Vibrational and Structural Behavior of l-Cysteine Ethyl Ester

Article pubs.acs.org/JPCA

Vibrational and Structural Behavior of L‑Cysteine Ethyl Ester Hydrochloride in the Solid State and in Aqueous Solution M. E. Defonsi Lestard,†,∥ S. B. Díaz,† M. Puiatti,‡,∥ G. A. Echeverría,§,∥ O. E. Piro,§,∥ A. B. Pierini,‡,∥ A. Ben Altabef,*,†,∥ and M. E. Tuttolomondo*,† †

INQUINOA-CONICET, Instituto de Química Física, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, San Lorenzo 456, T4000CAN Tucumán, R. Argentina ‡ INFIQC-CONICET, Instituto de Investigaciones en Fisicoquímica de Córdoba, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000 Córdoba, R. Argentina § Departamento de Física, Facultad de Ciencias Exactas, Universidad Nacional de La Plata and Institute IFLP (CONICET, CCT-La Plata), C.C. 67, 1900 La Plata, R. Argentina S Supporting Information *

ABSTRACT: The aim of this work is to evaluate the vibrational and structural properties of L-cysteine ethyl ester hydrochloride (CE), and its electronic behavior mainly in relation to the action of the thiol and amine groups at different degrees of solvation. The crystal structure of CE was determined at room temperature by Xray diffraction methods. Infrared and Raman spectra were collected to compare the behavior of different functional groups in the molecule, both in the solid phase and in aqueous solution. Its UV and circular dichroism spectra were also measured in aqueous solution. The influence of an aqueous environment on the CE spectra was simulated by means of implicit (polarizable continuum model) and explicit (molecular dynamics, solute−solvent clusters) methods. Calculations in explicit and continuous solvent are of interest to explain the behavior of bioavailable sites in this medium. The study was completed by natural bond orbital analysis to determine the presence of hyperconjugative interactions.

■

INTRODUCTION L-Cysteine ethyl ester hydrochloride (CE) has been evaluated as a dietary sulfur amino acid supplement for increasing wool growth rate.1 L-Cysteine ethyl ester is used for the synthesis of nonsteroidal anti-inflammatory drugs (NSAIDs).2 The thiol group of cysteine takes part in a variety of biochemical reactions.3 The possible formation of a weak hydrogen bond at receptor sites is of considerable interest, as it might contribute to the biological response. Haas has showed that the bonding between the urea and cysteine ethyl ester is of relevance because the −NH−C− CO− group is the structural unit from which the α helix is constructed. Crystal structure studies show that urea can be hydrogen-bonded to the charged nitrogen atom of a single amino acid residue.4 The purpose of this work is to study the structural and vibrational behavior of CE in the solid state and in water solutions at different concentrations. The first X-ray study of this compound (at 120K) has been reported by Görbitz.5 We present here its room temperature crystal structure. There were no significant changes in the comparison between the two determinations. The compound is described as a chlorine salt of L-cysteine ethyl ester protonated at the amino group. The crystal is further stabilized by medium strength −NH3+···Cl− bonds and a weak SH···Cl− interaction. Görbitz5 has observed that both cysteine and its esters (CE and L© 2013 American Chemical Society

cysteine methyl ester·HCl (CM)) are present in the solid form +gauche which is maintained over successive dilutions. The vibration IR and Raman spectra were measured, both in the solid and in aqueous solution, to confirm the presence of the +gauche form as prevalent. Calculations were carried out for the structural determinations, which served not only to compare experimental data but also to choose the best level of calculations for later use in the study of electron density and the effects of hyperconjugation taking place in its main biologically active sites. Thus, the fully optimized geometry was obtained for L-cysteine ethyl ester using different theoretical methods (HF, MP2, B3LYP) and basis sets, in the isolated state and in water solution, the latter to explain the behavior of bioavailable sites in aqueous solution. There are two methods used nowadays for the latter purpose: (a) implicit solvent models,6−8 where the solvent is described as a continuous, dielectric, or conductor-like (IEF-PCM model) medium and (b) explicit solvent models, where quantum chemical calculations are carried out for a cluster containing the molecule of interest surrounded by solvent molecules.9,10 Received: September 16, 2013 Revised: December 5, 2013 Published: December 5, 2013 14243

dx.doi.org/10.1021/jp409252d | J. Phys. Chem. A 2013, 117, 14243−14252

The Journal of Physical Chemistry A

Article

As Benesch and Benesch11 have reported, when the carboxyl group is further modified, as in cysteine ester and the cysteinyl peptides, its acid strengthening effect (particularly on the ammonium group) becomes even greater because the electrostatic, but not the inductive effect, is modified. As a result, the acid strength of the ammonium group becomes actually greater than that of the −SH group. This behavior was studied by circular dichroism measurements at different molar concentrations, and by Raman spectroscopy, complemented with the calculation in continuous and explicit solvent. Different methods could be employed to explain molecular properties related with the structure of compounds. Natural bond orbital (NBO)12 analysis could help to determine the presence of hyperconjugative interactions. Besides, the study of the topological properties of several bond and nonbonding critical points has been useful to analyze molecular electrostatic potential (MEP).13 MEP is suitable to inspect the recognition mechanism operating between a receptor drug and the enzyme− substrate because two species primarily recognize each other through their potentials. It is through this potential that the molecule is first “seen” by another reacting chemical species. MEP is extensively used to interpret and predict the behavior of a wide variety of chemical systems in electrophilic and nucleophilic reactions involved in the study of biological processes and hydrogen type interactions.

dispersion spectrum of the solid was measured in the 3500−50 cm−1 interval with a Thermo Scientific DXR Raman microscope. Raman data were collected using a diode-pump, solid state laser of 532 nm (5 cm−1 spectral resolution), a confocal aperture of 25 μm pinhole, and a 10× objective. Water solutions of CE at different concentrations (from 3 M to 3 × 10−2 M) were measured with the Thermo Scientific DXR Smart Raman spectrometer. The solid sample was placed on gold-coated sample slides and the liquid sample was placed in a glass cuvette. To achieve a sufficient signal-to-noise ratio, 100 expositions of 2 s each were accumulated during the measurements with the laser power maintained at 10 mW. UV-Circular Dichroism Spectroscopy. The circular dichroism spectra were collected with a J-815 circular dichroism and UV/vis absorbance spectrometer for solutions at concentrations of 10−1, 10−3, and 10−4 M with a path length of 1 mm.

