Combined Utilization of 1H NMR, IR, and Theoretical Calculations To

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A Combined Utilization of H NMR, IR and Theoretical Calculations to Elucidate the Conformational Preferences of Some L-Histidine Derivatives Carolyne Brustolin Braga, and Roberto Rittner J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.6b12515 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 4, 2017

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The Journal of Physical Chemistry

A Combined Utilization of 1H NMR, IR and Theoretical Calculations to Elucidate the Conformational Preferences of Some LHistidine Derivatives Carolyne B. Bragaa,* and Roberto Rittnera a

Chemistry Institute, University of Campinas, P.O. Box 6154, 13083-970, Campinas, SP, Brazil.

1 ACS Paragon Plus Environment


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Abstract The conformational preferences of amino acids and their derivatives have been subject of many investigations, since protein folding pathways that determine three-dimensional geometries are primarily restricted by the conformational space of each amino acid residue. Here we systematically describe the conformational behavior of L-histidine methyl ester (His-OMe) and its N-acetylated derivative (Ac-His-OMe) in isolated phase and in solution. To this end, we employed spectroscopic techniques (1H NMR and IR), supported by quantum chemical calculations. Initially, the energetically favourable conformers, their energies and structural properties obtained by density functional theory (DFT) and MøllerPlesset perturbation theory (MP2) calculations in isolated phase and in solution via the implicit solvation model IEF-PCM were presented. Next, experimental 3JHH spin-spin coupling constants obtained in different aprotic nonpolar and polar solvents were faced with the theoretically predicted ones for each conformer at IEF-PCM/ωB97X-D/EPR-III level. A joint analysis of these data allowed the elucidation of the conformational preferences of the compounds in solution. Infrared data were also employed as complement to estimate the His-OMe conformer populations. Finally, the Quantum Theory of Atoms in Molecules (QTAIM), the Non Covalent Interactions (NCI) and the Natural Bond Orbitals (NBO) analyses were used to determine the intramolecular interactions that govern the relative conformational stabilities.


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1. Introduction The different stable spatial arrangements that a molecule can assume due to the rotation of its single bonds, named conformations or conformers, have a critical effect on their physicochemical properties. Thus, the study of the conformational preferences of acyclic and alicyclic compounds is of great interest for physical organic chemists, biochemists, spectroscopists, etc. In particular, extensive structural research has been conducted on the conformational equilibrium of amino acids and small peptides,1-5 in an attempt to elucidate their dynamic role in proteins or polypeptides formation, since the spatial arrangements of the latter are intrinsically related to their corresponding biological function.6-8 A complete understanding regarding the way these biomacromolecules fold would result in a significant impact on humanity, such as the development of more specific drugs for diseases that claim millions of lives annually. Among the different studies related to the conformational behavior of amino acids, those in their natural solid-state have been the most extensively addressed over the years.910

However, a negative aspect of these reports is due to the fact that amino acids in crystals

exhibit a bipolar zwitterionic structure [+H3N-CH(R)-COO-] with very different characteristics from those of the neutral one [H2N-CH(R)-COOH] that occurs in polypeptide chains. For example, the intermolecular interactions via hydrogen bondings lead to a considerable stability of the zwitterion when compared to the neutral structure, and it is difficult to determine the inherent conformational preferences and corresponding intramolecular interactions of the bare molecule. As in crystals, amino acids in aqueous medium are also known to adopt this zwitterionic form over a wide pH range.9,11,12 Nowadays, rotational and vibrational spectroscopy have been employed for investigating amino acids in gas phase, where they exhibit a neutral form free of disturbing



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agents like solvent or neighbour molecules.13-15 Although the high melting points, very low vapor pressures and thermal instabilities of amino acids constitute a barrier to their gas-phase studies, significant instrumental improvements have been recently made to vaporize them, in order to obtain important information about these protein building blocks. However, only a limited number of studies have focused on isolated amino acids and relevant structural aspects concerning these systems remain unknown, mainly when dealing with amino acid containing more complex side chains, such as histidine, which is proved to be difficult to characterize due of its physical properties.16 As a good approximation to the electronic environment of an amino acid residue in a protein chain and also in order to circumvent the experimental limitations above mentioned, we have performed systematic studies about the conformational behavior of amino acid methyl esters (R-OMe) and their N-acetylated derivatives (Ac-R-OMe), where R = amino acid, employing spectroscopic methods (NMR and IR) in conjunction with theoretical calculations.17-21 Since these compounds do not form zwitterions and are soluble in organic solvents, they can be studied in solution by using these powerful methodologies, such as the NMR spectroscopy. This alternative is capable of providing more relevant information about the conformational preferences of amino acid residues in condensed media than gas-phase study. Furthermore, while the conformational preferences of amino acids are often interpreted in terms of intramolecular hydrogen bondings (IHBs),13,15,16 we have demonstrated that the stabilities of the most stable conformers of their derivatives are controlled by a balance between hyperconjugative and steric effects. Surprisingly, the formation of IHBs represents only a secondary effect in their conformational preferences. It is therefore of interest to investigate thoroughly all these intramolecular interactions.


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As a further step in our research to understand the conformational behavior of -amino acids derivatives and the intramolecular interactions responsible for conformational stability in both isolated phase and solution, we now report an investigation of some L-histidine derivatives (Scheme 1). Histidine (His) is one of the twenty proteinogenic amino acids and plays a pivotal role in many relevant biological processes due to its unique molecular structure22 composed by an aromatic imidazole moiety in its side chain. Thus, 1H NMR, IR and theoretical calculations of electronic structure were employed to search the most stable conformers of His-OMe and Ac-His-OMe (Scheme 1) and to assess the conformational preferences observed. We also used the Quantum Theory of Atoms in Molecules (QTAIM), the Non Covalent Interactions (NCI) and the Natural Bond Orbitals (NBO) analyses to interpret the obtained results in terms of intramolecular interactions (IHB, steric hindrance and hyperconjugation).

