Fluorinated tropane alkaloids generated by directed biosynthesis in transformed root cultures of Datura stramonium

Article abstract of DOI:10.1039/a902731i

2′-, 3′- and 4′-Fluorophenyl-(RS)-lactic acids were administered to transformed root cultures of Datura stramonium to determine their abilities as substrates for incorporation into the phenyllactoyl and tropoyl ester moieties of the tropane alkaloids, littorine and hyoscyamine respectively. In the event, all of the fluorinated phenyllactates generated the corresponding fluorinated littorine and hyoscyamine analogues. The efficiency of conversion for the isomerisation of the fluorinated littorines (F-lit) to fluorinated hyoscyamines was 3′-F-lit > 4′-F-lit ? 2′-F-lit.

Full text of DOI:10.1039/a902731i

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Fluorinated tropane alkaloids generated by directed biosynthesis
in transformed root cultures of Datura stramonium
a
b
a
a
b
David O’Hagan,* Richard J. Robins,* Marina Wilson, Chi W. Wong, Marc Berry
a
and Ioannis Zabetakis
a
Department of Chemistry, University of Durham, Science Laboratories, South Road,
Durham, UK DH1 3LE
Laboratoire d’Analyse Isotopique et Electrochimique de Metabolismes, CNRS UPRES-A
b
6
4
006, Université de Nantes, Département de Chimie, 2 rue de la Houssinière,
4322 Nantes cedex 03, France
Received (in Cambridge) 6th April 1999, Accepted 3rd June 1999
2
Ј-, 3Ј- and 4Ј-Fluorophenyl-(RS)-lactic acids were administered to transformed root cultures of Datura stramonium
to determine their abilities as substrates for incorporation into the phenyllactoyl and tropoyl ester moieties of the
tropane alkaloids, littorine and hyoscyamine respectively. In the event, all of the fluorinated phenyllactates generated
the corresponding fluorinated littorine and hyoscyamine analogues. The efficiency of conversion for the isomerisation
of the fluorinated littorines (F-lit) to fluorinated hyoscyamines was 3Ј-F-lit > 4Ј-F-lit ӷ 2Ј-F-lit.
3
Introduction
to hyoscyamine in an intriguing biosynthetic isomerisation.
The mechanism of the rearrangement is unclear at present
but our working hypothesis envisages the removal of the 3-pro-
R) hydrogen by the putative isomerase to generate either
The origin of the tropic acid ester moiety of the tropane
alkaloids hyoscyamine 2 and scopolamine 3 (Scheme 1) has
4
(
a carbocation or radical at the benzylic C-3 position, and this
intermediate undergoes rearrangement to a product carb-
ocation or radical as shown in Scheme 2.
5
In a recent oxygen-18 isotopic labelling study it was demon-
Scheme 1
1
been the subject of biosynthetic interest for many years,
particularly as the tropate moiety was shown over twenty
2
years ago to arise from an intramolecular rearrangement of a
3
phenylalanine derived metabolite. Recently it was discovered
that littorine 1, the tropanyl ester (R)-phenyllactic acid is the
substrate for this rearrangement and that littorine is converted
Scheme 2
J. Chem. Soc., Perkin Trans. 1, 1999, 2117–2120
2117

View Article Online
strated that ~25–29% of the oxygen from the hydroxy oxygen at
C-2 of littorine is lost in the rearrangement to hyoscyamine.
