Investigation of the transition metal ions (Co2+ , Mn2+ ) complexation process with pravastatin by high-resolution NMR spectroscopy and MD methods

Investigation of the transition metal ions (Co2+ , Mn2+ ) complexation process with pravastatin by high-resolution NMR spectroscopy and MD methods | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of the transition metal ions (Co2+ , Mn2+ ) complexation process with pravastatin by high-resolution NMR spectroscopy and MD methods T. R. Islamov, O. V. Aganova, A. R. Yulmetov, A. S. Tarasov, F. Kh. Karataeva, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7027909/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2025 Read the published version in BioNanoScience → Version 1 posted 8 You are reading this latest preprint version Abstract The paper studies the complexation of pravastatin with transition metal ions (Co 2+ , Mn 2+ ) using high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) methods. It was shown that the presence of transition metal ions leads to changes in 1 H, 13 C NMR chemical shifts of CH 2 -14, CH-15, CH 2 -16 groups of pravastatin. MD modeling for systems of pravastatin aqueous solutions with cobalt and manganese ions confirms the experimental assumption about the most probable area of metal ions near the pravastatin molecule. NMR spectroscopy pravastatin Co2+ Mn2+ molecular dynamics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Coronary heart disease (CHD) remains one of the causes leading to death worldwide, despite significant advances in diagnosis and treatment. In recent decades, there has been an increase in the incidence of CHD, especially in developing countries, due to lifestyle changes and an increase in the prevalence of risk factors such as obesity and hypertension [1–3]. Elevated level of low-density lipoprotein cholesterol (LDL-C) is a key risk factor for the development of coronary heart disease (CHD), as they contribute to the deposition of cholesterol in the arterial walls and the formation of atherosclerotic plaques. Statins are one of the drugs most common classes used to lower LDL-C levels. The main problem with these drugs is their low efficiency according to clinical studies [4,5]. It is known that the physicochemical properties of organometallic substances or coordination compounds formed by organic compounds and transition metals differ significantly from the physicochemical properties of the ligands included in the organometallic compounds [6,7]. In this regard, the use of modern NMR methods to determine the structure and analyze the properties of organometallic or coordination compounds based on derivatives of statins and some transition elements, as well as the study of their complexation, is of particular importance. To confirm the assumption about the most probable area of the metal ion site in the system based on derivatives of statins and some transition metal ions (Co 2+ , Mn 2+ ), additional MD modeling can be performed. To create new drugs that must have certain predetermined properties, it is important to know the spatial structure of the object [8]. The NMR method is also extremely sensitive to the interactions of organic substances with transition metal ions. 2. Materials and methods 2.1. Materials The initial sample of the study was a solution of pravastatin in deuterated water (D 2 O). To decrease the solvent signal and to recreate an organic environment, D 2 O was used for this research. Pravastatin from «Sigma-AldrichRus» company was used without additional purification. The structural formula of pravastatin is shown in Fig. 1 . The CoCl 2 and MnCl 2 salts were used to prepare aqueous solutions of pravastatin to study the complexation of pravastatin with Co 2+ and Mn 2+ ions. The initial salt was initially dissolved in D 2 O and then added to the pravastatin-D 2 O sample by titration with calculation of the concentration of Co 2+ , Mn 2+ ions in an ampoule. All samples were prepared in standard 5-mm NMR tubes. Concentrations of the substances were 22.0 mM (pravastatin) and 2.2, 4.4 mM (CoCl 2 , MnCl 2 ) and dissolved in aqueous solution (95% D 2 O + 5% H 2 O, pH = 7.3). The solution volume for all samples were 0.5 ml. 2.2. Methods Registration of 1D, 2D NMR spectra of pravastatin in D 2 O at a temperature of 308 K with and without addition of Co 2+ , Mn 2+ ions were carried out using pulsed NMR spectrometers Bruker Avance II-500 NMR (500 MHz ( 1 Н), 125.76 MHz ( 13 С)) and Bruker Avance III-700 NMR (700 MHz ( 1 Н), 175 MHz ( 13 С)). The spectrometers are equipped with a 2 H lock system to stabilization of magnetic field. 1 H NMR spectra were recorded using 90° pulses, the delay between pulses was 2 s, the spectral width was 10 ppm and a minimum of 16 scans. 