Conformational preference of glycinamide in solution: An answer derived from combined experimental and computational studies

Article abstract of DOI:10.1016/j.jmgm.2013.09.007

Conformational problems are often subtle but very important in controlling many intricate features in chemistry and biochemistry. We have performed the conformational analysis of glycinamide using NMR experiments and computational studies. ¹H NMR experiments suggest the prevalence of intramolecular hydrogen bonded conformation of glycinamide (2B) in acetonitrile, whereas, non-intramolecular hydrogen bonded conformation 2A is favoured in dimethylsulfoxide. The NOESY experiments carried out for glycinamide in DMSO-d₆, showed stronger NOE interaction of the NH_a-atom of amide group with CH₂ than that of NH_b-atom confirming the presence of conformer 2A. DFT calculations performed with explicit DMSO molecules also suggested a clear preference for the conformer 2A. The molecular dynamics simulations performed with the explicit DMSO molecules also showed that the intermolecular hydrogen bonding exists between the solvent and solute molecules to stabilize the conformer 2A. The present study sheds light on the debate of conformational preference of neutral glycinamide in the present literature.

Full text of DOI:10.1016/j.jmgm.2013.09.007

Accepted Manuscript
Title: Conformational Preference of Glycinamide in Solution:
An Answer Derived from Combined Experimental and
Computational Studies
Author: Bishwajit Ganguly Manoj K. Kesharwani Nikola
´
Basaric Eringathodi Suresh Abul Kalam Biswas Kata
´
Mlinaric-Majerski
PII:
S1093-3263(13)00162-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jmgm.2013.09.007
JMG 6338
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Journal of Molecular Graphics and Modelling
Received date:
Revised date:
Accepted date:
4-4-2013
14-8-2013
11-9-2013
´
Please cite this article as: B. Ganguly, M.K. Kesharwani, N. Basaric, E.
´
Suresh, A.K. Biswas, K. Mlinaric-Majerski, Conformational Preference of
Glycinamide in Solution: An Answer Derived from Combined Experimental and
Computational Studies, Journal of Molecular Graphics and Modelling (2013),
http://dx.doi.org/10.1016/j.jmgm.2013.09.007
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Conformational Preference of Glycinamide in Solution: An Answer Derived
from Combined Experimental and Computational Studies
Bishwajit Ganguly,*,a Manoj K. Kesharwani,a Nikola Basarić,b Eringathodi Suresha, Abul Kalam
Biswasa and Kata Mlinarić-Majerski*,b
a Analytical Discipline and Centralized Instrument Facility, Central Salt & Marine Chemicals
Research Institute (Council of Scientific and Industrial Research) Bhavnagar, Gujarat, India-
364 002
bDepartment of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta
54, P.O. Box 180, 10 002, Zagreb, Croatia
*Corresponding Authors. Fax: (+91)-278-2567562
E-mail: ganguly@csmcri.org and majerski@irb.hr
Abstract:
Conformational problems are often subtle but very important in controlling many
intricate features in chemistry and biochemistry. We have performed the conformational analysis
of glycinamide using NMR experiments and computational studies. 1H NMR experiments
suggest the prevalence of intramolecular hydrogen bonded conformation of glycinamide (2B) in
acetonitrile, whereas, non-intramolecular hydrogen bonded conformation 2A is favoured in
dimethylsulfoxide. The NOESY experiments carried out for glycinamide in DMSO-d6, showed
stronger NOE interaction of the NHa-atom of amide group with CH2 than that of NHb-atom
confirms the presence of conformer 2A. DFT calculations performed with explicit DMSO
molecules also suggested a clear preference for the conformer 2A. The molecular dynamics
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simulations performed with the explicit DMSO molecules also showed that the intermolecular
hydrogen bonding exists between the solvent and solute molecules to stabilize the conformer 2A.
The present study sheds light on the debate of conformational preference of neutral glycinamide
in the present literature.
Keywords: Glycinamide; Solvent effect; NMR; DFT; Conformer stability
1. Introduction
The amide functional groups are of considerable interest to researchers due to their
presence from chemistry to biochemistry. This is a fundamental linkage in building blocks of
biomolecules i.e. protein and other kinds of bioactive molecules [1,2]. Naturally occurring
polypeptide chains, found in proteins, are heteropolymers of α-amino acid residues linked
together by peptide bonds and are dependent on the conformational preference of the amide
groups [1,2]. Glycinamide is a simple derivative of glycine and is the simplest amide, which is
important to form complexes and an appropriate model compound for N-terminal amino acids in
peptides.