One rationale for this leakage of oxygen-18 is the intermediacy
of aldehyde 4 formed after collapse of the product carbocation,
which would be vulnerable to loss of the isotope by exchange
with the aqueous medium. The aldehyde would then require to
be reduced by a dehydrogenase to generate littorine. In order
to explore the littorine to hyoscyamine rearrangement further,
Table 1 Relative ratios of littorine (lit), hyoscyamine (hyo), 2Ј-, 3Ј- and
Ј-fluorolittorines (F-lit) and 2Ј-, 3Ј- and 4Ј-fluorohyoscyamines (F-
hyo) after feeding experiments with 2Ј-, 3Ј- and 4Ј-fluorophenyllactates
to D. stramonium root cultures. Values in the Table are ratios of two
metabolites derived from GC integration values after feeding experi-
ments worked up on days 11, 14 and 17
4
Precursor fed
Day 11
Day 14
Day 17
2
Ј-, 3Ј-, and 4Ј-fluorophenyllactates were administered to root
Control
2Ј-F-pla
hyo/lit
hyo/lit
F-hyo/F-lit
F-lit/lit
F-hyo/hyo
hyo/lit
F-hyo/F-lit
F-lit/lit
F-hyo/hyo
hyo/lit
F-hyo/F-lit
F-lit/lit
F-hyo/hyo
2.72
2.97
0.09
0.26
0.01
3.07
1.15
0.13
0.05
2.60
0.40
0.24
0.04
4.07
3.08
0.14
0.28
0.01
3.12
1.26
0.11
0.05
2.73
0.42
0.23
0.04
3.93
3.07
0.08
0.20
0.01
3.02
1.60
0.09
0.05
3.19
0.62
0.22
0.04
cultures of Datura stramonium. It was envisaged that these
substrates, where each of the aromatic hydrogen atoms is in
turn replaced by a fluorine atom, would be sufficiently similar
to phenyllactate to be assimilated by the cells and become
esterified to the corresponding fluorinated littorines. However
the particular focus of this study was to test whether these
fluorinated littorines would act as substrates for isomerisation
to the equivalent fluorinated hyoscyamines. From classical
substituent effects it was anticipated that the position of the
fluorine would influence the stability of the putative inter-
mediate benzylic carbocation or radical involved in the isomer-
isation process.
3
Ј-F-pla
4Ј-F-pla
Table 2 In vivo conversions (% values) of fluorolittorines to fluoro-
hyoscyamines after biotransformations of F-phenyllactates in D. stram-
onium root cultures in experiments worked up after days 11, 14 and
Results and discussion
Racemic samples of 2Ј-, 3Ј- and 4Ј-fluorophenyllactic acids
were prepared by diazotization of the corresponding fluorin-
ated phenylalanines in aqueous acidic conditions (Scheme 3).
1
7. The conversions are percentage values relative to the endogenous
conversions of littorine to hyoscyamine in the same experiments. The
data emerge from Table 1 as (F-hyo/F-lit)/(hyo/lit) × 100
Day 11
Day 14
Day 17
2
3
4
Ј-F-lit to 2Ј-F-hyo
Ј-F-lit to 3Ј-F-hyo
Ј-F-lit to 4Ј-F-hyo
3
37
15
4.5
40
15.3
2.6
53
19.4
ϩ
of M = 307, characteristic of monofluorinated analogues of
littorine and hyoscyamine. The fluorinated littorines were
unambiguously identified by co-elution on GC and identical
fragmentation patterns in GC-MS, with the appropriate syn-
thetic reference compound. In all of the analyses, the fluor-
inated littorines proved to be the earlier eluted of the two new
peaks. The latter peak was then assigned to the fluorinated
hyoscyamine analogue.
The relative abundances of these compounds from each of the
three experiments is presented in Table 1. For the three fluoro-
phenyllactates studied, both fluorolittorines and fluorohyo-
scyamines were generated in vivo. The F-lit/lit ratios in Table 1
give an approximate measure of the ‘incorporation’ of exogen-
ous fluorophenyllactic acid into the littorine pool (lit ϩ F-lit).
For 2Ј- and 4Ј-fluorophenyllactates the ‘incorporation’ levels
reach about 25%. For 3Ј-fluorophenyllactate the incor-
poration level is lower at about 10%. On this basis, 3Ј-fluoro-
phenyllactate appears to be a less efficient substrate for the
esterification with tropine to generate the fluorinated littorine,
however the levels of 3Ј-fluorolittorine in the extracts will be
attenuated by the much higher in vivo conversions of 3Ј-F-lit to
3Ј-F-hyo relative to the other two isomers of F-lit (see below).
