Study of a Conformational Equilibrium of Lisinopril by HPLC, NMR, and DFT

The isomerization of lisinopril has been investigated using chromatographic, NMR spectroscopic, and theoretical calculations. The NMR data, particularly the NOEDIFF experiments, show that the major species that was eluted first is the trans form. The proportion was 77% and 23% for the trans and cis, respectively. The thermodynamic parameters (ΔH, ΔS, and ΔG) were determined by varying the temperature in the NMR experiments. The interpretations of the experimental data were further supported by DFT/B3LYP calculations.


Introduction
Lisinopril, N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline, belongs to a class of antihypertensive agents which inhibit the angiotensin-converting enzyme (ACE) to control blood pressure [1]. The active parts of ACE inhibitors are peptide derivatives containing C-terminal proline residues. Like other proline-containing peptides, lisinopril exists as an equilibrium mixture of cis and trans isomers, with respect to the proline amide bond ( Figure 1) [2,3]. Under unstrained conditions most peptide bonds adopt the trans isomeric form, mainly because of the weaker steric repulsion between hydroxyl and carboxyl group effects in the molecule when compared to the cis. The trans form in lisinopril was shown to be the preferred isomer and biologically active [4][5][6]. The assignment separation of cis and trans form of lisinopril has been carried out by HPLC [5,[7][8][9][10][11][12], CZE [13][14][15][16], and NMR spectroscopy [2,[17][18][19][20][21][22]. The latter technique is a powerful tool and has been widely applied for structural and stereochemical characterization of amino acids, oligo-and polypeptides [23][24][25][26]. The cis-trans isomerization of peptide bonds is a slow process on the NMR time scale under normal conditions at ambient temperature due to the high barrier resulting from the C-N partial double bond character. NMR spectroscopy has therefore been successfully used to study the cis-trans isomerization process of lisinopril in solution [25,27].
In this paper, we report on the isomerization of lisinopril using a combination of HPLC, NMR spectroscopy, and theoretical approaches. The effect of temperature on the cis-trans isomerization process of lisinopril was investigated in order to determine different thermodynamic parameters (Δ , Δ , and Δ ).

Experimental
2.1. Samples. Lisinopril was kindly provided from Solvay Pharmaceuticals.
The mobile phase was prepared by first preparing a solution of 0.02 M KH 2 PO 4 , adjusting its pH to 2 with phosphoric acid and finally mixing the solution with an organic modifier (acetonitrile, methanol, and THF). The mobile phases were always filtered using 0.45 m membrane filter (Supelco) and degassed by sonication.

Chromatography.
Liquid chromatographic analyses were performed using a Hewlett Packard 1100 HPLC system equipped with a photo diode array UV detector set at 215 nm. Injection was performed using an autoinjector. A Supelco LC 18 (5 m) column (250 × 4.6 mm I.D) and a guard column (20 × 4.6 I.D) both from Supelco (Bellefonte, PA, USA) were used. The pH of mobile phase buffers was adjusted by means of a Schott model CG 825 pH meter (Germany). All measurements were made on lisinopril in CD 3 CN/D 2 O (1/9) solution. The variable temperature NMR spectra were acquired using the same instrument. Probe temperatures (±0.5 K) were measured with a calibrated digital thermocouple. Samples were allowed to equilibrate for 10 min at each temperature before recording the spectrum.

Computational Details.
Density functional theory (DFT) calculations were carried out on the cis and trans isomers of lisinopril with the Gaussian 03W suite of programs [28], with the nonlocal hybrid functional denoted as B3LYP [29]. Then basis sets used were zeta 6-31+G * [30][31][32][33][34], doubly polarized with diffuse functions on all the atoms. The geometries of both the cis and trans isomers were optimized using an analytical gradient. The harmonic vibration frequencies of the different stationary points of the potential energy surfaces (PES) have been calculated at the same level of theory in order to identify the local minima as well as to estimate a corresponding zero-point vibrational energy (ZPE).

HPLC Study.
The study of the cis/trans equilibrium of lisinopril by HPLC demonstrates that chromatographic conditions such as flow rate, temperature, pH, and organic modifier have an important effect on peak shape and retention time of lisinopril.
It appears that the separation of the two isomers of lisinopril can be achieved using a mobile phase consisting of a mixture of 20 mM phosphate buffer [pH 7]-acetonitrile (90/10; v/v), a column temperature of 279 K, and flow rate of 2 mL/min with retention time 1 = 3.49 min and 2 = 4.55 min. However, a higher temperature is required for the elution of lisinopril as a single sharp peak at 2.76 min ( Figure 2). This is because it was found that an elevated temperature led to deterioration in the separation of the two isomers.
Moreover, at 328 K lisinopril was eluted as a narrow single peak due to the high isomerization rate of the two isomers. On the other hand, at low temperature the two isomers were resolved almost completely indicating that the interconversion rate had slowed down.
From ambient temperature chromatograms, the isomer trans/cis ratio was integrated to be 76/24. This result is similar to those reported earlier demonstrating that high temperature was useful for elution of proline-containing substances as a single peak [7,11,35]. Conversely, a low temperature is known to have a potential effect on the separation of isomers [5,9,[36][37][38][39].