Besides its importance in peptides and proteins, glycinamide has also been used to
examine the habit of rock salt [3]. The computational results revealed the relative significance of
conformations of glycinamide towards the morphology of salt crystals [3]. Two stable
conformers of glycinamide discussed in the literature are given in Scheme 1 [4,5].
< Scheme 1 >
Sulzbach et al. have performed the quantum chemical study to examine the
conformational behaviour of glycinamide. The ab initio quantum chemical calculations revealed
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that the glycinamide conformer with torsional angle ψ(4,3,2,1) = 165° (2A) is a global minimum
and 1.3 kcal/mol lower than conformer that has ψ(4,3,2,1) = 2° (2B) (Scheme 1) [4]. The structural
assignments of 2A and 2B were carried out with the NMR calculations as structures of peptides
in solution are obtained by two- and three-dimensional Fourier-transform (FT) NMR
experiments [6]. Recently, Bu and co-workers have performed extensive study towards
understanding the conformational behaviour of glycinamide using density functional theory in
both the gas and aqueous phases [5]. The B3LYP/6-311++G** calculated results suggested that
the glycinamide conformer 2B is energetically preferred than 2A in both the mediums. They
have found similar results with single point energy calculations at higher-level computations,
including the MP2, MP3, MP4SDQ, and CCSD(T) methods employing the aug-cc-pVDZ basis
set. The calculations performed by Bu et al. confronted the earlier predictions of conformational
preference of glycinamide [4]. However, it has been observed that in the aqueous phase the
energy difference between the two conformers is smaller than the gas phase calculated results
[5].
The influence of glycinamide conformers on the crystal morphology of NaCl crystals was
apparent in the aqueous phase calculations [3]. The experimental observations suggest that
glycinamide is not a habit modifier for sodium chloride crystals. The inability of glycinamide as
a habit modifier of rock-salt is envisaged due to the predominant presence of conformer 2A in
the aqueous medium [3]. It is also known that the solution behaviour comprises a much greater
challenge in the conformational analysis at the molecular level [7,8]. Therefore, these results
prompted us to re-examine the conformational behaviour of glycinamide in solvent, which,
however, is a subject of debate in the literature.
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The hydrogen bonding between the solute and the solvent molecules can control the
relative stability of the conformers. Such interactions alter the conformational equilibria
dramatically in polar solvents [7-10]. The influence of hydrogen bonding of polar solvent
molecules with the solutes is not considered in the continuum solvation model calculations.
Glycinamide has polar functional groups which can participate in hydrogen bonding with the
polar solvent molecules. Therefore, it is important to examine the influence of hydrogen bonding
of solvent molecules on the stability of glycinamide conformers. The calculations with single
explicit water molecule have been reported,[11] in which the water molecule is interacting with
only the amide oxygen, but it lacks the interaction of solvent molecule with the polar hydrogens
of glycinamide. In the present study, we have examined the influence of solvent molecules on
the conformational behaviour of glycinamide with extensive NMR study in DMSO-d6 and
CD3CN followed by molecular dynamics study using the same solvent molecules. The solvent
DMSO is known to form hydrogen bonds, whereas, CD3CN will participate less in such
interactions with the substrate molecules [12-14].
2. Methodology
2.1. Experimental Details
Methods: 1H and 2D NOESY spectra were recorded on a Spectrometer at 300 or 600
MHz. NMR spectra were measured in DMSO-d6, CD3CN or C6D6 using tetramethylsilane as a
reference. The mixing time used in the NOESY experiments for glycinamide hydrochloride (1)
and glycinamide (2) in DMSO was 0.3 s. The mixing time in the NOESY experiments for
glycinamide (2) in CH3CN was 0.1, 0.3, 0.5, and 0.9 s. 1H and 13C NMR spectra were recorded
on a Bruker Spectrometer at 300 or 600 MHz. All NMR spectra were measured in deuterated
solvents using residual non-deuterated solvent signal as a reference.
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Materials: Glycinamide hydrochloride (1) is commercially available, whereas
glycinamide (2) was prepared by deprotonation of 1. In a flask (50 mL) was placed glycinamide
hydrochloride (1, 80 mg, 0.72 mmol), K2CO3 (2 g, 145 mmol) and anhydrous CH3CN (50 mL).
The slurry was stirred over 4 hours at rt. Insoluble salts were filtered off, and from the remaining
solution solvent was removed on a rotary evaporator to afford glycinamide (30 mg, 56 %).