(a)

(b)

Scheme 1. Studied compounds: (a) L-histidine methyl ester (His-OMe) and (b) N-acetyl-Lhistidine methyl ester (Ac-His-OMe).

2. Experimental and Theoretical Methods 2.1. Preparation of the compounds His-OMe was commercially available as a hydrochloride salt (His-OMe • 2HCl) and was deprotonated using sodium methoxide in absolute methanol, while the N-acetylated derivative (Ac-His-OMe) was prepared from the esterification of commercial N(-acetyl5 ACS Paragon Plus Environment


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L-histidine

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hydrate (Ac-His-OH • H2O) using anhydrous methanol and thionyl chloride. The

detailed procedures are described in the Supporting Information.

2.2. 1

NMR Spectra

H NMR spectra were recorded on a Bruker Avance III spectrometer operating at

600.17 MHz for 1H. Spectra were obtained using solutions of ca. 15 mg in 0.7 mL of deuterated solvents (CDCl3, CD2Cl2, CD3CN and DMSO-d6, according to the solubility of the compound) and all chemical shifts were referenced against internal TMS. Measurements were carried out at 5 mm TBI probe, at temperature of 25 oC. The typical conditions used were: from 16 to 32 transients (depending on solute solubility), spectral width around 6.0 kHz, and 64 k data points, giving an acquisition time of ca. 6 s. The free induction decays (FID) were zero-filled to 128 k, providing a digital resolution of about 0.09 Hz/point. 1H NMR spectra were provided in the Supporting Information (Figures S1 - S6).

2.3.

Infrared Spectra

Infrared spectra for His-OMe were acquired using samples with concentrations of ca 0.03 mol L-1 in solvents of different polarities (CHCl3, CH2Cl2, CH3CN and DMSO), which were dried and purified following standard methods and stored over freshly prepared molecular sieves. The IR spectra were recorded on a Shimadzu FTIR Prestige-21 spectrometer continuously purged with dry nitrogen gas during the measurements. All spectra were acquired at 1 cm-1 resolution and averaged using 64 scans. A CsF cell was used for DMSO, while a NaCl cell was employed for the other solvents, both liquid cells with optical path of 0.5 mm to register the carbonyl stretching band in the fundamental region (1800-1600 cm-1). The spectra were analyzed with the GRAMS AI spectroscopy software suite23 and the overlapped carbonyl bands were deconvoluted by means of the curve fitting 6 ACS Paragon Plus Environment

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procedure. The populations of the different conformers were estimated from the area of each component of the resolved carbonyl doublet.

2.4.

Computational Details

The low energy His-OMe conformers were initially selected through 3D-potential energy surfaces (PES). Aiming to reduce the computational cost of the calculations, the arrangements of the backbone [CH3-O-C(O)-CH(NH2)-] of the six less energetic conformers (I, III, IV1, IV2, V1 and V2) previously optimized for L-alanine methyl ester (Ala-OMe)17 were used as starting points. To this end, a methyl hydrogen atom (side chain) of each AlaOMe conformer was replaced by the CH2-imidazole group, giving rise to the side chain of L-histidine

and, consequently, to six His-OMe geometries. Thus, the six PES showed in

Figure S7 in the Supporting Information were built by simultaneous scanning the 1 [C-CC-C(O)] and 2 [(C=)N-C-C-C)] dihedral angles (Figure 1) of these new geometries from 0° to 360° in steps of 10° at the B3LYP/cc-pVDZ level. It is noteworthy that at this step the

[nN-N-C-C(O)] and [N-C-C=O] dihedral angles (previously optimized during Ala-OMe conformational investigation) were kept fixed to preserve the optimized geometry of the backbone. Based on the location of the valleys in the six PES, a total of 34 local minima unique conformers of the His-OMe have been located in our calculations.



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Figure 1. Analysed dihedral angles: (a) 1 [C-C-C-C(O)] and 2 [(C=)N-C-C-C)] for HisOMe and (b) θ [C-C(=O)-N-C] for Ac-His-OMe.

Then, optimization and frequency calculations without geometric restrictions were carried out for the 34 His-OMe minima by using the B3LYP,24 B3LYP-D3,25 CAMB3LYP,26 M05-2X,27 M06-2X,28 B97-D29 and B97X-D30 DFT functionals with the augcc-pVTZ basis set and zero-point energy (ZPE) correction. The lack of negative harmonic vibrational frequencies confirmed that all conformers are energy minima (Figure S8 in the Supporting Information). Although these geometries are theoretically possible, some of the optimized conformers do not present significant contribution to the conformational equilibrium of the isolated compound and including the solvent effect (conformers with relative energy above 1.5 kcal mol-1 are not expected to exist considerably). Then, Table S1 shows a complete comparison of the energies, relative energies, main dihedral angles and dipole moments obtained for the six lowest energy His-OMe optimized conformers, in isolated phase, using the different methods above mentioned, with the aug-cc-pVTZ basis set.31 The B97X-D/aug-cc-pVTZ level showed the smallest mean absolute deviation (MAD) from MP2/aug-cc-pVTZ31,32 single point energy calculations (optimized geometries at the MP2/aug-cc-pVDZ) and, hence, it was used in all subsequent calculations. The conformer populations were estimated following Boltzmann distribution. Also, the same frequency calculations were used to evaluate thermodynamic corrections affording Gibbs free energies at ambient, standard temperature and pressure for each conformer. 8 ACS Paragon Plus Environment

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Conformers of Ac-His-OMe in isolated phase were searched from lowest energy geometries previously found for His-OMe, where one of the hydrogen atoms of the amino group in His-OMe geometry was replaced by the N-acetyl group. Then, the potential energy curves (PEC) were scanned at the B3LYP/cc-pVDZ level by varying the θ [C-C(=O)-N-C] dihedral angle (Figure 1) in steps of 10° from 0 to 360°. Two stereoisomers (cis and trans) were found in each PEC. All the conformers found were fully reoptimized and their frequencies were calculated with Gibbs free energy correction, at the B97X-D/aug-ccpVTZ level of theory. The geometries of the studied compounds were also fully optimized by using an implicit solvent model, specifically the IEF-PCM [Integral Equation Formalism variant of the Polarizable Continuum Model]33 in aprotic solvents of different dielectric constants, at the B97X-D/aug-cc-pVTZ level. Also, using these optimized structures in solution, the 3

