Revealing the Conformational Preferences of Proteinogenic Glutamic

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A: Molecular Structure, Quantum Chemistry, and General Theory

Revealing the Conformational Preferences of Proteinogenic Glutamic Acid Derivatives in Solution by H NMR Spectroscopy and Theoretical Calculations 1

Weslley G. D. P. Silva, Cláudio Francisco Tormena, and Roberto Rittner J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.8b02523 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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The Journal of Physical Chemistry

Revealing the Conformational Preferences of Proteinogenic Glutamic Acid Derivatives in Solution by 1H NMR Spectroscopy and Theoretical Calculations

Weslley G. D. P. Silvaa,b, Cláudio F. Tormenaa and Roberto Rittnera*

a

Chemistry Institute, University of Campinas, Campinas, São Paulo 13083-970, Brazil b

Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada

*Corresponding author: [email protected]

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Abstract The conformational preferences of proteinogenic glutamic acid esterified (GluOMe) and N-acetylated (AcGluOMe) derivatives have been determined in solution for the first time. Theoretical calculations at the ωB97X-D/aug-cc-pVTZ made possible the assignment of six and eight stable conformers for GluOMe and AcGluOMe, respectively. The conformational equilibrium of the studied compounds was evaluated in different organic solvents using a combination of the integral equation formalism polarizable continuum model (IEF-PCM) and 1H NMR spectroscopy data. The results showed that the conformational equilibrium of both derivatives change in the presence of solvent. According to the Quantum Theory of Atoms in Molecules (QTAIM), Non-Covalent Interactions (NCI) and Natural Bond Orbitals (NBO) analyses, the conformational preferences observed for GluOMe and AcGluOMe are not dictated by the presence of a specific interaction, but due to a combination of hyperconjugative and steric effects.


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1.

INTRODUCTION A complete understanding of the structures assumed by amino acids is of interest

in a variety of research fields, since they provide relevant information on the geometries adopted by macromolecules of biological relevance.1,2 Among the 20 proteinogenic amino acids entering into the composition of peptides, glutamic acid, or glutamate (Glu), has been under intense investigation due to its important role in the central nervous system and for being responsible for sensory information, motor coordination, emotions and cognition.3 Moreover, this amino acid is also used in the alimentary industry as an additive responsible for “umami taste”.4 The geometries of glutamic acid and its derivatives have been the subject of study by a few theoretical and experimental methodologies.1,3,5–9 The most recent and detailed conformational study of Glu was performed by Penã et al.1 using a combination of Fourier transform microwave (FTMW) spectroscopy with laser ablation (LA) and molecular beams (MB) in the LA-MB-FTMW technique. The investigation allowed the characterization of five different conformers of Glu in the gas-phase and their stabilities have been explained by the presence of intramolecular hydrogen bonding (IHB). On the other hand, to the best of our knowledge, no experimental studies dealing with the conformational analysis of Glu or its derivatives in solution have been reported. The lack of information about the geometries of Glu in solution can be related to the experimental difficulties presented by amino acids in general. In solution, instead of the neutral form, amino acids exhibit a bipolar zwitterionic structure (+H3N-CHR-COO-) stabilized by intermolecular hydrogen bonding formed with the solvent, which affects their conformational equilibrium. Furthermore, glutamic acid also has a relatively long side chain, which gives rise to many low energy conformers, making the theoretical



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calculations challenging. An alternative, which has been shown to be useful in the investigation of amino acid derivatives in solution, is the study of amino acids esterified and N-acetylated derivatives.10–14 These derivatives are soluble in most of organic solvents and thus, their conformational equilibrium can be studied by 1H NMR spectroscopy. Moreover, some studies have shown that the identification of N-acetyl amino acid compounds in body fluids can be associated to inborn metabolism errors and neurological diseases.15–17 In this sense, knowing the structures adopted by these derivatives, as well as the effects ruling their conformational preferences is relevant. On the basis of the aforementioned, this work presents the first conformational study of glutamic acid esterified (GluOMe) and N-acetylated (AcGluOMe) derivatives (Figure 1) in solution using a combination of 1H NMR spectroscopy and theoretical calculations. The stereoelectronic effects governing the conformational preferences of the studied compounds were evaluated using quantum topological methods by means of the Quantum Theory of Atoms in Molecules (QTAIM)18 and the Non-Covalent Interactions (NCI)19 analyses and orbital based Natural Bond Orbital (NBO)20 methodology.