2.2. Computational Details
The two-dimensional potential energy surfaces were constructed by varying the O-C-C-N
and C-C-N-H torsional angle for rotation of 180° with increments of 10° in both gas phase and
solvent medium using Becke3 Lee Yang Parr functional and 6-311++G** basis set [15-17]. The
default polarizable continuum solvation model (PCM) ware employed for the solvent
calculations [18-22]. The default UFF radii were used for the PCM calculations, which
incorporate explicit hydrogen atoms. The desired conformers were further optimized with same
method in respective solvation medium. A hybrid approach of explicit and implicit continuum
model was also used to optimise both conformers of glycinamide (2A and 2B) with six
molecules of solvent (DMSO). Single point calculations were performed at MP2/6-311++G**
level using B3LYP optimized geometries. Two dimensional potential energy scan were
performed using Gaussian 03 program, whereas, all other calculations were performed using
Gaussian 09 program [23,24].
The molecular dynamic simulations were performed using density functional theory
method employing DMol3 suit program. The LDA/PWC/DND methods were employed for
dynamics calculations [25-27]. The simulation cell was a periodically repeated cubic box of side
length 15 Å. The conductor-like screening solvation model (COSMO) was employed to
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incorporate the continuum dielectric constant environment for DMSO (ε = 46.7) [18,28]. The
whole simulations were performed with the canonical NVT ensemble and temperature is
controlled at 25°C and 80°C with the Nosé-Hoover chain thermostat. In each case, initially the
simulations were performed for 1ps with a time step of 2fs. For the case of conformer 2A,
simulations were further extended upto 2ps. The statistics were collected every 2fs for all
simulations.
3. Results and Discussion
Initially, extensive computational search was performed to examine the influence of
dielectric constant of dimethylsulfoxide (DMSO) and acetonitrile on the relative stabilities of
glycinamide conformers. The conformational changes associated with the rotation of C−C bond
and C−N(amine) bond were analyzed by constructing a two-dimensional potential energy scan
using B3LYP/6-311++G** method [15-17] in both the gas phase and implicit solvent mediums
(DMSO and acetonitrile) (Fig. 1 and Fig. S1, Supporting Information). To construct the potential
energy surface representing the effect of the internal rotation---the C−C and C−N(amine) bonds
were allowed to rotate 180° with increments of 10° in all the studied mediums. The lowest
energy forms of 2A and 2B were chosen from potential energy surfaces in each medium and
further optimized without any constraint using B3LYP/6-311++G** methods in the respective
medium. Additionally, highest energy conformers were chosen from potential energy surfaces
and further considered for transition state optimizations at the same level of theory in each
studied medium. Single point calculations were performed to compute the energies at MP2/6-
311++G** level using B3LYP/6-311++G** optimized geometries. Gas phase calculated results
show that conformer 2B is 2.1 kcal/mol more stable than 2A at B3LYP/6-311++G**, which is
similar to that reported by Bu and coworkers [5]. However, the energy difference decreases by
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~0.6 kcal/mol in both DMSO and acetonitrile mediums. Similarly, the calculated activation
barrier for the interconversion of these two conformers decrease by 1.2 kcal/mol in solvents
compared to the barrier calculated in the gas phase (Fig. S2, Supporting Information). MP2/6-
311++G** calculations also predicted similar results as observed with B3LYP/6-311++G**
level of theory. DFT and ab initio calculations suggest that the stability of 2A in polar solvent
increases compared to the gas phase calculations. Nevertheless, 2B is the most stable conformer
in the gas and solvent phase.
<Figure 1>
To examine the influence of hydrogen bonding towards the conformational preference of
glycinamide, additional calculations have been performed using both implicit and explicit
solvent medium. Such hydrogen bonding interactions were examined with 6 DMSO molecules.