JHH spin-spin coupling constants (SSCC) were computed at the B97X-D/EPR-III and

BHandH/EPR-III theoretical levels for the representation of the hydrogen atoms. Since theoretically predicted SSCC are very sensitive to the method of calculation and the basis set, B97X-D and BHandH functionals were selected because generally produce fairly good results for a large variety of SSCC involving H and C atoms, while EPR-III34 basis set was developed and optimized for the calculation of the Fermi contact (FC), which is usually the most important component of SSCC. The aug-cc-pVTZ basis set was used instead of EPRIII for representing the remaining oxygen and nitrogen atoms. Finally, fully optimized geometries from B97X-D/aug-cc-pVTZ calculations were used to run Natural Bond Orbital (NBO)35 calculations on the same theoretical level. The keyword NOSTAR was included in the NBO input calculations to assess the importance of hyperconjugative and steric interactions in stabilizing the conformers. Also, Quantum Theory of Atoms in Molecules (QTAIM)36 and Non Covalent Interactions (NCI)37 methods 9 ACS Paragon Plus Environment


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were carried out on the electron densities obtained from B97X-D/aug-cc-pVTZ optimized conformers to indicate possible intramolecular hydrogen bondings. The PES, PEC, optimization, frequency, IEF-PCM, 3JHH SSCC and NBO calculations were carried out in the Gaussian09 program package, Revision D.01,38 while QTAIM and NCI analysis employed the AIMALL39 and NCIPLOT 3.040 programs, respectively.

3. Results and Discussion

3.1. 1

L-histidine

methyl ester

H NMR spectra of His-OMe were acquired in aprotic solvents with different dielectric

constants (spectra are displayed in the Figures S1 - S4 of the Supporting Information) in order to determine the conformational preferences of this compound in solution. The 3JHaHb spin-spin coupling constants were determined, as well as the corresponding chemical shifts, and their values are shown in Table 1. The methylene protons Hb1 and Hb2 are diastereotopic and, therefore, exhibit distinct chemical shifts and coupling constants.

Table 1. Experimental 1H NMR chemical shifts (δ, in ppm, related to TMS) and 3JHaHb coupling constants (in Hz)a for the His-OMe obtained in solvents of different dielectric constants (ε). Solvent

ε

δHa

δHb1

δHb2

CDCl3 CD2Cl2 CD3CN DMSO-d6

4.8 9.1 37.5 46.7

3.73 3.78 3.67 3.56

3.04 3.09 2.93 2.81

2.84 2.88 2.81 2.70

a

3J

HaHb1

4.32 4.40 5.07 5.77

3J

HaHb2

8.01 7.91 7.36 7.05

Error in measurements of J = ± 0.05 Hz.

The data in Table 1 show that the 3JHaHb1 and 3JHaHb2 coupling constants vary depending on the solvent. There is an increase in the 3JHaHb1 values of about 1.5 Hz from CDCl3 to 10 ACS Paragon Plus Environment

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DMSO-d6, whereas the opposite behavior is observed for the 3JHaHb2 values, i.e. a decrease, and the difference between the less and more polar solvents is approximately 1.0 Hz. Another important observation is related to the fact that both 3JHaHb coupling constants have very different values in nonpolar solvents (4.3 and 8.0 Hz, respectively), but their values become closer to each other with the increase in the solvent polarity. Therefore, this result suggests that the His-OMe conformer populations are affected by the solvent effect. Based on well-known Karplus relationship41 and considering also that the rotational isomerism of His-OMe is constituted by geometries in the forms a, b and c (Figure 2), it is expected that conformers with the arrangements b and c exhibit larger values of 3JHaHb2 and 3

JHaHb1, respectively, than the corresponding coupling constants for the geometries a. This is

because conformers b and c have one anti relationship between the hydrogen atoms Ha and Hb, while the conformation a presents only gauche dispositions between these atoms. Thus, it can be suggested that the experimental variations in the 3JHaHb values with the increase of the dielectric constant values would be mainly due to increase of the population of the conformers c with respect to the other two arrangements, justifying the simultaneous increase of 3JHaHb1 and decrease of 3JHaHb2.

a

b

c

Figure 2. Newman projections representing the three possible arrangements (a, b and c) of the side chain in the studied histidine derivatives, resulting from rotation around the CCbond.



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Aiming to elucidate the NMR results, theoretical calculations of electronic structure were carried out. According to our calculations, 34 conformers were identified to compose the His-OMe conformational equilibrium, but of these, only six (Figure 3) are representative (with relative energies below 1.5 kcal mol-1) in isolated phase (i.e. vacuum) and in solution (using the implicit model IEF-PCM). The Gibbs free energies and populations of these six conformers are shown in Table 2. Due to the several His-OMe conformations with very close energies, besides the letters a, b and c, referring to the arrangements presented in Figure 2, the conformers were labeled I to VI in order of increasing B97X-D/aug-cc-pVTZ energy in isolated phase (Table 2).

Ia

IIa

IIIc

IVa

Vb

VIb

Figure 3. Spatial representations of the most stable conformers of His-OMe, optimized at

B97X-D/aug-cc-pVTZ level.


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Table 2. Relative Gibbs free energies (Grel, in kcal mol-1) and populations (P, in %) in isolated phase and in different solvents (IEF-PCM implicit solvation model) for the HisOMe conformers, calculated for the optimized geometries at B97X-D/aug-cc-pVTZ level. The dipole moments (in Debye) in isolated phase are also shown.

