10412
J. Phys. Chem. B 2000, 104, 10412-10418
Hydration Effect on Molecular Conformation of Oxyphenonium Bromide Noriaki Funasaki,* Hiroshi Yamaguchi, Seiji Ishikawa, and Saburo Neya Kyoto Pharmaceutical UniVersity, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan ReceiVed: January 4, 2000; In Final Form: September 6, 2000
The C-H COSY, H-H COSY, and NOESY spectra of 2-[(cyclohexylhydroxyphenylacetyl)oxy]-N,N-diethylN-methylethanaminium bromide (OB), an antiacetylcholine drug, in D2O are recorded, and calculations of molecular mechanics, molecular dynamics, and molecular hydrophobic and hydrophilic surface areas are carried out for the prediction of its solution structure. From the assignments of all protons and carbons of OB, it is established that a pair of the corresponding protons and carbons of the cyclohexyl group are magnetically nonequivalent with one another. On the basis of quantitative analysis of this nonequivalence by the ring current effect due to the phenyl group and the NOESY spectrum, the solution structure of the OB ion is estimated; the cyclohexyl and phenyl groups are spatially close to one another because of hydrophobic interactions. The structure that has the largest hydrophilic surface area and a very small hydrophobic surface area under the condition of no interatomic collision is very close to this NMR structure. Molecular mechanical calculations that take into consideration the solvation energy estimated from molecular surface areas would predict a more reasonable conformation of the OB ion in the aqueous solution. The molecular dynamics simulations provide information about the flexibility and conformations of OB; the internal rotations of the cyclohexyl and phenyl groups are rather restricted and these groups are spatially close to one another. The estimated solution structure serves for the understanding of the pharmacological action and the interaction between OB and cyclodextrin.
Introduction Recently, we have investigated the binding of the antiacetylcholine drug, 2-[(cyclohexylhydroxyphenylacetyl)oxy]N,N-diethyl-N-methylethanaminium bromide (oxyphenonium bromide) with cyclodextrins by ultraviolet absorption spectroscopy1 and ion selective potentiometry.2 The bitter taste of oxyphenonium bromide (OB) was depressed by the addition of these cyclodextrins and this suppression was successfully predicted by ultraviolet absorption spectroscopy1 and ion selective potentiometry.2 To understand this suppression mechanism, we need to determine the three-dimensional structure of OB. The chemical structure of OB (Figure 1) is similar to that of acetylcholine CH3COOCH2CH2N+(CH3)3; two methyl groups of acetylcholine are replaced by two ethyl groups in OB and three protons of the acetyl group are substituted by the phenyl group, the cyclohexyl group, and the hydroxyl group. From this analogy between OB and acetylcholine, we can suggest that these compounds competitively bind to the active sites of the acetylcholine receptor and acetylcholine esterase.3 The strengths of antiacetylcholinergic action and ligand affinity to these proteins will be remarkably influenced by the hydrophobicity of such substituent groups. Generally, bitter compounds are hydrophobic.4,5 We have shown that the hydrophobicity of a compound correlates with its molecular conformation.6-9 Thus, information about the three-dimensional structure of OB would shed some light on its bitter taste. Modern nuclear magnetic resonance (NMR) techniques, and the NMR chemical shift induced by the ring current effect will be used for the determination of the conformation of OB.10-17 Molecular mechanics or empirical force field methods provide * To whom correspondence should be addressed. Fax: +81-75-5954762. E-mail:
[email protected].
Figure 1. Labeling of the hydrogen atoms of S-OB and definitions of the rotational angles of the cyclohexyl and phenyl groups around the chiral carbon atom.
