Experimental and Molecular Modeling Studies on the Complexation of Chromium(III) with the Angiotensin-Converting Enzyme Inhibitor Captopril

Captopril (CPT) is an inhibitor of angiotensin I converting enzyme, used as a medication for the treatment of people with high blood pressure, renal insufficiency, and cardiovascular diseases. It inhibits the angiogenesis process, vasoconstriction, and tumor metastasis. Some metal–captopril complexes exhibit antimicrobial activities. In the current work, the formation of the CrIII–CPT complex was studied spectrophotometrically and potentiometrically in aqueous solution. Kinetics of CrIII–CPT complex formation was spectrophotometrically studied over the pH range 3.20–4.20, at an ionic strength of 0.3 M at 30–50 °C. CrIII–CPT complex formation was potentiometrically studied at 25 °C, where ligand protonation constants and complexes’ overall stability constants were calculated. UV–vis absorption spectra were executed to confirm the complex formation. Density functional theory and molecular dynamics simulation were performed to search the geometries of the CrIII–CPT complex. Atoms in molecules and interaction region indicator calculations are used to investigate intermolecular interactions for the formation of CrIII–CPT complex. The antimicrobial activity of the CPT ligand and CrIII–CPT complex on the prevention and control of environmental pathogenic bacteria, as tested on both Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli) via agar disc diffusion method, assess the ability to use as an antimicrobial agent. CPT had shown good antimicrobial activity against both types of bacteria, which had increased slightly the zone of inhibition in Cr-CPT that indicates the increased efficacy due to Cr(III) antimicrobial activity via its oxidative damage to the bacterial cell wall. No previous study tested the CPT antimicrobial activity against Gram-positive ones such as S. aureus


■ INTRODUCTION
Chromium(III) complex studies have a significant importance because of the high stability and biological activity of these complexes, especially that with amino acids which can be used as enzymatic labels. 1 Chromium is a fundamental nutritional metal that plays a role in carbohydrate, protein, and fat metabolism by potentiating the action of insulin. 2−5 Chromium deficiency has contributed to cardiovascular disease, type 2 diabetes, and metabolic syndrome. 6−8 Captopril (CPT) is an L-proline derivative (nonessential amino acid). CPT is an inhibitor for angiotensin I converting enzyme that prevents vasodilator prostaglandin degradation, by inhibiting vasoconstriction and simulating systemic vasodilation, inhibiting the production of angiotensin II, whose levels are elevated in patients with high blood pressure. 9,10 For that reason, CPT is used as a medication for the treatment of patients with hypertension, renal insufficiency, and cardiovascular diseases. 11−14 CPT is an antioxidant and can reduce oxidative stress, which is often involved in the pathogenesis of arterial hypertension, and also decreases the cardiac inflammation associated with arterial hypertension. 15−17 Captopril represents probable therapeutic choices to stop testicular injury and dysfunction resulting from cadmium toxicity due to its anti-inflammatory and antiapoptotic activities. 18 Also, it is effective for patients with insulin-dependent diabetes who have established nephropathy and retinopathy. 19,20 CPT inhibits the growth of some types of tumors; it hinders cell proliferation in a variety of human cell types including the neuroblastoma cell line; lung fibroblasts; and mammary ductal, renal, and esophageal carcinoma cells. 21−25 Captopril intake decreases the amount of adipose tissue, increases the level of angiotensin-(1−7) in plasma, which activates phosphorylation of hormonesensitive lipase, and may be a good candidate for weight control. 26 The maximal absorption of captopril would take place across the lipid membranes of the buccal mucosa at low pH values (pH 3 and pH 4). 27 The influence of pH on the rate of degradation of captopril in the phosphate−citrate buffer has been studied. 28 The reaction rate increased with pH and sharply so above pH 4 and, consequently, below pH 4, and deionized water was the appropriate vehicle for preparing the captopril dosage form. Lately, attention has been paid to the coordination of CPT with metal ions, the complexation of Ag(I) with captopril has been studied. The new complex formed, CPT−Ag(I), showed antimicrobial activity against different types of bacteria. 29 Here, kinetics and the formation of the Cr III −CPT complex were studied. The ligand protonation constants and complex overall stability constants were calculated potentiometrically. The proposed structure of the complex was computationally confirmed using density functional theory (DFT) and molecular dynamics (MD) simulation. The interaction between chromium(III) and CPT was investigated using the atoms in molecules (AIM) theory. The interaction region indicator (IRI) calculation is used to reveal chemical bonding and weak interaction. The ligand and metal complex have been screened for their microbiological activities against some kinds of bacteria (Gram-positive and Gram-negative).