Conformer



Isolated phase

CHCl3

CH2Cl2

CH3CN

DMSO

Grel

P

Grel

P

Grel

P

Grel

P

Grel

P

Ia

5.32

0.00

42.8

0.00

55.3

0.00

54.6

0.00

42.9

0.00

41.8

IIa

2.95

0.16

32.6

0.85

13.2

0.91

11.8

0.63

14.8

0.60

15.1

IIIc

4.26

0.87

9.9

0.99

10.4

0.75

15.2

0.42

21.2

0.39

21.6

IVa

4.58

1.19

5.7

0.92

11.7

1.24

6.8

1.12

6.4

1.10

6.5

Vb

5.87

1.25

5.2

1.82

2.5

1.83

2.5

1.60

2.9

1.58

2.9

VIb

4.25

1.43

3.8

1.23

6.9

1.06

9.1

0.76

11.8

0.73

12.1

Two conformers of His-OMe, Ia and IIa, predominate in isolated phase with populations of about 43 and 33%, respectively. They have very similar geometries and differ essentially in the orientation of the imidazole ring (Figure 3). In turn, the theoretical results taking into account the solvent effect (IEF-PCM) show that the conformer populations vary according to the dielectric constant of the solvent, as expected, and the conformers Ia and IIIc are the most affected by such a change. Unlike the isolated phase, the conformational equilibrium of this compound in solution becomes dominated by Ia, which presents considerable population values (about 55% in less polar solvents with the decrease to 42% in more polar solvents). Moreover, the conformer IIIc is the most stabilized in polar solvents (from 10.4% in CHCl3 to 21.6% in DMSO). Therefore, these results suggest that the hypothesis proposed above is correct, i.e. stabilization at least one conformer c in more polar solvents, and it seems to be directly related to what was observed experimentally. It is worth noting that these six amino ester conformers exhibit good agreement with the previously reported conformers for the correspondent amino acid.3,42,43 It is well-established that the experimental NMR coupling constants measured for a molecular system in fast interconversion among its stable conformers are given by the



weighted averages of the molar fractions (i) and coupling constants for the individual conformers (3JHaHb,i) in its equilibrium according to the Eq. (1).44, 45 Thus, considering that the i and 3JHaHb,i values are readily calculated with quantum mechanical calculations,46 they can be used to interpret the experimental 3JHaHb values. 3

JHaHb = Σ (i x 3JHaHb,i)

(1)

The coupling constants for all His-OMe conformers (3JHaHb,i) and the respective contribution of each one of these conformers (i x 3JHaHb,i) to the total 3JHaHb were calculated in the four solvents under study, at the ωB97X-D/EPR-III and BHandH/EPR-III theoretical levels. Since very similar results were obtained in two tested levels, in the Figures 4a and 4b are depicted the data for the former (see results at the BHandH/EPR-III in Figures S9a and 9b of the Supporting Information). Figure 4a shows that the values of both calculated 3JHaHb1,i and 3JHaHb2,i coupling constants were constants in all solvents and, therefore, they are not a direct indication of the experimental variations. However, the estimated contributions of each conformer (i

x

3

JHaHb,i) to the total 3JHaHb (Figure 4b) exhibited variations with the

change in the medium polarity and could explain the experimental results of the Table 1. (a)

12 10 CHCl3

6

CH2Cl2

4

CH3CN

2

2

DMSO

0

0

HaHb2,i

8

8 6

3J

3J

HaHb1,i

10

4

Ia

(b)

IIa

IIIc

IVa

Vb

Ia

VIb

2,5

IIa

IIIc

IVa

Vb

VIb

3,0

i x 3JHaHb2,i

i x 3JHaHb1,i

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2,0 1,5 1,0 0,5

2,5 CHCl3

2,0 1,5

CH2Cl2

1,0

CH3CN

0,5

DMSO

0,0

0,0 Ia

IIa

IIIc

IVa

Vb

VIb

Figure 4. (a) Individual 3JHaHb,i spin-spin

Ia

IIa

IIIc

IVa

Vb

VIb

coupling constants and (b) ηi x 3JHaHb,i

conformational contributions for the total 3JHaHb coupling constants, calculated for each His14 ACS Paragon Plus Environment

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OMe conformer at IEF-PCM/ωB97X-D/EPR-III level in different solvents. The values are presented in Hz.

In less polar solvents, the experimental 3JHaHb1 value of 4.3 Hz is in fair accordance with the low calculated relative value of this coupling constant for the conformer Ia (3.4 Hz in CDCl3, shown in the left graph of the Figure 4a). It is worth noting that the conformer Ia presents the highest contribution to the total 3JHaHb1 (see the left graph of the Figure 4b). On the other hand, in more polar solvents, the stabilization of the conformer IIIc combined with its high 3JHaHb1,i value (about 11.2 Hz) makes it the largest contributor to the experimental 3

JHaHb1 value. Moreover, it is verified in the Figure 4b (left graph) an increase in the

contribution of the conformer IIIc of nearly 1.5 Hz, ranging from CDCl3 to DMSO-d6, which is in full agreement with the corresponding experimental change (Table 1). A reasonable correlation between the experimental and theoretical data is also verified for the 3JHaHb2 values (compare the right graphs of the Figures 4a and 4b with the data of the Table 1). Thus, the joint analysis of the experimental NMR 3JHaHb (Table 1), calculated 3JHaHb,i for each conformer (Figure 4a) and the conformational contributions for the 3JHaHb (Figure 4b) allowed elucidating the conformational preferences of His-OMe in solution, i.e. that the experimental 3JHaHb variations from CDCl3 to DMSO-d6 are mostly due to the concomitant stabilization of the conformer IIIc and destabilization of the conformer Ia. IR spectroscopy was also applied as a complement to assess the solvent effect on the His-OMe conformational equilibrium. IR spectra were acquired in different solvents (CHCl3, CH2Cl2, CH3CN and DMSO), and the experimental carbonyl absorption regions, as well as the corresponding mathematically separated bands are displayed in Figure 5. In order to compare the experimental and theoretical (Table 3) results, the conformers were divided into two groups (A and B), according to the proximity in their calculated vibrational



frequencies of the carbonyl stretching. Since the conformers Ia and IIIc are those that present the largest population changes between the less and more polar solvents (Table 2 and 3), and also considering that they are the main components of each group (Table 3), it can be again noted an excellent accordance between experiment and theoretical calculations.

