)
0.003
0.005
+0.008
+0.017
+0.030
+0.016
-0.603
-0.608
0.131
0.121
48.72
48.72
H(25)
Trp7
Trp8
H(25)
H(22)
0.007
0.009
+0.022
+0.029
+0.020
+0.066
-0.606
-0.585
0.129
0.129
48.15
45.43

Table 3
Total energy (EFULL), Lewis energy (ELewis) and hyperconjugative energy (Ehyper) for
Trp-OMe conformers (in kJ mol-1) at the B3LYP/aug-cc-pVDZ theoretical level.
Conformer
Trp 1
0.00
Trp 2
Trp 3
Trp 4
4.22
Trp 5
4.81
Trp 6
5.35
Trp 7
5.43
Trp 8
5.52
ΔEtotal
2.17 3.85
ΔELewis 61.36 52.25 14.63 21.15 0.00
ΔEhyper. 66.13 54.88 15.59 21.74 0.00
48.66 18.60
48.11 17.89
49.87
49.12