Four DMSO molecules were engaged in H-bonding with four –N-H hydrogens of glycinamide
unit and the rest 2 DMSOs interacted with the oxygen atom of the carbonyl group (Fig. 2). To
achieve the intermolecular hydrogen bonding interactions between DMSO and the solute system,
solvent molecules were placed in such a way to minimize the steric effects and to gain maximum
H-bonding interactions. These calculations were performed using B3LYP/6-311++G** level of
theory employing PCM solvation model. The single point calculations performed with MP2/6-
311++G**//B3LYP/6-311++G** level of theory suggest that the conformer 2A is energetically
more stable by 5.4 kcal/mol compared to the conformer 2B (Fig. 2). MP2 energies corrected with
B3LYP zero point vibrational energy values also showed the similar preference for the stability
of glycinamide conformers (Table S1, Supporting Information). These results showed the
importance of hydrogen bonding between solute and solvent molecules in controlling the
conformational preferences, which is different from the implicit solvent model results [5]. It is
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likely that DMSO might be contaminated with water in the experimental studies. Hence, we have
further examined the interaction of water molecules with glycinamide analogous to DMSO in the
dielectrics of DMSO medium. The calculated results with MP2/6-311++G**//B3LYP/6-
311++G** level of theory suggest for the preference of conformer 2A, similarly as with the
DMSO molecules (Fig. S3, Supporting Information). Further, to account the influence of
dispersion forces on relative energy of glycinamide conformers, additional calculations were
performed using dispersion corrected functional B3LYP-D [29]. B3LYP-D//B3LYP/6-
311++G** calculated results also favor 2A as energetically more stable conformer than
conformer 2B by 3.4 kcal/mol in DMSO medium (Table S1, Supporting Information).
To segregate the strength of interaction energies between the amine and amide –N-H
hydrogens with DMSO molecules, additional calculations have been performed. B3LYP/6-
311++G** optimized geometries of glycinamide interacting with 6 DMSO molecules were
utilized for this purpose. To examine the interaction energy of DMSO molecules with amine
hydrogens of glycinamide, other 4 DMSO molecules were detached without perturbing the
geometry. In a similar fashion, interaction energy of 2 DMSO molecules with amide hydrogens
was also calculated. The calculated results suggest that interaction of DMSO molecules with
amide hydrogens are 4.9 and 2.0 kcal/mol stronger than the corresponding interactions with
amine hydrogens for 2A and 2B, respectively (Fig. S4, Supporting Information). The
computational results though predicted the conformational preferences of glycinamide; however,
an experimental verification is important at this point. We have performed 1H NMR studies on
the conformational equilibria of glycinamide with DMSO-d6 and CD3CN. Furthermore, the
interaction of solvent molecules with the glycinamide conformers was explored with molecular
dynamics study.
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<Figure 2>
1H NMR Study
To probe the conformational behaviour of glycinamide in solution, initially, a NMR study
was performed on glycinamide hydrochloride (1) (Scheme 1). Glycinamide is commercially
available in the form of glycinamide hydrochloride. 1H NMR spectrum of 1 was recorded in
DMSO-d6, which shows three well resolved signals in low magnetic field (characterized by the
+
ratio of intensities 3:1:1) corresponding to the hydrogens of the NH3 and two amide -CONH2
H-atoms (at 7.86 and 7.47 ppm). Assignation of the chemical shifts was based on the assignation
in the 1H NMR of formamide and is presented in Table 1 [30]. In the NOESY spectrum of 1, a
stronger NOE interaction is observed between the NHa-atom and CH2, than between the NHb-
atom and CH2. This finding supports the conformation of 1 as shown in Fig. 1. Further, the
single x-ray crystal structure of 1 was determined in our laboratory (CCDC number: CCDC
838431), which also showed the same conformation as derived by the NMR results (Scheme 1
and Fig. S5, Supporting Information). These results were encouraging for further evaluation of
glycinamide conformers 2 in solution. Glycinamide hydrochloride 1 was neutralized to
glycinamide 2 following a procedure mentioned in the experimental section. 1H NMR spectra of
2 were taken in solvents of different polarity and at different temperatures. In addition, NOESY
spectra were also recorded in DMSO-d6 and CD3CN at different mixing times.
In the 1H NMR spectrum of 2 in DMSO-d6, two amide NH signals are present at 7.24 and
6.94 ppm, whereas the amine NH2 signal appears at higher field at 1.62 ppm. As it can be seen
(Table 1), deprotonation of the ammonium group in the molecule shifted signals of the amide
and the methylene H-atoms in the 1H NMR spectrum to the higher magnetic field for ~0.5 ppm.
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This shielding is in agreement with the change of the inductive effect of the substituent (from
ammonium to amino group) [31].