(a)

3.0

Experimental spectrum Sum of deconvoluted bands Group A Group B

2.5

0.5

Absorbance

Absorbance

(b)


0.7 0.6

0.4 0.3 0.2

2.0 1.5 1.0 0.5

0.1 0.0

0.0 1760

1740

1720

1760

1700

(c)

(d)


0.6

1.6

1720

1700


1.4 1.2

Absorbance

0.5

1740

Wavenumber (cm-1)

Wavenumber (cm-1)

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 34

0.4 0.3 0.2 0.1

1.0 0.8 0.6 0.4 0.2

0.0

0.0 1770

1760

1750

1740

1730

1720

1710

1770

1760

Wavenumber (cm-1)

1750

1740

1730

1720

1710

1700

Wavenumber (cm-1)

Figure 5. His-OMe carbonyl stretching region in (a) CHCl3, (b) CH2Cl2, (c) CH3CN and (d) DMSO, showing the IR experimental spectrum and the corresponding deconvoluted bands.


Page 17 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2


Table 3. Experimental and calculated vibrational frequencies (υ, in cm-1) and relative populations (P, in %) of the His-OMe carbonyl stretching band in different solvents. Theoretical values were obtained at IEF-PCM/ωB97X-D/aug-cc-pVTZ level. CHCl3 Conformer

Group υC=Oa

Ia IIa Vb VIb IIIc IVa

Conformer Ia IIa Vb VIb IIIc IVa

3 4 5

A

B

Group

A

B

Calculated Pcalcb PTotalc

1805.4 1804.1 1805.3 1804.0 1796.1 1788.2

55.3 13.2 2.5 6.9 10.4 11.7

Experimental υC=Oa Pexpb

77.9

1737.8

80.4

22.1

1726.1

19.6

υC=O 1798.8 1798.5 1798.8 1798.9 1791.5 1782.8

CH3CN Calculated Experimental υC=O Pcalc PTotal υC=O Pexp 1792.4 1792.6 1792.2 1793.6 1787.3 1777.6

42.9 14.8 2.9 11.8 21.2 6.4

72.4

1741.2

69.4

27.6

1733.8

30.6

υC=O 1791.8 1792.1 1791.7 1793.2 1786.9 1777.2

CH2Cl2 Calculated Experimental Pcalc PTotal υC=O Pexp 54.6 11.8 2.5 9.1 15.2 6.8

78.0

1738.7

79.3

22.0

1730.2

20.7

DMSO Calculated Experimental Pcalc PTotal υC=O Pexp 41.8 15.1 2.9 12.1 21.6 6.5

71.9

1738.6

69.6

28.1

1730.1

30.4

a

Calculated frequencies values are overestimated with relation to the experimental values due to anharmonicity effects; however, the relationship among the frequencies of conformers is not affected. b Population of each conformer. c Sum of the populations of the conformers corresponding to a specific group. d The experimental population of each carbonyl doublet component was estimated through the corresponding area.

6



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Page 18 of 34

7

In order to get a detailed comprehension of the intramolecular effects responsible for

8

the observed conformational preferences in isolated phase and in solution, the QTAIM, NCI

9

and NBO analyses were employed. It is expected that the presence of the polar imidazole

10

ring in the side chain of histidine increases the number of possible intramolecular

11

interactions.

12

Firstly, the presence of an intramolecular hydrogen bonding (IHB) in the conformers

13

and its influence on the relative energies were investigated both in isolated phase and in

14

solution. According to the molecular graphs (MGs) obtained by QTAIM (Figure 6a), it was

15

found a bond critical point (H-BCP) and a bond path (H-BP) related to the IHB only for the

16

conformers Ia, IVa and Vb. Although many hydrogen bonding combinations were possible,

17

like a N-H…O-type IHB between backbone atoms, these three conformers are stabilized by

18

a N(17)...H-N-type IHB, which occurs between one of the hydrogen atoms of the amino group

19

and the hydrogen bond-acceptor nitrogen of imidazole ring. Similar results were also

20

attained by NCI (Figure 6b) and NBO methods. Regarding the NBO calculations, the

21

conformers Ia, IVa and Vb are the only ones that exhibit the LP1(N17) → *N5-H7

22

hyperconjugative interaction.


Page 19 of 34

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Ia

IIa

IIIc

IVa

Vb

VIb

(a)

HBCP = 0.0141 au 2HBCP = +0.050 au ε = 0.1669 au



sign(λ2)ρ = -0.0142

sign(λ2)ρ = -0.0149

(b)

sign(λ2)ρ = -0.0141

sign(λ2)ρ = -0.0073

sign(λ2)ρ = -0.0146

sign(λ2)ρ = -0.0137

23

Figure 6. (a) QTAIM molecular graphs for the His-OMe conformers. The electron density (HBCP), Laplacian of the electron density (2HBCP)

24

and elipticity (ε) values at the hydrogen bond BCP are indicated for each case. The bond paths (BP, dotted lines), the bond critical points (BCPs,

25

green dots) and the ring critical points (RCPs, red dots) are also shown. (b) NCI plots of the reduced density gradients (RDG) versus sign (λ2)ρ

26

for the His-OMe conformers. The sign(λ2)ρ values corresponding to the IHB peak are given when this interactions occurs. All the representations

27

were obtained from electron densities of the optimized geometries at B97X-D/aug-cc-pVTZ level.