For the conversion of fluorolittorines to fluorohyoscyamines
the incorporations are all low, and in the range 1 to 5% (see
F-hyo/hyo levels in Table 1). To assess which of the fluorinated
littorines was most efficiently converted to a fluorinated hyoscy-
amine, the F-hyo/F-lit ratio was evaluated as a percentage of
the endogenous hyo/lit ratio in each experiment. This was
judged to offer the most accurate measure of the efficiency of
conversion of F-lit to F-hyo from the available data. These
relative % conversions are presented in Table 2. It emerges that
the efficiency of the biotransformation of the fluorolittorines to
fluorohyoscyamines is in the order 3Ј-F-lit > 4Ј-F-lit ӷ 2Ј-F-lit.
For 3Ј-F-lit and 4Ј-F-lit there is an increase in the level of con-
version from day 11 to day 17 whereas for 2Ј-F-lit the conver-
sions are sufficiently low that this trend is not apparent. The
Scheme 3 i, NaNO , cHCl, H O; ii, dry HCl(g), tropine.
2
2
It was also important for the study to prepare each of the
corresponding fluorinated littorines to use as reference com-
pounds for GC-MS analysis. These fluorinated littorines were
3
prepared in an identical manner to that described previously
for the synthesis of littorine from phenyllactic acid and tropine.
The fluorinated phenyllactic acids were administered at a
final concentration of 0.1 mmol to transformed root cultures of
6
Datura stramonium, growing in a nutrient medium. This con-
centration was selected following a recent study in our labor-
atory where it was demonstrated that supplementation with
7
phenyllactate at this concentration stimulated littorine levels
by approximately 35%. Higher concentrations produced
no additional increase in littorine levels and this level of
supplementation proved to be optimal. Cultures were fed in
separate experiments with the fluorophenyllactate isomers on
day 7. They were then worked up on days 11, 14 and 17 and
6
the tropane alkaloids isolated as previously described and
8,9
the alkaloid mixtures analysed by GC-MS. Analysis of the
chromatographs revealed, as expected, littorine and hyoscy-
amine production as well as two additional but resolvable peaks
indicating compounds closely related to these alkaloids. In the
three cases studied, these two new peaks have molecular ions
2
118
J. Chem. Soc., Perkin Trans. 1, 1999, 2117–2120

View Article Online
1
very low conversion of 2Ј-F-lit to 2Ј-F-hyo can most probably
be attributed to the proximity of the fluorine atom to the benz-
ylic reaction centre for the isomerisation and clearly the induct-
ive influence of fluorine will be greatest at the 2-position. The
results however do not reveal a progressive inductive effect for
substitution at the other sites as 3Ј-F-lit emerged as a better
substrate than 4Ј-F-lit for the isomerisation. From classical
studies on fluorine substituent effects, the intermediacy of a
benzylic carbocation or radical is predicted to derive greater
stabilisation from fluorine at the 4Ј-position over the 3Ј-
position due to both inductive and mesomeric conjugative
effects. However the relative incorporations of 3Ј- and 4Ј-
fluorophenyllactates into hyoscyamine show the presence of a
contrary effect which is not so straightforward to interpret.
Perhaps significant differences in binding affinities to the iso-
merase between isomers are also contributing to this overall
profile. To unravel this problem, it will be necessary to assess the
kinetics of these fluorinated substrates with a purified isomer-
ase, if and when such a system is forthcoming.
72.6 (C-2), 40.6 (C-3), 163.2 (d, J = 242.6 Hz, C-4Ј), 134.8 (d,
CF
4
3
JCF2= 2.9 Hz, C-1Ј), 132.3 (d, J = 8.0 Hz, C-2Ј, C-6Ј), 115.7
CF
(d, JCF = 21.3 Hz, C-3Ј, C-5Ј); δ (CD OD) Ϫ119.6 (m, 1F,
F
3
ϩ
ϩ
Ar-F); m/z (EIϩ) 184 (M , 6.55%), 166 (M Ϫ 18, 20.97), 109
(M Ϫ 75, 100); ν 3417, 2950, 1702, 1600, 1507, 1445, 1417,
1363 and 1228 cm (Calcd. mass for C F H O : 184.053573.
Found: 184.053398).