13 C NMR spectra were recorded using 90° pulses, the delay between pulses was 2 s, the spectral width was 200 ppm and a minimum of 100 scans. Chemical shifts were determined relative to TMS (tetramethylsilane). The TOPSPIN 3.0 software was used to obtain and process the spectra. The assignment of spectral lines in NMR spectra was performed by using high resolution NMR spectroscopy methods of 1D, 2D experiments [9]. The program GROMACS was used for MD calculations of the pravastatin and Co 2+ , Mn 2+ ions interaction in an aqueous solution [10,11]. The initial configuration with the beginning energy was minimized using the most responsive descent algorithm and the low energy threshold was less than 1000 kJ/mol*nm. Then, each optimized molecular system was equilibrated in the canonical ensemble (NVT) at 300 K using the Berendsen thermostat with the coupling constant τ = 0.1 ps for 200 ps. Subsequently, the molecular system was equilibrated at a constant temperature and constant pressure (T = 300 K and P = 1 bar) for 300 ps using the thermostat with τ = 0.1 ps and the Parrinello-Rahman barostat with τ = 3 ps and a pressure of 45*10 − 6 bar. Next, the resulting molecular system was simulated in the NPT ensemble (T = 300 K and P = 1 bar) without any constraints. Periodic boundary conditions were used in the MD simulations. Electrostatic interactions were handled using the Ewald particle mesh method using CHARM conditions. The various simulations were run for at least 250 ns, and snapshots of the trajectories were taken every 2 ns [12]. 3. Results and discussions 3.1. NMR experiments of pravastatin with Co 2+ ions in deuterated water solution The complete assignment of the 1 H NMR spectrum of the studied compound was carried out using two-dimensional NMR experiments and based on the analysis of signal multiplicity, characteristic chemical shifts and integral values [9,13]. Figure 2 shows the 1 H NMR spectra of an aqueous solutions of pravastatin with cobalt ions at following molar concentration ratios (statin/ions): pure pravastatin (a), pravastatin with Co 2+ ions at ratios of 10/1 (b) and pravastatin with Co 2+ ions at ratios of 5/1 (c). with Co 2+ ions at statin/ions ratios 10/1 (b), 5/1 (c) in an aqueous solution (95% D 2 O + 5% H 2 O) at T = 308 K, * - solvent signal. Depending on the presence of the Co 2+ ions in solution, a number of differences are observed in the 1 H NMR spectra (Fig. 2 ): the proton signals of C H 2 -14, C H -15 and C H 2 -16 groups changes very much in intensities, and also in chemical shifts towards higher ppm; and at statin/ions ratio 5/1 (Fig. 2 c) the C H -15 signal disappears. We also observed similar changes in another case [13]. However, changes in other proton signals in the spectra are insignificant. An assumption about spatial proximity of the paramagnetic Co 2+ ions leads to a decrease in the relaxation time of protons was made. In this case, broadening of the spectral signals was observed. Moreover, there are no characteristic changes in the spectrum that usually occur during the formation of a chemical bond. Thus, this indicates that the interaction between pravastatin and Co 2+ ions has the character of a rapid exchange state. All 1 H NMR chemical shifts changes are presented in Table 1 . Table 1 Some chemical shifts changes in 1 H NMR spectra of pravastatin with Co 2+ ions at statin/ions ratios 1/0 (a), 10/1 (b), 5/1 (c) in an aqueous solution. a b c CH 2 -14, ppm 1.68 1.72 1.76 CH-15, ppm 4.14 4.26 not registered CH 2 -16, ppm 2.42 / 2.37 2.55 2.79 The heteronuclear 1 H– 13 C HSQC NMR spectra of an aqueous solution of pravastatin with various Co 2+ ions molar concentrations are shown in Fig. 3 . The changes in HSQC NMR spectra are consistent with 1 H NMR experiments: the proton signal of C H -15 group shifts toward lower fields (towards higher ppm) (Fig. 2 a – 4.15 ppm, Fig. 2 b – 4.26 ppm, Fig. 2 c – not registered). However, there is no change for the carbon-13 signal of C H-15 group is observed. Moreover, there is a slight shift towards higher ppm for the carbon-13 signal of C H 2 -14 group is observed: 43.4/43.8/44.6 ppm (Fig. 3 ). And the proton signal of C H 2 -16 group becomes very broad upon addition of the cobalt ions (Fig. 2 c). Thus, the assumption about the localization of the cobalt ions in the area of CH 2 -14, CH-15 and CH 2 -16 groups of molecule is confirmed. 3.2. NMR experiments of pravastatin with Mn 2+ ions in deuterated water solution 1 H NMR spectra of an aqueous solutions of pravastatin with manganese ions at following molar concentration ratios (statin/ions): pure pravastatin (a), pravastatin with Mn 2+ ions at ratios of 10/1 (b) and pravastatin with Mn 2+ ions at ratios of 5/1 (c) showed in Fig. 4 . Depending on the presence of the Mn 2+ ions in solution, a number of differences are observed in the 1 H NMR spectra (Fig. 4 ): the proton signals of C H 2 -14, C H -15 and C H 2 -16 groups becomes very broad and shift towards higher ppm (Table 2 ). And in case of the statin/ions ratio of 5/1, the proton signal of C H -15 group is disappears. Changes in other proton signals in the spectra are insignificant. Table 2 Some chemical shifts changes in 1 H NMR spectra of pravastatin with Mn 2+ ions at statin/ions ratios 1/0 (a), 10/1 (b), 5/1 (c) in an aqueous solution. a b c CH 2 -14, ppm 1.63 1.65 1.67 CH-15, ppm 4.13 4.15 4.20 CH 2 -16, ppm 2.41 2.45 2.59 The proton signals of C H -15 and C H 2 -16 groups disappears with an increase in the concentration of Mn 2+ ions in the solution (Fig. 4 c). Based on the data obtained from one-dimensional and two-dimensional experiments, in this case a similar assumption about the localization site of the manganese ions was made like it was for cobalt ions. 3.3. Molecular dynamics of pravastatin with Co 2+ , Mn 2+ ions in deuterated water solution To confirm the assumption of the most probable area of the metal ion in the system, molecular dynamics simulations of pravastatin with cobalt and manganese ions in aqueous solution were carried out. The model system consisted of 20 statin molecules with the addition of Co 2+ or Mn 2+ ions. The statin/ions ratios were chosen similarly to those in the experiment: 10/1 and 5/1 for both ions, i.e. 2 and 4 