■ EXPERIMENTAL SECTION
Chemicals. In experiments, chromium(III) nitrate nonahydrate (Merck, Germany) was used as a source of Cr 3+ ions. Captopril (EIPICO, Egypt) was used as a ligand. Sodium acetate and acetic acid (Merck, Germany) were prepared and used as buffer solutions. Sodium nitrate (Fisher, USA) solution was used to maintain the ionic strength of the solutions constant during course of the reaction. NaOH and HNO 3 were from Merck.
Apparatus and Procedures of Potentiometric Titrations. A Metrohm 702 titroprocessor provided with a 700 dosino buret and a 728 magnetic stirrer was used to achieve the potentiometric titrations. The titroprocessor was connected to a computer, and the titration and data acquisition were controlled by Vesuv, version 3.0, software. The pH titrations were executed in a double-walled glass vessel connected with a thermostated water bath. The ionic strength of the solution was maintained constant at 0.1 M by adding the required concentration of sodium nitrate solution as a supporting electrolyte. The pH meter was calibrated with standard buffer solutions (pH 4.0 and 7.0) before and after each series of pH measurements, and pK w = 13.77 at 25°C.
Each solution was allowed to equilibrate for about 20 min at 25 ± 0.1°C preceding the titration and frequently at least three times and used for fitting. The data were recorded at constant volume increments of 0.05 mL, forming real-time titration curves. The solution of sodium hydroxide was standardized with standard potassium hydrogen-phthalate. No calculations have been executed after precipitation.
Calculations of Ligand Protonation Constants and Cr III -Ligand Stability Constants. The potentiometric data and the species distribution diagram (SDD) of the formed complexes in the solution were analyzed with the Hyperquad 2008 30 package to compute captopril protonation constants and Cr III −ligand formation constants. The SDD is a powerful visualization tool for the accurate assessment of all species present in solution and their concentrations as a function of pH. This program simplifies visual interpretations of refinement, which helps in obtaining the best fit for the titration data. The program calculates formation constants from potentiometric data by a linear least-squares curve-fitting analysis. The stability constants are reported as β MLH , where M, L, and H designate Cr III , CPT, and H + , respectively.
Calculations of complex stability which gave the best fit to the experimental data with Hyperquad are determined by minimizing the error squares sums of the potentials: where w i represents a statistical weight assigned to each point of the titration curve and pH obs and pH calc refer to the measured and the calculated pH values, respectively. The calculated pH was calculated automatically by Hyperquad during the refinement cycle of the calculated complexation model. In the Hyperquad software, the minimizing error squares were expressed as a sigma parameter. The sigma parameter measures the goodness of statistical fitting of the experimental to calculated model. Additionally, it was expected that the standard deviations of the calculated overall formation constants (logβ pqr ) should be less than the 0.5 log unit. Kinetic Studies. The reaction rate was determined by observing the products' absorbance as a function of time. UV− vis absorption spectra of Cr III −CPT complex formation was spectrophotometrically monitored for a fixed period of time with a UV−vis Jasco 530 spectrophotometer.
Kinetic studies were executed via mixing thermostated solutions of captopril at the desired pH with Cr III . The pH measurements were executed with a Jenway pH meter fitted with a glass calomel electrode. Complexation of Cr III −CPT was followed at 572 nm using a thermostated Jenway 6315 spectrophotometer. Pseudo-order conditions were preserved where there was at least a 10-fold excess of captopril to Cr III .