nonexperimental predictions of stable molecular geometry.18-20 However, the effect of solvation energy on the total potential energy merits further investigation.18-20 A rapid method for calculations of this energy is to utilize solvent-accessible surface areas.1819,21-26 Water-accessible surface areas are used to predict aqueous solubility, oil/water partition coefficients,6 chromatographic retention times,6-8 critical micelle concentrations,9,27 host/guest complexation,28 membrane permeability,29 and molecular conformations.26-28,30 In the present work, we determine the chemical shifts of the OB protons and carbons from the one- and two-dimensional spectra in a D2O solution and estimate the solution structure of OB on the basis of these data and molecular modeling calculations. The chemically equivalent protons of the cyclohexyl group are magnetically nonequivalent to each other. This
10.1021/jp000018w CCC: $19.00 © 2000 American Chemical Society Published on Web 10/14/2000
Hydration Effect on Oxyphenonium Bromide nonequivalence is used for the determination of the molecular conformation of OB on the basis of calculations of the ring current effect, molecular mechanics, molecular dynamics, and interatomic distances. The molecular surface area, which reflects the hydration effect, is also used to predict the solution structure of OB. The solution conformation would be better explained by molecular mechanics complemented with the hydration energy term. Experimental Section Materials. Commercial samples of 99% deuterium oxide (Aldrich) and the racemic mixture of R- and S-OB (Sigma) were used as received. This sample of OB gave a single peak on a reversed-phase high-performance liquid chromatogram. Tetramethylammonium chloride (Nacalai Tesque, Kyoto) was of guaranteed grade. NMR Measurements. All NMR experiments were carried out for a 60 mmol dm-3 (mM) OB deuterium oxide solution at 309.7 ( 0.1 K, unless specified. This temperature was chosen for comparison with our experiments of the bitter taste of OB.1,2 The chemical shifts of the OB protons were determined in the OB concentration range from 1 to 60 mM. The proton chemical shifts (δ) were referenced to the internal signal of tetramethylammonium chloride at δ ) 3.176 ppm.31 1H NMR spectra were obtained with a JEOL Lambda 500 spectrometer at 500 MHz and the data processing was performed with standard software. All obtained one-dimensional spectra were deconvoluted with Nuts NMR data-processing software (Acorn NMR Inc.). The 1H-1H shift correlated spectrum (H-H COSY) and 13C1H shift correlated spectrum (C-H COSY) were obtained at delay times of 93.0 µs and 17.4 µs and 16 and 64 transients, respectively, with the JEOL standard pulse sequences. Twodimensional phase-sensitive nuclear Overhauser effect spectroscopy (NOESY) were performed at 500 MHz with the JEOL standard pulse sequences; the data consisted of 8 transients collected over 2048 complex points. A mixing time of 400 ms and a 90° pulse width of 11.0 µs were used. The NOESY data set was processed by applying an exponential function in both dimensions and zero-filling to 2048 × 2048 real data points prior to the Fourier transformation. Small cross-peaks were neglected because their magnitude was close to that of noise. The NOESY spectrum was also determined with a Varian Inova 400 spectrometer at 400 MHz; data consisted of 8 transients collected over 1024 complex points. A mixing time of 500 ms was used. Small cross-peaks were collected because their magnitude was larger than that of noise. The one-dimensional proton NMR spectra were obtained at 275.2 and 353.2 K with this spectrometer at 400 MHz. Molecular Mechanics Calculations. Molecular mechanics calculations of the OB ions were performed with the Molecular Simulation Insight II/Discover (98.0) on a Silicon Graphics Octane workstation. The Discover III CVFF force field was used for energy minimization.32 All calculations, unless specified, were performed in the presence of water. The starting structure of the OB ion was generated by the manual-drawing routine in Insight II. The counterion (Br-) of OB was neglected. The partial atomic charges and the structure of OB in vacuo were obtained by optimization using the Discover III module of Insight II, and potential types were derived from the CVFF force field. The optimized structure of OB was then soaked in a 2 nm layer of water (975 water molecules per OB molecule), and the total potential energy of this system was minimized. This minimization produced little changes in the structure. Initial torsion angles, φ1 and φ2, of the C1(cyclohexyl)-C* bond and