ions, respectively. Then the system was placed in a 10x10x10 nm periodic cubic cell with the addition of 32284 water molecules. This choice was also based on the experiment. The CHARMM36 force field was used for parameterization. The list of distances between the atoms of each statin and all ions for each simulation step was first compiled. Then the radial distribution function (RDF) graphs of Co 2+ and Mn 2+ ions relative to the location of statin protons were obtained to analyze the dynamics of binding of pravastatin with cobalt and manganese ions (Fig. 5 ). Based on the RDF graphs in Fig. 5 , Co 2+ ions have one maximum at distances 0.66 nm for C H -15 and 0.63 nm for C H 2 -16 atoms, which indicates a high probability of finding the cobalt ions in solution at the specified distances near these atoms. This arrangement of ions can be explained by the presence of fragment of COOH group with a large dipole moment in this part of the molecule. And the ion is surrounded by a solvate shell of water molecules. Therefore, hydrogen bonds are formed between the ion and the hydroxyl groups of statin when the ion and statin are located close to each other. The graphs characterizing the location of Mn 2+ ions have a more complex appearance - they have two high maximum, which indicates two possible areas of localization of ions near the statin molecule (Fig. 5 ). The first maximum is at the distance of 0.38 nm, which means that the solvate shell is destroyed and Mn 2+ ions are bound directly to pravastatin atoms, forming a stronger complex. Thus, the localization of Co 2+ and Mn 2+ ions predominantly occurs near the protons of C H 2 -14, C H -15, C H 2 -16 groups of pravastatin, which confirms by experimental results. The most probable sites of Co 2+ and Mn 2+ ions in an aqueous solution with pravastatin are schematically shown in Fig. 6. 4. Conclusion NMR study and MD modeling revealed the presence of interaction between pravastatin and transition metal ions (Co 2+ , Mn 2+ ). Moreover, stronger complex formation is observed in the case of the manganese ions. A more detailed arrangement of Co 2+ and Mn 2+ ions near the protons of C H 2 -14, C H -15, C H 2 -16 groups of pravastatin molecule was also determined by changes in experimental 1 H, 13 C NMR chemical shifts and MD simulation. Declarations Conflict of interest: The authors declare that they have no conflicts of interest. Financial interests The authors declare they have no financial interests. Non-financial interests none. Funding: This work is financially supported by Russian Science Foundation (Project № 25-13-00042). Author Contribution T.R.: Investigation, Visualization, Writing-Original draft preparation. O.V.: Methodology, Investigation, Validation. A.R.: Investigation, Modeling, Validation. A.S.: Investigation, Writing-Reviewing and Editing. F.Kh.: Validation, Writing-Reviewing and Editing. V.V.: Resources, Conceptualization, Writing-Reviewing and Editing, Supervision. All authors reviewed the manuscript. Acknowledgement The authors express their gratitude to the federal center for collective use of Kazan Federal University for the provided equipment. References Shi H, Xia Y, Cheng Y et al (2024) Global burden of ischaemic heart disease from 2022 to 2050: projections of incidence, prevalence, deaths, and disability-adjusted life years. European Heart Journal – Quality of Care and Clinical Outcomes. https://doi.org/10.1093/ehjqcco/qcae049 Pastena P, Frye JT, Ho C et al (2024) Ischemic cardiomyopathy: epidemiology, pathophysiology, outcomes, and therapeutic options. 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Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2025 Read the published version in BioNanoScience → Version 1 posted Editorial decision: Revision requested 19 Aug, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers invited by journal 08 Jul, 2025 Editor assigned by journal 08 Jul, 2025 Submission checks completed at journal 08 Jul, 2025 First submitted to journal 02 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7027909","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482661575,"identity":"27673b52-2cfa-4d1d-a00c-59f33988582f","order_by":0,"name":"T. R. 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Klochkov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIie3PMWvCQBTA8RcCl+U06wuCn+GVgCj0wxiEcwk4FIpTSQg8J+ks/RLtIh1zCHER50qXuHTvUsjWWMSCcMnqcP/pcdyPxwOw2W4xDwDHgADSTcBJxvULA8ybiHshzpnIAmDXRv6GC0HVTGjjHj+O8XAGUqdl9a76dPhSpWaYUW4iIhxGaxwlnTS7W+7ikD7VlmoyejWQIIMB1oTAd7jn8DxavUy5981Api1B5v1ckdWWUTcQ35XnLZ30ROLoGUXRRh5Ot5CQur6FVehLNaV8jxQYiPAWb4dq/US+nOiy4klfeMWgzB/vqWsg//Zqxpb/Ddxms9ls8At8U1YJxFOH3AAAAABJRU5ErkJggg==","orcid":"","institution":"Kazan Federal University","correspondingAuthor":true,"prefix":"","firstName":"V.","middleName":"V.","lastName":"Klochkov","suffix":""}],"badges":[],"createdAt":"2025-07-02 09:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7027909/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7027909/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12668-025-02159-z","type":"published","date":"2025-09-27T15:58:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86412046,"identity":"974f6a63-a711-49a6-8e3e-754a629c6b5e","added_by":"auto","created_at":"2025-07-10 10:46:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12995,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of pravastatin.