RS)-3-(3Ј-Fluorophenyl)lactic acid
ϩ
max
Ϫ1
9
1
9
3
10
(
The above procedure for (RS)-3-(4Ј-fluorophenyl)lactic acid
was repeated using (RS)-3-(3Ј-fluorophenyl)alanine (4.83 g, 26.4
mmol) to yield a yellow oil which crystallised after addition of
chloroform to give the product as a white crystalline solid (1.21
g, 25%). Mp 106–107 ЊC; δ (CD OD) 7.27 (dt, J = 6.8 Hz,
= 7.6 Hz, 1H, H-5Ј), 7.07 (d, J = 7.6 Hz, 1H, H-6Ј), 7.02
d, J = 10.0 Hz, 1H, H-2Ј), 6.93 (t, J
H-4Ј), 4.34 (dd, J = 4.4 Hz, J = 8.0 Hz, 1H, -CH), 3.10 (dd,
J = 4.0 Hz, J = 14.0 Hz, 1H, -CHH), 2.91 (dd, J = 8.0 Hz,
J = 14.0 Hz, 1H, -CHH); δ (CD OD) 176.8 (C-1), 164.1 (d,
4
H
3
3
HF
3
J
HH
3
HH
3
(
= 8.8 Hz, 1H,
HH
HF, HH
3
3
3
2
1
2
3
C
3
3
In summary it is demonstrated that 2Ј-, 3Ј- and 4Ј-fluoro-
phenyllactates are esterified with tropine in D. stramonium root
cultures and that the resultant fluorinated littorines are isomer-
ised to the corresponding fluorinated hyoscyamines. The
relative efficiencies of these conversions relate both to electronic
effects and most probably binding affinities to the relevant
enzymes.
JCF = 243.8 Hz, C-3Ј), 141.7 (d, JCF = 7.3 Hz, C-1Ј), 130.8
3
4
(
d, J = 8.0 Hz, C-5Ј), 126.5 (d, J = 2.6 Hz, C-6Ј), 117.3 (d,
CF CF
2
2
JCF = 21.3 Hz, C-2Ј), 114.2 (d, J = 22.4 Hz, C-4Ј), 72.4 (C-2),
CF
4
(
3
1.2 (C-3); δ (CD OD) Ϫ116.6 (m, 1F, Ar-F); m/z (EIϩ) 184
F 3
ϩ ϩ ϩ
M , 3.37%), 166 (M Ϫ 18, 32.26), 109 (M Ϫ 75, 100); ν
max
Ϫ1
428, 2940, 1722, 1585, 1486, 1450, 1429, 1324, 1240 cm
(
Calcd. mass for C F H O : 184.053573. Found: 184.053243).
9
1
9
3
Experimental
(
RS)-3-(2Ј-Fluorophenyl)lactic acid
General details
The above procedure for (RS)-3-(4Ј-fluorophenyl)lactic acid
was repeated using (RS)-(3)-2Ј-fluorophenylalanine (5.09 g,
IR spectra were recorded on a Perkin-Elmer F.T. 1720X or 1600
spectrometer. Mass spectra were recorded on a VG Analytical
2
7.8 mmol) to yield a yellow oil which crystallised after
addition of chloroform to give the product as a white crystalline
solid (0.957 g, 19%). Mp 80–81 ЊC; δ (CD OD) 7.29–7.32 (m,
1
13
7
070E Organic mass spectrometer. H and C NMR spectra
1
were recorded on a Varian Gemini 200 MHz ( H at 199.975
MHz, C at 50.289 MHz) and Varian VXR 400(S) ( H at
H
3
13
1
1
4
H, Ar-H), 7.20–7.24 (m, 1H, Ar-H), 7.02–7.09 (m, 2H, Ar-H),
3 3 3
13
19
.34 (dd, J = 4.4 Hz, J = 7.6 Hz, 1H, -CH), 3.17 (dd, J = 3.2
3
99.952 MHz, C at 100.577 MHz) instruments and F NMR
19
2
3
2
Hz, J = 14.0 Hz, 1H, -CHH), 2.91 (dd, J = 8.4 Hz, J = 13.2
spectra were recorded on a Varian VXR 400(S) ( F at 376.35
Hz) instrument. Chemical shifts are quoted relative to TMS
Hz, 1H, -CHH); δ (CD OD) 176.9 (C-1), 71.6 (C-2), 34.9 (C-
C
3
1
3
3
), 162.8 (d, J = 244.1 Hz, C-2Ј), 133.1 (d, J = 4.2 Hz,
(
Me Si) in CDCl or CD OD. GC-MS were recorded on a VG
CF
3
CF
4
3
3
2
C-6Ј), 129.5 (d, J = 8.0 Hz, C-4Ј), 125.8 (d, J = 15.7 Hz,
TRIOS-1S mass spectrometer (VG Masslab Ltd., Manchester)
fitted with a Hewlett Packard 5890 series II gas chromatograph
CF
CF
4
2
C-1Ј), 125.0 (d, J = 3.0 Hz, C-5Ј), 116.0 (d, J = 22.1 Hz, C-
CF
CF
ϩ
3
2
2
Ј); δ (CD OD) Ϫ120.4 (m, 1F, Ar-F); m/z (EIϩ) 184 (M ,
F 3
ϩ ϩ
.47%), 166 (M Ϫ 18, 23.19), 109 (M Ϫ 75, 100); ν
950, 1703, 1604, 1507, 1450, 1418, 1362, 1230 cm (Calcd.