NMR Studies.
The structure of lisinopril ( Figure 3) shows 21 carbon atoms with two sets of two chemically equivalent carbons describing the ortho-and metapositions on the aromatic ring. So, we expect to observe 19 signals in 13 C NMR spectra. However, the obtained spectra showed the doubling of all signals confirming the existence of the two isomers ( Figure 4).
In addition, the 1 H NMR spectra of lisinopril in CD 3 CN/D 2 O at 298 K ( Figure 5(a)) show two sets of triplets of unequal intensities. The multiplicity of each signal set reflected first from the interaction of H58 with H25 and H26, giving the two signals in the 3.8-4.1 ppm region, and second from the interaction of H43 with H45 in the 4.1-4.4 ppm region. The same spectrum recorded at 333 K ( Figure 5(b)) shows a better separation of the two signals at 4.1 ppm.
These isomers are assigned to a cis-trans equilibrium of the rotation around the amide bond. As described earlier, it is worth noting that this equilibrium appears to be slow on the NMR time scale at ambient temperatures [37,40]. Using the area of resonance signals of proton 58 (3.8-4.4 ppm), the isomer ratio was integrated to be 77/23 at 298 K. The result obtained in a separate experiment recorded at a probe temperature of 298 K is consistent with that determined by HPLC at the same temperature.
We conclude that the major conformer in the 1 H NMR spectrum of lisinopril corresponds to the first eluted peak in the HPLC chromatogram at ambient temperature, which exists in a higher proportion. A similar study demonstrated this correspondence in the case of ramiprilat [4], enalaprilat [5], and perindopril [39] in different solvents.
It is well known that the NOE effect ( ) between two dipole-dipole interacting nuclei is inversely proportional to the distance ( ) 6 between the irradiation site ( ) and the measured one ( ), respectively [41], according to the following formula: ( ) = (1/ 6 ). In addition, when H 48 ( = 4.45 ppm) is irradiated, we observe at H 58 a NOE ( = 34%) in the major conformer and a NOE ( = 10%) in the minor conformer ( Figure 6). This shows that the NOE in the minor conformer for the H43/H58 is more important than the NOE in the major conformer, which implies that in the major conformer, the distance between the nuclei is higher than the one in the minor conformer    ( 48-58 ) maj . (2) Consequently, the examination of the molecular structure of each conformer of lisinopril confirmed that the distance 43-58 in the cis conformer is indeed smaller than the distance in the trans form. The 1 H NMR intensities suggest that the major conformer is the trans form and the minor conformer is the cis form. The nuclei of the s-trans conformer are more deshielded than those of s-cis conformer, in agreement with literature results [41,42] Remarkably, the equilibrium cis to trans isomerization was enthalpically and entropically favored in this condition. Consequently, the decrease in the temperature expected a displacement of the conformational equilibrium to the strans conformer. The latter is stabilized by hydrogen bonding between the carbonyl (40) and the hydrogen of hydroxyl group OH of the acid function (56). This result is in agreement with other studies reported on a similar product such as enalapril [5].

Theoretical Calculations.
In order to confirm the NMR data obtained for the cis/trans isomerization of lisinopril, the geometrics of the two conformers were fully optimized at the DFT/B3LYP level of theory using 6-31++G * basis set. The structures have been identified as local minima on the singlet potential energy surfaces (PES) (Figure 8). Optimized values of selected geometrical parameters are listed in Table 1. The potential energy difference between the two isomers of lisinopril was 11.397 kJ/mol indicating the stability of trans over the cis isomer. This difference is in good agreement with experimental data that the trans is the majorities form.
As shown in Table 1, in the trans conformer the interaction between H56-O40 ( H56-O40 = 1.717Å) would be stronger than that in the cis ( H56-O40 = 3.857Å), thus generating a sharp reduction of the valence angle C53-O55-H56 ( = 103.1 ∘ in the trans form and = 2.3 ∘ in the cis form) and a strong variation on dihedral angle O40-C39-C41-C50 ( = 176.6 ∘ in the trans form and 2.6 ∘ in the cis form), indicating possible existence of some hydrogen bond interactions which would be more favored in the trans than in cis isomers.
This stability of trans form over the cis form is further confirmed by the charge density between the same atoms (Δ -= 0.08, Δ -= 0.02). The strong interaction between O21-H56 atoms ( H56-O21 = 1.799Å) in the scis conformer triggers a modification of the dihedral angle O21-H56-O55-C53 ( = 156.8 ∘ ) which may explain the low stability of the cis form. On the other hand, the low interaction between H56-O21 ( H56-O21 = 4.816Å) in the strans conformer yields a change in the dihedral angle ( = 3.9) so the atoms H56 and O40 were far from each other and the trans was more stable. It is worth noting that there are two hydrogen bonds, one between atoms H56-O40 and another between H55-O21, but the stability was determined by the first bond because 6 International Journal of Analytical Chemistry the distance is shorter ( O21-H56 = 1.799Å). In addition, there are strong intramolecular interactions in the trans conformer ( = 10.6 D) than in the cis form ( = 3.9 D) indicating a higher stability of the conformer trans.
The distance between the nuclei in both conformers (cis and trans) was compared. It is revealed that in the cis conformer, H43 is close to H58 (4.058Å), while H48 is at a much greater distance from H58 (6.612Å). On the other hand, in the trans conformer H43 is further away from H58 (4.471Å), while H48 is at much greater distance from H58 (6.225Å). This is consistent with the results obtained with NOE difference experiments giving an enhancement of 8% and 21% in the first irradiation and 34% and 10% in the second irradiation.

Conclusions
Lisinopril exists individually as a mixture of cis-trans isomers in solution. The two isomers could be easily distinguished by HPLC, H NMR, C NMR, and 1 H-1 H NOE spectra. The results of the various investigations by NMR and those of the theoretical study are in favor of assigning trans form to the major isomer present under the conditions used in the HPLC studies. Additionally, a relative to successful combination of International Journal of Analytical Chemistry 7 molecular modeling studies with experimental spectroscopic assays was used in order to elucidate the molecular bases.