<Table 1>
To test if there is an intramolecular and/or intermolecular hydrogen bonding in
glycinamide in DMSO-d6, 1H NMR spectra were recorded at elevated temperatures. The increase
of temperature should disrupt the intermolecular hydrogen bonds (formed with the solvent
molecules), whereas such an influence is expected to be milder on the intramolecular hydrogen
bonds. At 60°C, the chemical shifts of the amide NH signals come closer with the resonances at
7.04 and 6.73 ppm (both signals of the amide NH shifted for ~0.2 ppm). Further increase in
temperature to 80°C resulted in appearance of these two amide NH signals as one very broad
singlet at 6.8 ppm, due to the cleavage of the intermolecular H-bonds with DMSO-d6, and free
rotation around -CO−N bond. Lowering the temperature gives the same appearance of the
spectrum, which was observed before heating the sample. These results suggest that glycinamide
presumably does not form the intramolecular H-bonding in DMSO-d6 solution.
1H NMR spectrum of glycinamide was also taken in CD3CN, which should participate
less in intermolecular hydrogen bonds with the amide-H atoms of 2 and therefore should not
affect the intramolecular H-bonding, if present in the solution. The 1H NMR spectrum of 2 in
CD3CN was characterized by the presence of two very broad amide NH singlets at δ 6.84 and
5.75 ppm, whereas CH2 appeared as a singlet at 3.19 ppm. Unfortunately, the amine NH2 signal
was not seen due to the overlapping with the signal of water and/or fast exchange with the
protons from water. The difference in chemical shift for the amide NHa atom in the protonated 1
and non-protonated form 2 is negligible in CD3CN, whereas the signal of the NHb atom upon
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deprotonation shifted to the higher field for ~0.5 ppm. However, in the DMSO-d6 both protons
shifted for ~0.5 ppm. This finding suggests that Ha proton is hydrogen bonded, most probably to
the amine lone pair (Scheme 1, conformer 2B). Thus, shielding due to the deprotonation of the
ammonium, and deshielding due to the hydrogen bonding gives negligible total effect in the
chemical shift of the NHa atom. Additionally, comparison of the 1H NMR spectrum of acetamide
with the spectrum of glycinamide in CD3CN demonstrates that the signal of the amide NHb atom
is present at the same chemical shift, whereas glycinamide NHa signal appears at lower magnetic
field (Δδ ~0.8 ppm). Consequently, it is inferred that glycinamide in CD3CN is present in the
form of conformer 2B. To verify this assumption, 1H NMR spectra were recorded also at
different temperatures. Increase in the temperature resulted in line broadening in 1H NMR
spectrum and a larger shift of the amide NHa signal (0.25 ppm) to the higher field than NHb
signal (0.1 ppm). This finding additionally indicates that Ha atom is hydrogen bonded. The H-
bond weakens with the increase in temperature and results in a larger shift to the higher magnetic
field. A similar trend as seen in CD3CN was observed by taking spectrum of 2 in benzene-d6,
with a larger difference in the chemical shifts of the amide Hb atom (compared to DMSO).
<Figure 3>
The NMR titration performed with DMSO-d6 also supports the existence of the
intramolecular H-bond between NHa and the amine lone pair in 2. To the solution of 2 in
CD3CN, small portions of DMSO-d6 were added and after each addition spectra were taken. It
was inferred that a small amount of DMSO-d6 would affect first the chemical shift of the non-
hydrogen bonded NH-atom, whereas a larger amount of DMSO-d6 is necessary to break the
intramolecular hydrogen bond and shifts the signal of the H-bonded atom. As expected (Fig. 4),
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addition of a small amount of DMSO-d6 resulted in pronounced shifting (Δδ=0.51) of the NHb
and a small shifting of the NHa signal (Δδ=0.16).
The 1H NMR spectrum of 1 was also recorded in CD3CN, but because of a very low
solubility, the spectrum was of poor quality. However, characteristic signals corresponding to the
amide-H atoms were observed at 6.85 and 6.35 ppm. By comparing 1H NMR spectra of 1 in
DMSO-d6 and CD3CN it can be seen that DMSO-d6 deshielded the amide NH signals for ~1
ppm, whereas CH2 H-atoms were shielded for ~0.6 ppm. This finding also supports the
conclusion that DMSO-d6 forms intermolecular H-bondings with the amide H-atoms.
Further, the confirmation for the possible prevalence of conformer 2A in DMSO was
obtained from the NOESY spectra. In the NOESY experiment for 2 in DMSO-d6, we have
observed stronger NOE interaction between the NHa-atom and CH2, than between the NHb-atom
and CH2. On the other hand, in the NOESY experiment carried out for 2 in CD3CN and with
different mixing time (0.1 s, 0.2 s, 0.3 s, 0.5 s, 0.9 s) no NOE interaction between the amide NH-
atoms and CH2 was observed. These observations additionally support the finding obtained by
DMSO-d6 titration that glycinamide in CD3CN most likely exists in the intramolecular hydrogen-
bonded form (conformer 2B). On the other hand, in DMSO-d6 1 and 2 are probably in the same
conformation, or there is a free rotation around the single bond between -C=O and CH2.