Figure 1 Structures of the studied compounds: L-Glutamic Acid Dimethyl Ester (GluOMe) and N-acetyl-L-Glutamic Acid Dimethyl Ester (AcGluOMe).


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2.

2.1

EXPERIMENTAL AND THEORETICAL METHODS

Syntheses of the compounds The esterified derivative (GluOMe) was commercially available (Sigma-Aldrich)

as a hydrochloride salt (GluOMe.HCl) and it was deprotonated using activated zinc dust, whereas the N-acetylated derivative (AcGluOMe) was obtained by the esterification of the corresponding N-acetyl-L-glutamic acid (Sigma-Aldrich) in the presence of anhydrous methanol and thionyl chloride. The detailed procedures12,21 are described in the Electronic Supporting Information (SI).

2.2

NMR spectra 1

H and 13C NMR experiments were recorded on a Bruker Avance III spectrometer

operating at a frequency of 600.17 MHz for 1H and 150.91 MHz for 13C. NMR spectra were acquired using solutions of ca 10 mg in 0.7 mL of deuterated solvent (CDCl3, CD2Cl2, CD3CN, DMSO-d6) referenced to internal TMS. The 1H NMR spectrum of AcGluOMe was also recorded in D2O referenced to internal DSS. Moreover, two-dimensional NMR experiments (COSY, HSQC and HMBC) were also used to unequivocally characterize the structures of the synthesized compounds. Typical acquisition and processing conditions are shown in the NMR spectra provided in the Figures S1-S18 in the SI.

2.3

Computational Details All possible conformers of GluOMe and AcGluOMe were generated using the

Marvin Sketch 16.10.10 program (ChemAxon version 6.1) and their geometry optimized using the MMFF9422 force field method. The search was limited to 50 conformers. After, the possible geometries previously generated with the MMFF94 method were fully 5 ACS Paragon Plus Environment


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optimized at the ωB97X-D/aug-cc-pVDZ23,24 level of theory. Next, the geometries that yielded relative energies smaller than 2.0 kcal mol-1 were re-optimized in both isolated phase and in solution (CHCl3, CH2Cl2, CH3CN and DMSO) according to the Integral Equation

Formalism

Polarizable

Continuum

Model

(IEF-PCM)25

at

the

ωB97X-D/aug-cc-pVTZ23,24 level of theory. Frequency calculations with zero-point energy (ZPE) corrections were also carried out to guarantee that imaginary frequencies were absent. The conformational search gave rise to six and eight stable conformers for GluOMe and AcGluOMe, respectively. Spin-spin coupling constants (3JHH) were calculated for each conformer of GluOMe and AcGluOMe in the IEF-PCM solvent model using the ωB97X-D functional with EPR-III26 (for C and H atoms) and aug-cc-pVTZ (for O and N atoms) basis sets. All calculations were carried out using the Gaussian 09 program.27 Possible intramolecular interactions governing the stability of the studied compounds were evaluated using Natural Bond Orbital (NBO) analysis at the ωB97X-D/aug-cc-pVTZ level of theory and the Quantum Theory of Atoms in Molecules (QTAIM) and Non-Covalent Interactions (NCI) topological methodologies. These analyses were carried out using the NBO 5.9,28 AIMALL29 and NCIPLOT30 programs, respectively. 3.

3.1

RESULTS AND DISCUSSION

Nomenclature The conformers of GluOMe and AcGluOMe were named using a Roman numeral

followed by a letter. The numeral represents the order of stability of the compounds in chloroform, while the letters (a, b or c) denote the relationship between the side and main chains. The three possible arrangements adopted by the conformers of the studied 6 ACS Paragon Plus Environment

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compounds are shown in the Newman projections of Figure 2. In geometry a, the hydrogen Ha is synperiplanar (syn), or gauche, to the hydrogens (Hb1 and Hb2) of the vicinal CH2 group bonded to the asymmetric center, while in geometries b and c, Ha is antiperiplanar (anti) to Hb2 and Hb1, respectively. Additionally, for the conformers of AcGluOMe, besides the above-mentioned nomenclature, the designation syn-anti was also used to indicate the position of the amide linkage with respect to the C(O)OMe group of the backbone. The geometries of GluOMe and AcGluOMe, as well as their relative energies and geometric parameters at the ωB97X-D/aug-cc-pVTZ level are shown in the Figures S19-S20 and Tables S1-S2 in the SI, respectively.