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7027909/v1/31f76cdec8a326742cf08755.png"},{"id":86412068,"identity":"ced28c71-4563-4117-bcff-e5b05ca3edd3","added_by":"auto","created_at":"2025-07-10 10:46:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":160853,"visible":true,"origin":"","legend":"\u003cp\u003ewith Co\u003csup\u003e2+\u003c/sup\u003e ions at statin/ions ratios 10/1 (b), 5/1 (c) in an aqueous solution (95% D\u003csub\u003e2\u003c/sub\u003eO + 5% H\u003csub\u003e2\u003c/sub\u003eO) at T = 308 K, * - solvent signal. NMR spectra (\u003csup\u003e1\u003c/sup\u003eH, 500 MHz) of pure pravastatin (a) and pravastatin\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7027909/v1/409541c3786b71709d11a887.png"},{"id":86412047,"identity":"53d8cc19-293c-4c28-add0-f5de7b19680e","added_by":"auto","created_at":"2025-07-10 10:46:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":198111,"visible":true,"origin":"","legend":"\u003cp\u003eThe fragment of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC NMR spectra (\u003csup\u003e1\u003c/sup\u003eH, 500 MHz, \u003csup\u003e13\u003c/sup\u003eС, 125.76 MHz) of pravastatin with Co\u003csup\u003e2+\u003c/sup\u003e ions at statin/ions ratios 1/0 (red color cross-peaks), 10/1 (blue color cross-peaks), 5/1 (purple color cross-peaks) in aqueous solution (95% D\u003csub\u003e2\u003c/sub\u003eO + 5% H\u003csub\u003e2\u003c/sub\u003eO) at T = 308 K.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7027909/v1/64b504adf4c0cdfe2af4216c.png"},{"id":86413355,"identity":"81adc7a4-b470-4694-974c-80baae638065","added_by":"auto","created_at":"2025-07-10 10:54:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":156513,"visible":true,"origin":"","legend":"\u003cp\u003eNMR spectra (\u003csup\u003e1\u003c/sup\u003eH, 700 MHz) of pure pravastatin (a) and pravastatin with Mn\u003csup\u003e2+\u003c/sup\u003e ions at statin/ions ratios 10/1 (b), 5/1 (c) in an aqueous solution (95% D\u003csub\u003e2\u003c/sub\u003eO + 5% H\u003csub\u003e2\u003c/sub\u003eO) at T = 308 K, * - solvent signal.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7027909/v1/ad3daed9ffbbe0362b931081.png"},{"id":86412049,"identity":"62af2ec3-da39-402f-bc05-568b44961582","added_by":"auto","created_at":"2025-07-10 10:46:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94361,"visible":true,"origin":"","legend":"\u003cp\u003eRDF graphs for C\u003cu\u003eH\u003c/u\u003e-15 and C\u003cu\u003eH\u003c/u\u003e\u003csub\u003e2\u003c/sub\u003e-16 protons with Co\u003csup\u003e2+\u003c/sup\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e ions obtained from MD simulation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7027909/v1/48bed608902c124269d29a05.png"},{"id":86412052,"identity":"f11960eb-2516-43e1-b41c-954972f30e29","added_by":"auto","created_at":"2025-07-10 10:46:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":479339,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the most probable sites of Co\u003csup\u003e2+\u003c/sup\u003e (a) and Mn\u003csup\u003e2+\u003c/sup\u003e (b) ions in an aqueous solution with pravastatin.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7027909/v1/c7513ca37c4874269ed72bc9.png"},{"id":92430665,"identity":"87bc0afc-2b89-49af-9651-98baea8cedbb","added_by":"auto","created_at":"2025-09-29 16:07:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1854590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7027909/v1/609096d5-042a-4ecf-9319-720e0af2b8ee.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of the transition metal ions (Co2+ , Mn2+ ) complexation process with pravastatin by high-resolution NMR spectroscopy and MD methods","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCoronary heart disease (CHD) remains one of the causes leading to death worldwide, despite significant advances in diagnosis and treatment. In recent decades, there has been an increase in the incidence of CHD, especially in developing countries, due to lifestyle changes and an increase in the prevalence of risk factors such as obesity and hypertension [1\u0026ndash;3]. Elevated level of low-density lipoprotein cholesterol (LDL-C) is a key risk factor for the development of coronary heart disease (CHD), as they contribute to the deposition of cholesterol in the arterial walls and the formation of atherosclerotic plaques.\u003c/p\u003e\u003cp\u003eStatins are one of the drugs most common classes used to lower LDL-C levels. The main problem with these drugs is their low efficiency according to clinical studies [4,5].\u003c/p\u003e\u003cp\u003eIt is known that the physicochemical properties of organometallic substances or coordination compounds formed by organic compounds and transition metals differ significantly from the physicochemical properties of the ligands included in the organometallic compounds [6,7]. In this regard, the use of modern NMR methods to determine the structure and analyze the properties of organometallic or coordination compounds based on derivatives of statins and some transition elements, as well as the study of their complexation, is of particular importance. To confirm the assumption about the most probable area of the metal ion site in the system based on derivatives of statins and some transition metal ions (Co\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e), additional MD modeling can be performed.\u003c/p\u003e\u003cp\u003eTo create new drugs that must have certain predetermined properties, it is important to know the spatial structure of the object [8]. The NMR method is also extremely sensitive to the interactions of organic substances with transition metal ions.