Conformational Exploration of Enflurane in Solution and in a

Oct 13, 2015 - Enflurane is a fluorinated volatile anesthetic, whose bioactive conformation is not known. Actually, a few studies have reported on the...
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Conformational Exploration of Enflurane in Solution and in a Biological Environment Laize A. F. Andrade,† Josué M. Silla,† Susanna L. Stephens,‡ Kirk Marat,‡ Elaine F. F. da Cunha,† Teodorico C. Ramalho,† Jennifer van Wijngaarden,*,‡ and Matheus P. Freitas*,† †

Department of Chemistry, Federal University of Lavras, 37200-000, Lavras, MG, Brazil Department of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

‡

S Supporting Information *

ABSTRACT: Enflurane is a fluorinated volatile anesthetic, whose bioactive conformation is not known. Actually, a few studies have reported on the conformations of enflurane in nonpolar solution and gas phase. The present computational and spectroscopic (infrared and NMR) work shows that three pairs of isoenergetic conformers take place in the gas phase, neat liquid, polar, and nonpolar solutions. According to docking studies, a single conformation is largely preferred over its isoenergetic isomers to complex with the active site of Integrin LFA-1 enzyme (PDB code: 3F78), where the widely used anesthetic isoflurane (a constitutional isomer of enflurane) is known to bind. Weak hydrogen bonding from an electrostatic interaction between the CHF2 hydrogen and the central CF2 fluorines was not found to rule the conformational isomerism of enflurane. Moreover, intramolecular interactions based on steric, electrostatic, and hyperconjugative effects usually invoked to describe the anomeric effect are not responsible for the possible bioactive conformation of enflurane, which is rather governed by the enzyme induced fit.

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as anesthetics, such as isoflurane4 and sevoflurane,5 only a few studies have been devoted to examine the interactions governing their conformational stabilities. While weak hydrogen bond C−H···F−C and hyperconjugative interactions have been indicated as dictating forces of the conformational isomerism of enflurane,1 classical steric and electrostatic interactions were found to govern the conformational stability of sevoflurane rather than hyperconjugation.6 The conformational preferences of isoflurane have been accounted for in terms of the generalized anomeric effect (hyperconjugation).7 Actually, these interactions are meaningful for the gas phase and solution conformations, while the bioactive conformation of enflurane is not known and, therefore, the role of the above-mentioned intramolecular interactions in enflurane in a biological environment has not been put to the test yet. Usually, intermolecular ligand−enzyme interactions overcome intramolecular interactions in the substrate as driving forces of the conformational equilibrium in a biological environment. For example, while the gauche effect caused by intramolecular interactions, namely, hyperconjugation and electrostatic effects, can rule the bioactive conformation of diphenhydramine,8 the most stable geometry for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in the gas phase and solution does not match its bioactive conformation complexed with the active site of TIR1 ubiquitin ligase enzyme.9 Thus, this study aims to analyze the intramolecular interactions governing the conformational

INTRODUCTION Enflurane (2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroetane) is a fluorinated volatile anesthetic that undergoes rotational isomerization around the dihedral angles φ1, φ2, and φ3 of Figure 1. Four stable conformers were identified in a carbon

Figure 1. Enflurane (2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroetane) and its three torsional angles.

tetrachloride solution and in an argon matrix using infrared spectroscopy, and electrostatic interactions between the H atom of the CHF2 group and the central F atoms were asserted to stabilize these conformations, in addition to hyperconjugation effects.1 More recently, six stable conformers were computationally found for enflurane that, in fact, are three pairs of conformers with the conformers in each pair differing only by the φ3 dihedral.2 Because of the very small rotational barrier separating the conformers of each pair, the experimental detection of all six stable conformers is a difficult task and, therefore, only three conformers can be identified.3 Despite some efforts to find the main conformations of enflurane, as well as in other important fluorinated ethers used © 2015 American Chemical Society

Received: August 19, 2015 Revised: September 23, 2015 Published: October 13, 2015 10735

DOI: 10.1021/acs.jpca.5b08087 J. Phys. Chem. A 2015, 119, 10735−10742

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Table 1. Relative Free Gibbs (Gas/Cyclohexane/Acetonitrile), Full Electronic (EFULL),b Lewis (ESTERIC and EELECTROSTATIC), and non-Lewis (EHYPERCONJUGATION) Energies (kcal mol−1), for All Conformations of Enflurane in the Gas Phase, According to ωB97X-D/6-311++g(d,p) Calculationsa

a

conformers

ΔG (298 K)

EFULL

EHYPERC.