28 29 30



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Page 20 of 34

31

These three applied methodologies also indicate that the conformer Vb present the

32

strongest IHB, followed by IVa and Ia. QTAIM uses the electron density (HBCP) and the

33

Laplacian of the electron density (2HBCP) values at the H-BCP to distinguish bond strength

34

and the highest values of these topological parameters occur for Vb (Figure 6a). For NCI,

35

the more negative is the value of electronic density multiplied by the sign of the second

36

Hessian eigenvalue (λ2) in the graphs of Figure 6b the more stabilized is the conformer by

37

IHB; therefore, the absolute energy of this interaction for Ia, IVa and Vb is 8.85, 8.91 and

38

9.35 kcal mol-1, respectively. In turn, the NBO LP1(N17) → *N5-H7 hyperconjugation energy

39

is 1.84, 1.84 and 1.99 kcal mol-1 for Ia, IVa and Vb, respectively. The occurrence of small

40

energy values together with an interaction strength tendency different from the expected

41

conformer stability order (that is, the Ia presenting the strongest IHB and the opposite for

42

the conformer VIb), suggest that the presence of IHB in the stable His-OMe conformers

43

plays a secondary role on the its conformational preferences.

44

The role of steric and hyperconjugative effects for the total Gibbs free energy of a

45

system could also be estimated from the NBO analysis (Table 4). The data indicate that there

46

are no trends in the hyperconjugative or steric contribution values that allow to explain the

47

observed stability order, as in isolated phase as in solution. Instead, it is an interplay between

48

these effects that governs the His-OMe conformational preferences. For example, in

49

solution, as the solvent polarity has increased (from CHCl3 to DMSO), the stable conformer

50

Ia showed an energy variation related to steric repulsions higher than to the corresponding

51

(stabilizing) energy variation associated to hyperconjugations, explaining its destabilization

52

in polar solvents. However, although the relative total energy of this conformer has

53

decreased with relation to the other conformers, it remained as the most stable even in

54

DMSO. In an opposite way, the conformer IIIc presented a higher stabilizing character than


Page 21 of 34

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55

the destabilizing one, that is, the hyperconjugative effects have overcome the steric

56

repulsions, resulting in its stabilization in polar solvents.

57 58 59 60 61

Table 4. Relative total Gibbs free energies of the system (ΔGtot),a,b,c relative energy of the steric (ΔGLewis)a,b,d and hyperconjugative (ΔGhyper)a,b,e interactions for the His-OMe conformers, in isolated phase and taking into account the solvent effects (IEF-PCM implicit solvation model in CHCl3 and DMSO), calculated at ωB97X-D/aug-cc-pVTZ level. Conf.

62 63 64 65 66

ΔGtot Isol.

ΔGLewis

CHCl3 DMSO

Isol.

ΔGhyper

CHCl3 DMSO

Isol.

CHCl3 DMSO

Ia

0.00

0.00

0.00

0.82

1.53

2.60

1.77

3.05

3.13

IIa

0.16

0.85

0.60

3.66

4.20

4.49

4.16

4.03

3.94

IIIc

0.87

0.99

0.39

0.00

0.21

0.78

0.20

0.44

1.21

IVa

1.19

0.92

1.10

4.45

4.23

4.93

3.66

4.62

4.08

Vb

1.25

1.82

1.58

4.56

4.57

4.81

4.08

3.59

3.56

VIb

1.43

1.23

0.73

0.71

0.00

0.00

0.00

0.00

0.00

a

Relative energy in kcal mol-1. b Thermodynamic corrections to Gibbs free energy, available in frequency calculations, were included. c Smaller ΔGtot values lead to more stable conformers. d Higher ΔGLewis values lead to more destabilized conformers by steric and electrostatic effects. e Higher ΔGhyper values lead to more stabilized conformers by hyperconjugative effects.

67 68

In Table S2 are presented the most energetic hyperconjugative interactions, which are

69

the main responsible for the stabilization of the His-OMe conformers. The highest-energy

70

hyperconjugation for all the conformers, *N17=C19 → *C16=C18, occurs between antibonding

71

orbitals. This unusual interaction is possible due to the high occupancy of *N17=C19 orbital,

72

caused by conjugation on the aromatic imidazole ring, and evidenced by the high energy of

73

LP1(N20) → *N17=C19 and C16=C18 → *N17=C19 interactions. Furthermore, as expected, among

74

the orbital interactions with the most considerable energy values are those involving as

75

donors the lone pairs of the nitrogen and oxygen atoms of the compound, as well as those

76

related to the resonance of the imidazole ring. However, NBO analysis indicates that only

77

subtle energy differences occur for the His-OMe conformers, but no particular interaction 21 ACS Paragon Plus Environment


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Page 22 of 34

78

can be attributed to the relative stability order. Indeed, this makes it clear that is a joint effect

79

between hyperconjugations and steric repulsions that determines the His-OMe

80

conformational behavior.

81 82

3.2.

N-acetyl-L-histidine methyl ester

83

The only difference between His-OMe and Ac-His-OMe is the additional presence of

84

an amide group instead of the amino. Thus, it is expected that this new important connection

85

in the N-acetylated derivative significantly changes the Ac-His-OMe conformational

86

behavior in comparison with His-OMe due to the restricted rotation around the amide C-N

87

linkage.

88

Analogously to the His-OMe study, initially the Ac-His-OMe conformational

89

preferences in solution were evaluated by using 1H NMR spectroscopy. The experimental

90

3

91

CD3CN and DMSO-d6 (Table 5), the only tested aprotic solvents in which the compound

92

could be solubilized. Despite the small difference between the dielectric constants of the

93

used solvents, it is verified a variation in the 3JHaHb1 values (from 4.38 Hz in CD3CN to 5.25

94

Hz in DMSO-d6). This result suggests that the conformer populations are dependent on the

95

medium polarity.

JHaH coupling constants were measured as well as the chemical shifts determined in both

96 97 98 99 100 101


Page 23 of 34

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102 103 104

105


Table 5. Experimental 1H NMR chemical shifts (δ, in ppm, related to TMS) and 3JHaHb coupling constants (in Hz)a for the Ac-His-OMe obtained in solvents of different dielectric constants (ε).

a

Solvent

ε

δH(N)

δHa

δHb1

δHb2

CD3CN DMSO-d6

37.5 46.7

8.01 8.58

4.59 4.56

3.24 3.13

3.16 3.03

3J

HaHb1

4.38 5.25

3J

HaHb2

9.48 9.26

3J

HaH(N)

7.74 7.70

Error in measurements of J = ± 0.05 Hz.

