Modeling of biliverdin reduction process: regio-specificity and H-bonding

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 β, γ, and δ have been obtained. It has been shown that γ-OEBR has two configurational isomers (named γ₁ and γ₂), 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, β, γ₁, and δ-OEBR are of similar stability whereas, at higher level of theory, γ₁-OEBR is less stable than others. Moreover, γ₂-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 γ₁- and γ₂-isomers. A conformational analysis show that an energy barrier of 25 kcal mol⁻¹ must be surmounted for γ₂ 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 borohydride (NaBH₄). Chemical reduction of OEBV with NaBH₄ was followed in CDCl₃ and CD₃OD solutions and the product was characterized by ¹H NMR and UV–Vis spectroscopy. The results show that γ₂-isomer as the major product, forms along with γ₁ via an equilibrium tautomerization reaction.