J. Phys. Chem. B, Vol. 104, No. 44, 2000 10413 C(phenyl)-C* bond were usually changed at an interval of 30°. Energy minimization was performed using the conjugate gradients method to a derivative of 0.001 kcal mol-1 with Insight II′ default values (van der Waals’ cut off distance ) 0.95 nm and electrostatic cut off distance ) 0.95 nm). The heat of formation of the OB ion was calculated using the MOPAC module of Insight II with MNDO electrostatic potential. The optimized structure of OB in vacuo was also determined. Molecular Dynamics Calculations.33 Molecular dynamics calculations of the OB ion were carried out on the basis of the structure (structure N) estimated by NMR. Its atomic charges were calculated by the MOPAC module with MNDO electrostatic potential. The periodic boundary condition was applied on a 3-nm cubic cell at which OB was centered. The cell was soaked with 872 water molecules and contained total atoms of 2676. The potential types of each atom were derived from the CVFF force field. The potential energy of the system was minimized. The initial velocities of atoms were given by the Maxwell-Boltzmann distribution at 10 K. Simulated annealing of the system was carried out for 1 ps at 600 K and for 1 ps at 450 K. Then molecular dynamics simulations were performed under a constant volume of 27 nm3 and a constant density of 0.9887 g cm-3 (the NVT ensemble). Temperature was maintained at 309.5 K by the direct velocity scaling method. The dynamics simulations were performed every 1 fs during 1000 ps, and the equilibration of temperature and total energy was attained at ca. 800 ps. The data trajectory was sampled every 1000 fs, and the last 200 ps of the trajectory was used for analysis. The time average structure of OB was obtained with the conformation command of the analysis module. The cut off distance for van der Waals and electrostatic forces was 1.6 nm. Calculations of Molecular Surface Areas. As already reported,9,28 the Bondi radii (r) were employed for calculations of water-accessible molecular surface areas; rH (aliphatic) ) 0.120 nm, rH (aromatic) ) 0.100 nm, rO (ether) ) 0.152 nm, rO (carbonyl) ) 0.150 nm, rC (aromatic) ) 0.177 nm, rC (aliphatic) ) 0.170 nm, and rN (aliphatic) ) 0.155 nm.34 The water radius of rW ) 0.14 nm was employed for calculations of water-accessible molecular surface areas. The groups of carbonyl, hydroxyl, ether oxygen, and ammonium nitrogen were regarded as hydrophilic groups and the other groups of OB were as hydrophobic groups. All atomic surface areas of OB were calculated, and then from the summation of these areas over the hydrophobic and hydrophilic groups, the hydrophobic and hydrophilic areas, SO and Sw, were calculated, respectively. The energy-optimized structure was used for these molecular surface area calculations, although the torsion angles, φ1 and φ2, were changed over 360°. These torsion angles were changed at an interval of 30°, though locally at an interval of 10°. Because two chemically nonbonded atoms cannot approach within their Bondi radii, such structures were regarded as being unstable . Results Assignments of Protons and Carbons. Figure 2 shows the 500 MHz C-H COSY spectrum of 60 mM OB in D2O and the chemical shifts of all protons and carbons are shown in Table 1. The assignments of these atoms have been established by C-H COSY (Figure 2), H-H COSY (data not shown), and NOESY (Figures 3 and 4) spectra. The cyclohexyl protons in Figure 2 were assigned on the basis of the number of protons and the spin-spin splitting patterns and the paired protons of the ortho- and meta- positions are indistinguishable, respectively. The protons except for the cyclohexyl protons are assigned in reference to propantheline bromide12 and similar compounds.10-17
10414 J. Phys. Chem. B, Vol. 104, No. 44, 2000
Funasaki et al.
Figure 3. 400 MHz NOESY spectrum of 60 mM OB in the region of phenyl and cyclohexyl protons, where all diagonal peaks were deleted. Figure 2. 500 MHz C-H COSY spectrum of 60 mM OB in the cyclohexyl group region (C2 to C6), together with assignments.
