Chemical synthesis of bilirubin glucuronide conjugates

In dimethylformamide, the two carboxyl groups of bilirubin react with the bifunctional coupling agent, carbonyldiimidazole, to form bilirubin diimidazole, which was isolated and crystallised. The bilirubin diimidazole, termed “activated bilirubin”, was shown to react spontaneously with primary alcohols to form diesters of bilirubin. After addition of the tetrabutyl ammonium salt of glucuronic acid, compounds with chromatographic mobilities similar to those of bilirubin mono- and diglucuronides from bile were formed. Bilirubin diglucuronides were isolated by barium precipitation followed by solvent extraction. The bilirubin diglucuronides were considered to be a mixture of α and β glucuronides esterified at positions 1, 2, 3, or 4 of glucuronic acid because the compound(s) was resistant to hydrolysis with glucuronidase and gave multiple spots by chromatography after diazotization with ethyl anthranilate. The model compounds lauryl glucuronides were synthesized similarly; the most polar product by chromatography had identical chromatographic mobility to synthetic lauryl l-d-glucuronide prepared by reductive debenzylation of lauryl (benzyl (2,3,4-tri-O-benzyl))-d-glucuronide. It is concluded that bilirubin-1-di-β-d-glucuronide can be synthesized when suitable protecting groups for the 2, 3, and 4 hydroxyl groups of glucuronic acid become available.