■

THEORETICAL METHODS Quantum chemical calculations were performed using the Gaussian 0317 and 0918 programs. To find relevant conformers of L-cysteine ethyl ester in solution an exploration was performed with the VConf program by using molecular mechanics.19 Geometry optimization was achieved for all the conformers obtained from this search using the DFT hybrid functional B3LYP,20,21 with the 6-311G(d)22 and 6-311++G(d,p)23,24 basis sets within the polarizable continuum model (IEF-PCM)25 to account for the solvent effects on the isomerism. All calculations were spin restricted and of the frozen-core type. The vibration wavenumbers were calculated from both numeric and analytic second derivatives to confirm that optimized structures corresponded to minima on the potential-energy surface (SEP). Experimental frequencies were compared with the ones calculated at the B3LYP/6-311++G(d,p) level for all atoms except sulfur for which a 6-311++G(3df,3pd) basis set was used. The atomic displacements given by the Gaussian program for each vibration mode were used to disclose the nature of the molecular vibrations qualitatively and, for that purpose, the corresponding data were represented graphically using the GaussView program.26 Natural bond orbital (NBO) calculations were performed at the B3LYP/6-311++G(d,p) level using the NBO 3.0 code as implemented in Gaussian 03. The MEPs obtained by the calculations were visualized with the Molekel program.27 Molecular dynamics (MD) simulations were run with the AMBER (11) program28 at 300 K under periodic boundary conditions. Nonbonded interactions were calculated using a cutoff of 10.0 Å. Electrostatic interactions were computed with the particle-mesh Ewald summation procedure29 and all hydrogen covalent bonds were constrained using the SHAKE algorithm.30 The trajectories obtained with NAMD were analyzed with VMD31 and the AmberTools programs.28 For building the system, CE was solvated with TIP3P water32 within 10 Å around the molecule, forming a rectangular box with a total size of 34.2 × 30.5 × 31.4 Å3. The construction of the CE unit used in the MD was achieved with the antechamber module, using the GAFF force field33,34 and employing the restricted ESP (RESP) charges35,36 obtained from a single point HF/6-31G* quantum chemical calculation of the B3LYP/6-31+G* optimized structures. Single point explicit solvent calculations were made at the B3LYP/6-311++G(d,p) level for the geometries taken from representative MD frames keeping the water molecules of the first solvation shell (∼3.5 Å around the ester moiety).37

■

EXPERIMENTAL METHODS ethyl ester·HCl was purchased from Sigma-Aldrich (purity not less than ≥98.5% (RT)). Its purity was confirmed by FTIR spectroscopy. X-ray Diffraction Data. The measurements were performed on an Oxford Xcalibur Gemini, Eos CCD diffractometer with graphite-monochromated Cu Kα (λ = 1.541 84 Å) radiation. Xray diffraction intensities were collected (ω-scans with ϑ- and κoffsets), integrated, and scaled with the CrysAlisPro14 suite of programs. The unit cell parameters were obtained by leastsquares refinement (based on the angular settings for all collected reflections with intensities larger than seven times the standard deviation of measurement errors) using CrysAlisPro. Data were corrected empirically for absorption employing the multiscan method implemented in CrysAlisPro. The structure was solved by direct methods with SHELXS-9715 and the molecular models were refined by the full-matrix least-squares procedure on F2 with SHELXL-97.16 The hydrogen atoms were located in a difference Fourier map phased on the heavier atoms. All H-atoms but the one of the sulfhydryl group were positioned on a stereochemical basis and refined with the riding model. The methylene and methyl H-positions were optimized by treating them as rigid groups that were allowed to rotate during the refinement around the corresponding O−C bond so as to maximize the residual electron density at the calculated positions. As a result, both groups, CH2 and CH3, converged to staggered conformations. The sulfhydryl hydrogen was refined at its found position with an isotropic displacement parameter. Crystal data and structure refinement results are summarized in Table S1 of the Supporting Information. Crystallographic structural data were deposited at the Cambridge Crystallographic Data Centre (CCDC). Any request to the Cambridge Crystallographic Data Centre for this material should quote the full literature citation and the reference number CCDC 957759. IR and Raman Spectroscopy. The CE infrared absorption spectrum was recorded in KBr pellets within the 4000−400 cm−1 range using a Perkin-Elmer GX FTIR instrument and the Raman L-Cysteine

14244



■

Article

RESULTS AND DISCUSSION Crystallographic Structural Data. The room temperature (298 K) crystal structure of CE is shown in the ORTEP38 view of Figure 1 and the corresponding bond distances and angles are

Table 1. CE Hydrogen Bond Distances in the Solid State and Calculated Ones for CE Isolated and in Water Solution (PCM) d(D−H)

D−H

N1−H1(av) 0.890 1.000 1.030 S−H 1.285 1.346 1.348

X-ray (298 K) isolateda IEF-PCMa X-ray (298 K) isolateda IEF-PCMa a

d(H···Cl)

d(D···Cl)

2.350 3.046 3.540

3.167 3.060 3.034

2.499 2.520 4.560

3.764 3.648 5.111

B3LYP/6-311++G(d,p).

