Fig. 2 30.72 MHz 2H{[1H]} NMR spectrum of rosmarinine 13 in
CHCl3 derived from [3,4-2H5]-2-aminobutanoic acid 11. The signal at δ
7.25 is natural abundance 2H in CHCl3.
Fig. 3 30.72 MHz 2H{[1H]} NMR spectrum of senecionine 13 in
CHCl3 derived from [3,4-2H5]-2-aminobutanoic acid 11. The signal at δ
7.25 is natural abundance 2H in CHCl3.
ties of the 13C NMR resonances were determined using DEPT
spectra with pulse angles of θ = 90Њ and θ = 135Њ. IR Spectra
were obtained on either a Perkin-Elmer 983 spectrophotometer
or a Philips PU 9800 FTIR spectrophotometer. Elemental
analyses were performed using a Carlo-Erba 1106 elemental
analyser. TLC was carried out on Merck Kieselgel G plates of
0.25 mm thickness, eluting with CHCl3–MeOH–conc. NH3
(85:14:1). Alkaloids were visualised with Dragendorff’s
reagent.13
0.7 deuterium into C-13 and C-20, respectively. This rules out
carbonyl groups at C-13 and C-20 as intermediates in the bio-
synthetic pathway. Loss of 0.5 of the deuterium at C-13 and
C-20 would be consistent with previous work using tritium-
2
labelled compounds7 but H isotope effects and lack of preci-
sion of the 2H integrals could account for this discrepancy.
It is clear from these results for rosmarinine 3 produced by S.
pleistocephalus, that the C-3 and C-4 positions of 2-amino-
butanoic acid are exclusively and equally incorporated intact
into the two halves of the senecic acid 6, labelling solely the
C-13 and C-19 alkaloid positions of the right hand portion and
the C-20 and C-21 positions of the left hand C5 unit.
The two precursors were also fed to Senecio vulgaris root
cultures and senecionine was extracted and purified by pre-
parative TLC. Although the incorporation of the precursor 10
into senecionine was comparable to that obtained for ros-
marinine 3, less alkaloid was obtained and signal to noise ratios
in the 13C NMR spectrum were poorer. However, in the 13C
NMR spectrum of senecionine, doublets could be observed
around the signals at δ 11.1, 15.1, 38.4 and 134.3 with coupling
constants of 36, 42, 36 and 42 Hz, respectively. Again, the
amounts of label in each half of senecic acid were approxi-
mately equal.
Diethyl 2-ethyl-2-acetamidomalonate 8
Ethyl iodide (0.38 cm3, 0.756 g, 4.85 mmol) was added dropwise
to a solution of sodium (0.110 g, 4.78 mmol) and diethyl
acetamidomalonate (0.944 g, 4.35 mmol) in absolute ethanol
(20 cm3). After addition was complete the solution was heated
at reflux for 1 h. The mixture was then cooled to room tem-
perature and the solution was decanted from the sediment. The
ethanol was removed in vacuo. Recrystallisation of the crude
product gave 8 as white crystals (0.878 g, 75%); Rf 0.53
(EtOAc); mp 82 ЊC (from 95% aqueous acetone) (lit.,12 83 ЊC)
(Found: M+, 245.1267; C, 53.4; H, 7.8; N, 5.8. C11H19NO5
requires M, 245.1263; C, 53.88; H, 7.76; N, 5.71).
After feeding [3,4-2H5]-2-aminobutanoic acid 11 to S. vul-
garis the 2H NMR spectrum showed three peaks at δ 0.91, 1.81
and 5.78 corresponding to labels at H-19, H-13 plus H-21 and
H-20, respectively (Fig. 3). The ratios of the relative intensi-
ties (approximately 3:3.5:0.5) indicate that there is equal
incorporation into the two methyl groups and ca. 0.5 deuterium
is lost from C-13 and C-20. The specific incoporation of H was
0.5%. These experiments confirmed our findings using
rosmarinine.
(±)-2-Aminobutanoic acid hydrochloride 9
Diethyl 2-ethyl-2-acetamidomalonate (0.904 g, 3.69 mmol) and
concentrated hydrochloric acid (35 cm3, 37%) were heated at
reflux for 4 h. The reaction mixture was then cooled and con-
centrated in vacuo. The residue was washed with acetone,
filtered and the filtrate was evaporated to dryness. Recrystal-
lisation gave 9 as white crystals (0.407 g, 79%); mp >230 ЊC
(from 95% ethanol) (Found: C, 34.25; H, 7.0; N, 10.1. C4H10-
NO2Cl requires: C, 34.41; H, 7.17; N, 10.04%).
2
Stable isotopes have been incorporated into senecic acid in S.
pleistocephalus and S. vulgaris and the first complete labelling
patterns for necic acids have been obtained. These labelling
patterns show conclusively that in the two Senecio species
senecic acid 6 is formed from two units of 2-aminobutanoic
acid. The C-3 and C-9 positions of senecic acid are generated
from the C-3 of 2-aminobutanoic acid, and the C-8 and C-10
diacid positions were originally the C-4 of the amino acid. It
has also been shown that there is equal incorporation into both
halves of the necic acid. More information on the biosynthesis
of necic acids may now be produced by use of other precursors
labelled with stable isotopes.
(±)-[3,4-13C2]-2-Aminobutanoic acid 10
The procedure was repeated as described above but with [13C2]-
iodoethane: δH (D2O) 0.81 (3 H, dm, J 130, 13CH3), 1.76 (2 H,
dm, J 130, 13CH2), 3.76 (1 H, m, CH); δC 9.3 (d, J 34, 13CH3᎐
13CH2), 24.2 (d, J 34, 13CH2᎐13CH3).
(±)-[3,4-2H5]-2-Aminobutanoic acid 11
The procedure was carried out as previously described but
using [2H5]iodoethane: δD (H2O) 1.20 (3 D, br s, CD3), 2.16 (2 D,
br s, CD2).
Feeding experiments with labelled (±)-2-aminobutanoic acid
hydrochloride
Experimental
Senecio pleistocephalus S. Moore plants were obtained from the
Royal Botanic Garden, Edinburgh, and were propagated by
stem cuttings and grown in five inch pots in a standard compost
in a greenhouse. The labelled amino acids were dissolved in
distilled water and fed by the wick method.
General details
Mps were measured on a Kofler hot-stage apparatus and are
uncorrected. EI Mass spectra were obtained with AEI MS 12 or
902 spectrometers. NMR Spectra were recorded with a Bruker
WP200-SY spectrometer operating at 200 MHz (δH ), 50.3 MHz
(δC) or 30.72 MHz (δD ). J Values are given in Hz. The multiplici-
(a) (±)-[3,4-13C2]-2-Aminobutanoic acid 10 (11 mg) was fed
to three six-month-old plants over four days. After a further ten
J. Chem. Soc., Perkin Trans. 1, 1997
679