Differences in the formation of mesobiliviolin and glaucobilin and d- and i-urobilins.

In separate communications (1, 2), a method is described for the approximate analysis of mixtures of the urobilin group as to proportions of clor i-urobilins or Z-stercobilin. This is an adaptation of the FeClrHCl and heat procedure as first employed by Fischer and Niemann (3) for the preparation of mesobiliviolin from mesobilirubinogen, and later by Legge (4), in analyzing mixtures of I-stercobilin and i-urobilin (Formula I in Fig. 1). The adaptation referred to depends on the observation (1, 2, 5) that under the conditions described, d-urobilin is converted mainly to glaucobilin (mesobiiiverdin) while i-urobilin yields a mixture of mesobiliviolin and glaucobilin. The former is invariably present in sufficient proportion that the ratio of absorption at 560 mp/650 rnp provides an approximate measure of the initial proportions of iand d-forms in the mixture. E-Stercobilin under the same conditions does not yield either mesobiliviolin or glaucobilin; hence there is no increase in absorption at 560 or 650 rnp and strong absorption persists at a maximum of 492 rnh, though generally diminished as compared with the starting intensity. Previous analysis of crystalline d-urobilinogen agreed with the formula C&H42N406, and for the corresponding d-urobilin CWH~O~ N406 (6, 7). This has recently been confirmed by Gray and Nicholson (8) who have described evidence suggesting that the d-urobilin molecule contains one of the vinyl groups characteristic of biliiubm and dihydrobilirubin. They suggest Formula I (Fig. 1) as most likely for d-urobilin. It was unexpected that a compound of this structure should so readily yield glaucobilin, in which both vinyl groups have been replaced by ethyl groups. Crystalline glaucobilin (V, Fig. l), prepared by FeCls-HCl and heat treatment of d-urobilin has been shown to be identical with the glaucobilin obtained from mesobilirubin or mesobilirubinogen (2). The latter is readily dehydrogenated first to i-urobilin (I) then MBV (IV) or its isomer, MBR.1 Gray and Nicholson (8) have described evidence that d-urobilin is converted to MBV or MBR by a series of proton transfers from the double bonds of the pyrrol nucleus A or D, depending upon whether the single vinyl group, thus converted to an ethyl group, is in position 2 or 8. In Fig. 1 position 2 is assumed. These considerations left unanswered the finding (1, 2, 5) that despite a considerable MBV formation both from d-urobilin and i-urobilin as a result of heating with FeC13 and methanolHCl, further conversion to glaucobilin proceeds more rapidly