106 107

According to the results obtained from theoretical calculations (Table 6) at B97X-

108

D/aug-cc-pVTZ level, trans-VIIIa (Figure 7) is the dominant conformer for the isolated

109

compound with a population of almost 100%. Despite its greater stability persists in polar

110

solvents such as CH3CN and DMSO, the energy difference of trans-VIIIa with respect to

111

the other conformers decreases and its population is reduced to about half that obtained for

112

the isolated compound. In contrast, the other three conformers (Figure 7) with arrangements

113

b (Figure 2), which are insignificant in isolated phase, become representative in these polar

114

media. Thus, at a first glance, the theoretical calculations are in good accordance with the

115

experimental data, confirming that the solvent exerts a substantial effect on the Ac-His-OMe

116

conformational equilibrium, as well as it was obtained for His-OMe.

117 118 119 120 121 122 23 ACS Paragon Plus Environment


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123 124 125 126

Page 24 of 34

Table 6. Relative Gibbs free energies (Grel, in kcal mol-1) and populations (P, in %) in isolated phase and in different solvents (IEF-PCM implicit solvation model) for the Ac-HisOMe conformers, calculated for the optimized geometries at B97X-D/aug-cc-pVTZ level. The dipole moments (in debyes) in isolated phase are also shown. Isolated phase Grel P

Conformer



trans-VIIIa trans-Vb trans-VIb trans-IXb

4.40 2.96 3.24 6.57

0.00 2.14 2.81 3.19

96.1 2.6 0.8 0.4

CH3CN

DMSO

Grel

P

Grel

P

0.00 0.85 0.59 0.53

49.4 11.9 18.4 20.3

0.00 0. 80 0.20 0.49

41.4 10.7 29.6 18.2

127 trans-VIIIa

trans-Vb

trans-VIb

trans-IXb

128

Figure 7. Spatial representations of the most stable Ac-His-OMe conformers, optimized at

129

B97X-D/aug-cc-pVTZ level.

130 131

The individual 3JHaH,i coupling constants for each conformer were theoretically

132

determined at B97X-D/aug-cc-pVTZ level (Figure 8a) and they are constant in the

133

different solvents. Also, the contributions of each conformer to the total 3JHaH (Figure 8b)

134

were estimated and they can explain the experimental variation induced by the solvent (Table

135

5), especially that one for 3JHaHb1, which presents the greatest change. Since this experimental 24 ACS Paragon Plus Environment

Page 25 of 34

136

variation is of about 0.9 Hz (Table 5), it is noted that the contribution ηi x 3JHaHb1 value of

137

the conformer trans-VIb from CD3CN to DMSO-d6 (Figure 8b) corresponds to this change.

138

Again, NMR and theoretical results are in good agreement. The individual 3JHaH,i coupling

139

constants and the respective conformational contributions of each conformer for the 3JHaH

140

were also calculated at BHandH/aug-cc-pVTZ level and they are presented in the Figures

141

S10a and 10b of the Supporting Information.

i x 3JHaHb1,i

3J HaHb1,i

4 3 2 1 0

1,5 1,0 0,5 0,0

trans-VIIIIa trans-Vb

trans-VIb

trans-IXb


trans-VIb

trans-IXb


trans-VIb

trans-IXb


trans-VIb

trans-IXb

12

i x 3JHaHb2,i

3J

HaHb2,i

10 8 6 4 2 trans-VIIIIa trans-Vb

trans-VIb

2,0

1,0

trans-IXb

10

3,0

8

2,5

i x 3JHaH(N),i

HaH(N),i

3,0

0,0

0

3J

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6 4 2 0

2,0 1,5 1,0 0,5 0,0

trans-VIIIIa trans-Vb CH3CN

trans-VIb

trans-IXb

DMSO

CH3CN

(a)

DMSO

(b)

coupling constants and (b) ηi x 3JHaH,i

142

Figure 8. (a) Individual 3JHaH,i spin-spin

143

conformational contributions for the total 3JHaH coupling constants, calculated for each Ac-

144

His-OMe conformer at IEF-PCM/ωB97X-D/EPR-III level in different solvents. The values

145

are presented in Hz.

146 147

Next, the presence of an IHB in the Ac-His-OMe conformers was investigated by

148

QTAIM, NCI and NBO analyses in order to identify the responsible effect for the higher 25 ACS Paragon Plus Environment


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Page 26 of 34

149

stability of the trans-VIIIa, in the different media, as well as its destabilization and

150

simultaneous stabilization of the conformers b as the solvent polarity is increased. The

151

QTAIM molecular graphs (Figure S11a) show that only the most stable conformer forms an

152

IHB (an unusual IHB). However, this interaction is instable due to its high elipticity value

153

(ε = 5.2318 au) at the respective BCP and, therefore, the IHB is not relevant to the stability

154

of the trans-VIIIa. The NCI plot of the reduced density gradient (RDG) versus the sign (λ2)ρ

155

(Figure S11b) for the trans-VIIIa also indicates a very weak interaction between the H(-C)

156

and O atoms, since repulsions are dominant in comparison to this IHB. In addition, NCI

157

method (Figures S11b and S11c) shows a weaker N-H...O-type IHB in the conformers trans-

158

Vb and trans-VIb, forming an instable five-membered ring, as well as the absence of this

159

interaction in trans-IXb, confirming that IHB is not responsible for the observed

160

conformational preferences. None of these IHB interactions was detected by NBO analysis.