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eThe initial sample of the study was a solution of pravastatin in deuterated water (D\u003csub\u003e2\u003c/sub\u003eO). To decrease the solvent signal and to recreate an organic environment, D\u003csub\u003e2\u003c/sub\u003eO was used for this research. Pravastatin from \u0026laquo;Sigma-AldrichRus\u0026raquo; company was used without additional purification. The structural formula of pravastatin is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe CoCl\u003csub\u003e2\u003c/sub\u003e and MnCl\u003csub\u003e2\u003c/sub\u003e salts were used to prepare aqueous solutions of pravastatin to study the complexation of pravastatin with Co\u003csup\u003e2+\u003c/sup\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e ions. The initial salt was initially dissolved in D\u003csub\u003e2\u003c/sub\u003eO and then added to the pravastatin-D\u003csub\u003e2\u003c/sub\u003eO sample by titration with calculation of the concentration of Co\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e ions in an ampoule.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAll samples were prepared in standard 5-mm NMR tubes. Concentrations of the substances were 22.0 mM (pravastatin) and 2.2, 4.4 mM (CoCl\u003csub\u003e2\u003c/sub\u003e, MnCl\u003csub\u003e2\u003c/sub\u003e) and dissolved in aqueous solution (95% D\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;5% H\u003csub\u003e2\u003c/sub\u003eO, pH\u0026thinsp;=\u0026thinsp;7.3). The solution volume for all samples were 0.5 ml.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Methods\u003c/h2\u003e\u003cp\u003eRegistration of 1D, 2D NMR spectra of pravastatin in D\u003csub\u003e2\u003c/sub\u003eO at a temperature of 308 K with and without addition of Co\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e ions were carried out using pulsed NMR spectrometers Bruker Avance II-500 NMR (500 MHz (\u003csup\u003e1\u003c/sup\u003eН), 125.76 MHz (\u003csup\u003e13\u003c/sup\u003eС)) and Bruker Avance III-700 NMR (700 MHz (\u003csup\u003e1\u003c/sup\u003eН), 175 MHz (\u003csup\u003e13\u003c/sup\u003eС)). The spectrometers are equipped with a \u003csup\u003e2\u003c/sup\u003eH lock system to stabilization of magnetic field. \u003csup\u003e1\u003c/sup\u003eH NMR spectra were recorded using 90\u0026deg; pulses, the delay between pulses was 2 s, the spectral width was 10 ppm and a minimum of 16 scans. \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded using 90\u0026deg; pulses, the delay between pulses was 2 s, the spectral width was 200 ppm and a minimum of 100 scans. Chemical shifts were determined relative to TMS (tetramethylsilane). The TOPSPIN 3.0 software was used to obtain and process the spectra. The assignment of spectral lines in NMR spectra was performed by using high resolution NMR spectroscopy methods of 1D, 2D experiments [9].\u003c/p\u003e\u003cp\u003eThe program GROMACS was used for MD calculations of the pravastatin and Co\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e ions interaction in an aqueous solution [10,11]. The initial configuration with the beginning energy was minimized using the most responsive descent algorithm and the low energy threshold was less than 1000 kJ/mol*nm. Then, each optimized molecular system was equilibrated in the canonical ensemble (NVT) at 300 K using the Berendsen thermostat with the coupling constant τ\u0026thinsp;=\u0026thinsp;0.1 ps for 200 ps. Subsequently, the molecular system was equilibrated at a constant temperature and constant pressure (T\u0026thinsp;=\u0026thinsp;300 K and P\u0026thinsp;=\u0026thinsp;1 bar) for 300 ps using the thermostat with τ\u0026thinsp;=\u0026thinsp;0.1 ps and the Parrinello-Rahman barostat with τ\u0026thinsp;=\u0026thinsp;3 ps and a pressure of 45*10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e bar. Next, the resulting molecular system was simulated in the NPT ensemble (T\u0026thinsp;=\u0026thinsp;300 K and P\u0026thinsp;=\u0026thinsp;1 bar) without any constraints. Periodic boundary conditions were used in the MD simulations. Electrostatic interactions were handled using the Ewald particle mesh method using CHARM conditions. The various simulations were run for at least 250 ns, and snapshots of the trajectories were taken every 2 ns [12].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussions","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.1. NMR experiments of pravastatin with Co\u003csup\u003e2+\u003c/sup\u003e ions in deuterated water solution\u003c/h2\u003e\u003cp\u003eThe complete assignment of the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of the studied compound was carried out using two-dimensional NMR experiments and based on the analysis of signal multiplicity, characteristic chemical shifts and integral values [9,13]. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the \u003csup\u003e1\u003c/sup\u003eH NMR spectra of an aqueous solutions of pravastatin with cobalt ions at following molar concentration ratios (statin/ions): pure pravastatin (a), pravastatin with Co\u003csup\u003e2+\u003c/sup\u003e ions at ratios of 10/1 (b) and pravastatin with Co\u003csup\u003e2+\u003c/sup\u003e ions at ratios of 5/1 (c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ewith Co\u003csup\u003e2+\u003c/sup\u003e ions at statin/ions ratios 10/1 (b), 5/1 (c) in an aqueous solution (95% D\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;5% H\u003csub\u003e2\u003c/sub\u003eO) at T\u0026thinsp;=\u0026thinsp;308 K, * - solvent signal.