TABLE 1: Observed Chemical Shifts of the Protons and Carbons of OB proton
Ho Hm Hp Hc Hd He Hf Hg H1a H2a H2e H3a H3e H4a H4e H5a H5e H6a H6e
δP (ppm)
7.611 7.469 7.404 4.545a 3.573a 3.214 1.216 2.826 2.422 1.369 1.491 1.271 1.815 1.120 1.658 1.245 1.675 0.991 1.339
carbon
δC (ppm)
CdO C* Co Cm Cp Cc Cd Ce Cf Cg C1 C2
175.06 141.53 128.16 130.78 130.40 59.44 59.11 57.67 7.47 47.48 44.37 27.68
C3
26.36
C4
26.17
C5
26.12
C6
25.93
a These protons do not consist of a simple A B spin system in the 2 2 one-dimensional proton spectrum at 309.7 K.36-39
Because the present sample of OB is the racemic mixture of S-OB and R-OB, these isomers could give a pair of spectra slightly different in chemical shift.15,35 Actually, their signals were not resolved at all. All carbons of OB chemically bound with protons are assigned from the cross-peaks in the C-H COSY spectrum. The spectral patterns of aromatic protons, cyclohexyl protons, and ethylene protons Hc and Hd depended on the temperature.36-39 The cyclohexyl protons of OB can be assigned from the C-H COSY, H-H COSY, and NOESY spectra. In the 13C NMR spectrum, it is notable that this group consists six separate peaks, regardless of four kinds of chemically corresponding carbons. From the chemical structure of OB, it is expected that the carbon and proton signals at the lowest field in the cyclohexyl group are carbon C1 bonding the chiral carbon C* and proton H1 bonding carbon C1, respectively. In the C-H COSY spectrum, the C1 carbon correlates with a single proton, proton H1. This proton signal is split into a triplet with spin-spin vicinal coupling constants of 3J ) 12 Hz and the 1:2:1 intensity
Figure 4. 500 MHz NOESY spectrum of 60 mM OB in the cyclohexyl group region (δ ) 0.9-2.5 ppm), where all diagonal peaks were deleted.
distribution and each of these triplet peaks is split further into a triplet with spin-spin vicinal coupling constants of 3J ) 2.5 Hz and the 1:2:1 intensity distribution. Proton H1 bonds to the axial position of carbon C1 because the splitting pattern of the proton H1 signal is explained on the basis of vicinal spin-spin coupling constants of 3J1a2a ) 3J1a6a ) 12 Hz and 3J1a2e ) 3J1a6e ) 2.5 Hz. Furthermore, the proton H1a signal has large and median cross-peaks with protons Ho and Hm in the NOESY spectrum, indicating that these protons are spatially close to one another. Proton H6e has large cross-peaks with protons Ho and H6a in the NOESY spectrum and is a doublet with 2J6a6e ) 12 Hz. The proton H6a near δ ) 1 ppm is a quartet with geminal and vicinal spin-spin coupling constants of 2J6a6e ) 3J6a1a ) 3J6a5a ) 12 Hz and the 1:3:3:1 intensity distribution and each of the quartet peaks is split further into a doublet with a vicinal coupling constant of 3J6a5e ) 2.5 Hz. In the C-H COSY spectrum, each of protons H6a and H6e has cross doublet peaks with carbon C6. The signal of proton H2a near δ ) 1.4 ppm appears to be a quartet with 2J2a2e ) 3J2a1a ) 3J2a3a ) 12 Hz and an intensity
Hydration Effect on Oxyphenonium Bromide
J. Phys. Chem. B, Vol. 104, No. 44, 2000 10415
TABLE 2: Chemical Shift Differences between the Paired Protons of the Cyclohexyl Group, Heats of Formation, and Hydrophobic and Hydrophilic Molecular Surface Areas for Six Probable Conformations struct
φ1 (deg)
φ2 (deg)
observed Ew Ev N Dw Sc Sn
-44 -52 -64 -44 0 -60
68 62 102 59 120 100
6e-2e
∆δ (ppm) 6a-2a 5e-3e
5a-3a
-0.152
-0.378
-0.026
-0.140
-0.234 -0.756 -0.150 -0.255 0.283 -0.099
-0.120 -0.191 -0.350 -0.086 0.466 -0.325
-0.013 -0.112 -0.021 -0.024 0.012 -0.013
-0.083 -0.167 -0.122 -0.079 0.080 -0.110