ESTERIC

EELEC.

a a′ b b′ c c′ d e f g h i j k l m n o p q r s t

0.0/0.0/0.0 0.1/0.3/0.3 0.6/0.4/0.2 − /0.3/0.3 0.6/0.4/0.2 0.3/0.1/0.2 1.9/2.1/2.1 2.7/2.5/2.2 2.7/2.8/2.5 3.3/3.4/3.3 4.8/4.3/2.6 5.7/5.5/4.9 2.2/2.1/2.0 2.3/2.1/1.8 2.6/2.4/3.8 2.5/2.3/2.1 5.5/5.4/5.7 5.4/2.3/2.6 1.7/1.8/1.9 2.8/2.6/2.2 3.5/3.2/3.3 2.5/1.7/2.6 6.6/6.3/6.3

0.0 (0.0) 0.0 (0.0) 0.1 (0.2) −(0.2) 0.2 (0.4) 0.2 (0.4) 1.4 2.1 2.1 2.5 4.0 4.4 2.0 1.3 1.9 1.9 4.0 4.2 1.5 2.0 2.4 1.9 5.6

−2.0 −2.2 −2.1 − −3.0 −3.1 0.0 −6.6 −0.4 −4.2 −4.0 −5.6 −2.1 −1.5 −6.2 −6.2 −6.8 −6.0 −1.3 −6.6 −4.3 −0.4 −3.7

13.4 14.0 13.0 − 11.4 11.9 1.9 7.9 5.1 1.8 12.2 4.4 6.0 1.4 6.0 2.7 5.5 12.7 7.1 5.0 4.5 1.8 8.2

−11.4 −11.8 −10.8 − −8.2 −8.6 −0.5 0.8 −2.6 4.9 −4.2 5.6 −1.9 1.4 2.1 5.4 5.3 −2.5 −4.3 3.6 2.2 0.5 1.1

The results in implicit solvents are shown in SI. bResults obtained at the MP2/6-311++g(d,p) level are given in parentheses for a−c′.

Figure 2. Three pairs of low-energy conformers for enflurane.

with resolution enhancement (Lorentz to Gauss transformation) and zero filling, resulting in line widths of ca. 0.3 Hz and a digital resolution of 0.09 Hz/pt. Calculated spectra were generated using a reasonable set of starting parameters, and assignments were made between the transitions in the calculated spectra and the peaks the experimental spectra. The program was allowed to iteratively adjust the shifts and couplings until convergence was achieved. Typical RMS deviation between the experimental and calculated spectrum was 0.3 Hz, while statistical uncertainties in the coupling constants were below 0.05 Hz and are given in the Supporting Information. A high correlation coefficient (typically 0.8 to 0.9) was observed between 2JH2,F4and 2JH2,F5. With few exceptions, other correlation coefficients were below 0.1. Enflurane has 3 rotatable bonds and, considering 3 possible conformations for every one of them (gauche, anti, and gauche′), gives 27 initial structures. The 27 possible conformations of enflurane were optimized using DFT/ωB97X-D12 and MP213 methods and the 6-311++G(d,p)14 Pople’s basis set, but only 23 different conformers converged to energy minima. Frequency calculations at the DFT level were employed to guarantee that the minima obtained were not saddle points. These calculations were performed for the gas phase and implicit solution (cyclohexane and acetonitrile) using the conductor-like polarizable

stability of enflurane and, subsequently, examine their roles in the possible bioactive conformation of enflurane in the Integrin LFA-1 enzyme, predicted using docking studies.