Computation Details. Density functional theory calculations were used to support our experimental results. The Gaussian 09W program 31 was used to predict the molecular structure and energies of the Cr III −CPT complex. DFT geometric optimization was executed in the gas phase using the B3LYP method with basis set 6-31+G (d). The correction to zero-point vibration energy was considered in the calculations of the Cr III −CPT binding energy.
MD simulation of the formation of the Cr III −CPT complex in aqueous solution was carried out by using the Materials Studio (BIOVIA, 2017) package. Two MD simulation systems were studied: one contains five molecules of Cr III ions and five molecules of captopril ions in 1000 H 2 O molecules, and the other contains five Cr III ions and 10 captopril ions in 1000 water molecules. All molecules were placed randomly in the simulation box. These systems are simulated with NPT ensemble under pressure (0.1 MPa) for 100 ps, followed by the NPT ensemble for 1000 ps at 25 ± 0.1°C. The MD simulation was computed with the COMPASS II force field, and its charges were assigned. The temperature and pressure of the simulation systems were controlled by a Noséthermostat and Berendsen barostat, respectively. The convergence tolerance quality of optimization was ultrafine. The summation method of van der Waals interaction and electrostatic forces were atom-based and Ewald, respectively. The g(r)-RDF tool was used to calculate the radial distribution functions.
Additionally, we used AIM theory 32 to investigate topological properties within the optimized structure of the Cr III −CPT complex at the B3LYP/6-31+G(d) level. The total electron density (ρ), its Laplacian (∇ 2 ρ), and the electronic energy density (H) at a corresponding bond critical point (BCP) are employed to identify the nature of chemical bond interactions. In general, ρ > 0.20 au is in a covalent interaction, and < 0.20 au is in a coordinate and closed-shell interaction (ionic, van der Waals, hydrogen bonding, etc.). The Laplacian of electron density, which is the trace of the Hessian, also determines the interaction type. A negative sign for the Laplacian of electron density signifies covalent interactions, and its positive sign stands for coordinate and closed-shell interactions. Furthermore, the electronic energy density (H) composed of electronic potential energy density (V) and electronic kinetic energy density (G) at a BCP is a more appropriate index to understand interactions. The sign of electronic energy density at a BCP determines whether the interaction is a closed shell interaction (H > 0) or a covalent and coordinate interaction (H < 0).
Also, IRI calculations are used to reveal chemical bonding and weak interaction in a Cr III −CPT system. The AIM and IRI calculations were implemented in Multiwfn version 3.8. 33 Isosurface maps were rendered by VMD 1.9.4 software. 34 Antimicrobial Activity Testing of Zn−Al LDH/GA Using a Disc-Diffusion Assay. Fifty discs of a Whitman filter paper of standard size (80 mm diameter) were used then kept in six screw-capped bottles, and to ensure sterilization, the screw-capped bottles were placed in a hot air oven for 30 min at 150°C. The sterilized discs were impregnated overnight with the tested concentrations of both CPT and Cr-CPT at concentrations of 5 mg/mL. The paper disks were soaked at concentrations of 5 mg/mL. Isolated bacteria at a concentration of 10 8 CFU mL −1 were diluted in normal saline tubes according to a McFarland reference of 0.5. Thereafter, 100 μL diluted tubes were inoculated on Mueller−Hinton agar plates. The impregnated discs were placed aseptically with sterile forceps on Mueller−Hinton agar plates. All agar plates were inculcated at 37°C for 24 h. Then, the inhibition zone diameter of all tested bacteria was determined via 2-fold serial dilution. This method was done according to CLSI 35 and Perez et al. 36 The standard strain S. aureus (ATCC 25923), indicative for Gram-positive bacteria, and E. coli (ATCC 25922), indicative for the Gram-negative ones, were used in this study.