(
(
Hewlett Packard Inc., Fort Collins, USA) and a DB-17 column
J&W Scientific, Folsom, USA) was used for separation. The
3416,
max
Ϫ1
three monofluorinated (RS)-phenylalanines were purchased
from the Aldrich Chemical Company (Gillingham, Kent, UK)
or Lancaster Synthesis Ltd., Morecambe, Lancashire, UK.
Root cultures of Datura stramonium L. D15/5 were grown as
previously described.
Alkaloids were extracted and analysed by GC-MS essentially
mass for C F H O : 184.053573. Found: 184.053840).
9
1
9
3
Tropanyl (RS)-3-(4Ј-fluorophenyl)lactate (4Ј-F-littorine)
6
Rigorously dried (RS)-3-(4Ј-fluorophenyl)lactic acid (390 mg,
2.1 mmol) and tropine (270 mg, 1.9 mmol) were mixed
8
,9
as described previously.
intimately under N in the solid phase. The mixture was heated
2
to 130 ЊC and a current of dry HCl gas was passed periodically
over the reaction for 5 h and the reaction mixture was allowed
to cool to ambient temperature. The product was dissolved as
completely as possible in 0.05 M H SO (10 ml), filtered and the
(
RS)-3-(4Ј-Fluorophenyl)lactic acid
Conc. HCl (6.9 ml, 3 equiv.) was added to a solution of (RS)-3-
4-fluorophenyl)alanine (4.98 g, 27.2 mmol) in water (260 ml),
(
2
4
and the reaction mixture stirred until homogenous. The solu-
filtrate was treated with 10% aqueous ammonium hydroxide
tion was then cooled to 0 ЊC and NaNO (3.90 g, 56.5 mmol, 2
until it was basic, and then the organics were extracted
2
equiv.) was added portion-wise over 4 h maintaining 0 ЊC. The
reaction was left to come to ambient temperature and stirred
for a further 48 h. The solution was concentrated under reduced
pressure and extracted into ether (3 × 150 ml). The com-
into chloroform (4 × 15 ml), the extracts dried (MgSO ) and
4
concentrated under reduced pressure to give a pale yellow solid,
4
(367 mg, 57%). Mp 77–79 ЊC; δ (CDCl ) 7.20 (dd, J = 5.6
H
3
HF
3
3
Hz, J = 8.8 Hz, 2H, H-2Ј, H-6Ј), 6.99 (t, J
= 8.8 Hz,
HH
HF, HH
3
3
bined organic extracts were dried over MgSO , filtered and
2H, H-3Ј, H-5Ј), 5.06 (t, J = 5.2 Hz, 1H, H-3), 4.36 (dd, J = 4.8
Hz, J = 7.2 Hz, 1H, -CH), 3.10 (dd, J = 4.8 Hz, J = 14.0 Hz,
1H, -CHH), 3.10–3.18 (br, 2H, H-1, H-5), 2.95 (dd, J = 6.8 Hz,
J = 14.0 Hz, 1H, -CHH), 2.30 (s, 3H, NMe), 2.12–2.26 (m, 2H,
4
3
3
2
evaporated under reduced pressure to yield a yellow oil.