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EXPERIMENTAL AND THEORETICAL METHODS Enflurane (99% purity) was purchased from SynQuest Laboratories and used without further purification. The infrared spectrum for the neat liquid enflurane (capillary film using NaCl windows) was acquired in a Shimadzu IR Affinity-1 FT spectrometer at room temperature (ca. 298 K), using 64 scans and resolution of 4 cm−1. The 1H and 19F NMR spectra of enflurane were acquired on a Bruker Avance III 500 spectrometer, operating at 500.13 and 470.54 MHz, respectively, at varying temperatures. Samples were prepared as ca. 10 mg mL−1 cyclohexane-d12 and CD3CN solutions, using hexafluorobenzene as the internal reference for the 19F NMR experiments. In order to simplify the analysis, spectra were first recorded and analyzed with 1 H decoupling, and these values used as starting parameters for the complete 1H coupled spectrum. The fluorine atoms in enflurane are strongly coupled, and reliable coupling constants cannot be obtained by simple first order analysis. The spectra were analyzed with the NUMMRIT10 simulation module of the SpinWorks11 NMR processing package. Spectra were processed 10736

DOI: 10.1021/acs.jpca.5b08087 J. Phys. Chem. A 2015, 119, 10735−10742

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Table 4. Experimental Coupling Constants 3J (Hz) for the Enflurane in a Variety of Temperatures and Cyclohexane/ Acetonitrile Solvents cyclohexane Parameters 3

JF1,F2exp. 3 JF1,F3exp. 3 JH1,F2exp. 3 JH1,F3exp.

acetonitrile

278 K

298 K

233 K

253 K

278 K

298 K

−11.7 −11.6 4.2 4.5

−11.6 −11.4 4.4 4.5

−11.4 −11.3 4.1 4.2

−11.3 −11.3 4.3 4.1

−11.1 −11.0 4.4 4.4

−11.0 −10.9 4.5 4.4

impact of the interaction between the molecular target and the studied conformations. The crystal structure of enflurane inside the integrin LFA-1 active site was obtained from the Protein Data Bank (PDB code: 3F78)20 and used for the docking procedure and alignment of the 27 conformations of enflurane. The calculation of the docking energies of the rigid conformation inside the integrin active site was performed using the software Molegro Virtual Docker (MVD).21 This program is able to predict the most likely conformation of how a ligand will bind to a macromolecule.22,23 In summary, the MolDock scoring function was used to superimpose the enflurane conformations onto a template molecule (isoflurane).

Figure 3. IR spectra for the neat liquid of enflurane, indicating the three pairs of isoenergetic conformations by Gaussian deconvolution (population, according to the relative intensities scaled by the calculated IR intensities: 37% for a and a′, 32% for b and b′, and 31% for c and c′).

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continuum model (C-PCM) described by Tomasi and coworkers.15 Natural bond orbital analysis (including NBODEL NOSTAR and NBO STERIC)16,17 was carried out over the optimized geometries at the ωB97X-D/6-311++G(d,p) level in order to evaluate the steric, electrostatic, and hyperconjugation contributions for the conformational equilibrium of enflurane. NMR coupling constant calculations in the gas phase and implicit cyclohexane and acetonitrile solvents were performed at the BHandH/EPR-III18 level for C, O, F, and H using the 6-311++g(d,p) basis set for Cl. Calculations were all performed using the Gaussian 09 program.19 Finally, docking calculations were performed in order to understand the physical−chemical

RESULTS AND DISCUSSION Upon rotation of the three dihedral angles in enflurane, five highly stable conformations were identified at the ωB97X-D/ 6-311++G(d,p) level in the gas phase, increasing to six lowenergy conformers at the MP2/6-311++G(d,p) level and in implicit solvents for both levels (Table 1 and Supporting Information). In fact, three pairs of conformers have been obtained, in which very low energy barriers separate the conformers of each pair and these are the corresponding small geometrical differences owing only to φ3. Indeed, similar results were found by Melikova et al.2 at the MP2/6-311++G(df,pd)

Table 2. Theoretical (Gas Phasea) and Experimental (Neat Liquid) Vibrations for the Main Conformers of Enflurane (a−c′)

a

vibrations

a

a′

b

b′

c

c′

Exp.