Cl···HS and does not involve the CO group as acceptor, where the NH3+ and the thiol group act as proton donors. Taking into consideration the conformationally relevant degrees of freedom of the molecule and to get information about its equilibrium conformational distribution a stochastic exploration of the SEP was performed. From this search 36 geometries were obtained and their electronic energy minima computed at the B3LYP/6-311G(d) level in a continuous solvent (SCRF calculations), see Table S8 of the Supporting Information. The conformations within 8 kJ/mol of the most stable one were selected for further refinement and frequency calculations at the B3LYP/6-311++G(d,p) level in continuous solvent. As a result, six different conformations characterized as local minima were obtained (Figure 2). The structural results obtained in these calculations are reported in Table 2. At 298 K the three most populated conformations are +gauche,syn,anti (g+,s,a),+gauche,syn,−gauche (g+,s,g−), and −gauche,syn,anti (g−,s,a). The conformations g+,s,a and g+,s,g− for CE have almost the same energy, separated by a very low energy difference (0.10 kJ mol−1), arising from the rotation in ±88° (B3LYP/6-311++G(d,p) calculation) around the O(2)− C(1E) bond (Figure 1). In the solid phase CE adopts a co nf or mat ion remark ably sim ilar to t hat o f th e +gauche,syn,gauche (g +,s,g) conformer, with an N(1)− C(1A)−C(1B)−S angle of 80.5° (g+), Table S6 of the Supporting Information. According to our DFT calculations this angle has a value of 57.5−58.7° similar to that of 60° previously reported.5 To understand this behavior, we show in Figure S1 of the Supporting Information the potential energy variation as a function of the dihedral angle N(1)−C(1A)− C(1B)−S; this curve shows the two enantiomeric forms (g+ and g−) 12.6 kJ mol−1 apart in energy. The same calculation was carried out under a continuum solvent with an energy difference of 1.59 kJ mol−1 (Figure S2, Supporting Information).41 In solution, the chloride is far apart due to the formation of solvent separated ion pairs. These NH3+ and SH sites are thus stabilized. This spectroscopically observed behavior is confirmed by calculations performed in continuous solvent (Table 1) and in MD simulations with explicit solvent. Figure 3 shows the MD simulated behavior of the N···Cl distances when discrete solvation effects are taken into account. UV-Circular Dichroism Spectroscopy. UV and CD data for L-cysteine ethyl ester hydrochloride at different concentrations of water solutions and in the solid state were measured at 298 K. CE has an absorption maximum, in aqueous solution, at 218 nm for concentrations 10−4 and 10−3 M and at 235 nm for a concentration of 10−1 M (Figure 4), whereas L-cysteine shows maximum absorption at 199 nm.

Figure 1. View of L-cysteine ethyl ester hydrochloride X-ray structure showing the labeling of the non-H atoms and their displacement ellipsoids at the 30% probability level. H-bonds are indicated by dashed lines. Symmetry operations: (i) x + 1, y, z; (ii) −x + 2, y + 1/2, −z + 1/2.

listed in Table S2 of the Supporting Information. To within experimental accuracy, it is coincident with the low temperature (120 K) structure reported previously.5 In fact, the rms separation between homologous non-H atoms in the best least-squares structural fitting, calculated by the Kabsch’s procedure,39 is 0.030 Å. Therefore, the covalent structure of HSCH2CH(NH3)COOC2H5 will not be discussed here any further. The crystal is stabilized by medium strength −N(sp3)− H3+···Cl− bonds involving all three H-atoms [N···Cl distances from 3.129 to 3.244 Å and N−H···Cl angles from 152.7 to 157.4°] and a weak SH···Cl interaction [d(H···Cl) = 2.50(5) Å, d(S···Cl) = 3.764(1) Å, and ∠(S−H···Cl) = 167(4)°]. The Hbonding structure is shown in Figure 1 and corresponding bond lengths and angles detailed in Table S7 of the Supporting Information. Computational Structural Results. Starting from the X-ray structure the conformation of isolated CE molecule was optimized by employing MP2 and DFT with all the basis sets used. The calculations predicted a preferred +gauche,syn,gauche conformation (Figure 1), in agreement with the experimental results. Calculated geometric parameters for CE are listed in Table S2 of the Supporting Information, where they are compared with the corresponding X-ray structural data at 1205 and 298 K. As found for the related compound CF3C(O)SCH2CH3,40 the inclusion of extra polarization functions (beyond a single d-function) is necessary to accurately predict the bond lengths in this type of molecule. The most sensitive parameters of this orbital description are the C−S and S−H bonds, which were shortened by 0.08 and 0.01 Å, respectively, upon replacing the 6-311++G(d,p) basis set with 6-311+ +G(3df,3pd). An additional geometry optimization was performed with the 6-311++G(d,p) basis set on all atoms except sulfur, for which a 6-311++G(3df,3pd) basis set was used. This produced a geometry close to both the experimental structure and to that calculated using the 6-311++G(3df,3pd) basis set, hence demonstrating that only the polarization of the basis set on the sulfur is critical for obtaining accurate bond lengths in these types of structures. No major structural differences were observed when the theoretical results were compared with the experimental X-ray diffraction data (Table S2 of the Supporting Information). Data for intramolecular hydrogen bonds are given in Table 1. The solid structure presents only intermolecular bonds NH···Cl and 14245



Article

Figure 2. Six different conformations for CE characterized by local minima obtained with B3LYP/6-311++G(d,p).