Figure 2 Three possible arrangements (a, b and c) adopted by the most stable conformers of GluOMe and AcGluOMe.

3.2

L-Glutamic Acid Dimethyl Ester (GluOMe)

3.2.1 Conformational equilibrium of GluOMe The population of each conformer of GluOMe was grouped into the three possible arrangements (a, b or c) and the contribution of each form for the conformational equilibrium of GluOMe is shown in Table S3. These populations are derived from ΔG energies obtained at the ωB97X-D/aug-cc-pVTZ level of theory for the isolated compound and in different solvents (IEF-PCM). According to the theoretical calculations (Error! Reference source not found.3), conformers b are favored in the conformational 7 ACS Paragon Plus Environment


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equilibrium of GluOMe in all different studied media, representing more than 80.0% of the conformational equilibrium of GluOMe. No significant variations in the population of conformers b are observed when the polarity of the solvent is increased from CHCl3 to DMSO and thus, the theoretical calculations indicate that the conformational equilibrium of GluOMe is not sensitive to the solvent effects. To evaluate the influence of the solvent on the conformational equilibrium of GluOMe, 1H NMR spectroscopy data and experimental and theoretical 3JHH coupling constants values were examined. The advantage of using 1H NMR spectroscopy in conformational analysis studies such as the one conducted here, is the dependence that exists between the 3JHH and the dihedral angle value of two coupled hydrogen atoms. Based on the well-known Karplus- type31 equations, hydrogen atoms in an anti relationship are expected to have higher 3JHH values than hydrogens in a gauche relationship. Moreover, due to this dependence between 3JHH and dihedral angles, changes in the 3JHH values upon media variation are an indication that dihedral angles are also varying and thus, the conformational equilibrium of this compound is sensitive to solvent effects.32 As the observed 3JHH,obs coupling constants, obtained in 1H NMR spectra, represent a weighted average of the contribution of each conformer present in the conformational equilibrium, the calculated 3JHH,calc values represent the sum of the individual 3Ji coupling constant multiplied by the relative population, or molar fraction (ni), of each conformer i present in the equilibrium, as shown in Equation 1:32 3

𝐽𝐻𝐻,𝑐𝑎𝑙𝑐 = ∑ 𝑛𝑖

×

3

𝐽𝑖 (1)

In fact, the calculated 3JHH,calc values (Error! Reference source not found.4) for GluOMe confirm the predominance of conformers b in all different media, evidenced by the two different values of 3JHaHb1,calc (~ 10.0 Hz) and 3JHaHb2,calc (~ 3.0 Hz). These 8 ACS Paragon Plus Environment

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different values represent, respectively, the anti and gauche couplings that are present in the conformers b. Moreover, in 1H NMR spectra, the signal of Ha is observed as a triplet in CDCl3, CD2Cl2 and CD3CN with two similar experimental 3JHH,obs of about 6.0 Hz (Error! Reference source not found.S4) that represent the average of the calculated ones. However, in DMSO-d6, instead of two similar 3JHH,obs values, the signal of Ha is characterized as a doublet of a doublet with experimental 3JHaHb1,obs and 3JHaHb2,obs values of 6.4 and 7.0 Hz, respectively. A comparison between the multiplicity of hydrogen Ha in the different solvents is depicted in Figure 3.



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Figure 3 1H NMR spectra of GluOMe in different solvents (CDCl3, CD2Cl2, CD3CN and DMSO-d6). In the spectra, chemical shifts are given in ppm, whereas in the highlighted signal of Ha, in Hz. Typical acquisition and processing data are shown in the individual spectra in the Figures S2-S5 in the SI. Although theoretical calculations indicate the conformational equilibrium of GluOMe is not very sensitive to solvent effects, the variations observed in the signal of Ha upon solvent change show that the conformational equilibrium of GluOMe changes when the solvent polarity increases from CDCl3 to DMSO. As the theoretical calculations are consistent with the results obtained in CDCl3, CD2Cl2 and CD3CN and the level of theory (ωB97X-D/ aug-cc-pVTZ) used in the present study has presented coherent results


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for similar studied systems,10–12 the differences observed between theoretical and experimental data in DMSO can possibly be attributed to the use of an implicit solvent model in the study. It is likely that the implicit solvent model does not consider some specific interaction that occurs in GluOMe when the solvent is DMSO-d6.