βC5FF βC5FF, βC5FO, ωC3OF ωC3FF, βCCF υCCl, υasCCCl, υC3FF, ωC5FF υC5O, βCCH υC1F, γCCH υasCOC, υasC3FF, υC5FF, ωCCH γCCH, υC3O tCOC, tCCH γC5H γC1H, γC5H, υCC,

574.9 646.0 778.2 842.8 898.5 1066.8 1113.4 1149.4 1243.4 1298.5 1393.3 1409.3

574.6 643.1 779.8 842.2 899.0 1067.1 1111.1 1151.4 1241.8 1299.8 1382.0 1405.5

572.7 665.0 764.2 827.1 905.0 1088.4 1123.2 1145.4 1223.5 1295.3 1391.8 1411.5

566.5 654.1 761.3 823.8 901.2 1078.3 1117.5 1152.1 1216.6 1288.1 1376.9 1409.5

550.6 648.7 710.6 853.2 921.2 1095.3 1127.9 1156.4 1192.1 1326.3 1392.4 1410.7

557.4 651.3 713.4 853.2 920.8 1097.0 1114.1 1148.5 1195.3 1315.1 1396.5 1407.4

569 644 766 827 901 1072 1121 1150 1265 1294 1371 1402

For b′, the values are given in implicit cyclohexane.

Table 3. Theoretical Coupling Constants 3J (Hz) for the Conformers a−c′ in Implicit Cyclohexane/Acetonitrile Solvents cyclohexane

acetonitrile

3

J

a

a′

b

b′

c

c′

Meana

a

a′

b

b′

c

c′

Meana

JF1,F2 JF1,F3 3 JH1,F2 3 JH1,F3

−13.8 −14.5 −0.7 11.8

−14.0 −14.5 −0.9 11.4

−17.8 −9.3 13.3 −2.7

−17.8 −9.6 12.2 −2.6

−5.5 −14.8 −0.9 1.4

−6.3 −14.9 −1.0 1.5

−11.6 −11.7 3.0 3.8

−13.9 −14.8 −0.8 11.8

−14.0 −14.5 −0.9 11.9

−18.0 −8.4 12.4 −2.5

−18.1 −8.7 12.3 −2.6

−4.5 −14.8 −1.1 1.4

−5.2 −14.9 −1.1 1.4

−10.3 −11.6 2.3 3.5

3 3

a3

J weighted by the calculated % of conformer population. 10737

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Table 5. Gauche Effect-Like Hyperconjugative Interactions for Conformers a−c′ at the ωB97X-D/6-311++g(d,p) Level

Conf.

σCF2→σ*CCl

σCH1→σ*CF3

σCO→σ*CF1

σCCl→σ*CF2

σCF3→σ*CH

σCF1→σ*CO

σCF2→σ*CH1

σCF1→σ*CF3

σCCl→σ*CO

a a′ b b′ c d e f g h i j k l m n o p q r s t Conf.

1.50 2.99 0.00 1.19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.70 1.52 1.54 1.55 1.55 1.41 σCH1→σ*CF2

4.78 4.77 0.00 0.71 0.73 0.00 0.00 0.00 0.54 0.61 0.00 0.68 0.68 0.76 0.73 0.69 3.86 4.47 4.49 3.94 4.19 3.82 σCF3→σ*CF1

1.10 1.11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.88 1.19 1.19 1.01 1.03 0.83 σCO→σ*CCl

1.14 1.14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.50 2.82 2.79 2.67 2.85 2.01 σCF1→σ*CF2

0.57 0.56 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.63 0.58 0.56 0.59 0.57 0.00 σCCl→σ*CF3