Recrystallisation of the initial product from chloroform gave
3
2
(
(
5
RS)-3-(4Ј-fluorophenyl)lactic acid as a white crystalline solid
4
1.25 g, 25%). Mp 100.5–101.5 ЊC; δ (CD OD) 7.26 (dd, J
=
H-2 , H-4 ), 1.96–2.04 (m, 2H, H-6 , H-7 ), 1.74–1.85 (m, 2H,
H
3
HF
e
e
e
e
3
3
2
.6 Hz, J = 8.0 Hz, 2H, H-2Ј, H-6Ј), 6.98 (t, J
= 8.8
H-7 , H-6 ), 1.67 (t, J = 14.0 Hz, 2H, H-4 , H-2 ); δ (CDCl )
HH
HF, HH
a
a
a
a
C
3
3
3
1
Hz, 2H, H-3Ј, H-5Ј), 4.30 (dd, J = 4.4 Hz, J = 8.0 Hz, 1H,
173.2 (C᎐O), 162.0 (d, JCF3= 245.3 Hz, C-4Ј), 132.0 (d,
᎐
3
2
4
-
CH), 3.07 (dd, J = 4.4 Hz, J = 14.0 Hz, 1H, -CHH), 2.88 (dd,
JCF2= 3.4 Hz, C-1Ј), 130.9 (d, J = 7.9 Hz, C-2Ј, C-6Ј), 115.3
CF
3
2
J = 8.0 Hz, J = 14.0 Hz, 1H, -CHH); δ (CD OD) 177.0 (C-1),
(d, J = 21.3 Hz, C-3Ј, C-5Ј), 71.3 (C-HЈ), 69.3 (C-3), 59.7
C
3
CF
J. Chem. Soc., Perkin Trans. 1, 1999, 2117–2120
2119

View Article Online
(
(
(
C-1, C-5), 40.3 (NMe), 39.6 (C-HHЈ), 36.4 (C-2, C-4), 25.4
C-6, C-7); δ (CDCl ) Ϫ116.4 (m, 1F, Ar-F); m/z (EIϩ) 307
sterile solution (1 ml) of (RS)-3-(2Ј-fluorophenyl)lactic acid
Ϫ3
(54.3 mmol dm ) dissolved in MeOH was fed on day 7 at a
F
3
ϩ
ϩ
ϩ
Ϫ3
M , 25.02%), 140 (M Ϫ 167, 15.06), 124 (M Ϫ 183, 100);
final concentration of 0.1 mmol dm in the medium. Two
Ϫ1
νmax 2944, 1730, 1601, 1508, 1448, 1418, 1218 cm (Calcd. mass
flasks each of roots were harvested and freeze-dried after days
for C F H NO : 307.158372. Found: 307.158504).
11, 14 and 17. The freeze-dried roots (day 11, 0.6 g; day 14,
17
1
22
3
0
.6 g; day 17, 0.6 g) were ground with acid-washed sand and
Ϫ3
Tropanyl (RS)-3-(3Ј-fluorophenyl)lactate (3Ј-F-littorine)
extracted into H SO (10 ml, 0.05 mol dm ) by stirring for
2 4
2
0 min. The aqueous extract was made basic with 35% NH3
The above procedure for 4Ј-F-littorine was repeated using
previously prepared (RS)-3-(3Ј-fluorophenyl)lactic acid (201
mg, 1.1 mmol) and tropine (155 mg, 1.1 mmol). The product
was purified on a Hyflow column (10 g) containing phosphate
solution and then filtered through Hydromatrix and eluted
with CHCl –MeOH (20:1). The eluent was evaporated under
reduced pressure to give a brown oil (day 11, 7 mg; day 14,
8
3
mg; day 17, 10 mg) containing the alkaloids and this extract
Ϫ3
buffer (0.5 mol dm ) at pH 6.6, eluting with successive volumes
was subjected to GC-MS analysis.