3.2.2 Evaluation of the stereoelectronic effects ruling the conformational preferences of GluOMe The most stable conformers of GluOMe (Figure S19) exhibit at least one amine hydrogen oriented towards the oxygen of the carbonyl group of the backbone and thus, some interactions involving these atoms are expected. It was shown that intramolecular hydrogen bonding (IHB) NH…O=C, the so-called type I IHB, that occurs between the -NH2 and the α-COOH groups is responsible for the conformational preferences of free and protonated glutamic acid, and also other aliphatic α-amino acids.1,9,33,34 Furthermore, IHB NH…O=C interactions are also known for contributing to (or explaining) the stabilization of some dipeptides and proteins.14,35,36 Although the α-COOH of free glutamic acid has been substituted by an ester (α-COOMe) in GluOMe, the presence of the type I IHB NH…O=C, which is illustrated in the Figure 4 for the conformers Ib and IIa of GluOMe, is also expected. Consequently, NBO, QTAIM and NCI analyses were carried out to investigate the effects responsible for the conformational preferences of GluOMe.



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Figure 4 Representation of the possible type I IHB NH…O=C in the conformers Ib and IIa of GluOMe. Although only these two conformers are illustrated, other conformers of GluOMe can also exhibit this interaction. According to the QTAIM analysis (Figure S21), no bond path (BP) or bond critical point (BCP) is observed between the amine hydrogens and the carbonyl oxygen of the α-COOMe group, indicating that there is no type I IHB in the conformers of GluOMe. In contrast, the NCI analysis (Figure S22) shows the presence of a weak NH…O=C attractive interaction in all conformers of GluOMe, visualized by the presence of a bluer isosurface and a trough with λ2 < 0 (ρ between 0.01 and 0.02 au) in the NCI isosurface and in the graph of the reduced density gradient (S) versus sign (λ2)ρ, respectively. The values of ρ found for the GluOMe derivative are in agreement with the values recently reported for the conformers of protonated glutamic acid, which were also obtained based on NCI analysis.9 The presence of this weak NH…O=C attractive interaction is confirmed by the NBO method (Table S5), evidenced by the presence of the LP2(O)→σ*NH hyperconjugative interaction. However, the absolute values of the second order perturbation energies related to these interactions are small (< 0.4 kcal mol-1) and although the type I IHB plays a major role in the stabilization of free and protonated glutamic acid, for the GluOMe derivative, the same trend is not observed. One could also 12 ACS Paragon Plus Environment

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consider the effects of electrostatic interactions to the stabilization of GluOMe. Although the system does not present any formal charges and the study was carried out in solution, coulombic interactions can also play a role in the stabilization of polar molecules with close intramolecular contacts, such as the GluOMe derivative discussed here. The investigation of the contributions of steric (ELewis) and hyperconjugative (EHyper) effects to the total energy of the system, also obtained from NBO calculations (Table S5), shows that the conformers of GluOMe, which are more stabilized due to hyperconjugative effects are also more destabilized by steric repulsion. Therefore, the conformational preferences of GluOMe are explained by a combination of hyperconjugative and steric effects.

3.3

N-Acetyl L-Glutamic Acid Dimethyl Ester (AcGluOMe)

3.3.1 Conformational equilibrium of AcGluOMe The AcGluOMe derivative differs from GluOMe due to the presence of an amide group instead of the amine group. Most of the conformers of AcGluOMe (Figure S20) present the amide linkage anti to the C(O)OMe group of the backbone, which follows the same trend observed for other N-acetylated amino acid derivatives previously studied in the literature.10,11,12 Contrary to what was observed for the conformers of GluOMe, no conformers a are observed for AcGluOMe and thus, the conformational equilibrium of this compound is governed by only conformers that present arrangements b and c. According to the theoretical calculations (Table S6), in isolated phase and CHCl3, conformers c are more favored than conformers b and represent approximately 60.0% of the total population of AcGluOMe. On the other hand, in more polar solvents, such as CH2Cl2, CH3CN and DMSO, the opposite is observed and the conformers b account for



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approximately 60.0% of the conformational equilibrium. The inversion observed in the populations of conformers b and c when the solvent dielectric constant changes from that of CHCl3 to DMSO indicates that the conformational equilibrium of AcGluOMe is sensitive to solvent effects. As for the GluOMe derivative, the influence of the solvent in the conformational equilibrium of AcGluOMe was also investigated using experimental and theoretical 1H NMR data (Table S7). The calculated 3JHaHb1,calc and 3JHaHb2,calc values confirm the predominance of conformers c in CHCl3, evidenced by the greater value of 3JHaHb1,calc (8.2 Hz) in comparison to 3JHaHb2,calc (6.7 Hz). When the polarity of the solvent is varied from CHCl3 to DMSO, 3JHaHb1,calc has its value decreased from 8.2 to 5.6 Hz, while the 3

