Biosynthesis of yatein in Anthriscus sylvestris.

Article abstract of DOI:10.1039/b304411d

Little is known about the biosynthesis of yatein, in spite of its importance as a typical heartwood lignan and a key biosynthetic intermediate of the antitumor lignan podophyllotoxin. The present study, based on individual administration of [13C]phenylalanine and deuterium labelled lignans and simultaneous administration of two distinct lignans labelled with deuterium atoms to Anthriscus sylvestris, established the two independent branch pathways from matairesinol, one to afford yatein via thujaplicatin, 5-methylthujaplicatin, and 4,5-dimethylthujaplicatin and the other to bursehernin via pluviatolide. The latter pathway did not lead to yatein, eliminating the presence of a metabolic grid from matairesinol to yatein.

Full text of DOI:10.1039/b304411d

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Biosynthesis of yatein in Anthriscus sylvestris†
Norikazu Sakakibara, Shiro Suzuki, Toshiaki Umezawa* and Mikio Shimada
Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan
Received 23rd April 2003, Accepted 27th May 2003
First published as an Advance Article on the web 20th June 2003
Little is known about the biosynthesis of yatein, in spite of its importance as a typical heartwood lignan and
a key biosynthetic intermediate of the antitumor lignan podophyllotoxin. The present study, based on individual
administration of [13C]phenylalanine and deuterium labelled lignans and simultaneous administration of two distinct
lignans labelled with deuterium atoms to Anthriscus sylvestris, established the two independent branch pathways
from matairesinol, one to afford yatein via thujaplicatin, 5-methylthujaplicatin, and 4,5-dimethylthujaplicatin and
the other to bursehernin via pluviatolide. The latter pathway did not lead to yatein, eliminating the presence of
a metabolic grid from matairesinol to yatein.
Introduction
An antitumor lignan, podophyllotoxin, has been successfully
used as the starting material for semi-synthetic anti-cancer
drugs, etoposide and teniposide.1–6 Podophyllotoxin is isolated
from herbaceous perennial Podophyllum plants.5,7,8 However,
due to the limited supply of the plants, much attention has been
focused on the availability and biosynthesis of the lignan.1,3,5,8
In the 1980s, Dewick and co-workers reported a series of
detailed feeding experiments with radio-labelled precursors and
revealed the pathways from yatein (11) to podophyllotoxin and
its congeners via deoxypodophyllotoxin (=desoxypodophyllo-
toxin, anthricin).9–12 Next, they showed that matairesinol (2)
is metabolized to podophyllotoxin and proposed that (11) was
the possible intermediate in the conversion of (2) to podo-
phyllotoxin.13 During the last decade, significant progress has
been made in the biosynthetic studies of lignans, and the bio-
synthesis of matairesinol (2) from coniferyl alcohol has been
established.1,14 Recently, several enzymes involved in the
transformation of deoxypodophyllotoxin to podophyllotoxin,
β-peltatin, and 5-methoxypodophyllotoxin have been iso-
lated.15–17 These data allow us to draw the outline of
biosynthetic pathways for podophyllotoxin derivatives as in
Fig. 1. Briefly, the dimerization of coniferyl alcohol, followed
by three metabolic steps gives rise to matairesinol (2). Then,
matairesinol (2) is transformed to yatein (11) which is in turn
converted to podophyllotoxin congeners. However, the pathway
from matairesinol (2) to yatein (11) remains unknown.
Besides the role for the precursor of podophyllotoxin, yatein
(11) is also of importance as a heartwood lignan.18 Heartwood
is the colored region of the inner part of tree trunks, and its
formation is a metabolic event specific to woody plants, as it
does not operate in herbaceous plants. Recently, metabolic
engineering of trees has been developed significantly. For
example, a transgenic aspen (Populus tremuloides) with less
lignin and higher cellulose contents has been successfully
produced by Chiang and co-workers.19 These developments were
Fig. 1 A proposed biosynthetic pathway from coniferyl alcohol to
achieved with the aid of the knowledge of herbaceous plant
molecular biology. Now, it is necessary to exploit knowledge of
previous reports. Broken arrow: established in this report.
molecular mechanisms for metabolic events specific to woody
podophyllotoxin. 2: matairesinol, 11: yatein. Solid arrow: established in
plants in order to accelerate metabolic engineering of trees.
the molecular mechanisms of their biosynthesis can be a clue to
help us elucidate heartwood formation mechanisms.