Molecular Dynamics Study
The conformational equilibria of glycinamide explored with the 1H NMR spectra suggest
that the preference of 2A in DMSO-d6 is due to the intermolecular hydrogen bonding at room
temperature. We have performed molecular dynamics (MD) simulations to examine the
dynamical nature of glycinamide 2A in DMSO. The MD simulations will provide the effect of
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temperature on the rotation along O-C-C-N bond and the extent of interaction of DMSO
molecules with glycinamide’s amide hydrogen (Ha). The amide hydrogen Ha is crucial in
governing the conformations of glycinamide as this hydrogen participates in the hydrogen
bonding with the amine nitrogen in conformer 2B (Scheme 1).
The molecular dynamic calculations were performed for glycinamide conformer 2A
interacting with DMSO molecules with local spin density approximation with the Perdew-Wang
correlational (LDA/PWC) method employing DMol3 suite program [25-27]. We used a DND
double numerical basis set which is comparable to the 6-31G* basis set. Cubic box with side
lengths of 15 Å were used as simulation cells. The COSMO solvation model was employed to
incorporate the continuum dielectric constant environment for DMSO (ε = 46.7) [18,28]. The
whole simulations were performed with the canonical NVT ensemble and temperature is
controlled at 25°C and 80°C with the Nosé-Hoover chain thermostat. In each case, the
simulations were initially performed for 1ps with a time step of 2fs (Fig. S6, Supporting
Information), which were further extended to 2ps (Fig. 4). The longer simulations performed for
the glycinamide conformer 2A yields very similar information that we have received with the 1
ps. At both temperatures, the simulations were started with the conformer 2A of glycinamide
surrounded by 10 molecules of DMSO and all NH hydrogens of glycinamide are hydrogen
bonded with DMSO oxygens. The representative configuration of glycinamide surrounded by 10
DMSO molecules is given in supporting information (Fig. S7, Supporting Information).
The LDA/PWC/DND simulated results show that the change in the orientation of
conformer 2A with DMSO molecules is similar up to 0.2 ps at both 25°C and 80°C. However, at
a longer time step i.e., 0.26 ps the change in the O-C-C-N torsional angle was found to be
maximum (~82°) at 25°C. However, at 0.44 ps the O-C-C-N torsional angle reduces to 41° and
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then starts fluctuating between 40° to 60° till 0.64 ps. The O-C-C-N torsional angle at 25°C
appears to revert to its original situation after 0.64 ps and then further starts to rotate in opposite
direction and reaches upto ~40° and fluctuates between 13° to -40° only. The change in the O-C-
C-N torsional angle with the time steps was also reflected in the distance between the
glycinamide nitrogens (Fig. 4). The N…N distance of the glycinamide molecule becomes closer
with the increase in O-C-C-N torsional angle. During the simulation, the intermolecular Ha…O1
hydrogen bond remains conserved though slight fluctuations were noticed (1.514 Å to 2.654 Å),
however, from plot it is clear that most of the time this distance is below 2.0 Å (Fig. 4). The MD
simulation results at 25°C suggest that the rotation along O-C-C-N bond is possible for the
conformer 2A, however, such rotations do not perturb the intermolecular hydrogen bonding
interactions between the DMSO oxygen and the amide hydrogen (Ha) as suggested in the 1H
NMR studies (vide supra).
<Figure 4>
The interaction of DMSO molecules with glycinamide 2A simulated at 80°C shows that
the O-C-C-N torsional angle rotates to maximum (~82°) at the time step of 0.27 ps. Further, the
O-C-C-N torsional angle fluctuates between 85° to 60° upto time step of 0.54 ps, which,
however reverts to the initial position (0°) and again starts to rotate after 0.72 ps. Further, at the
time step of 0.9 ps, O-C-C-N torsion angle again rotates to ~64° and then fluctuation has been
observed between 28° to 53° up to 1.22 ps. The disruption in the intermolecular hydrogen
bonding between 2A and DMSO was observed as the distance varied from 1.503 Å to 3.160 Å
upto time steps of 0.79 ps (Fig. 4). After 0.79 ps, the DMSO molecule interacting with Ha
hydrogen atom starts to move away and another DMSO molecule approaches to Ha atom (Fig.
