Contents lists available at ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids Magnesium and calcium reveal diferent chelating efects in a steroid compound: A model study of prednisolone using NMR spectroscopy Kathleen D. Carillo a,b,c,1 , Danni Wu c,1 , Su-Ching Lin c , Shen-Long Tsai d , Jiun-Jie Shie c , Der-Lii M. Tzou c,e, ⁎ a International Graduate Program, SCST, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC b The Department of Applied Chemistry, National Chiao-Tung University, Hsinchu 30013, Taiwan, ROC c Institute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC d Chemical Engineering Department of NTUST, Taipei 10607, Taiwan, ROC e Department of Applied Chemistry, National Chia-Yi University, Chia-Yi 60004, Taiwan, ROC ARTICLE INFO Keywords: Prednisolone NMR Cation chelation Conformational change Calcium Magnesium ABSTRACT In this work, we used high resolution NMR spectroscopy to investigate metal cation chelation by the steroidal drug 1,4-pregnadiene-11β,17α,21-triol-3,20-dione (Prednisolone; abbreviated as Prd). Prd/MgCl 2 and Prd/ CaCl 2 mixtures were prepared at eight diferent molar ratios. Using two-dimensional 1 H/ 13 C heteronuclear correlation spectroscopy, we were able to resolve most of the 1 H signals, except those at 1.4–1.55 ppm, where signals for H15β, H16α and Me-19 are superimposed. The chelation sites were determined by the cation con- centration-dependent 13 C signals. Both ring A and ring D of Prd were found to be involved in Mg 2+ chelation, whereas only ring A was involved in Ca 2+ chelation. The dihedral angles deduced from the 3 J H-H coupling constants indicated that ring D of Prd might undergo rather small, but diferent, distortions in the presence of Mg 2+ and Ca 2+ . Additionally, using the continuous variation method, we deduced that the stoichiometric ratios of the Prd/Mg 2+ and Prd/Ca 2+ complexes were 1:1 and 2:1, respectively. All of the evidence led us to conclude that the Prd/Mg 2+ and Prd/Ca 2+ complexes are mediated by diferent chelating mechanisms. 1. Introduction Magnesium ions (Mg 2+ ) participate in glucose metabolism and ac- tivity of respiratory enzymes, and also act as an indispensable cofactor. For example, Mg 2+ plays a catalytic role in protein synthesis and is involved in the formation of acetylcholine and the metabolism of fatty acids. Mg 2+ also aids in the control of blood sugar levels and blood pressure in humans and, together with potassium, calcium and sodium ions, maintains the excitability of the muscular nervous system and the normal structure and function of the myocardium. Mg 2+ , acting as a Ca 2+ antagonist, directly infuences cardiac muscle contraction because Mg 2+ is able to bind to troponin C and calmodulin in competition with Ca 2+ [1]. It has, however, been reported that long-term treatment with corticosteroids, such as prednisone, prednisolone and deltasone, might reduce Mg 2+ levels in patients [2]. Calcium ions (Ca 2+ ) play a pivotal role in many physiological processes in mammals and Ca 2+ levels are, therefore, tightly regulated [3]. Ca 2+ signaling is used throughout the life cycle of an organism, in functions such as cell proliferation, metabolism and cell death [4–6]. Ca 2+ is known to interact with a variety of proteins, including calmo- dulin [7–11], estrogen receptor α [12] and prothrombin [13]. When Ca 2+ interacts with calmodulin, the Ca 2+ /calmodulin complex acts as a Ca 2+ sensor that modulates specifc functions in humans [14]. The Ca 2+ binding site in calmodulin, which is situated within the 12-re- sidue loop of a 29-residue helix-loop-helix domain, incorporates side- chain oxygen atoms and a carboxyl group from the main chain, as well as a bidentate ligand [15]. Ca 2+ has been reported to bind in a similar manner to steroidal molecules, such as cortisone and hydrocortisone, where the α-hydroxyl ketone moiety serves as a bidentate chelator for Ca 2+ [16]. Prednisolone (Prd, Scheme 1) is a corticosteroid used for the symptomatic treatment of infammatory and autoimmune conditions, such as asthma, rheumatoid arthritis and ocular infammation [17–21], and in acute lymphoblastic leukemia [22] and Kawasaki disease [23,24]. It has been hypothesized that the electron-donating groups, i.e., ketone, hydroxyl group and α-hydroxy ketones, of Prd are https://doi.org/10.1016/j.steroids.2019.108429 Received 20 December 2018; Received in revised form 16 May 2019; Accepted 17 June 2019 ⁎ Corresponding author at: Institute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC. E-mail address: Tzougate@gate.sinica.edu.tw (D.-L.M. Tzou). 1 These authors contributed equally. Steroids 150 (2019) 108429 Available online 21 June 2019 0039-128X/ © 2019 Elsevier Inc. All rights reserved. T