1.38 1.37 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.47 1.25 1.32 1.49 1.43 1.32 σCO→σ*CH1

0.00 0.00 0.57 0.00 0.00 0.60 0.57 0.56 0.57 0.60 0.59 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 σCF2→σ*CF1

0.00 0.00 1.56 0.00 0.00 1.40 1.46 1.45 1.37 1.26 1.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 σCF3→σ*CCl

0.00 0.00 2.65 0.00 0.00 2.56 2.58 2.78 2.81 2.71 2.86 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 σCH1→σ*CO

a a′ b b′ c d e f g h i j k l m n o p q r s t

0.00 0.00 4.73 0.52 0.51 4.37 4.48 4.21 4.12 4.05 3.93 0.54 0.62 0.00 0.51 0.83 0.66 0.59 0.51 0.77 0.62 0.00

0.00 0.00 1.15 0.00 0.00 1.25 1.20 1.21 1.21 1.41 1.28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 1.54 0.00 0.00 1.48 1.61 1.44 1.48 1.30 1.29 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 1.54 1.56 0.00 0.00 0.00 0.00 0.00 0.00 1.39 1.36 1.45 1.45 1.16 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 2.99 2.98 0.00 0.00 0.00 0.00 0.00 0.00 2.76 2.66 2.81 2.80 2.58 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.54 0.54 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 1.19 1.20 0.00 0.00 0.00 1.21 0.00 0.00 1.23 1.27 1.22 1.20 1.44 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 1.53 1.52 0.00 0.00 0.00 0.00 0.00 0.00 1.52 1.63 1.54 1.55 1.62 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.52 3.96 3.96 0.00 0.52 0.00 0.00 0.00 0.62 3.87 4.19 3.89 3.88 4.78 0.00 0.00 0.00 0.00 0.00 0.68

the C−Cl stretching mode for each pair with nearly the same intensity (Figure 3 and Table 2 for frequency assignments). Self-association is not expected to be an important effect, since no hydrogen bonding is expected and, therefore, the neat liquid should perform like a solvent with dielectric constant between cyclohexane and acetonitrile (which were indeed tested in the NMR experiments) and similar to chloroform.

level. Summed up, these three pairs of conformers, namely, a, a′, b, b′, c, and c′ (Figure 2), have estimated conformational population around 85% in gas phase, polar, and nonpolar solvents, according to the Boltzmann distribution (the results in implicit solvents are shown in SI). The predominance of these three nearly isoenergetic pairs of conformers even in the neat liquid is confirmed by infrared spectroscopy, which shows 10738