Table 2. Dihedral Angles, Free Energies, and Relative Population for CE dihedral angles conformer

NCCS

HSCC

NCCO

COCC

ΔΔGa/kJ mol−1

% populationb

+gauche,syn,anti +gauche,syn,−gauche +gauche,syn,+gauche +gauche,syn,anti −gauche,syn,anti anti,syn,gauche

58.7 58.6 57.6 58.0 −55.3 −155.3

−106.9 −105.7 −106.4 −103.0 110.1 −70.3

−170.8 −171.2 −169.0 18.2 168.3 −165.9

177.5 −91.9 89.7 179.6 −174.9 88.7

0 0.10 2.80 5.43 1.59 11.95

34.0 33.0 11.0 3.8 18.0 0.3

B3LYP/6-311++G(d,p), IEFPCM(water). bThe population was calculated according to a Boltzmann distribution: % popi = e−ΔGi/RT/∑nk=1e−ΔGk/RT × 100%. a

Figure 3. N---Cl distance vs time during MD simulation in the presence of explicit solvent (H2O). Figure 4. UV-circular dichroism spectra of CE in water solution at different concentrations.

When these results are compared with those for L-cysteine, it turns out that the introduction of an α-ester group in cysteine results in an increase in the acid strength of the ammonium group because the inductive effect of the carbonyl group outweighs the electrostatic effect of the previously existent negative charge. This brings the ionization of the ammonium group into the range of that of the −SH group, although the ammonium group still

remains the stronger acid. In the solid state, the presence of the chloride ion linked by hydrogen bonds to NH and SH sites leads to a further increase in the acid strength of these ammonium and thiol group sites. This effect becomes weaker when the 14246



Article

In the PCM calculations dealing with continuous solvent, the molecule is immersed in a cavity of given potential. Therefore, the electrostatic potential value is overestimated when compared with the explicit solvent where the water molecules are arranged in certain places and the electrostatic interaction is diminished by affinity and steric reasons. In the positive regions of V(r) we found a maximum value of 0.144 au on the H-atoms of the NH3+ and S−H groups. When the molecule is solvated, V(r) values increase (0.085 to 0.144) at both sites of high electron concentration, being the negative charged centers the most affected ones. Vibrational Spectroscopy. The bands observed in the solid state IR absorption and in the solid and water solution Raman dispersion spectra were interpreted in terms of the substance normal modes of vibrations. A tentative assignment of bands was assisted by the calculated mode frequencies and also by comparison with the spectra of related amino acids, especially cysteine.46−50 Experimental (solid state) and calculated (isolated molecule) frequencies and their assignments are collected in Table S9 of the Supporting Information. Representative spectra are illustrated in Figure 6 (IR and Raman spectra of the solid) and Figures 7−9 (Raman spectra of the solid and of water solutions at different concentrations).

concentration of the solution is diminished, a fact reflected in a pH increment. That is why in the CD data we observed the peak at 235 nm in the more concentrated solution. The position of the absorption maximum of these compounds does not remain constant. In fact, we observe shifts from 235 to 218 nm upon decreasing amino acid concentration (Figure 4). This behavior has its counterpart in the UV spectra. Benesch and Benesch11 calculated the four microscopic constants, namely KA, KB, KC, and KD from the variation with pH of the ultraviolet absorption of cysteine in the region between 230 and 240 nm (Scheme 1). Scheme 1

CD and UV results indicate the preferred enantiomeric form in consecutive dilutions, but they also give us information about the ionization degree of the amino acid at the studied concentration range. The UV and CD spectra lead us to think that at concentrations on the order of 10−1 to 10−2 M (corresponding pH values in the 2.8−3.8 range) the HSRNH3+ ↔ HSRNH2 equilibrium prevails, whereas at lower concentrations, the HSRNH2 ↔ S−RNH2 equilibrium prevails. Molecular Electrostatic Potential (MEP) Maps. The MEP map was explored primarily for predicting hydrogen bonding interactions and potential sites of biological recognition.42−45 The emphasis of these studies has been on negative regions of V(r). Here, the molecular electrostatic potentials of isolated CE, in continuum and explicit solvent, are depicted in Figure 5a,b. Each of these molecules has several possible reactive sites and V(r) calculations have provided insights into this matter.

Figure 6. FTIR (upper) and Raman (lower) spectra of solid state ethyl cysteine·HCl.

The relative sensibility of the Raman equipment made it necessary to work at relatively high concentrations, so that we only dealt here with water solutions higher than 0.03 M. The CE serial dilutions were measured from a concentration of 3 M down to 0.03 M. Figure 8 shows the spectra of the some representative dilutions (Table S10 of the Supporting Information). DFT calculations reproduced the normal vibration wavenumbers with the following root-mean-square deviations (RMSD) for each basis set: 80.77 cm−1 for 6-31G(d), 71.20 cm−1 for 6-311G(d,p), and 70.49 cm−1 for 6-311++G(d,p). The B3LYP/6-311++G(d,p) results were used for the vibrational analysis. In the Figures 7−9 we show the calculated spectrum for the molecule solvated in continuum and in explicit solvent. The B3LYP/6-311++G(d,p) calculations in explicit solvent correspond to the +gauche,syn,anti representative geometry taken from the MD simulation (obtained after clustering according the rms similarities, after 180 ps of DM simulation).37 On the basis of the B3LYP/6-311G(d,p) calculations three conformations of g+ geometry (with respect to the HS−CH2−

Figure 5. Calculated 3D-contour map of CE [au] electrostatic potential: (a) isolated; (b) continuous solvent.

The MEP was calculated at the B3LYP/6-31++G** level from molecular geometries optimized at the same level of theory. The results are shown in the color-coded graphs of Figure 5a,b where the palette ranges from deep blue (for the most positive potential) to deep red (for the most negative potential). As expected, the negative regions are associated to Cl− and CO with three local minima, with values close to −0.140, −0.127, and −0.012 au for the continuous solvent, explicit solvent and isolated state, respectively. The increase in the values of the negatively charged molecule of CE in water is primarily due to the solvation of the Cl ion. 14247



Article

Figure 7. Experimental (A and C) and calculated for the g+sa conformer (B and D) Raman spectra in the S−H and C−S stretching regions of ethyl cysteine·HCl in the solid state and in water solution. Experimental spectra of solutions at different concentrations are presented.