Although nothing is known about the molecular mechanisms,
the facts that biosynthesis of lignans, norlignans and related
compounds are involved in heartwood formation implies that
Anthriscus sylvestris20–26 is a good source of yatein (11) and
deoxypodophyllotoxin, both of which are effective precursors
of podophyllotoxin.9–11 In addition, angeloyl podophyllotoxin
has been isolated from this species.27 The high content of yatein
(11) and deoxypodophyllotoxin as well as the good growth
behaviour stimulated us to employ A. sylvestris as a plant
† A part of this work was performed as a part of the R & D Project of
Industrial Science and Technology Frontier Program supported by
NEDO (New Energy and Industrial Technology Development
Organization).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 7 4 – 2 4 8 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Fig. 2 A proposed biosynthetic pathway of yatein in Anthriscus sylvestris. 1: secoisolariciresinol, 2: matairesinol, 3: thujaplicatin, 4: 5-
methylthujaplicatin, 5: 4,5-dimethylthujaplicatin, 6: pluviatolide, 7: 5-hydroxypluviatolide, 8: 4-demethylyatein, 9: bursehernin, 10: 5-demethylyatein,
11: yatein. Solid arrow: proposed biosynthetic pathway in Anthriscus sylvestris. Broken arrow: physiologically insignificant pathway.
material for the study of yatein biosynthesis, and we have
recently reported the survey of lignans in this species.26
The conversion of matairesinol (2) to yatein (11) involves
four steps: 5-hydroxylation, dual methylation at C4OH and
C5OH, and methylenedioxy bridge formation at C3Ј and
C4Ј (Fig. 2). Many possible orders of their occurrence can be
envisaged suggesting that a metabolic grid might be present in
the transformation. The aim of the present study is to identify
the physiologically important biosynthetic pathway from
matairesinol (2) to yatein (11).
thujaplicatin (4) (TMS ether): m/z (EI) 532 (Mϩ, 100%),
239 (84.1), 209 (26.0); 4,5-dimethylthujaplicatin (5) (TMS
ether): m/z (EI) 474 (Mϩ, 100%), 209 (71.0), 181 (56.0)] and
retention times on GC with those of chemically synthesized
authentic samples. In addition, yatein (11) and deoxypodophyl-
lotoxin were isolated from the MeOH extracts and identified by
comparison of the 1H-NMR and mass spectrum (data not
shown) with those of the literature data.28
Feeding experiments
Herein, we report the administration of [13C]phenylalanine
and a series of deuterium labelled lignans to A. sylvestris, and
discuss the biosynthetic pathway from matairesinol (2) to yatein
(11) in this species.