■ RESULTS AND DISCUSSION Protonation Constants. Captopril protonation constants were computed with Hyperquad from the potentiometric pH profile of the CPT solutions in the absence of chromium(III) ions. Raw data for each titration were treated with a nonlinear least-squares refinement, where optimized Hyperquad fitted the model titration curve in Figure 1a (line curve) to the experimental data (symbols) based on a least-squares analysis of K a1 and K a2 . There is a very good match of the calculated model for the experimental titration curve. The overall protonation constants, β n , of CPT can be described as where K 1 ...K 2 define the stepwise CPT dissociation constants. The overall (log β pqr ) and successive (log K) protonation constants of CPT as calculated by the Hyperquad software are listed in Table 1. The symbols p, q, and r were used as the coefficients for Cr III , protons, and ligands, respectively, to designate the stoichiometry associated with the possible equilibria in solution. The pK a values for the two ionizable protons of CPT were calculated as 3.85 and 9.68. They are fairly consistent with the reported data in the literature, 37 where the pK a values are 3.48 and = 9.68. Cr III −CPT stability constants The overall formation constants (log β pqr ) for the systems containing the metal ion Cr III and CPT as ligand, with a molar ratio of Cr III to CPT 1:1 and 1:2.5, were computed from the where p, q, and r are the coefficients for Cr III , H + , and captopril, respectively, and the square-bracket symbol refers to molar concentration. The p, q, and r values are zero or positive integers. The formation constant of the hydroxo complex was taken into account and represented by a negative value for q in Table 1. The value of the Cr III −CPT complex's overall stability constant is high, showing that CPT forms strong complexes with the Cr III ion. Calculation of the stepwise formation constants (log K) was executed to show the strength of bonding between Cr III and captopril. The values of log K Cr(CPT) and log K Cr(CPT) 2 are 8.34 and 6.87, respectively, where log K Cr(CPT) = log β101 and log K Cr(CPT) 2 = log β102 − log β101. The value of log K Cr(CPT) > log K Cr(CPT) 2 shows that the chromium ion binds more strongly with one captopril molecule.
The species distribution diagram of the Cr(CPT) x (OH) y system at a 1:2.5 metal-to-ligand molar ratio as a function of pH and is presented in Figure 1b. The analysis of SDD for the Cr(CPT) x (OH) y system shows that the formation of [Cr-CPT] + starts from pH 2.4 and dominates in the solution at pH 5 and begins to be negligible at pH 7.0 onward. [Cr-(CPT) 2 ] + species start to form at pH 3.6, are at a maximum at pH 5.88, and begin to be negligible at pH 7.7 onward. The formation of [Cr(CPT)(OH) 3 ] begins to be significant from pH 6 onward, reaching a maximum concentration of 100%, at pH = 7.87.
Kinetics Study on the Complexation of Cr III with CPT. Cr III −captopril complex formation is confirmed using UV−vis absorption. The Cr III −CPT absorption spectra at a particular pH value are presented in Figure 2. It shows that, after the mixing of CPT with Cr III , the absorbance increased with time over the whole wavelength range and that the two peaks of chromium nitrate at λ = 580 and 410 were shifted to λ = 572 and 408; the color changes from green to violet, proving the complexation between Cr III and CPT.
The rate of the reaction was followed under pseudo-firstorder conditions. Values of the observed pseudo-first-order rate constants, k obs , were determined from the slopes of plotting −ln(A ∞ − A t ) vs time, t, where A indicates the measured absorbance and the subscripts refer to the time of reaction. The absorbance (A ∞ ) was obtained after completion of the reaction. These plots were linear for more than 97% of the reaction progress.
Variations  Table 2; the results indicate that the reaction is first-order dependent on the concentration of Cr III , as there is no significant change in the values of k obs when the concentration of Cr III was varied, as described by eq 4:    Under constant reaction conditions and various temperatures, the pH effect on the reaction rate was studied in the 3.20−4.20 pH range ( Table 3). The obtained results indicate that the rate of the reaction increases with decreasing the hydrogen ion concentration. Table 2 show that k obs is unaffected by changing the ionic strength of the medium in range of 0.1−1.0 M, indicating that the reaction is proceeding between charged (Cr(H 2 O) 5 (OH) 2+ ) and uncharged species (captopril).