161

In Table 7, ΔGLewis obtained from NBO calculations show that trans-VIIIa is the

162

conformer with the less pronounced steric repulsion effect and this is very important for its

163

highest stability in isolated phase and in solution. Although this geometry presents three

164

bulky groups targeted to the same region of the space (Figure 2), they are disposed as far as

165

possible from each other – like a “T”. This occurs mainly because this conformer has the

166

smallest deviation of the dihedral angle ψ [N-C-C=O] from 0º (Table S3). Consequently,

167

trans-VIIIa is the conformer that presents the less accentuated stabilization due to

168

hyperconjugative effects. From Table 7 it is also possible to verify that the contribution of

169

hyperconjugative effects overcomes the steric repulsion for trans-Vb as the dielectric

170

constant of the solvent increases and, as a result, its relative energy decreases with relation

171

to trans-VIIIa. These outcomes show that, in general, the balance between steric and

172

hyperconjugative effects determines the total conformational relative energy order (ΔGtot) in


Page 27 of 34

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173

both isolated phase and solution, i.e. the more balanced are the values between ΔGLewis and

174

ΔGhyper, the less energetic is the Ac-His-OMe conformer.

175 176 177 178 179

Table 7. Relative total Gibbs free energies of the system (ΔGtot),a,b,c relative energy of the steric (ΔGLewis)a,b,d and hyperconjugative (ΔGhyper)a,b,e interactions for the Ac-His-OMe conformers, in isolated phase and taking into account the solvent effects (IEF-PCM implicit solvation model in CH3CN and DMSO), calculated at ωB97X-D/aug-cc-pVTZ level. Conf.

180 181 182 183 184 185

ΔGtot Isol.

ΔGLewis

CH3CN DMSO

Isol.

ΔGhyper

CH3CN DMSO

Isol.

CH3CN DMSO

trans-VIIIa

0.00

0.00

0.00

0.00

0.00

0.00

0.84

0.00

0.00

trans-Vb

2.14

0.81

0.77

3.01

0.26

0.12

1.79

1.72

1.90

trans-VIb

2.81

0.59

0.20

1.71

2.07

1.84

0.00

3.48

2.94

trans-IXb

3.19

1.23

0.50

6.66

3.75

4.90

4.96

5.06

5.22

a

Relative energiy in kcal mol-1. b Thermodynamic corrections to Gibbs free energy, available in frequency calculations, were included. c Smaller ΔGtot values lead to more stable conformers. d Higher ΔGLewis values lead to more destabilized conformers by steric and electrostatic effects. e Higher ΔGhyper values lead to more stabilized conformers by hyperconjugative effects.

186

Finally, the energies of the most significant orbital interactions for each Ac-His-OMe

187

conformer are presented in Table S4. The data show that all the conformers are mainly

188

stabilized by interactions involving the nitrogen and oxygen nonbonding electron pairs as

189

donors. There are also another two hyperconjugations with high energies for this compound:

190

N16=C18 → *C15=C17 and C15=C17 → *N16=C18. These interactions have energy values that

191

slightly vary among the conformers and as previously stated, it is an interplay between the

192

sum of all the hyperconjugative contributions (Ghyper), including the lowest energy ones not

193

presented in Table S4, together with the overall contribution from steric repulsions that

194

governs the conformational preferences of this compound. However, it is worth to highlight

195

that despite the trans-VIIIa presenting the smallest stabilizing effect due to



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Page 28 of 34

196

hyperconjugation (Table 7), an important contribution from LP1(N5) → *C23-O28 appears to

197

contribute to its highest stability.

198 199

4. Conclusions

200

We provided a detailed insight into the conformational preferences of two L-histidine

201

derivatives both in isolated phase and in solution. The experimental results obtained by 1H

202

NMR spectroscopy show that the conformational equilibria of the studied compounds are

203

sensitive to the solvent effects. Furthermore, the theoretical calculations of electronic

204

structure successfully and accurately predict the experimental conformational preferences,

205

showing that the selected theoretical levels are reliable in the description of the

206

conformational behavior of these amino acid derivatives.

207

Our results also show that an interplay between hyperconjugation and steric effects

208

accounts in determining the conformational energies of the studied histidine derivatives as

209

in isolated phase as in solution. Furthermore, contrary to the expected for amino acid

210

derivatives, IHB does not represent a significant role on the conformational preferences of

211

His-OMe and Ac-His-OMe.

212

The exchange of the carboxyl group of amino acids by a methyl ester group in His-

213

OMe as well as the inclusion of the N-acetyl in Ac-His-OMe, which are soluble in organic

214

solvents, is an approximation capable of providing significant information about the

215

conformational preferences of amino acid residues in condensed medium. Thus, we hope the

216

outcomes of this work may help to elucidate the conformational behavior of their amino acid

217

analogues in more complex biological systems.

218 219 220 28 ACS Paragon Plus Environment

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221

Associated Content

222

Supporting Information

223

The Supporting Information is available free of charge on the ACS Publications website at

224

DOI 10.1021/acs.jpca.xxxxxxx

225

Detailed procedures for preparation of the compounds; 1H NMR spectra for the two

226

studied compounds; Potential energy surfaces of His-OMe; Comparison of the

227

energies, populations and other relevant structural parameters for the His-OMe

228

conformers in several theoretical levels; Individual 3JHaH,i coupling constants for each

229

His-OMe and Ac-His-OMe conformer, calculated at IEF-PCM/BHandH/EPR-III

230

level; QTAIM and NCI molecular graphs for the Ac-His-OMe conformers (PDF).

231 232

Author Information

233

Corresponding Author

234

*E-mail: [email protected]

235

Notes

236

The authors declare no competing financial interest.

237 238

Acknowledgements

239

The authors thank a grant #2014/25903-6 from São Paulo Research Foundation (FAPESP)

240

for financial support of this work and for a scholarship (to C.B.B. #2012/18567-4). Thanks

241

also to Conselho Nacional de Pesquisa (CNPQ) for a fellowship (to R.R.).

242 243 244 245 29 ACS Paragon Plus Environment


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246

References

247

1.

Page 30 of 34

Comez, L.; Lupi, L.; Morresi, A.; Paolantoni, M.; Sassi, P.; Fioretto, D. More is

248

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