Small scale feeding experiments. Nine lignans labelled with
deuterium atoms at the methoxy groups, ( )-[3-OC2H3]matai-
resinols (2-d3), ( )-[3-OC2H3]thujaplicatins (3-d3), ( )-[3,5-
(OC2H3)2]-5-methylthujaplicatins (4-d6), ( )-[3,5-(OC2H3)2]-
4,5-dimethylthujaplicatins (5-d6), ( )-[3-OC2H3]pluviatolides
(6-d3), ( )-[3-OC2H3]-5-hydroxypluviatolides (7-d3), ( )-[3,5-
Results
(OC2H3)2]-4-demethylyateins
(8-d6),
( )-[3,4-(OC2H3)2]-
Lignans in Anthriscus sylvestris
bursehernins (9-d6), and ( )-[3,4-(OC2H3)2]-5-demethylyateins
(10-d6), were fed individually to young shoots of A. sylvestris
(0.4 mg of each lignan to one shoot). Results of GC-MS
analysis of the MeOH extracts obtained following the adminis-
tration are summarized in Tables 1–3. Table 1 shows the
molecular ion and important daughter ion regions of
unlabelled authentic yatein (11) and of this lignan obtained
following the administration: unlabelled yatein (11) gives a
molecular ion at m/z 400 and principal daughter ions at m/z
181 and 135 which are derived from benzylic cleavage of A-
and B-rings (Fig. 2), respectively. When the lignans labelled
Recently, we have surveyed lignans occurring in Anthriscus
sylvestris:26 GC-MS analysis of the MeOH extracts of the plant
showed the presence of lignans, lariciresinol, secoisolariciresi-
nol (1), matairesinol (2), pluviatolide (6), yatein (11), hinokinin,
nemerosin, and deoxypodophyllotoxin. Additionally, the exist-
ence of small amounts of bursehernin (9) was suggested by
mass chromatography.26 Further GC-MS analysis in the present
study indicated the presence of 5-methylthujaplicatin (4) and
4,5-dimethylthujaplicatin (5) in the MeOH extracts, which
were identified by comparing their mass spectra [5-methyl-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 7 4 – 2 4 8 5
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Table 1 Mass tables of molecular-ion and principal daughter-ion regions of lignan (11) obtained following administration of 13C- or deuterium-
labelled precursors into A. sylvestris.
Yatein (11)
Relative intensity (%)
Administrated labelled compounds
m/z Unlabelled [13C6]Phe 1-d3
2-d3
38.2b
3-d3
42.6b
4-d6
47.0b
13.3
5.2
2.5
1.2
0.7
1.3
1.0
0.4
5-d6
39.9b
13.6
3.3
2.6
1.0
0.0
0.0
0.3
0.9
6-d3
33.5b
12.4
3.3
1.4
0.7
0.3
0.6
0.0
0.4
7-d3
46.3b
16.0
8.7
2.8
3.8
1.5
1.4
0.1
0.5
8-d6
42.9b
12.7
5.4
2.1
1.9
0.5
0.2
0.2
0.6
9-d6
30.3b
10.6
4.1
2.1
3.4
0.0
0.0
2.4
1.2
10-d6
1-d5 ϩ 3-d3 1-d5 ϩ 6-d3
135
136
137
138
139
140
141
142
143
30.6b
13.5
4.7
3.1
1.3
1.0
0.7
1.1
37.2b
12.7
5.2
2.0
1.6
43.4b
16.3
6.7
2.7
2.1
0.4
0.4
0.3
0.9
36.6b
13.9
6.2
3.6
1.0
0.0
0.0
0.0
0.0
41.8b
12.9
6.6
41.1b
13.1
5.4
14.1
3.5
1.6
0.9
0.0
0.0
0.2
0.7
13.1
4.7
2.0
2.5
1.0
1.1
0.1
0.9
1.9
1.5
2.2
1.0
1.6
0.5
0.7
4.5b
1.4
1.5
0.4
0.5
0.3
0.4
1.0
0.8
0.8
181 100.0a
100.0a
58.5
7.9
100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a
100.0a
54.4
7.6
182
183
184
185
186
187
188
189
400
401
402
403
404
405
406
407
408
412
413
414
48.9
5.6
1.2
2.2
0.8