(
50 ml) of light petroleum, ether and chloroform, to give 3Ј-F-
littorine (51 mg, 15%) as a pale yellow gum. δ (CDCl ) 7.22–
H
3
3
Feeding (RS)-3-(3Ј-fluorophenyl)lactic acid to Datura
stramonium root cultures
7
.29 (m, 1H, Ar-H), 6.90–7.02 (m, 3H, Ar-H), 5.05 (t, J = 4.8
3 3
Hz, 1H, H-3), 4.37 (dd, J = 4.4 Hz, J = 6.8 Hz, 1H, -CH), 3.11
3
2
(
dd, J = 4.4 Hz, J = 14.4 Hz, 1H, -CHH), 3.11–3.16 (br, 2H,
The above procedure for (RS)-3-(2Ј-fluorophenyl)lactic acid
was repeated for (RS)-3-(3Ј-fluorophenyl)lactic acid. The
freeze-dried roots obtained (day 11, 0.5 g; day 14, 0.6 g; day 17,
0.5 g) yielded the alkaloid extract as a brown oil (day 11, 14 mg;
day 14, 8 mg; day 17, 7 mg) which was subjected to GC-MS
analysis.
3
2
H-1, H-5), 2.96 (dd, J = 7.2 Hz, J = 14.0 Hz, 1H, -CHH), 2.29
(
s, 3H, NMe), 2.14–2.24 (m, 2H, H-2 , H-4 ), 1.98–2.04 (m, 2H,
e
e
H-6 , H-7 ), 1.79 (d, J = 8.0 Hz, 2H, H-7 , H-6 ), 1.66 (t,
e
e
a
a
2
1
3
2
J = 16.4 Hz, 2H, H-4 , H-2 ); δ (CDCl ) 173.4 (C᎐O), 163.0 (d,
JCF = 245.7 Hz, C-3Ј), 139.2 (d, J = 7.5 Hz, C-1Ј), 130.1 (d,
JCF = 8.3 Hz, C-5Ј), 125.3 (d, J = 2.3 Hz, C-6Ј), 116.6 (d,
JCF = 21.0 Hz, C-2Ј), 114.0 (d, JCF = 20.9 Hz, C-4Ј), 71.4
a
a
C
3
᎐
3
CF
4
CF
2
Feeding (RS)-3-(4Ј-fluorophenyl)lactic acid to Datura
stramonium root cultures
(
C-H), 69.5 (C-3), 60.0 (C-1, C-5), 40.5 (NMe), 40.4 (C-HH),
3
1
1
1
3
6.6 (C-2), 36.5 (C-4), 25.7 (C-6, C-7); δ (CDCl ) Ϫ113.8 (m,
F 3
ϩ ϩ
The above procedure for (RS)-3-(2Ј-fluorophenyl)lactic acid
was repeated for (RS)-3-(4Ј-fluorophenyl)lactic acid. The freeze-
dried roots obtained (day 11, 0.5 g; day 14, 0.6 g; day 17, 0.6 g)
yielded the alkaloid extract as a brown oil (day 11, 10 mg;
day 14, 9 mg; day 17, 10 mg) which was subjected to GC-MS
analysis.
F, Ar-F); m/z (EIϩ) 307 (M , 19.62%), 140 (M Ϫ 167, 13.92),
ϩ
24 (M Ϫ 183, 100); ν 2948, 1740, 1620, 1592, 1492, 1452,
max
Ϫ1
254 cm (Calcd. mass for C F H NO : 307.158372. Found:
17
1
22
3
07.158538).
Tropanyl (RS)-3-(2Ј-fluorophenyl)lactate (2Ј-F-littorine)
The above procedure for 4Ј-F-littorine was repeated using (RS)-
Acknowledgements
3
-(2Ј-fluorophenyl)lactic acid (180 mg, 0.98 mmol) and tropine
(
138 mg, 0.98 mmol). The product was purified on a Hyflow
We thank the EPSRC for a studentship (C. W. W.) and the
CIBA Foundation for an ACE Award. We would also like to
acknowledge the EPSRC Mass Spectroscopy Service at the
University of Swansea for HRMS analysis.
