Bilirubin conjugates of human bile. Nuclear-magnetic-resonance, infrared and optical spectra of model compounds

Abstract

N.m.r., i.r. and optical spectra of model compounds were recorded. These were to help in elucidating the structures of the phenylazo derivatives of bilirubin conjugates isolated from human bile. Model compounds included commercial and human bile bilirubin, mesobilirubin, bilirubin dimethyl ester, dimethoxybilirubin dimethyl ester and the corresponding phenylazo derivatives. The phenylazo derivative of vinylneoxanthobilirubinic acid was also investigated. All compounds were of the type IXalpha, and no other isomer could be detected with the spectroscopic methods employed. The compounds crystallize as the lactams, except for dimethoxybilirubin dimethyl ester and its phenylazo derivative, which are held in the lactim ether configuration. With all other compounds no tautomeric forms other than the lactams could be detected, although small proportions of bilirubin must exist as the lactim. Bilirubin does not form a betaine, a structure that has been proposed by von Dobeneck & Brunner (1965) to explain the bathochromic shift of its optical spectrum as compared with the expected position of the absorption maximum at 420nm. However, this shift to 453nm can be explained on the basis of internal hydrogen bonds occurring between the carboxylic protons and the pyrrole rings of bilirubin, as proposed by Fog & Jellum (1963), and new evidence for such a bonding has been accumulated. The bilirubin sulphate described by Watson (1958), which is formed by treatment of bilirubin with concentrated sulphuric acid and acetic anhydride, was also investigated. The main product of this reaction was isolated as its phenylazo derivative, and was shown to be 3,18-di(ethylidene sulphate)-2,7,13,17-tetramethylbiladiene-ac-8,12-dipropionic acid. The reaction leading to this compound is an addition of sulphuric acid to the vinyl side chains of bilirubin according to Markownikoff's rule.

Animal Pigment Bilirubin Discovered in Plants

“…The 1 H NMR and 13 C NMR spectra of the isolated pigment matched published values of authentic bilirubin4,5,10 (Tables S1 and S2). Identification was further supported by 1 H NMR analysis of bilirubin standard (Aldrich).…”

Studies of the conformation of bilirubin and its dimethyl ester in dimethyl sulphoxide solutions by nuclear magnetic resonance

“…Partial assignments of the spectra of the compounds in DMSO solutions have been published (Kuenzle, 1970;Maruyama, 1978;Kaplan & Navon, 1980, 1981b. In the present study, we assigned the resonances of the NH and methyl protons on the basis of our previous assignment of these resonances in [2Hlchloroform solutions (Kaplan & Navon, 1981a) and NOE experiments.…”

On the assignment of the carbon‐13 NMR spectrum of bilirubin

“…The VBA prepared in this way was characterized through its azo-pigment, which was prepared from a sample of the recrystallized product by coupling with phenyl diazonium chloride according to the procedure of Kuenzle.3 The proton NMR spectrum of the azopigment was then measured and was found to be identical with the spectrum reported for the azopigment A,,' prepared from an authentic sample of VBA. 3 Bilirubin dimethyl ester was prepared from bilirubin and diazomethane as described by Kuenzle.3…”