0.9
1.8
2.2
70.1c
19.8
3.7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
56.8
7.4
51.2
7.0
0.0
1.5
0.9
0.9
1.0
2.1
68.9c
19.1
5.5
2.5
1.3
0.4
0.0
0.2
0.0
0.0
0.0
0.0
55.3
6.9
55.6
6.3
1.0
53.2
6.7
1.5
0.3
0.9
61.9
6.7
1.4
0.1
0.4
0.1
1.9
2.7
75.2c
18.6
5.1
1.6
0.6
0.5
0.5
0.0
0.4
0.0
0.0
0.0
61.3
8.3
62.7
8.2
2.3
54.9
8.3
1.8
0.0
0.0
0.0
0.0
2.9
76.6c
16.6
4.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
60.2
10.7
1.2
53.3
7.0
0.8
10.3a
5.0
11.7a
6.7
19.3a
11.1
2.1
11.8a
7.4
2.0
1.3
1.0
0.6
0.0
1.6
2.1
1.0
0.9
1.1
1.7
0.5
2.1
1.0
0.1
0.0
8.2a
3.7
3.5a
2.3
17.7a
7.9
16.9a
9.9
4.2a
1.3
2.0
12.0a
8.6
12.3a
8.8
2.4
2.7
1.2
0.6
2.3
2.9
1.8
2.5
3.1
2.8
4.6
1.8
3.2
78.0c
20.2
4.8
74.2c
21.7
6.7
71.7c
19.8
4.2
73.7c
21.4
4.6
67.0c
20.6
3.8
93.7c
22.2
4.5
92.1c
23.0
4.6
88.9c
21.9
6.3
75.2c
21.9
4.6
76.4c
21.2
5.3
1.4
0.0
9.0c
2.4
7.8c
3.2
1.9
1.6
1.8
0.8
16.1c
4.6
2.1
1.7
1.3
0.0
10.3c
4.4
1.6
2.0
1.1
0.7
0.7
0.3
0.6
0.0
0.7
1.6
1.0
1.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.3c
1.8
3.4c
2.3
3.9c
1.5
12.9c
4.5
2.5c
1.6
13.3c
4.7
9.3c
3.0
0.6
1.3
0.8
0.0
0.8
0.0
0.0
0.0
0.0
0.0
0.0
0.9
0.0
0.4
0.0
0.4
0.2
0.1
0.4
1.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
13.0c
1.6
0.4
0.5
0.0
0.0
0.3
0.0
0.0
0.0
0.0
[13C6]Phe: [ring-13C6]phenylalanine, 1-d3: ( )-[3-OC2H3]secoisolariciresinols, 2-d3: ( )-[3-OC2H3]matairesinols, 3-d3: ( )-[3-OC2H3]thujaplicatins, 4-d6:
( )-[3,5-(OC2H3)2]-5-methylthujaplicatins, 5-d6: ( )-[3,5-(OC2H3)2]-4,5-dimethylthujaplicatins, 6-d3: ( )-[3-OC2H3]pluviatolides, 7-d3: ( )-[3-OC2H3]-
5-hydroxypluviatolides, 8-d6: ( )-[3,5-(OC2H3)2]-4-demethylyateins, 9-d6: ( )-[3,4-(OC2H3)2]bursehemins, 10-d6: ( )-[3,4-(OC2H3)2]-5-demethyl-
yateins. a Ions formed after benzylic cleavages of A-ring. b Ions formed after benzylic cleavages of B-ring. c Molecular ions. Bold-faced: extra ions due
to incorporation of 13C or deuterium atoms.
with six deuterium atoms at the two methoxy groups on the
A-ring (Fig. 2), ( )-[3,5-(OC2H3)2]-5-methylthujaplicatins
(4-d6), ( )-[3,5-(OC2H3)2]-4,5-dimethylthujaplicatins (5-d6),
( )-[3,5-(OC2H3)2]-4-demethylyateins (8-d6), and ( )-[3,4-
(OC2H3)2]-5-demethylyateins (10-d6), were administrated, the
extra ions at m/z 406 and 187 were observed in the mass spectra
of (11), while in the feeding of the lignans with one OC2H3
group and three deuterium atoms, ( )-[3-OC2H3]thujaplicatins
(3-d3) and ( )-[3-OC2H3]-5-hydroxypluviatolides (7-d3), extra
ions at m/z 403 and 184 were observed. These results unequivo-
cally indicate the conversion of (3-d3), (4-d6), (5-d6), (7-d3),
(8-d6), and (10-d6) to yatein labelled with three or six deuterium
atoms (11-d3 or 11-d6). However, no evidence of isotope
incorporation into yatein (11) was observed when ( )-[3-
OC2H3]matairesinols (2-d3), ( )-[3-OC2H3]pluviatolides (6-d3),
and ( )-[3,4-(OC2H3)2]bursehernins (9-d6) were administrated
(Table 1). Similarly, Tables 2 and 3 show mass spectral data of
the other lignans obtained following the administration.
