Mass-spectrometric structure elucidation of dog bile azopigments as the acyl glycosides of glucopyranose and xylopyranose

Abstract

1. The structures of the alpha(2)- and alpha(3)-azopigments, prepared by diazotization of dog bile with ethyl anthranilate, were shown by mass spectrometry and g.l.c. to correspond to azobilirubin beta-d-xylopyranoside and azobilirubin beta-d-glucopyranoside respectively. 2. Both azopigments consist of a mixture of two methyl vinyl isomers having structures (IIIa) and (IIIb) for the alpha(2)-azopigment and structures (IVa) and (IVb) for the alpha(3)-azopigment. Separation of methyl vinyl isomers was obtained by t.l.c. or column chromatography performed on the acetylated azopigments. Hydrolysis of the less polar acetates derived from components (IIIa) and (IVa) gave rise to the azopigment (Ia), whereas hydrolysis of the more polar acetates derived from components (IIIb) and (IVb) gave rise to the azopigment acid (Ib). The positions of methyl and vinyl substituents in compounds (Ia) and (Ib) were assigned on the basis of their n.m.r. spectra. 3. Molecular ions in the mass spectra of the trimethylsilyl and acetyl derivatives of the azopigments indicated the presence of a pentose and a hexose conjugating sugar. 4. The ester functions linking the sugars to the propionic acid side chain of azobilirubin were demonstrated by ammonolysis and identification of the amide of azobilirubin as the aglycone derivative. 5. The sugar moieties were shown to occur as xylopyranose (alpha(2)) and glucopyranose (alpha(3)), bound at C-1, by application of a sequence of reactions performed on a micro-scale. The sugar hydroxyl groups were acetylated and the 1-acyl aglycone removed selectively by treatment with hydrogen bromide in acetic acid. Hydrolysis of the 1-bromo sugar acetates followed by acetylation afforded the alpha- and beta-xylopyranose tetra-acetates and alpha- and beta-glucopyranose penta-acetates, identified by a combination of g.l.c. and mass spectrometry. 6. The validity of this degradation scheme was confirmed (a) by g.l.c.-mass spectrometry identification of the alpha- and beta-1-propionyl derivatives of glucopyranose tetra-acetate, obtained from the alpha(3)-azopigment after final reaction with propionic anhydride; (b) by subjecting the acetates of alphabeta-glucopyranose, alphabeta-xylofuranose and alphabeta-glucofuranose to the same sequence of reactions.