CHR−NH3 dihedral) and only one of g− geometry should be observed in water at room temperature. On the basis of their relative free energies, a ratio g+:g− of ∼4:1 is estimated. Both conformers were found in the MD simulations of CE in the presence of discrete water molecules, the g+:g− ratio estimated with this procedure being ∼1.5:1, Figure S3 (Supporting Information). The conformationally averaged Raman spectrum, presented in Figure 9, could be obtained by summing the population-weighted spectra of g+ and g− conformers calculated by using B3LYP/6-311G(d,p) frequencies and intensities considering Lorentzian band shapes (γ = 2 cm−1). The Raman spectrum of the 1.3 M solution unambiguously demonstrates the presence of g+and g− conformers by the resolution of the twisting of the CH2(SH) group at ∼1280−1320 cm−1, as indicated by the simulated profiles shown in Figure 9. The predicted conformational splitting for this mode is in good agreement with the observed splitting in the solution Raman spectrum. On the other hand, in the solid state only one peak is observed in this region, due to the presence of only the g+ conformer. NH3+ Modes. The bands at 3473 and 3428 cm−1 in the solid state IR spectrum are assigned to the antisymmetric and symmetric modes of the NH3+ group, respectively. These bands appear in the saturated aqueous solution Raman spectrum at 3248 cm−1 (see Table S10 of the Supporting Information). Two bands should also appear for the antisymmetric NH3+ bending modes calculated at 1628 and 1537 cm−1. The corresponding bands in the Raman spectrum of the solid appear at 1602 and 1576 cm−1 (Table S9 (Supporting Information), Figure 6). The water solution Raman spectrum shows only one broad band centered at 1627 cm−1 corresponding to the water bending mode. This behavior is reproduced in the explicit solvent calculation, which shows a single broad band at 1642

cm−1. The NH3+ rocking modes were assigned by taking into account the theoretically predicted mode frequencies and also the observed spectrum of the cysteine compound.43,44 Thus the bands located at 1151 and 992 cm−1 in the Raman spectrum were assigned to the rocking modes of the NH3+ group, which appear around 1140−994 cm−1 in related molecules.47,48 The first band in aqueous solution is observed at 1165 cm−1 with dramatically decreased intensity. In the Raman spectrum of the aqueous solutions a weak band is observed at 1064 cm−1, which can be assigned to a rocking mode of the NH2 group (Figure 8B). In the theoretical Raman spectrum derived from the geometry of the NH2RSH molecule (using the continuous solvent approximation), this band is calculated at ∼1033 cm−1 (Figure 8A and Figure S4, Supporting Information). This behavior suggests that the band at 1064 cm−1 could be associated with an uncharged amino group. The presence of this band was also reported by Garfinkel and Edsall47 for an aqueous cysteine hidrochloride solution at 9−11 pH interval. These results reinforce the hypothesis that the equilibrium shifts to the nonionic form with increasing dilution. CH2 and CH3 Modes. Seven well-defined bands appear in the solid state Raman spectrum within the 2999−2882 cm−1 range. These are assigned to the expected CH2 and CH3 stretching modes. The bands centered at 2958 and 2946 cm−1 are assigned to the CH2 symmetric stretching modes, the most intense band in the spectrum being the solid state Raman counterpart. In the water solution’s Raman spectrum, the above modes are observed as a weak band in the 2975−2882 cm−1 interval. The previously reported spectra of CF3CO2CH2CH3 were used as a guide for mode assignment of other bands.49 The solid state Raman band appearing at 1496 cm−1 is assigned to the CH2(CH3) bending mode, which in turn appears at 1472 cm−1 in 14248



Article

decreases and are red-shifted (1160−1122 cm−1) (Table S10, Supporting Information). SH and C−S Modes. The band centered at 2483 cm−1 in the Raman spectrum of the solid is assigned to the S−H stretching mode. This band appears at higher frequency (2578 cm−1) in the Raman spectrum of the substance in solution because solvation of the Cl− ion disrupt the hydrogen bonding between the H of the molecule SH group and the chlorine atom of HCl. This band is sensitive to the degree of dilution, and it can be seen that with decreasing concentration it is blue-shifted (Table S10 in the Supporting Information). Thus, in these aqueous concentrations only the un-ionized form of the thiol group can be observed. Not only the frequency but also the intensity of this band are important to assert the degree of ionization of the thiol group at different concentrations. This study was carried out by following the procedure proposed by Elson and Edsall50 for cysteine. In particular, we considered two strong bands: the S−H band at ∼2578 cm−1 and the C−S band at ∼684 cm−1. The intensity of the latter appears to be independent of the concentration and pH, and therefore it serves as a convenient internal standard for determining the relative intensity of the 2578 cm−1 band as it decreases with decreased concentrations of the solutions (increasing the pH). At 0.12 M concentration where we may assume the sulfhydryl group to be un-ionized, the peak height ratio, ISH/ICS = 1.359 ± 0.01. We denote this ratio as A0 and the other ratio as A. Then the fractional ionization of the S−H group, αS−H, may be taken as αSH = 1 − (A /A 0)

(1)

In Figure10 we show αSH as a function of concentration. It is to be noted that for the range of concentrations dealt with here, the Figure 8. Calculated for the g+sa conformer (A) and experimental (B) Raman spectra of ethyl cysteine·HCl in the solid state and in water solution. Experimental spectra of solutions at different concentrations are presented.