( )-[3,5-(OC2H3)2]-4,5-dimethylthujaplicatins
(5-d6)
[2H6]yatein (11-d6) (Table 1)
( )-[3-OC2H3]pluviatolides (6-d3)
[2H3]bursehernin (9-d3)
(Table 3)
( )-[3-OC2H3]-5-hydroxypluviatolides (7-d3)
[2H3]-4-
demethylyatein (8-d3) (Table 3), [2H3]-5-demethylyatein (10-d3)
(Table 3), and [2H3]yatein (11-d3) (Table 1)
( )-[3,5-(OC2H3)2]-4-demethylyateins (8-d6)
(11-d6) (Table 1)
[2H6]yatein
[2H6]yatein
( )-[3,4-(OC2H3)2]-5-demethylyateins (10-d6)
(11-d6) (Table 1)
Large scale feeding experiments. The following compounds
were administered to A. sylvestris in the same manner as the
small scale experiments, but scaled up ten times (4 mg of each
compound to 10 shoots): [ring-13C6]phenylalanine, ( )-[3-
OC2H3]secoisolariciresinols (1-d3), ( )-[3-OC2H3]matairesinols
(2-d3), ( )-[3-OC2H3]pluviatolides (6-d3), and ( )-[3,4-
(OC2H3)2]bursehernins (9-d6). GC-MS analysis confirmed the
Thus, these data demonstrate the conversions of the labelled
precursors into several lignans summarized as follows:
( )-[3-OC2H3]matairesinols (2-d3)
[2H3]pluviatolide (6-d3)
transformation [(2-d3)
(6-d3) and (9-d3); and (6-d3) (9-d3)]
(Table 3), and [2H3]bursehernin (9-d3) (Table 3)
( )-[3-OC2H3]thujaplicatins (3-d3)
as in the small scale experiments (data not shown). In addition,
the conversions listed below were detected by GC-MS analysis.
[2H3]-5-methyl-
thujaplicatin (4-d3) (Table 2), [2H3]-4,5-dimethylthujaplicatin
(5-d3) (Table 2), and [2H3]yatein (11-d3) (Table 1)
[ring-13C6]phenylalanine
[13C12] and [13C6]matairesinol (2-
13
C
and 13C6) (Table 2), [13C12] and [13C6]-5-methylthujaplicatin
12
( )-[3,5-(OC2H3)2]-5-methylthujaplicatins (4-d6)
[2H6]-4,5-
(4-13C12 and 13C6) (Table 2), [13C12] and [13C6]-4,5-di-
methylthujaplicatin (5-13C12 and 13C6) (Table 2), [13C12] and
[13C6]pluviatolide (6-13C12 and 13C6) (Table 3), [13C12]
dimethylthujaplicatin (5-d6) (Table 2), and [2H6]yatein (11-d6)
(Table 1)
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 7 4 – 2 4 8 5
2476

Table 2 Mass tables of molecular-ion and principal daughter-ion regions of lignans (2), (4) and (5) obtained following administration of 13C- or deuterium-labelled precursors into A. sylvestris
Matairesinol (2) (TMS ether)
5-Methylthujaplicatin (4) (TMS ether)
4,5-Dimethylthujaplicatin (5) (TMS ether)
Relative intensity (%)
Relative intensity (%)
Relative intensity (%)