JHaHb1,calc increases from 6.7 to 9.1 Hz, confirming the predominance of conformers b in

more polar solvents. The observed coupling constants 3JHaHb1,obs and 3JHaHb2,obs can assume one of two different values (approximately 8.0 or 5.0 Hz) in the solvents studied. In this way, the greater value of 3JHH refers to the anti, while the smaller represents the gauche coupling, which are observed between hydrogen Ha and hydrogens Hb1 and Hb2. Contrary to the theoretical calculations, experimentally is not possible to distinguish between hydrogens Hb1 and Hb2, since the hydrogens that are anti or gauche to hydrogen Ha will always have the same chemical shift in the 1H NMR spectra. In this manner, considering only the experimental values of 3JHH,obs, one could erroneously assume that the conformational equilibrium of AcGluOMe is not affected by the solvent change since in the 1H NMR spectrum of AcGluOMe in each solvent, two 3JHH,obs of about 8.0 and 5.0 Hz are observed. However, with the use of quantum mechanical calculations, it is possible to conclude that the conformational equilibrium of AcGluOMe changes upon solvent change. In this case, the coincidence in the values of 3JHH,obs in the different solvents can be explained by the 14 ACS Paragon Plus Environment

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proportional inversion in the populations of conformers b and c when the polarity of the solvent is changed from CDCl3 to DMSO-d6. Experimental 1H NMR data of AcGluOMe were also recorded in aqueous solution. The values of 3JHaHb1,obs and 3JHaHb2,obs in D2O (Table S7) are similar to those obtain in CD2Cl2, CD3CN and DMSO-d6 and thus, the results obtained in these solvents are a good approximation to the conformational equilibrium of AcGluOMe in aqueous solution.

3.3.2 Evaluation of the stereoelectronic effects ruling the conformational preferences of AcGluOMe The most stable conformers of AcGluOMe (Figure S20) exhibit the amide hydrogen oriented towards one of the two oxygen atoms of the C(O)OMe group of the backbone. Thus, as for the GluOMe derivative, the presence of an intramolecular interaction involving these atoms was also investigated in the conformers of AcGluOMe using QTAIM, NCI and NBO analyses. According to the QTAIM analysis (Figure S23), neither a BP nor a BCP regarding the IHB NH…O was characterized, indicating the absence of this interaction in the conformers of AcGluOMe. On the other hand, the analysis shows a BP and a BCP related to a weak IHB CH…O due to the formation of a 6-membered ring which occurs between a hydrogen atom of the CH2 group vicinal to the ester of the backbone and one of the oxygen atoms of the C(O)OMe group, in the conformers anti-Ic, anti-IIc and anti-Vc. To verify the stability of this CH…O interaction, the evaluation of some QTAIM parameters (Table S8) was carried out following the well-known Koch and Popelier37 (KP) criteria. Only the IHB CH…O exhibited by the conformer anti-Ic follows the KP criteria. 15 ACS Paragon Plus Environment


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In agreement with the QTAIM analysis, the NCI method (Figure S24) confirms the presence of the CH…O attractive interaction in the conformer anti-Ic. However, the NCI results also indicate the presence of this interaction in the conformers anti-IIc and anti-Vc, which can be visualized by the trough with ρ near 0.01 au and the presence of a greenish isosurface in the graph of S versus sign (λ2)ρ and in the NCI isosurface, respectively. This interaction appears in green in the NCI isosurface because it is a weak (van der Waals) interaction. A similar CH…O attractive interaction was recently reported in the literature9 for the most stable conformers of protonated glutamic acid. Although the QTAIM analysis does not show the presence of the NH…O interaction, as for the conformers of GluOMe, the NCI analysis indicates the presence of this interaction in most of the conformers of AcGluOMe, except syn-VIIb and syn-VIIIb. Similarly to the GluOMe derivative, the interaction can be visualized by the presence of a bluer isosurface and a trough with λ2 < 0 (ρ between 0.01 and 0.02 au) in the NCI isosurface and in the graph of S versus sign (λ2)ρ, respectively. NBO calculations (Table S9) confirm the presence of the NH…O interactions in the conformers of AcGluOMe, except syn-VIIb and syn-VIIIb, evidenced by the hyperconjugative LP(O) → σ*NH interaction. When the interaction involves the carbonyl oxygen (O6) of the ester, the calculations show that there is a better contribution of the LP2(O) to the charge transfer LP(O) → σ*NH. It is well known that an oxygen atom presents two different lone-pairs, the LP1(O) and LP2(O). Although the orbital LP2(O) is a better electron donor when compared with LP1(O), since LP2(O) is the higher energy p-type orbital whereas LP1(O) is the low energy rich in s-character orbital, when the amide hydrogen is oriented to the non-carbonyl oxygen (O7) of the ester, the lone pair LP1(O) plays a major role in the LP(O) → σ*NH interaction.38 This fact can be explained due to the presence of resonance structures in the ester and a better overlap between the 16 ACS Paragon Plus Environment