Results in
The dependence of k obs on hydrogen ion concentration can be described by the following equilibrium according to a value of K a of 1. 31  The form of chromium that reacts with CPT is [Cr-(H 2 O) 5 (OH)] 2+ since it is more reactive than [Cr(H 2 O) 6 ] 3+ species due to the existence of the hydroxyl group, which increases the water labilities due to its π-bond ability. 39,40 From the proposed mechanism (Scheme 1), the reaction rate is given by The suggested rate law presented in eq 9 is in agreement with the experimental law in eq 5, in which k′ = kK a . Using the value of K a obtained potentiometrically (K a = 3.26 × 10 −4 M), values of 10 2 k at T = 35°C calculated from eq 9 and Figure 3 are 3.66, 7.64, 12.14, and 22.7 s −1 , respectively, at 30, 35, 40, and 50°C.
Captopril reacts with [Cr(H 2 O) 5 (OH)] 2+ , developing the inner sphere complex Cr III −CPT in the rate determining step, where chromium forms a bond with CPT through the oxygen of the carboxylic group. The previous step is followed by a very rapid protonation equilibrium, which favors the aqua species, followed by another bond formation between Cr III and the second oxygen of ligand carboxylate group, forming the final complex.
Calculated thermodynamic activation parameters ΔH* and ΔS*, from an Eyring equation plot, were found to be 69.48 ± 9 kJ/mol and −108.74 ± 3 J/K mol, respectively. The negative entropy value and the positive enthalpy value support the associative mechanism. The same isokinetic temperature was acquired from the isokinetic plot for the [Cr(H 2 O) 6 ] 3+ with different ligands (Figure 4) backing the associative mechanism. For water substitution in [Cr(H 2 O) 6 ] 3+ by anthranilic acid, 41 tryptophan, 42 L-lysine, 43 glycine, 44 valine, 45 Figure 5 shows the optimized structure of the Cr III −CPT complex with the B3LYP method with the 6-31+G(d) basis set. Density functional theory calculations for the complexation between chromium and captopril ions demonstrated that the complexation takes place over the oxygen atoms of the captopril carboxylate group and chromium atom. The optimized structure shown in Figure 5 has no negative vibrational force constant, indicating that it is a ground state compound. The binding energy (ΔE bind ) for the Cr III −CPT complex equals −1178.3313 kcal/mol. MD simulation for the complexation of CPT with Cr III was simulated in water molecules to explore their complexation in aqueous solution. Figure 6 displays the MD snapshots taken at the end of the simulation of Cr III /CPT 1:1 and 1:2 systems. This figure shows that the coordination between Cr III and CPT takes place via the oxygen atoms of the captopril carboxylate group. A variety of hydrogen bond formations with four or more water molecules were detected at a distance of 0.35 nm from captopril.
The radial distribution function (RDF) obtained from the MD simulation provides further characterization of the possible interaction sites between different atoms to identify the complexation reaction. Figure 7 shows the RDF of Cr··· OC, Cr···O nitrate , Cr···S, Cr···OW, O nitrate ···HW, CO··· HW, and SH···OW, where HW and OW are hydrogen and oxygen atom of water, respectively. MD simulation of the Cr III −CPT system shows that the chromium atom forms three bonds: a strong coordinate bond with the oxygen atoms of the CPT carboxylate group as shown in Cr···OC RDF, which has a peak at 2.11 Å, with the oxygen atom of water molecules as shown in the Cr···OW at 2.39 Å and with oxygen atoms of nitrate as shown in the Cr···O nitrate at 2.15 Å. The intense peak between chromium and nitrate proposes the formation of the complex in neutral form.