Administrated labelled
compounds
Administrated labelled compounds
Administrated labelled compounds
m/z
Unlabelled
[13C6]Phe
1-d3
m/z
Unlabelled
[13C6]Phe
1-d3
3-d3
8.2b
m/z
Unlabelled
[13C6]Phe
1-d3
27.1b
15.0
3.6
33.1
22.7
3.2
3-d3
17.3b
3.5
0.0
15.8
9.9
0.0
4-d6
209
210
211
212
213
214
215
216
217
502
503
504
505
506
507
508
509
510
514
515
516
100.0a, b
29.3
8.4
1.6
0.4
0.0
0.3
0.9
21.9a, b
7.8
3.1
0.3
1.9
100.0a, b
28.3
9.5
209
210
211
212
213
214
215
216
217
239
240
241
242
243
244
245
246
247
532
533
534
535
536
537
538
539
540
544
545
546
54.4b
16.7
5.9
0.0
0.0
0.0
0.0
0.0
26.6b
6.1
2.5
7.3
5.4
16.3b
3.7
1.2
8.2
3.4
3.5
0.2
0.7
209
210
211
212
213
214
215
216
217
181
182
183
184
185
186
187
188
189
474
475
476
477
478
479
480
481
482
486
487
488
46.4b
25.2
6.2
1.3
0.0
1.5
0.0
1.7
30.8b
18.0
6.7
4.3
0.0
23.3b
15.4
0.0
12.6
10.4
0.5
2.0
4.0
2.0
7.4
2.2
0.0
0.0
0.0
9.9a
6.0
4.1
4.5a
3.7
1.7
0.0
7.0
0.0
14.2c
12.5
4.9
10.4c
4.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
71.4a
18.3
5.3
1.7
4.5
6.2
18.6a, b
4.7
0.0
0.0
1.6
11.4b
4.0
21.2b
5.3
2.9
1.4
1.8
0.0
13.5
0.0
4.9
3.0
2.7
2.8
0.0
5.2
0.2
2.0
0.8
0.0
58.4c
26.7
11.4
3.6
100.0c
46.7
20.6
9.5
28.8c
15.6
12.3
84.0c
33.4
13.0
0.0
83.0a
31.2
10.5
3.4
21.2a
11.9
3.4
31.3a
13.7
5.6
100.0a
73.1
8.7
74.0a
36.3
16.5
0.0
100.0a
68.0
11.2
24.2a
13.3
4.8
100.0a
55.0
12.2
42.6a
24.7
8.8
88.5a
78.5
17.7
3.5
1.3
0.2
10.0a
3.7
1.1
0.0
0.5
0.0
1.4
0.5
0.0
0.4
0.2
0.1
0.0
0.0
3.2
11.4
1.4
0.0
2.8
0.0
4.8
0.0
1.5
1.1
1.3
4.9
0.0
0.0
9.1c
9.4
10.0a
9.0
33.9a
21.7
0.0
1.3
2.4
3.0
2.9
9.3
30.0a
13.0
2.0
0.0
0.0
0.0
0.0
0.0
2.9
0.6
.
5.6
2.9
2.0
14.7
24.1c
16.7
0.0
9.4c
4.2
100.0c
46.5
21.5
2.1
100.0c
46.4
8.4
28.1c
14.1
14.9
35.1c
17.8
6.1
95.9c
35.8
11.3
1.5
100.0c
38.3
0.0
36.7c
14.2
10.3
99.1c
33.6
16.2
5.8
38.3c
27.0
8.6
2.6
0.0
0.0
0.0
9.0
0.0
12.3c
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
9.6c
8.3
0.0
0.0
0.0
0.0
0.0
0.0
20.5c
9.3
28.8c
15.1
0.0
1.9
0.0
1.4
0.0
0.0
2.5
20.3c
0.0
30.8c
5.5
0.0
0.0
0.0
0.0
3.1
0.0
[13C6]Phe: [ring-13C6]phenylalanine, 1-d3: ( )-[3-OC2H3]secoisolariciresinols, 3-d3: ( )-[3-OC2H3]thujaplicatins, 4-d6: ( )-[3,5-(OC2H3)2]-5-methylthujaplicatins. a Ions formed after benzylic cleavages of A ring. b Ions
formed after benzylic cleavages of B-ring. c Molecular ions. Bold-faced: extra ions due to incorporation of 13C or deuterium atoms.