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The Journal of Physical Chemistry A Quantitative conformational analysis using the C−Cl stretching mode in IR spectroscopy could not be performed in diluted solution because of solvent interference. Earlier studies have shown that similar conformational behavior to our findings for the neat liquid persists in nonpolar CCl4 solution1 and liquid Kr.2 Since solvent polarity can strongly affect the conformational equilibrium, particularly if conformers have different dipole moments, the conformational isomerism of enflurane was further analyzed using NMR spectroscopy in cyclohexane (nonpolar solvent) and acetonitrile (ε = 37.5) solution. Since F-2 and F-3 are diastereotopic fluorines (as well as F-4 and F-5), they are distinguishable by 19F NMR and both exhibit 3J coupling constants with the vicinal hydrogen (H-1) and fluorine (F-1). According to the well-known Karplus curve, the following 3J pattern is expected to be observed for each pair of isoenergetic conformers (see calculated 3J in Table 3): a and a′: two large 3JF‑1,F‑2/3; a large 3JH‑1,F‑2; a small 3JH‑1,F‑2. b and b′: a large 3JF‑1,F‑2; a small 3JF‑1,F‑3; a small 3JH‑1,F‑3; a large 3JH‑1,F‑2. c and c′: a large 3JF‑1,F‑3; a small 3JF‑1,F‑2; two small 3JH‑1,F‑2/3. If conformational changes appear upon change of solvents and temperature, the 3J should move toward the pattern corresponding to the pair of conformers whose population is increased after these changes. Table 4 shows that, experimentally, both 3JF,F and 3JH,F have averaged values of the six main conformers, while changes in temperature and solvents with different dielectric constants do not alter significantly these coupling constants. This indicates that all three pairs of isoenergetic conformers coexist in solution and their populations are not significantly affected by changes in temperature and solvent. The main difference among the pairs of low-energy conformers lies in the Cl−C−C−O dihedral angle (φ1). According to the hyperconjugative point of view, in agreement with the usual explanation for the gauche effect in 1,2-disubstituted ethanes,24 the good electron donor orbital σC−H1 is antiperiplanar to good electron acceptor orbitals σ*C−F2/3 or σ*C−O in all six main conformations of enflurane (the complete list of such interactions is given in Table 5). Thus, the similar energies of these conformers agrees with the hyperconjugation model. In addition, in terms of steric and electrostatic repulsion, the bulkier Cl and F-1 atoms are in a gauche relationship with the vicinal O and F atoms in all six main conformers, thus experiencing similar spatial interactions. Consequently, both Lewis and quantum-type interactions explain the nearly equivalent energies of a−c′. The C−C−O−C moiety (φ2) in enflurane assumes the anti orientation in all six low-energy conformers. It is worth mentioning that such a geometry coincides with the optimal orientation for a favorable hyperconjugative interaction nO → σ*C−F (central CF2 fluorines - F-2 and F-3), which usually describes the anomeric effect in carbohydrate models.25 The natural bond orbital (NBO) data of Table 6 confirms the important contribution (ca. 20 kcal mol−1) of this interaction to the stabilization of the low-energy conformers. However, Michalska et al.1 proposed an electrostatic interaction between the H atom of the CHF2 group and the F atoms of the central CF2 group, in addition to hyperconjugative interactions as secondary effects, as ruling forces of the orientation of these groups and, therefore, of the conformational equilibrium of enflurane. In this sense, the following discussion is focused on exploring experimental and theoretical approaches to feed the search for the possible interactions governing the conformational isomerism of enflurane, as well as to trace a parallel between these interactions and the bioactive conformation of enflurane.

Table 6. Selected Anomeric-Like Hyperconjugative Interactions Obtained by NBO Analysis at the ωB97X-D/6311++g(d,p) Level for Enflurane in the Gas Phase conformers

nO→σ*C2−F2

nO→σ*C2−F3

nO→σ*C3−F4

nO→σ*C3−F5

a a′ b b′ c c′ d e f g h i j k l m n o p q r s t

20.9 21.4 19.6 − 21.4 22.2 16.4 14.0 10.4 9.9 10.1 19.3 11.7 9.2 22.6 15.7 9.3 22.3 17.0 21.3 15.3 14.7 9.0

20.2 19.6 20.8 − 21.7 21.1 9.8 21.1 14.5 14.7 22.1 9.2 16.0 19.6 15.7 22.5 20.7 9.6 10.3 14.3 9.6 10.3 19.0

15.0 11.7 11.7 − 17.0 12.9 15.8 13.8 7.9 16.3 17.8 15.2 9.7 12.8 18.5 13.9 19.0 8.3 19.4 18.4 19.5 19.8 20.0

13.7 16.7 16.8 − 11.4 15.7 14.5 18.4 19.9 19.6 8.2 19.8 19.3 17.2 13.8 21.4 14.5 17.1 9.0 13.9 16.2 8.0 15.8

