Nuclear magnetic resonance studies of the conformation of bilirubin and its derivatives in solution

Abstract

Selective and non-selective spin–lattice relaxation times (T1), nuclear Overhauser enhancements (NOE), and rates of exchange of protons in bilirubin and related compounds in chloroform solutions were measured. The NOE observed between methyl and methine protons and between lactam and pyrrole NH protons indicate that the configuration within the pyrromethenone units is exclusively syn-Z. An NOE was also observed between the protons of the carboxylic acid and the lactam NH groups of mesobilirubin. The selective T1 relaxation times, NOE values of the protons, and correlation times calculated from carbon-13 relaxation data were used for the calculation of interproton distances. These distances, as well as the extremely slow rates of exchange of the pyrrole NH protons, establish the presence of hydrogen bonds between the carboxy-residues and the lactam and pyrrole NH groups of mesobilirubin in chloroform solutions. The T1 values, chemical shifts, and rates of proton exchange in bilirubin are similar to those of mesobilirubin, indicating that the same conformation occurs for the two compounds. The conformation of bilirubin dimethyl ester is very different from that of bilirubin and mesobilirubin. It is present as a dimer even at concentrations as high as 0.17M. The proton exchange rates of both the lactam and pyrrole NH groups of bilirubin dimethyl ester are slightly slower than in a model pyrromethenone compound.