ratio of 1:3:3:1, and each of the quartet peaks is composed of a doublet with 3J2a3e ) 2.5 Hz. The left part of this signal consists of peaks close in shape to those of proton H6a, though the right part overlaps with the H6e peaks. Proton H2a has crosspeaks with protons H1a, H6e, and H6a in the NOESY spectrum. Carbon C2 was assigned to be the peak bonding to protons H2a and H2e in the C-H COSY spectrum. Proton H2e near δ ) 1.5 ppm has cross-peaks with protons H1a, H2a, and H6e in the NOESY spectrum and consists of a fine-structured doublet with 2J2e2a ) 12 Hz. Proton H5a (δ ) 1.25 ppm) has cross-peaks with protons Ho, H1a, H2e, and H6a in the NOESY spectrum. The shape of this proton signal is close to that of proton H4a (shown below), though it overlaps with a large triplet of proton Hf. Proton H5e (δ ) 1.67 ppm) bonds to proton H5a and carbon C5 in the C-H COSY spectrum and has large cross-peaks with proton H5a both in the NOESY and H-H COSY spectra. Proton H3a (δ ) 1.27 ppm) has cross-peaks with protons H1a and H2e in the NOESY spectrum and the right part of the proton H3a signal is close in shape to that of the proton H4a (shown below) in the one-dimensional spectrum, though the left part overlaps with the proton H6e signal. Proton H3e (δ ) 1.8 ppm) has cross-peaks with protons H2e and H3a in the NOESY spectrum and appears to be a doublet with 2J3e3a ) 12 Hz. Both protons H3a and H3e bond to carbon C3 in the C-H COSY spectrum. In the NOESY spectrum, proton H4a (δ ) 1.1 ppm) has cross-peaks with protons H3e and H5e, and proton H4e has cross-peaks with protons H3e, H4a, and H5a. Proton H4a is a quartet and each of the quartet peaks is further split into a triplet. Protons H4a and H4e bond to carbon C4 in the C-H COSY spectrum. Although proton H4e overlaps with proton H5e, it consists of a doublet (2J4a4e ) 12 Hz) in the C-H COSY spectrum, as expected. Thus, for the C3-C5 protons, the axial proton signal consists of a quartet (3Jaa ) 2J ) 12 Hz and the 1:3:3:1 intensity distribution) and each of the quartet peaks is split into a triplet by vicinal spin-spin coupling with two equatorial protons (3Jae ) 2.5 Hz and the 1:2:1 intensity distribution). The equatorial proton signal consists of a broad doublet (2J ) 12 Hz and the 1:2:1 intensity distribution), and each of the doublet peaks is split into a multiplet by vicinal spin-spin coupling with four protons (3Jae ) 3Jee ) 2.5 Hz). These large separations by spinspin coupling (3Jaa ) 2J ) 12 Hz) appear to be cross-peaks in the two-dimensional spectra, for the case in which two or more peaks overlap with each other in the one-dimensional spectrum. The above assignments are consistent with the result of the H-H COSY spectrum (data not shown); all geminal protons, most vicinal protons, and a few allyl protons have cross-peaks in this spectrum. The chemical shifts of protons and carbons shown in Table 1 were determined from the one-dimensional, C-H COSY, H-H COSY, and NOESY spectra. The assignments and
SS
∆Hf (kJ/mol)
So (nm2/molecule)
Sw (nm2/molecule)
0.0767 0.4074 0.0011 0.0993 0.9512 0.0070
412.5 412.2 444.3 415.6 731.1 443.0
5.848 6.123 5.857 5.858 5.636 5.854
0.538 0.479 0.548 0.529 0.641 0.551
splitting patterns of the protons are consistent in both 400 and 500 MHz spectra. The chemical shift of the axial proton of OB is smaller than that of the paired equatorial proton, as is found for most of the cyclohexane derivatives (∆δ ) 0.13-0.51 ppm).17 The chemical shifts of the OB protons are independent of OB concentration up to 60 mmol dm-3, indicating that OB does not self-associate below this concentration. Because a critical micelle concentration (cmc) of OB is 108 mM,40 some concentration dependence of the chemical shift may be observed above this high concentration. Molecular Conformation of OB. Molecular mechanical calculations have been carried out in the presence of 975 water molecules for optimizing the structure of the OB ion, where the effect of the bromide ion was neglected. The bromide ion will completely dissociate from the OB ion to form an ionic atmosphere around the OB ion. The distribution of these ions in the aqueous solution will obey the Poisson-Boltzmann equation for the spherical symmetry.19,41 Molecular mechanical calculations of this system were carried out using the Discover force field32
Etotal )
Kr(r - req)2/2 + ∑ Kθ(θ - θeq)2/2 + ∑ bonds angles V [1 + cos(nφ - γ)]/2 + ∑ n dihedrals (AijRij-12 - BijRij-6 + qiqjD-1Rij-1) ∑ i