Hydrogen    Figure 8 shows the critical points (CPs) in the optimized structure of the Cr III −CPT complex. The topological parameters of the interaction between chromium and oxygen atoms of the captopril carboxylate group were analyzed from the optimized structure at the B3LYP/6-31+G(d) level (Table  4). It is observed that the values of ρ are lower than 0.2 au, ∇ 2 ρ > 0, and H < 0, indicating that the interactions between Cr III and CPT are coordinate bonds, where chromium ions form two coordinate bonds with the two oxygen atoms of the captopril carboxylate group. The topological analysis of electron density provides evidence for the existence of intramolecular interactions between Cr III and CPT. Figure 9 presents the isosurface map of IRI = 1.0 of the Cr III −CPT complex which reveals both covalent and noncovalent interaction regions. IRI isosurfaces successfully revealed the Cr−O bonds. The coordinate bonds between the Cr III and coordinated oxygen atoms of CPT are exhibited by blue IRI isosurfaces. The steric effect within the fivemembered pyrrole ring and the Cr−O−O ring can be identified by the red areas of the isosurfaces. The van der Waals interaction (vdW) due to the close contact between the same atoms can also be identified by the green part of the IRI isosurfaces. 46 Antimicrobial Activity Study. The antimicrobial activity of CPT and Cr-CPT had been tested against both Grampositive and Gram-negative bacteria via the agar disc diffusion method on treptone soya agar media. We confirmed the modest inhibitory activity of CPT and Cr III −CPT against both Gram-negative bacteria E. coli as previously reported in the literature 47 and Gram-positive S. aureus that showed a more wide zone of inhibition as shown in Figure 10. Moreover, the extent of inhibition was also affected and increased in Cr III − CPT than in CPT. As previous studies had reported, the CPT mechanism of action against antimicrobial activity inhibiting N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a metallohydrolase involved in the meso-diaminopimelate (mDAP)/lysine biosynthetic pathway necessary for lysine biosynthesis and for building the peptidoglycan cell wall of bacteria. 47 The zone of inhibition was slightly more in Grampositive strains and in Cr III −CPT. Therefore, CPT is a modest Figure 6. MD snapshots of CPT (dark gray), Cr III (gray), and water (red) molecules taken from simulations for Cr III + CPT + nitrate + sodium + water system (a) 1:1 Cr III /CPT and (b) 1:2 Cr III /CPT. antibiotic, inhibiting Gram-negative bacteria at high doses, but its mechanism of action or molecular target remains unknown. Finally, considering that DapE is a promising antibiotic target, the failure of a lead compound that inhibits DapE in vitro to show any measurable anti-DapE effect in bacteria provides a sobering reminder of the difficulty of translating in vitro data to effects in vivo, even in pure microbiological cultures as had been tested in previous studies. 47 The work described here shows that the Cr III −CPT complex is bactericidal for S. aureus and E. coli. Our results indicate that Cr(III) itself may be interesting to open new paths for metallo drug chemotherapy against different bacterial genera since some of these complexes have been found to exhibit remarkable antibacterial activities. Cr(III) addition to CPT    for complex formation had shown high antimicrobial activities against E. coli and S. aureus. In our biological experiments, by using Cr(III) we have observed high biological activity against Gram-negative and Gram-positive bacteria. The results showed that the Cr III −CPT complex used in the present work inhibits the growth of bacteria to a wider extent compared to CPT alone. 48

■ CONCLUSION
Here, the reaction between Cr III with CPT was spectrophotometrically and potentiometrically studied in aqueous solution, where [Cr(H 2 O) 5 OH 2+ ] is the reactive species. The reaction is first-order dependent on [Cr III ], increasing with decreasing hydrogen ion concentration and increasing the temperature. An associative mechanism is proposed for this reaction. The calculated values of CPT protonation constants (pK a = 3.85, 9.68) were fairly consistent with the data reported in the literature. The values of the stepwise stability constants calculated potentiometrically show that the Cr III −CPT complex is more stable than Cr III −CPT 2 . DFT, MD simulation, IRI, and topological analysis of electron density provides evidence for the existence of chemical bonds between Cr III and CPT, where chromium forms two coordinate bonds with CPT, proving the complexation between them. The Cr III −CPT complex had shown high antimicrobial activities against E. coli and S. aureus. It may be concluded that the Cr III −CPT complex used in the present work inhibits the growth of bacteria to a wider extent compared to CPT alone.