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LP1(O) and σ*NH orbitals. The main role of the LP1(O) orbital for intramolecular hydrogen bonding interactions was recently reported by Karas et al. for a series of substituted alcohols.39 This trend can be exemplified with the second order perturbation energy values of the LP(O) → σ*NH interactions obtained for conformers anti-Ic and anti-IIc (Figure 5). Conformer anti-Ic has the amide hydrogen oriented to O7 and only the LP1(O7) → σ*NH (0.59 kcal mol-1) interaction is observed. On the other hand, the conformer anti-IIc, which has the amide hydrogen oriented to the carbonyl oxygen (O6) experiences both LP1(O6) → σ*NH and LP2(O6) → σ*NH of 0.29 and 1.00 kcal mol-1, respectively. For conformer anti-IIc, it is evident the higher contribution of LP2(O6) for the LP(O) → σ*NH interaction.



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Figure 5 Orbital plots and second order perturbation energies, in kcal mol-1, of the LP(O) → σ*NH interactions presented by conformers anti-Ic and anti-IIc. The NBO calculations also confirmed the presence of the CH…O attractive interaction in some conformers of AcGluOMe, evidenced by the LP(O) → σ*CH hyperconjugative interactions. The energies for these interactions are also shown in Table S9.


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In general, both NH…O and CH…O interactions present small absolute values of energy and thus, they do not play a major role in the stability of the conformers of AcGluOMe. The investigation of the contributions of steric (E Lewis) and hyperconjugative (E Hyper) effects to the total energy of the system (Table S10) shows that not one specific interaction governs the conformational preferences of AcGluOMe, but rather, a combination of steric and hyperconjugative effects. 4.

CONCLUSIONS The use of 1H NMR spectroscopy combined with theoretical calculations allowed

the first conformational study of glutamic acid esterified and N-acetylated derivatives in solution. The use of theoretical and experimental 3JHH coupling constants showed that the conformational equilibrium of GluOMe is not particularly sensitive to solvent effects, whereas the conformers of AcGluOMe have their populations changed upon solvent change. Results obtained for AcGluOMe in aqueous solution were very similar to those obtained in CD2Cl2, CD3CN and DMSO-d6 and thus, the same conformational behaviour is expected for these studied media. NBO, QTAIM and NCI methodologies confirmed the presence of an attractive NH…O interaction in most of the conformers of GluOMe and AcGluOMe, which is in agreement with previous glutamic acid studies. However, unlike those of free and protonated glutamic acid, the conformational preferences of both derivatives are not explained by a specific interaction, but instead due to a combination of steric and hyperconjugative effects.



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ASSOCIATED CONTENT Supporting Information Detailed procedures for preparation of the compounds; NMR spectra for the studied compounds; Pictorial representation of the conformers of GluOMe and AcGluOMe, as well as their calculated energies, experimental and theoretical 3JHH coupling constants, natural bond orbitals results, QTAIM and NCI molecular graphs, and hyperconjugative interactions are provided in the Supporting Information file. Notes The authors declare no competing financial interest ACKNOWLEDGEMENTS The authors thank São Paulo Research Foundation (FAPESP, Grant 2015/085416 and #2016/24109-0) for the financial support of this work as well as for the scholarship to W.G.D.P.S. #2016/12005-5. Thanks also to Conselho Nacional de Pesquisa (CNPq) for a fellowship (to R.R. and C.F.T.).


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TOC Graphic


Revealing the Conformational Preferences of Proteinogenic Glutamic

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