\u003c/p\u003e\u003cp\u003eDepending on the presence of the Co\u003csup\u003e2+\u003c/sup\u003e ions in solution, a number of differences are observed in the \u003csup\u003e1\u003c/sup\u003eH NMR spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e): the proton signals of C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-14, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15 and C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-16 groups changes very much in intensities, and also in chemical shifts towards higher ppm; and at statin/ions ratio 5/1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) the C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15 signal disappears. We also observed similar changes in another case [13]. However, changes in other proton signals in the spectra are insignificant. An assumption about spatial proximity of the paramagnetic Co\u003csup\u003e2+\u003c/sup\u003e ions leads to a decrease in the relaxation time of protons was made. In this case, broadening of the spectral signals was observed. Moreover, there are no characteristic changes in the spectrum that usually occur during the formation of a chemical bond. Thus, this indicates that the interaction between pravastatin and Co\u003csup\u003e2+\u003c/sup\u003e ions has the character of a rapid exchange state. All \u003csup\u003e1\u003c/sup\u003eH NMR chemical shifts changes are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSome chemical shifts changes in \u003csup\u003e1\u003c/sup\u003eH NMR spectra of pravastatin with Co\u003csup\u003e2+\u003c/sup\u003e ions at statin/ions ratios 1/0 (a), 10/1 (b), 5/1 (c) in an aqueous solution.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e-14, ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCH-15, ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003enot registered\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e-16, ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.42 / 2.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe heteronuclear \u003csup\u003e1\u003c/sup\u003eH\u0026ndash;\u003csup\u003e13\u003c/sup\u003eC HSQC NMR spectra of an aqueous solution of pravastatin with various Co\u003csup\u003e2+\u003c/sup\u003e ions molar concentrations are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe changes in HSQC NMR spectra are consistent with \u003csup\u003e1\u003c/sup\u003eH NMR experiments: the proton signal of C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15 group shifts toward lower fields (towards higher ppm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea \u0026ndash; 4.15 ppm, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb \u0026ndash; 4.26 ppm, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec \u0026ndash; not registered). However, there is no change for the carbon-13 signal of \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH-15 group is observed. Moreover, there is a slight shift towards higher ppm for the carbon-13 signal of \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e-14 group is observed: 43.4/43.8/44.6 ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). And the proton signal of C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-16 group becomes very broad upon addition of the cobalt ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Thus, the assumption about the localization of the cobalt ions in the area of CH\u003csub\u003e2\u003c/sub\u003e-14, CH-15 and CH\u003csub\u003e2\u003c/sub\u003e-16 groups of molecule is confirmed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.2. NMR experiments of pravastatin with Mn\u003csup\u003e2+\u003c/sup\u003e ions in deuterated water solution\u003c/h2\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectra of an aqueous solutions of pravastatin with manganese ions at following molar concentration ratios (statin/ions): pure pravastatin (a), pravastatin with Mn\u003csup\u003e2+\u003c/sup\u003e ions at ratios of 10/1 (b) and pravastatin with Mn\u003csup\u003e2+\u003c/sup\u003e ions at ratios of 5/1 (c) showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDepending on the presence of the Mn\u003csup\u003e2+\u003c/sup\u003e ions in solution, a number of differences are observed in the \u003csup\u003e1\u003c/sup\u003eH NMR spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e): the proton signals of C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-14, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15 and C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-16 groups becomes very broad and shift towards higher ppm (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). And in case of the statin/ions ratio of 5/1, the proton signal of C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15 group is disappears. Changes in other proton signals in the spectra are insignificant.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSome chemical shifts changes in \u003csup\u003e1\u003c/sup\u003eH NMR spectra of pravastatin with Mn\u003csup\u003e2+\u003c/sup\u003e ions at statin/ions ratios 1/0 (a), 10/1 (b), 5/1 (c) in an aqueous solution.