Ϫ3
column (10 g) containing phosphate buffer (0.5 mol dm
at pH 6.6, eluting with successive volumes (50 ml) of light
petroleum, ether and chloroform to give 2Ј-F-littorine (42 mg,
)
1
4%) as a pale yellow oil. δ (CDCl ) 7.20–7.30 (m, 2H, Ar-H),
H 3
7
.00–7.11 (m, 2H, Ar-H), 5.01–5.10 (br, 1H, H-3 ), 4.40 (dd,
e
3 3
References
3
J = 5.2 Hz, J = 7.2 Hz, 1H, -CH), 3.17 (dd, J = 4.8 Hz,
J = 14.0 Hz, 1H, -CHH), 3.16–3.24 (br, 2H, H-1, H-5), 3.01
2
1
D. O’Hagan and R. J. Robins, Chem. Soc. Rev., 1998, 27, 207.
2 E. Leete, N. Kowanko and R. A. Newmark, J. Am. Chem. Soc.,
975, 97, 6826.
R. J. Robins, P. Bachmann and J. G. Woolley, J. Chem. Soc., Perkin
Trans. 1, 1994, 615.
3
2
(
dd, J = 7.2 Hz, J = 13.6 Hz, 1H, -CHH), 2.33 (s, 3H, NMe),
1
2.20–2.32 (m, 2H, H-2 , H-4 ), 1.96–2.08 (m, 2H, H-6 , H-7 ),
e e e e
2
1
1
3
.78–1.92 (m, 2H, H-7 , H-6 ), 1.75 (d, J = 14.4 Hz, 1H, H-2 ),
a
a
a
2
.65 (d, J = 14.4 Hz, 1H, H-4 ); δ (CDCl ) 173.4 (C᎐O), 161.2
4 N. C. J. E. Chester, K. Walker, D. O’Hagan and H. G. Floss, J. Am.
Chem. Soc., 1996, 118, 925.
a
C
3
1
3
(
(
d, J = 245.3 Hz, C-2Ј), 131.9 (d, J = 4.2 Hz, C-6Ј), 128.8
CF CF
3 4
5
C. W. Wong, J. T. G. Hamilton, D. O’Hagan and R. J. Robins, Chem.
Commun., 1998, 1045.
d, J = 8.4 Hz, C-4Ј), 124.1 (d, J = 3.1 Hz, C-5Ј), 123.4 (d,
CF CF
2
2
JCF = 15.6 Hz, C-1Ј), 115.3 (d, JCF = 22.1 Hz, C-3Ј), 70.3
6
R. J. Robins, A. L. Parr, E. G. Bent and M. J. C. Rhodes, Planta,
(
(
(
C-H), 68.9 (C-3), 59.9 (C-1, C-5), 40.0 (NMe), 36.1 (C-2), 35.8
C-4), 34.1 (C-HH), 25.3 (C-7), 25.1 (C-6); δ (CDCl ) Ϫ118.2
m, 1F, Ar-F); m/z (EIϩ) 307 (M , 23.67%), 140 (M Ϫ 167,
1
991, 183, 185.
F
3
7 I. Zabetakis, R. Edwards, J. T. G. Hamilton and D. O’Hagan, Plant
Cell Rep., 1998, 18, 341.
8 R. J. Robins, J. G. Wooley, M. Ansarin, J. Eagles and B. J.
Goodfellow, Planta, 1994, 194, 86.
ϩ
ϩ
ϩ
1
2.47), 124 (M Ϫ 183, 100); ν 2923, 1739, 1587, 1493, 1461,
max
Ϫ1
1
377, 1260 cm (Calcd. mass for C F H NO : 307.158372.
17
1
22
3
9
B. Drager, A. Portsteffen, A. Schaal, P. McCabe, A. C. J. Peerless
and R. J. Robins, Planta, 1992, 188, 581.
Found: 307.159058).
1
0 G. Kohnstam and D. L. H. Williams, The Chemistry of the Ether
Linkage, ed. S. Patai, Wiley-Interscience, New York, 1967, p. 81.
Feeding (RS)-3-(2Ј-fluorophenyl)lactic acid to Datura stramonium
root cultures
To six subcultured flasks each containing an initial innoculum
(
0.5 g) of fresh mass of roots in culture medium (50 ml), a
Paper 9/02731I
2
120
J. Chem. Soc., Perkin Trans. 1, 1999, 2117–2120