Biosynthesis of hyoscyamine involves an intramolecular rearrangement of littorine

Abstract

The direct biosynthesis of hyoscyamine 3 from littorine 1 has been demonstrated by feeding (RS)-phenyi[1,3-13C2]lactoyl[methyl-2H3]tropine (littorine 13) to transformed root cultures of Datura stramonium. In tropane alkaloids isolated 7 days later and examined by GC–MS, the labelling patterns of hyoscyamine 3 and apoatropine 4 indicated that the quintuply labelled precursor had been incorporated intact. Incorporations into the M + 5 peaks of hyoscyamine 16 and apoatropine 4 of 4.4 and 3.8% isotopic excess were found respectively. The % isotopic excesses in the M + 2 and M + 3 ions of these alkaloids indicate that a biosynthetic route involving the hydrolysis of the ester followed by re-esterification could only account for 0.2% isotopic excess in the observed M + 5 incorporation into hyoscyamine 16. Furthermore, the incorporation of (RS)-phenyl[1,3-13C2]lactoyl[methyl-2H3]tropine 13 into hyoscyamine 16 and apoatropine was not diminished in the presence of added tropine 6 or (RS)-phenyllactic acid. The retention of a high isotopic excess within hyoscyamine 16, coupled to the inability of either added tropine 6 or phenyllactic acid to dilute the extent of labelling, shows that hyoscyamine 3 is derived directly by the rearrangement of littorine 1. That this rearrangement is Intramolecular is shown by the high level of 13C spin-spin coupling observed in the 13C NMR spectrum of the derived hyoscyamine 16.