Modeling of biliverdin reduction process: regio-specificity and H-bonding Mansour Zahedi * , Mina Ghiasi, Nasser Safari * Department of Chemistry, Faculty of Sciences, Shahid Beheshti University, Evin, 19839-63113, Tehran, Iran Received 29 July 2004; accepted 20 October 2004 Available online 11 November 2004 Abstract Octa ethyl biliverdin (OEBV) has been employed as a model for natural biliverdin and its geometry has been optimized by using semiempirical (AM1, PM3), DFT, and hybrid ONIOM methods. Geometries and energetics of formation of octa ethyl bilirubin (OEBR) formed by reduction from OEBV via three carbon sites b, c, and d have been obtained. It has been shown that c-OEBR has two configurational isomers (named c 1 and c 2 ), which can convert to each other by internal 1,5-hydrogen shift. The results show that, within the accuracy level of semiempirical methods, all three isomers namely, b, c 1 , and d-OEBR are of similar stability whereas, at higher level of theory, c 1 -OEBR is less stable than others. Moreover, c 2 -isomer with one more of its pyrrole rings being aromatic can achieve a higher symmetry, and is the most stable among others by at least 5–6 kcal mol 1 based on various methods employed. It is interesting to note that the ridge-tile conformation, which has been confirmed for natural bilirubin was not observed for calculated geometries of c 1 - and c 2 -isomers. A conformational analysis show that an energy barrier of 25 kcal mol 1 must be surmounted for c 2 to obtain the ridge-tile geometry. OEBV was synthesized and purified from octa ethyl porphyrin iron (III) chloride, and was reduced to OEBR by sodium boro- hydride (NaBH 4 ). Chemical reduction of OEBV with NaBH 4 was followed in CDCl 3 and CD 3 OD solutions and the product was characterized by 1 H NMR and UV–Vis spectroscopy. The results show that c 2 -isomer as the major product, forms along with c 1 via an equilibrium tautomerization reaction. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Octa ethyl biliverdin; Reduction; Octa ethyl bilirubin; Ab initio calculations; H-bonding; Regio-specificity 1. Introduction Bilirubin a compound which is generated by metabo- lism of hemoglobin and other iron porphyrin containing enzymes in mammals, is an important compound both in terms of chemical as well as biological point of views. Biliverdin regio-specifically reduces to a yellow pigment, bilirubin, in the presence of bilirubin reductase just from c-position [1–4]. This compound is toxic and it has long been found that an excess amount of bilirubin from its natural doze of 1.93–3 mg/dl in human body can cause jaundice in addition to sever defects to central nervous system [5]. Bilirubin has attracted chemistsÕ attention due to close relation existing between its chemical and physico-biological properties. Several experimental and theoretical studies have shown the ridge-tile conforma- tion for both natural bilirubin [6–10] and bilirubin dian- ion [11]. Lightner et al. [12–14] have shown experimentally using NMR studies and Zahedi et al. [8] have proved by theoretical calculations that natural bilirubin has the ridge-tile conformation which is stabi- lized by internal hydrogen bonds (Fig. 1). So, besides the aforementioned regio-specificity of the enzymatic proc- ess to merely producing c-bilirubin, it seems plausible 0301-0104/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.chemphys.2004.10.032 * Corresponding author. Tel./fax: +98212401765. E-mail address: m-zahedi@cc.sbu.ac.ir (M. Zahedi). www.elsevier.com/locate/chemphys Chemical Physics 310 (2005) 179–187