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e-14, ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCH-15, ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e-16, ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe proton signals of C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15 and C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-16 groups disappears with an increase in the concentration of Mn\u003csup\u003e2+\u003c/sup\u003e ions in the solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Based on the data obtained from one-dimensional and two-dimensional experiments, in this case a similar assumption about the localization site of the manganese ions was made like it was for cobalt ions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Molecular dynamics of pravastatin with Co\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e ions in deuterated water solution\u003c/h2\u003e\u003cp\u003eTo confirm the assumption of the most probable area of the metal ion in the system, molecular dynamics simulations of pravastatin with cobalt and manganese ions in aqueous solution were carried out. The model system consisted of 20 statin molecules with the addition of Co\u003csup\u003e2+\u003c/sup\u003e or Mn\u003csup\u003e2+\u003c/sup\u003e ions. The statin/ions ratios were chosen similarly to those in the experiment: 10/1 and 5/1 for both ions, i.e. 2 and 4 ions, respectively. Then the system was placed in a 10x10x10 nm periodic cubic cell with the addition of 32284 water molecules. This choice was also based on the experiment. The CHARMM36 force field was used for parameterization.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe list of distances between the atoms of each statin and all ions for each simulation step was first compiled. Then the radial distribution function (RDF) graphs of Co\u003csup\u003e2+\u003c/sup\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e ions relative to the location of statin protons were obtained to analyze the dynamics of binding of pravastatin with cobalt and manganese ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on the RDF graphs in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Co\u003csup\u003e2+\u003c/sup\u003e ions have one maximum at distances 0.66 nm for C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15 and 0.63 nm for C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-16 atoms, which indicates a high probability of finding the cobalt ions in solution at the specified distances near these atoms. This arrangement of ions can be explained by the presence of fragment of COOH group with a large dipole moment in this part of the molecule. And the ion is surrounded by a solvate shell of water molecules. Therefore, hydrogen bonds are formed between the ion and the hydroxyl groups of statin when the ion and statin are located close to each other.\u003c/p\u003e\u003cp\u003eThe graphs characterizing the location of Mn\u003csup\u003e2+\u003c/sup\u003e ions have a more complex appearance - they have two high maximum, which indicates two possible areas of localization of ions near the statin molecule (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The first maximum is at the distance of 0.38 nm, which means that the solvate shell is destroyed and Mn\u003csup\u003e2+\u003c/sup\u003e ions are bound directly to pravastatin atoms, forming a stronger complex.\u003c/p\u003e\u003cp\u003eThus, the localization of Co\u003csup\u003e2+\u003c/sup\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e ions predominantly occurs near the protons of C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-14, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-16 groups of pravastatin, which confirms by experimental results. The most probable sites of Co\u003csup\u003e2+\u003c/sup\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e ions in an aqueous solution with pravastatin are schematically shown in Fig.\u0026nbsp;6.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eNMR study and MD modeling revealed the presence of interaction between pravastatin and transition metal ions (Co\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e). Moreover, stronger complex formation is observed in the case of the manganese ions. A more detailed arrangement of Co\u003csup\u003e2+\u003c/sup\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e ions near the protons of C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-14, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e-15, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-16 groups of pravastatin molecule was also determined by changes in experimental \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC NMR chemical shifts and MD simulation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFinancial interests\u003c/strong\u003e\u003cp\u003eThe authors declare they have no financial interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNon-financial interests\u003c/strong\u003e\u003cp\u003enone.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work is financially supported by Russian Science Foundation (Project № 25-13-00042).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.R.: Investigation, Visualization, Writing-Original draft preparation. O.V.: Methodology, Investigation, Validation. A.R.: Investigation, Modeling, Validation. A.S.: Investigation, Writing-Reviewing and Editing. F.Kh.: Validation, Writing-Reviewing and Editing. V.V.: Resources, Conceptualization, Writing-Reviewing and Editing, Supervision. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their gratitude to the federal center for collective use of Kazan Federal University for the provided equipment.