S8, Supporting Information). However, from the plot, it is clear that the Ha…O1 distance
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increases with the increase in O-C-C-N torsional angle (Fig. 4). The Ha…O1 distance was found
to be greater than 2.0 Å in most of the simulation period, which suggests that the intermolecular
hydrogen bond interrupts at the higher temperature, which is in accord to the observed 1H NMR
spectral results. The energy profiles obtained during the simulations at 25°C and 80°C show that
the glycinamide conformer 2A is energetically more stable when the intermolecular hydrogen
bonding is more intact compared to that of the situation when such interactions are disrupted at
elevated temperature (Fig. 4).
The molecular dynamic simulations were also carried out for glycinamide conformer 2B,
surrounded with 10 DMSO molecules, at both temperatures 25°C and 80°C for 1ps. The
simulation results show that at both temperatures, the intramolecular hydrogen bonding is
conserved between the amide hydrogen (Ha) and amine nitrogen atom (Fig. S9, Supporting
Information). During the simulation period, most of the time -NH–N distance are less than 2.4 Å,
which is within the intramolecular hydrogen bonding distance for -NH–N systems [32]. Further,
this intramolecular hydrogen bonding restricts the rotation along O-C-C-N torsional angle of
glycinamide conformer 2B. Smaller fluctuations were observed in O-C-C-N torsional angle
during the simulations at both temperatures. Interestingly, the variations in the energy of
glycinamide 2A and 2B conformers during 1ps simulations suggest that the conformer 2A is
more stabilized by the DMSO solvent molecules compared to the corresponding 2B conformer
during the simulations (Fig. S6 and S9, Supporting Information). This is in accord with the
results of quantum chemical calculations performed with DMSO molecules. Further, 1HNMR
results also indicate that the 2A conformer of glycinamide should predominantly prevail in
DMSO.
4. Conclusions
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Our study of conformational preference of neutral glycinamide reveals interesting results
to settle the debate on this topic. The computational study performed with DFT calculations by
Bu et al. showed that the intramolecular hydrogen bonded glycinamide conformer 2B is the
global minimum, which contradicted the results of conformations reported by Sulzbach et al.
similar to 2A [4,5]. In the absence of experimental results the conformation study of glycinamide
could not be established. We have customized in our study that both the conformers can exist in
different solvent mediums. The 1H NMR studies have shown that the conformer 2A prevails in
the DMSO solution, or there is a free rotation around the C-C bond. The quantum chemical
calculations with hybrid solvent environment and ab initio molecular dynamics simulations also
suggest that the conformer 2A is more stable than that of 2B in DMSO due to the involvement of
intermolecular hydrogen bonding with solvent molecules. On the other hand, in CH3CN
conformer 2B is likely to prevail in the solution. These results clearly suggest that the
conformations of glycinamide can be altered with the nature of the solvent. The molecular
dynamics simulations also supported the effect of intermolecular hydrogen bonding towards the
stability of the conformer 2A. Overall, these results showed the importance of solvent effects in
controlling the conformations and can have far reaching implications in many research problems.
Acknowledgements
This research was supported by CSIR, New Delhi, India (MSM) and the Ministry of
Science, Education and Sports of the Republic of Croatia (grant no. 098-0982933-2911). MKK
and AKB are thankful to UGC, New Delhi, India for awarding Fellowship. We thank the
reviewers for their comments and suggestions that have helped us to improve the paper.
Supplementary data
16
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B3LYP/6-311++G** optimized Cartesian coordinates and electronic energies of all stationary
points, crystal data of compound 1 and 1H NMR graphs.
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21
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Table 1.Chemical shifts in 1H NMR spectra of 1 and 2.