First, the weak hydrogen bonding hypothesis for the stabilization of the main conformers of enflurane (electrostatic interaction −CF2H···F2C−) was checked using three theoretical methods widely used to probe nonbonding interactions, namely, QTAIM (Quantum Theory of Atoms in Molecules),26 NCI (Non Covalent Interactions),27 and NBO (Natural Bond Orbital).17 The QTAIM approach describes hydrogen bonds using electronic density and its Laplacian to characterize bond paths between H-bound atoms. No bond paths were found for the six main conformations of enflurane, thus indicating the absence of hydrogen bond between the central fluorines and the hydrogen atom of the CHF2 group. However, on the basis of some controversial cases in the literature,28 in which a bond critical point (BCP) cannot be found in compounds where intramolecular hydrogen bond is expected, the NCI method was applied. The NCI method computes noncovalent interactions based on the analysis of regions where the electron density ρ(r) and the corresponding reduced gradients s(r) are low. For enflurane, the NCI isosurface shows a single circular volume between (F2)CH and the central CF2 fluorines due to the collapse of both regions of interaction in the intramolecular bonding, while the plot of reduced density gradient (RDG) vs sign(λ2)ρ illustrates the presence of two low-intensity (troughs do not reach RDG 0.0) peaks (Figure 4). The peak on the negative sign represents weak attractive interactions, while the other belongs to repulsive interactions (ring formation). These interactions are nearly equivalent in intensity and, therefore, the overall result of the −CF2H···F2C− approximation is neither attractive nor repulsive; thus, NCI supports the result obtained by QTAIM. NBO analysis corroborates the absence of a weak hydrogen bond in enflurane, since the corresponding interaction nF‑2/3 → σ*C−H2 was not observed. Indeed, H-bound fluorinated compounds are expected to exhibit through-hydrogen bond coupling constants (1hJ), such as in 2-fluorophenol29 and 10739

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Figure 4. NCI isosurfaces indicating some intramolecular noncovalent interactions for the most stable conformers of enflurane. The isosurfaces were constructed with RDG = 1.0 au and blue−red colors scaling from −0.02 au < sign(λ2)ρ < + 0.02 au. (λ2)ρ corresponds to the electronic density along the perpendicular plan of the interaction, characterized by the second derivative of the Hessian matrix (negative sign for attractive and positive sign for repulsive interactions).

8-fluoro-4-methyl-1-naphthol,30 but the 19F NMR spectrum for enflurane does not show any 1hJF,H(C) coupling constant, in agreement with the nearly zero calculated values for this spin− spin coupling constant (Supporting Information). Since hydrogen bonding between H-2 and the central CF2 fluorines was not confirmed to be the stabilizing interaction of conformers a−c′, NBO analysis was used to find out the intramolecular interactions governing the conformational equilibrium of enflurane. NBO analysis provides the following partitioning scheme to decompose the full energy of a system into non-Lewis (ENL, hyperconjugation) and Lewis-type (EL, steric + electrostatic) interactions, while the ESTERIC term can also be obtained using the STERIC keyword:

conformations. This behavior persists in implicit solvents (Supporting Information). Intuitively, the high steric hindrance in a−c′ comes from the eclipsed geometry along with the H2−C−O−C dihedral angle, which is accompanied by an electrostatic attraction that comes from the above-mentioned H2···F2/3 interaction. We now should understand if the intramolecular interactions operating in the gas phase and solution persist in a biological environment as ruling forces of the bioactive conformation of enflurane. Docking studies were performed to obtain the relative interaction energy of the enflurane conformations in the integrin LFA-1 active site, replacing the isoflurane structure in the complex available in the Protein Data Bank (PDB code: 3F78) with all possible enflurane conformers previously determined. The interaction energy between each enflurane atom and integrin (Supporting Information) was determined as the ligand− protein interaction energy, Einter:

E FULL = E NL + E L

E L = ESTERIC + E ELECTROSTATIC

Table 1 shows that the strong steric repulsion experienced by conformers a−c′ is nearly counterbalanced by stabilizing electrostatic interactions, in agreement with the NCI findings. Thus, non-Lewis interactions appear to dictate the high stabilization of these conformers relative to the remaining

E inter =

∑ i ∈ ligand

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⎡ qq ⎤ ⎢E PLP(rij) + 332.0 i j ⎥ ⎢ 4rij2 ⎥⎦ j ∈ protein ⎣