Figure 10. Fractional ionization αSH vs concentration. Figure 9. Calculated and Experimental Raman spectra of ethyl cysteine. HCl in water solution.

degree of ionization varies in the 0−0.14 interval and αSH decreases with decreasing concentration. This is consistent with the increased frequency observed for the SH stretching band. Hence at these concentrations only the un-ionized form of the thiol group can be detected. This pattern is seen when a calculation is performed for the isolated molecule where the SH band appears at 2630 cm−1, whereas for calculations based on the continuous and explicit solvent models, these modes appear at 2675 and 2700 cm−1, respectively (Figure 7), which amounts to a small shift in the

the CF3CO2CH2CH3 spectrum. The shoulders at 1496 and 1492 cm−1 are assigned to the CH3 antisymmetric deformation. The corresponding symmetric counterpart is assigned to a weak IR band at 1424 cm−1. The vibrations of the CH2(SH) group appear at 1448 cm−1 (bending), 1295 cm−1 (twisting), and 791 cm−1 (rocking) (Table S9, Supporting Information). The bands located at 1171 and 1124 cm−1 are assigned to the methyl rocking modes. The intensity of these bands in aqueous solution 14249



Article

Table 3. Relevant Hyperconjugative Interactions (kJ mol−1) of the HS−CH2−CH(NH3)−C(O)−O−CH2−CH3·HCl, Calculated at the B3LYP/6-311+G(d) Level H2O solution explicit interaction

isolated state

continuous

1 ps

180 ps

394 ps

n O(2) → σ* CO n O(2) → π* CO n O(2) → σ* C1EC2E n O(1) → σ* CO n O(1) → σ* C1AC1 n S → σ* C1BC1A n(1−3) Cl− → σ* NH n(1−3) Cl− → σ* SH n4 Cl− → σ* NH n4 Cl− → σ* SH n4 Cl− → σ* OHw n S → σ* OHw n N → σ* OHw σ SH → σ* OHw n Ow → σ* NH n Ow → σ* SH

37.1 215 17.09 122.2 85.82 23.99 9.74 20.77 113.24 4.51

38.5 222.3 17.22 122.0 85.52 19.31 15.13

37.5 210.5 19.65 127.3 85.65 24.49 7.69 13.8 53.42 14.4

32.7 210.3 20.05 119.0 80.46 2.67 15.52 4.72 22.86

6.35 143.9 22 180.0 93.84

198.84

131.33 0.54 1.05 0.3 52.66 4.72

frequency of vibration of the C−S bond from 686 cm−1 (solid) to 682 cm−1 (solution). CO and CO Stretching Modes. The very strong dispersion band observed at 1748 cm−1 in the solid state Raman spectrum was assigned to the CO stretching mode. This band appears about 1747 cm−1 in the water solution Raman spectrum (Table S10 of the Supporting Information). This red shift can be explained by the formation in solution of hydrogen bonds between the carbonyl CO group and the water solvent, which in turn leads to a decrease of the force constant of the CO bond in solution and consequently to a concomitant increase of the CO force constant. This is reflected in the observed increment from 1232 cm −1 (solid) to 1277−1278 cm−1 (solution) in the vibration frequency of the CO stretching mode, in agreement with calculations. The symmetric and antisymmetric O(2)−C(1E)−C(2E) stretching modes appear in the solid-state spectrum at 1020 and 863 cm−1, respectively. In aqueous solution the corresponding bands are red-shifted (Figures S5 and S6, Supporting Information)). The bands located at 758 and 626 cm−1 in the solid state spectrum are assigned to the CO out-of-plane and CO in-plane wagging modes, respectively. In aqueous solution the band intensity of these modes is considerably reduced without a significant change in the frequencies (Tables S9 and S10, Supporting Information). NBO Analysis. An NBO analysis was performed to investigate the conjugative and hyper-conjugative effects on the stabilization of the molecule (Table 3). As mentioned in the Introduction, the thiol group of cysteine takes part in a variety of biochemical reactions. To rationalize the thiol properties of CE, it is to be noted that the anomeric effect n Cl → σ* S−H, observed in the isolated molecule, does not exist in the water solution. These results agree with the NBO analysis of the S−H bond because there is an increase of the electronic population in the bonding orbital σ S−H along with a decrease in its antibonding counterpart σ* S−H (Table S11 of the Supporting Information), which involves a strengthening of the bond. These results agree with the frequency increase of the ν(S−H) in the water solution.

2.38 4.39 3.64 55.09 5.98

There was an increase in the hyper conjugation of the n Ow → σ* N−H as it undergoes solvation, with values greater than those observed for the interaction n Ow → σ* S−H. This would explain why the ρ NH3+ band disappears as the concentration decreases, as obviously this site is the first to be solvated. For the OCO group we observed an increase in the interaction n O → σ* CO and a decrease in the interaction n O → σ* CO in aqueous solution, which is reflected in the displacement of the ν(CO) and ν(CO) bands or signals. With these results, we infer that in aqueous solution there is an increased π-orbital delocalization of the OCO bonding structure. Furthermore, we observed an increase in the interaction n O(2) → σ* C(1E)−C(2E) with increasing dilution. This is reflected in a shift to higher frequencies of the asymmetric stretching mode O(2)−C(1E)−C(2E) both theoretically calculated and experimentally observed.

■

CONCLUSIONS Raman spectra of the solid state [HSCH2CH(NH3)COOC2H5] Cl salt and its aqueous solution at different concentrations were complemented with theoretical calculations of the electronic and vibrational structure of the substance. The frequency of the S−H stretching mode is observed to experience an appreciable blue shift in aqueous solution as compared to the solid. This is due to the fact that water solvation of Cl− ion in solution disrupts the weak S−H···Cl bond found in the solid giving rise to a strengthened S−H bond. Hence, in aqueous solution the thiol site, highly required in biological reactions, would remain open to undergo chemical reactions. The results from UV and CD spectroscopy suggest that at concentrations of the order of 10−1 to 10−2 M (pH = 2.8−3.8), the HSRNH3+ ↔ HSRNH2 equilibrium prevails. This agrees with the Raman spectroscopic results, where a band at 1064 cm−1 assignable to NH2 modes is observed. As observed in the simulated solvation dynamics, the Cl− ion moves away from the two molecular H-bond donors, namely, NH3+ and S−H groups, freeing them to form new and much weaker H-bonds with neighboring water molecules. This 14250