Compound / solvent
Ha
Hb
CH2
NH2
+
(NH3 )
(8.12)
1.62
1 / DMSO-d6
2 / DMSO-d6 rt
(Δδ)a
7.86
7.24
7.47
6.94
3.48
3.01
(0.62) (0.53) (0.47)
~7.04 ~6.73 3.03
(0.20) (0.21)
2 / DMSO-d6 60 °C;
1.59
(Δδ)b
2 / DMSO-d6 80 °C ;
(Δδ)b
~6.8
(0.44) (0.14)
~6.8
~3.1
-
-
-
1 / CD3CN rt
~6.85 ~6.35 3.61
(1.01) (1.12) (0.13)
~6.84 ~5.85 3.15
(0.01) (0.50) (0.46)
(0.40) (1.09) (0.15)
~6.59 ~5.75 3.18
(0.25) (0.10)
(Δδ)c
2 / CD3CN rt
(Δδ)a
(Δδ)c
2 / CD3CN 60 °C
-
-
-
-
-
(Δδ)b
2 / CD3CN rt
+ 12 µL DMSO
2 / CD3CN rt
+ 22 µL DMSO
2 / CD3CN rt
+ 42 µL DMSO
2 / CD3CN rt
+ 142 µL DMSO
(Δδ)d
~6.86 ~5.91 3.14
~6.89 ~5.97 3.14
~6.92 ~6.07 3.13
~7.00 ~6.36 3.10
(0.16) (0.51)
2 / C6D6 rt
~6.0
~4.8
2.70
-
-
Acetamide / CDCl3 rt
Formamide / D2O rte
Formamide / DMSO-d6
rte
~6.05 ~5.82 1.94
7.3
7.43
6.9
7.16
-
a The difference in chemical shift between protonated and non-protonated form
b The difference in chemical shift at rt and at elevated temperature
c The difference in chemical shift between DMSO-d6 and CD3CN
d The difference in chemical shift between CD3CN, and CD3CN with the addition of DMSO-d6
e The data taken from ref. 18.
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H
H
O
O
O
H
H
+
Cl-
base
1
Hb
4
N
N
Hb
Hb
N
3
3
2
N
N
H
4
ψ
2
ψ
1
Ha
N
Ha
Ha
H
1
2A
2B
H
Scheme 1. Glycinamide hydrochloride (1) and two stable conformers (2A and 2B) of
glycinamide.
23
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Legends of Figures:
Fig. 1. B3LYP/6-311++G** calculated two dimensional potential energy surface of glycinamide
in gas phase.
Fig. 2. B3LYP/6-311++G** optimized geometries and relative energies (kcal/mol) of
glycinamide conformer 2A and 2B interacting with six DMSO molecules in DMSO solvent
medium using PCM solvation model. MP2/6-311++G** calculated energies are given in
parentheses.
Fig. 3. Part of the 1H NMR spectra of glycinamide corresponding to the amide-H atoms recorded
in CD3CN (6), CD3CN with the increasing amounts of DMSO-d6 (from 5-2) and DMSO-d6 (1).
Fig. 4. LDA/PWC/DND calculated profile of variation in energy, O-C-C-N torsional angle, Ha-
O1 and N−N distance during molecular dynamics of 2ps, (a) at 25 °C temperature and (b) at 80
°C temperature.
24
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H
O
H
H
N
N
H
SC1
SC2
Fig. 1. B3LYP/6-311++G** calculated two dimensional potential energy surface of glycinamide
in gas phase.
25
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5
2.328
1
3
.
2
458
2.
2.087
2.114
2
.38
1
1.910
1.927
7
2
2.056
2
1
.
3
7
.
2
1
1.896
2.058
ΔE = 0.0 (0.0)
ΔE = 0.3 (5.4)
2B with 6 DMSO
2A with 6 DMSO
Fig. 2. B3LYP/6-311++G** optimized geometries and relative energies (kcal/mol) of
glycinamide conformer 2A and 2B interacting with six DMSO molecules in DMSO solvent
medium using PCM solvation model. MP2/6-311++G** calculated energies are given in
parentheses (Only selected hydrogens are shown for clarity purpose). [Gray = carbon; blue =
nitrogen; red = oxygen; yellow = sulphur; white = hydrogen]
26
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Fig. 3. Part of the 1H NMR spectra of glycinamide corresponding to the amide-H atoms recorded
in CD3CN (6), CD3CN with the increasing amounts of DMSO-d6 (from 5-2) and DMSO-d6 (1).
27
Page 27 of 30

S
S
3
O
4
O
H
O
H
S
2
O
N
Hb
N
Ha
1
O
S
(a)
(b)
Fig. 4. LDA/PWC/DND calculated profile of variation in energy, O-C-C-N torsional angle, Ha-
O1 and N−N distance during molecular dynamics of 2ps, (a) at 25 °C temperature and (b) at 80
°C temperature.
Graphical Abstract
Conformational Preference of Glycinamide in Solution: An Answer Derived from
Combined Experimental and Computational Studies
Bishwajit Ganguly, Manoj K. Kesharwani, Nikola Basarić, Eringathodi Suresh, Abul Kalam
Biswas and Kata Mlinarić-Majerski
28
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ACN
DMSO
29
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