∑

DOI: 10.1021/acs.jpca.5b08087 J. Phys. Chem. A 2015, 119, 10735−10742

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The Journal of Physical Chemistry A The EPLP term is a ‘piecewise linear potential’ using two different sets of parameters: one set for approximating the steric (van der Waals) term between atoms, and another stronger potential for hydrogen bonds. The second term describes the electrostatic interactions between charged atoms. It is a Coulomb potential with a distance-dependent dielectric constant given by D(r) = 4r. The numerical value of 332.0 adjusts the units of the electrostatic energy to kilocalories per mole. Our theoretical findings reported in Table 7 show that conformations a and a′ interact more strongly with integrin LFA-1 Table 7. Docking Results for the Conformers of Enflurane conformers

ΔΔEint (kcal mol−1)

a m p a′ f b′ b s o k r c c′ d h n l e q g t j i

0.00 0.98 1.98 2.06 3.15 3.97 4.07 7.15 9.13 11.83 11.31 12.65 12.79 14.05 14.97 16.95 17.02 21.53 35.12 40.52 41.63 103.47 131.61

Figure 5. Conformer a complexed with the active site of the Integrin LFA-1 enzyme. The proximity of the highlighted amino acids can influence the bioconformation of enflurane.

biological activities. Since the choice for the conformer used in 3D-QSAR is usually based on its stability in a biological-free environment (unless that docking studies are carried out for each conformer before the generation of molecular descriptors), which may not correspond to the bioactive conformation, the conformational screening step would be unnecessary in this case.

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CONCLUSIONS Enflurane exists predominantly as three pairs of conformers in the gas phase and solution. Despite the appearance of a weak electrostatic attraction between H2 and the central CF2 fluorines, this interaction is accompanied by steric effects and, therefore, hydrogen bonding cannot be characterized as the ruling force of the conformational equilibrium of enflurane. Hyperconjugation is proposed instead. However, intramolecular interactions are not strong enough to explain the possible bioactive conformation, which is rather governed by ligand−enzyme interactions, since a single conformer among the three pairs is much more stable in the active site of the Integrin LFA-1 enzyme.

than other conformations. The largest intermolecular energy changes were observed in oxygen, fluor-1, and chlorine atoms. In fact, there are three amino acids, Ile259, Glu301, and Leu302, surrounding these atoms, which can be responsible for increasing the potency of conformation a and a′. Table 7 shows that a major conformation of free enflurane (a) is probably preserved in the active site of the Integrin LFA-1 enzyme, while its five isoenergetic conformers are weakly complexed to the protein if compared to a, i.e., at least 2.0 kcal mol−1 less stable in the active site of the enzyme than a. From this, it is implicit that intramolecular interactions operating in a do not rule the conformation of enflurane in the biological environment and, therefore, the bioactive conformation is better described in terms of ligand−enzyme interactions. An inspection of the docked structure of a inside the active site of Integrin LFA-1 enzyme indicates the potential appearance of, e.g., hydrophobic intermolecular interactions with proximate amino acid residues Ile259, Glu301, and Leu302 (Figure 5), which possibly determine the bioconformation of enflurane. These findings can be useful, e.g., to decide whether to use conformation and alignmentbased methods when developing QSAR (Quantitative Structure− Activity Relationships) models. It is worth mentioning that most three-dimensional QSAR methods require exhaustive conformational screening and 3D-alignment steps prior to the generation of molecular descriptors, which are subsequently regressed against

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.5b08087. Standard coordinates, tables with energy and spectroscopic data, and NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]fla.br. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to FAPEMIG, CNPq, and CAPES for the financial support of this research, studentships (to L.A.F.A. and J.M.S.) and fellowships (to E.F.F.C., T.C.R., and M.P.F.), as well as to the Emerging Leaders in the Americas Program (ELAP) for a scholarship (to L.A.F.A.). This work is a collaborative research ́ project of members of the Rede Mineira de Quimica (RQ-MG) supported by FAPEMIG (Project: CEX - RED-00010-14). 10741

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The Journal of Physical Chemistry A

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