Article

(11) Benesch, R. E.; Benesch, R. The Acid Strength of the -Sh Group in Cysteine and Related Compounds. J. Am. Chem. Soc. 1955, 77, 5877− 5881. (12) NBO 4.0 Glendening, E. D.; Badenhoop, J. K.; Reed, A. D.; Carpenter. E. ; Weinhold, F. F. Theoretical Chemistry Institute, University of Wisconsin, Madison, WI, 1996. (13) Scrocco, E.; Tomasi, J. Electronic Molecular Structure, Reactivity and Intermolecular Forces: An Euristic Interpretation by Means of Electrostatic Molecular Potentials. Adv. Quantum Chem. 1978, 11, 115− 193. (14) CrysAlisPro, Oxford Diffraction Ltd., version 1.171.33.48 (release 15-09-2009 CrysAlis171.NET). (15) Sheldrick, G. M. SHELXS-97. Program for Crystal Structure Resolution.; University of Göttingen: Göttingen, Germany, 1997. See also: Sheldrick, G. M. Acta Crystallogr..1990, A46, 467−473. (16) Sheldrick, G. M.SHELXL-97. Program for Crystal Structures Analysis; University of Göttingen: Göttingen, Germany, 1997. See also: Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112−122. (17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; et al. GAUSSIAN 03,Revision D.01; Gaussian, Inc.: Wallingford, CT, 2004, http://www.gaussian.com (see Supporting Information for full citation). (18) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09, Revision B.01; Gaussian, Inc.: Wallingford, CT, 2009 (see Supporting Information for full citation). (19) Chang, C.-E.; Gilson, M. K. Tork: Conformational analysis method for molecules and complexes. J. Comput. Chem. 2003, 24 (16), 1987−1998. (20) Becke, A. D. Density-Functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648. (21) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron Density. Phys. Rev. B 1988, 37, 785−789. (22) Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A. Self-Consistent Molecular Orbital Methods. XX. A Basis Set For Correlated Wave Functions. J. Chem. Phys. 1980, 72, 650. (23) McLean, A. D.; McLean, A. D.; Chandler, G. S. Contracted Gaussian Basis Sets for Molecular Calculations. I. Second Row Atoms, Z=11−18. J. Chem. Phys. 1980, 72, 5639. (24) Hehre, W. J.; Schleyer, P. v. R.; Pople, J. A. Ab initio Molecular Orbital Theory; Wiley: New York, 1986. (25) Miertuš, S.; Scrocco, E.; Tomasi, J. Electrostatic Interaction of a Solute with a Continuum. A Direct Utilizaion of Ab Initio Molecular Potentials for the Prevision of Solvent Effects. Chem. Phys. 1981, 55, 117−129. (26) Nielsen, B.; Holder, A. J. GaussView, User’s Reference; GAUSSIAN Inc.: Pittsburgh, PA, 1997−1998. (27) Flúkiger, P.; Lúthi, H. P.; Portmann, S.; Weber, J. MOLEKEL 4.0; Swiss Center for Scientific Computing: Manno, Switzerland, 2000. (28) Case, D. A.; Darden, T. A.; T.E. Cheatham, I.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; et al. AMBER 11; University of California: San Francisco, 2010 (see Supporting Information for full citation) (29) Golosov, A. A.; Karplus, M. Probing Polar Solvation Dynamics in Proteins: A Molecular Dynamics Simulation Analysis. J. Phys. Chem. B 2007, 111, 1482−90. (30) Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of N-Alkanes. J. Comput. Phys. 1977, 23, 327−341. (31) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graph. 1996, 14, 33−38. (32) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926. (33) Wang, J.; Cieplak, P.; Kollman, P. A. How Well Does a Restrained Electrostatic Potential (Resp) Model Perform in Calculating Conforma-

behavior reflects itself in the Raman spectrum through the observed frequency blue shift of vibration modes associated with these groups. Another possible site for nucleophilic attack is the CO group. Comparing the vibrational behavior of the aqueous solution with the one in the solid, we can observe the frequency red shift of CO and CO stretching modes. This can be explained by an increase in the electron delocalization in the bonding structure of the OCO group, an effect observed both experimentally and theoretically.

■

ASSOCIATED CONTENT

S Supporting Information *

Figures and tables including crystal data and structure refinement results, calculated and experimental structures of CE, potential energy diagrams, torsion angle evolution, IR and Raman spectra, xyz geometries, full refs 17, 18, and 26, and a CIF file are available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*A. B. Altabef: tel, +54-381-4311044; fax, +54-381-4248169; email, [email protected]. *M. E. Tuttolomondo: tel, +54-381-4311044; fax, +54-3814248169; e-mail, [email protected]. Notes

The authors declare no competing financial interest. ∥ ́ Members of the Carrera del Investigador Cientifico, CONICET, R. Argentina.

■

ACKNOWLEDGMENTS We thank research grants from CIUNT (26D/411), CONICET (PIP 0629 and 1529), and by ANPCyT (PME06 2804 and PICT06 2315) of Argentina.

