Competitive Feeding Experiments with Tropic Acid Precursors in Datura

Abstract

Phenyl{ 1 -14C]alanine [31, phenyl[2-14C)pyruvate [4], and phenyl[ 1 -14C]lactate [51 were fed singly and in competition with unlabeled phenylalanine, phenyllactate, and phenylpyruvate to Datura stramonium. The activities recorded in the alkaloids hyoscyamine [11 and hyoscine (scopolamine) [21 suggested that phenyllactate [51 was the closest precursor for the tropic acid moiety of these alkaloids.Hyoscyamine [11, from plants fed with phenyl[2-14C}pyruvate [4l, was hydrolyzed, but the resultant tropic acid 7 contained only 21% of the activity, the remainder being located in the tropine. However, degradation of the tropic acid showed that the phenylpyruvate incorporated in a specific manner, although it is clearly not an efficient precursor.( -)-S-Tropic acid is the aromatic acid moiety of the important tropane ester alkaloids hyoscyamine [1] and hyoscine (scopolamine) (2], which are constituents of Datura spp. Its biosynthesis, which has aroused much interest, was first investigated in I960 when Leete (1) demonstrated that the label from phenyl[3-14C]alanine infiltrated into Datura stramoniumL. (Solanaceae) incorporated into C-2 of tropic acid [6], Underhill and Youngken (2) and Gross and Schutte (3) were able to confirm this result using intact D. stramonium and Datura metel isolated root cultures, respectively. The former workers reported that phenylacetic acid was also a good precursor but that the usual one-carbon sources (formate and serine), capable of extending the two-carbon side chain to three carbons, only labeled the tropane part of the ester alkaloids. Methionine and formaldehyde (1) also failed as additional one-carbon sources, but bicarbonate (4), although labeling other carbons, predominantly labeled the hydroxymethyl group of tropic acid, a result that favored the intermediacy of phenylacetate. However, it was later (5) demonstrated that all three carbons in the side chain of tropic acid [6} were derived from the side-chain carbons of phenylalanine [31 by means of a rearrangement (Scheme 1). The primary role of phenylalanine was confirmed when it was shown that after very careful purification, the alkaloids derived from phenylacetate feedings were devoid of activity 'Part 3 in the series "The Biosynthesis of Tropic Acid.' For Part 2, see Evans and Woolley ( 14). 2Dedicated to the memory of Professor Edward Leete.

Kinetic Study of Littorine Rearrangement inDaturainnoxiaHairy Roots by13C NMR Spectroscopy

“…This biosynthesis involves the conversion of ornithine into tropine in six steps 6 and of phenylalanine into phenyllactate in two steps. [7][8][9] Tropine and phenyllactate (Figure 1) then condense into littorine, as reported by Robins et al 10 The biosynthesis of hyoscyamine involves the molecular rearrangement of littorine. However, the reaction mechanism is still uncharacterized.…”

“…Recently, transformed root cultures were used to investigate the biosynthesis of tropane alkaloids. This biosynthesis involves the conversion of ornithine into tropine in six steps and of phenylalanine into phenyllactate in two steps. − Tropine and phenyllactate (Figure ) then condense into littorine, as reported by Robins et al The biosynthesis of hyoscyamine involves the molecular rearrangement of littorine. However, the reaction mechanism is still uncharacterized. − Although significant progress allows a better understanding of tropane alkaloid biosynthesis, little is known about its regulation.…”

“…Phenyl[1- 13 C]alanine was provided by Euriso-Top (St. Aubin, France) (99% specific enrichment). (RS)-Phenyl [1,[3][4][5][6][7][8][9][10][11][12][13] C2]lactic acid (80.9% specific enrichment) was kindly provided by J. G. Woolley (Faculty of Applied Sciences, De Monfort University, Leicester, UK).…”

Tropic acid ester biosynthesis in Datura stramonium and related species

“…This observation is readily rationalised if these compounds interconvert in vivo. The importance of phenyllactic acid as an intermediate was confirmed by feeding (R,S)-phenyl [1,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]lactate to D. stramonium transformed root cultures 7 or plants. 8 High incorporation was obtained and the observed spin-spin coupling of the two 13 C nuclei in the extracted hyoscyamine 7 and scopolamine 8 confirmed both the intramolecular rearrangement shown by Leete 3 and the putative role of phenyllactic acid in the formation of hyoscyamine.…”

“…In a definitive experiment twenty years later (Scheme 1) Leete demonstrated 3 in Datura innoxia plants that (R,S)phenyl [1,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]alanine was indeed incorporated intact into the tropate moiety of hyoscyamine. Importantly, this experiment demonstrated an intramolecular rearrangement where the two isotopes became contiguous in the resultant tropate.…”