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Shi H, Xia Y, Cheng Y et al (2024) Global burden of ischaemic heart disease from 2022 to 2050: projections of incidence, prevalence, deaths, and disability-adjusted life years. European Heart Journal \u0026ndash; Quality of Care and Clinical Outcomes. https://doi.org/10.1093/ehjqcco/qcae049\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Pastena P, Frye JT, Ho C et al (2024) Ischemic cardiomyopathy: epidemiology, pathophysiology, outcomes, and therapeutic options. Heart Failure Reviews 29(1): 287\u0026ndash;299. https://doi.org/10.1007/s10741-023-10377-4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Tsao CW, Aday AW, Almarzooq ZI et al (2023) Heart disease and stroke statistics\u0026ndash;2023 update: a report from the American Heart Association. Circulation 147(8): e93-e621. https://doi.org/10.1161/cir.0000000000001123\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Volpe M, Patrono C (2021) The cardiovascular benefits of statins outweigh adverse effects in primary prevention: results of a large systematic review and meta-analysis. European Heart Journal 42(44): 4518\u0026ndash;4519. https://doi.org/10.1093/eurheartj/ehab647\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Pham K, Mulugeta A, Lumsden A, Hyppӧnen E (2023) Genetically instrumented LDL\u0026ndash;cholesterol lowering and multiple disease outcomes: A Mendelian randomization phenome-wide association study in the UK Biobank. British Journal of Clinical Pharmacology 89(10): 2992\u0026ndash;3004. https://doi.org/10.1111/bcp.15793\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Kim HJ, Graham DW, DiSpirito AA et al (2004) Methanobactin, a copper-acquisition compound from methane-oxidizing bacteria. Science 305(5690): 1612\u0026ndash;1615. https://doi.org/10.1126/science.1098322\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Chaturvedi KS, Hung CS, Crowley JR et al (2012) The siderophore yersiniabactin binds copper to protect pathogens during infection. 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Chem. 26(16): 1701\u0026ndash;1718. https://doi.org/10.1002/jcc.20291\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Hess B, Kutzner C, Van Der Spoel D et al (2008) GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4(3): 435\u0026ndash; 447. https://doi.org/10.1021/ct700301q\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Shurshalova GS, Yulmetov AR, Sharapova DA et al (2020) Interaction of lovastatin with model membranes by NMR data and from MD simulations. Bionanoscience 10: 493\u0026ndash;501. https://doi.org/10.1007/s12668-020-00722-4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Tarasov AS, Rakhmatullin IZ, Blokhin DS et al (2023) (Gd\u003csup\u003e3+\u003c/sup\u003e) Complexation with oligopeptide (SFVG) and amyloid peptide (Aβ\u003csub\u003e13\u0026ndash;23\u003c/sub\u003e) in aqueous solution by NMR spectroscopy. Results in Chemistry 5(100762): 7. https://doi.org/10.1016/j.rechem.2023.100762\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NMR spectroscopy, pravastatin, Co2+, Mn2+, molecular dynamics","lastPublishedDoi":"10.21203/rs.3.rs-7027909/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7027909/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe paper studies the complexation of pravastatin with transition metal ions (Co\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e) using high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) methods. It was shown that the presence of transition metal ions leads to changes in \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC NMR chemical shifts of CH\u003csub\u003e2\u003c/sub\u003e-14, CH-15, CH\u003csub\u003e2\u003c/sub\u003e-16 groups of pravastatin. MD modeling for systems of pravastatin aqueous solutions with cobalt and manganese ions confirms the experimental assumption about the most probable area of metal ions near the pravastatin molecule.\u003c/p\u003e","manuscriptTitle":"Investigation of the transition metal ions (Co2+ , Mn2+ ) complexation process with pravastatin by high-resolution NMR spectroscopy and MD methods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 10:46:17","doi":"10.21203/rs.3.rs-7027909/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-19T04:18:37+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"307162892268057676375932970540781997486","date":"2025-07-31T08:07:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-09T02:39:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224087434457347211947128471726660081131","date":"2025-07-09T01:46:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-08T13:36:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-08T13:23:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-08T12:42:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-07-02T09:45:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b754b6a2-8db4-4eb6-b8d5-fbe60d95f567","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:04:14+00:00","versionOfRecord":{"articleIdentity":"rs-7027909","link":"https://doi.org/10.1007/s12668-025-02159-z","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2025-09-27 15:58:04","publishedOnDateReadable":"September 27th, 2025"},"versionCreatedAt":"2025-07-10 10:46:17","video":"","vorDoi":"10.1007/s12668-025-02159-z","vorDoiUrl":"https://doi.org/10.1007/s12668-025-02159-z","workflowStages":[]},"version":"v1","identity":"rs-7027909","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7027909","identity":"rs-7027909","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}