■

REFERENCES

(1) Radcliffe, B.; Hynd, P.; Benevenga, N.; Egan, A. Effect of Cysteine Ethyl Ester Supplements on Wool Growth Rate. Aust. J. Agric. Res. 1985, 36, 709−715. (2) Galanakis, D.; Kourounakis, A. P.; Tsiakitzis, K. C.; Doulgkeris, C.; Rekka, E. A.; Gavalas, A.; Kravaritou, C.; Charitos, C.; Kourounakis, P. N. Synthesis and Pharmacological Evaluation of Amide Conjugates of NSAIDS with L-Cysteine Ethyl Ester, Combining Potent Antiinflammatory and Antioxidant Properties with Significantly Reduced Gastrointestinal Toxicity. Bioorg. Med. Chem. Lett. 2004, 14, 3639−43. (3) Jocelyn, P. C. Biochemistry of the SH Group; Academic Press Inc.: New York, 1972; p 1−404. (4) Haas, D. J. Intermolecular Complexes. III. L-Cysteine Ethyl Ester Hydrochloride−Urea(1:1). Acta Crystallogr. 1965, 19, 860−861. (5) Görbitz, C. H. Hydrogen Bonding of Cysteine in the Solid Phase. Crystal and Molecular Structures of L-Cysteine Methyl Ester.HCl and L-Cysteine Ethyl Ester.HCl. Acta Chem. Scand. 1989, 43, 871−875. (6) Tomasi, J.; Persico, M. Molecular Interactions in Solution: An Overview of Methods Based on Continuous Distributions of the Solvent. Chem. Rev. 1994, 94, 2027−2094. (7) Tomasi, J.; Mennucci, B.; Cammi, R. Quantum Mechanical Continuum Solvation Models. Chem. Rev. 2005, 105, 2999−3093. (8) Cramer, C. J.; Truhlar, D. G. A Universal Approach to Solvation Modeling. Acc. Chem. Res. 2008, 41, 760−8. (9) Hopmann, K. H.; Ruud, K.; Pecul, M.; Kudelski, A.; Dracinsky, M.; Bour, P. Explicit Versus Implicit Solvent Modeling of Raman Optical Activity Spectra. J. Phys. Chem. B 2011, 115, 4128−37. (10) Kubelka, J.; Huang, R.; Keiderling, T. A. Solvent Effects on IR and VCD Spectra of Helical Peptides: DFT-Based Static Spectral Simulations with Explicit Water. J. Phys. Chem. B 2005, 109, 8231−43. 14251



Article

tional Energies of Organic and Biological Molecules? J. Comput. Chem. 2000, 21, 1049−1074. (34) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and Testing of a General Amber Force Field. J. Comput. Chem. 2004, 25, 1157−1174. (35) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Kollmann, P. A. Application of Resp Charges to Calculate Conformational Energies, Hydrogen Bond Energies, and Free Energies of Solvation. J. Am. Chem. Soc. 1993, 115, 9620−9631. (36) Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A.; WellBehaved, A. Electrostatic Potential Based Method Using Charge Restraints for Deriving Atomic Charges: The RESP Model. J. Phys. Chem. 1993, 97, 10269−10280. (37) For selecting the representative frames, the clustering tool from ptraj was employed. The frames were grouped according to the root mean square difference between the positions of the cysteine ethyl ester. After this analisys representative frames from the different clusters are obtained. For further details about the clustering tool see: Roe, D. R.; Cheatham, T. E. Ptraj and Cpptraj: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J. Chem. Theory Comput. 2013, 9, 3084−3095. (38) Johnson, C. K. ORTEP-II. A Fortran Thermal-Ellipsoid Plot Program; Report ORNL-5318; Oak Ridge National Laboratory: Oak Ridge, TN, USA, 1976. (39) Kabsch, W. A Solution for the Best Rotation to Relate Two Sets of Vectors. Acta Crystallogr. A 1976, 32, 922−923. (40) Lestard, M. E.; Tuttolomondo, M. E.; Wann, D. A.; Robertson, H. E.; Rankin, D. W.; Ben Altabef, A.; Conformational, A. Vibrational Study of CF(3)COSCH(2)CH(3). J. Chem. Phys. 2009, 131, 214303. (41) The ΔG0298 of the solvated species were employ for this calculations. When considering the ΔH0298 the energy difference between the conformers is 5.8 kJ/mol. (42) Politzer, P.; Daiker, K. C. The Force Concept in Chemistry. Models for Chemical Reactivity; Deb, D. M., Ed.; Van Nostrand Reinhold Co.: New York, 1981; Chapter 6. (43) Politzer, P.; Laurence, P. R.; Jayasuriya, K. Structure Activity Correlation in Mechanism Studies and Predictive Toxicology; McKinney, J., Ed.; Special issue of Environmental Health Perspectives; NIH: Washington, DC, 1985; Vol. 61, p 191. (44) Scrocco, E.; Tomasi, J. New Concepts II; Topics in Current Chemistry; Springer Verlag, Berlin, 1973; Vol. 42, p 95 (45) Politzer, P., Truhlar, D. G., Es. Chemical Applications of Atomic and Molecular Electrostatic Potentials; Plenum Press: New York, 1981. (46) Susi, H.; Byler, D. M.; Gerasimowicz, W. V. Vibrational Analysis of Amino Acids: Cysteine, Serine, B-Chloroalanine. J. Mol. Struct. 1983, 102, 63−79. (47) Garfinkel, D.; Edsall, J. T. Raman Spectra of Amino Acids and Related Compounds. VIII. Raman and Infrared Spectra of Imidazole, 4Methylimidazole and Histidine. J. Am. Chem. Soc. 1958, 80, 3823−3826. (48) Kaminski, M.; Kudelski, A.; Pecul, M. Vibrational Optical Activity of Cysteine in Aqueous Solution: A Comparison of Theoretical and Experimental Spectra. J. Phys. Chem. B 2012, 116, 4976−4990. (49) Defonsi Lestard, M. E.; Tuttolomondo, M. E.; Wann, D. A.; Robertson, H. E.; David, W. H.; Rankin, D. W. H.; Ben Altabef, A. Experimental and Theoretical Structure and Vibrational Analysis of Ethyl Trifluoroacetate, CF3CO2CH2CH3. J. Raman Spectrosc. 2010, 41, 1357−1368. (50) Elson, E. L.; Edsall, J. T. Raman Spectra and Sulfhydryl Ionization Constants of Thioglycolic Acid and Cysteine. Biochemistry 1962, 1, 1−7.

14252


Vibrational and Structural Behavior of l-Cysteine Ethyl Ester

Recommend Documents