Kinetic Study of Littorine Rearrangement inDaturainnoxiaHairy Roots by¹³C NMR Spectroscopy

Abstract

The kinetics of tropane alkaloid biosynthesis, particularly the isomerization of littorine into hyoscyamine, were studied by analyzing the kinetics of carbon-13 ((13)C) in metabolites of Datura innoxia hairy root cultures fed with labeled tropoyl moiety precursors. Both littorine and hyoscyamine were the major alkaloids accumulated, while scopolamine was never detected. Feeding root cultures with (RS)-phenyl[1,3-(13)C(2)]lactic acid led to (13)C spin-spin coupling detected on C-1' and C-2' of the hyoscyamine skeleton, which validated the intramolecular rearrangement of littorine into hyoscyamine. Label from phenyl[1-(13)C]alanine or (RS)-phenyl[1,3-(13)C(2)]lactic acid was incorporated at higher levels in littorine than in hyoscyamine. Initially, the apparent hyoscyamine biosynthesized rate (v(app)()hyo = 0.9 micromol (13)C.flask(-1).d(-1)) was lower than littorine formation (v(app)()litto = 1.8 micromol (13)C.flask(-1).d(-1)), suggesting that the isomerization reaction could be rate limiting. The results obtained for the kinetics of littorine biosynthesis were in agreement with the role of this compound as a direct precursor of hyoscyamine biosynthesis.

NMR analysis of plant nitrogen metabolism

“…Similarly, at an earlier stage in the pathway, 13 C NMR was used to identify the intermediates between N-methylpyrrolinium salt and tropinone, and to demonstrate that hygrine was not a direct precursor of tropine or tropine esters (Robins et al 1997). The kinetics of the pathway have also been analysed by 13 C NMR, and in a recent study of the incorporation of (R,S)-phenyl-[1,3-13 C 2 ] lactic into hyoscyamine in Datura innoxia root cultures, results were obtained that suggested that the conversion of littorine to hyoscyamine might limit the overall flux through the pathway (Lanoue et al 2002).…”

“…Label was observed in littorine (L1, L3) and hyoscamine (H1, H2), and the carbon-carbon splitting observed for H1 and H2 (J 1,2 $55 Hz) provided direct evidence for the intramolecular rearrangement that occurs between littorine and hyoscyamine. Adapted from Lanoue et al (2002) with the permission of the American Chemical Society. region of the spectrum, and the preferred strategy is to monitor signals in the much less crowded aromatic region of the spectrum (Hinse et al 2001(Hinse et al , 2003.…”

Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica

“…The elucidation and specific assignments of these compounds were in supporting information. Other known compounds are isolated as scopolamine3940 ( 1 ), anisodamine4142 ( 2 ), hyoscyamine4344 ( 3 ), anisodine31 ( 4 ), N-Caffeoylputrescine364546 ( 14 ), N1,N10-di-dihydrocaffeoylspermidine2147 ( 17 ), Scotanamine D21 (19), and N1-caffeoyl-N3-dihydrocaffeoyl spermidine48 ( 20 )…”

Alkaloids

“…The enzyme that catalyzes this rearrangement, which has been purified partially, seems to proceed via a radical mechanism using S-adenysylmethione as the source of an adenosyl radical (156). Labeling studies have been used to examine the mechanism of rearrangement (136) (157)(158)(159). Hyoscyamine 6β-hydroxylase (H6H) catalyzes the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine as well as the epoxidation to scopolamine (Fig.…”