O
Table 3 Mass tables of molecular-ion and principal daughter-ion regions of lignans (6), (8)–(10) obtained following administration of 13C- or deuterium-labelled precursors into A. sylvestris
Pluviatolide (6) (TMS ether)
4-Demethyl-yatein (8) (TMS ether)
Bursehernin (9)
Relative intensity (%)
5-Demethyl-yatein (10) (TMS ether
Relative intensity (%)
Relative intensity (%)
Relative intensity (%)
Administrated
labelled
Administrated
labelled
Administrated labelled
Administrated labelled
compounds
compounds
compounds
compounds
m/z
Unlabelled
[13C6]Phe
1-d3
11.8b
1.6
2.4
0.0
2.9
0.8
1.8
2-d3
8.1b
3.2
0.5
0.0
0.0
0.3
0.0
m/z
Unlabelled
7-d3
m/z
Unlabelled
[13C6]Phe
1-d3
2-d3
23.6b
6-d3
21.4b
6.8
3.1
0.0
0.2
0.0
0.2
m/z
Unlabelled
7-d3
135
136
137
138
139
140
141
142
143
209
210
211
212
213
214
215
216
217
428
429
430
431
432
433
434
435
436
440
441
442
21.7b
4.1
3.4
0.0
0.8
0.6
0.0
1.3
9.5b
5.6
0.2
3.7
0.0
0.1
1.0b
0.1
0.1
7.0a
3.2
0.4
0.9
1.0
0.0
7.0a
1.1
0.9
6.4c
4.3
3.5
0.0
1.4
0.0
3.7c
1.1
1.4
4.4c
2.6
0.9
135
136
137
138
139
140
141
142
143
239
240
241
242
243
244
245
246
247
458
459
460
461
462
463
464
465
466
470
471
472
20.0b
4.9
3.9
0.7
0.8
0.0
0.0
0.3
48.7b
15.0
6.2
3.8
2.3
4.1
2.3
2.9
0.0
0.0
0.0
0.0
19.6a
11.9
0.0
0.0
0.0
2.9
0.0
0.0
0.0
25.4c
8.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
135
136
137
138
139
140
141
142
143
151
152
153
154
155
156
157
158
159
370
371
372
373
374
375
376
377
378
382
383
384
26.2b
10.4
3.0
1.4
0.4
0.1
0.2
0.2
28.5b
14.2
7.0
2.9
0.9
13.7b
11.2
2.5
0.7
1.3
3.3
1.1
0.0
135
136
137
138
139
140
141
142
143
239
240
241
242
243
244
245
246
247
458
459
460
461
462
463
464
465
466
470
471
472
40.9b
7.2
2.4
0.5
0.6
0.4
0.4
0.0
17.7b
6.2
4.7
0.0
0.0
0.0
0.0
0.0
0.2
10.5
2.6
0.6
0.7
1.3
1.5
0.2
0.3
0.4
9.8b
3.9
0.3
0.0
0.0
1.0
0.6
0.0
0.3
0.5
3.5
0.0
0.4
0.7
100.0a
20.4
7.6
100.0a
30.1
10.0
57.0a
14.5
2.9
24.3a
16.2
5.7
100.0a
33.6
9.0
100.0a
17.8
1.9
47.4a
7.2
61.4a
12.0
7.4
5.5a
36.3a
6.8
87.7a
80.3
17.9
5.2
0.0
0.0
1.7
2.3
1.5
2.6
2.3
3.9
0.0
2.9
0.8
0.0
2.5
100.0a
19.0
5.1
2.1
1.1
0.3
0.8
0.4
0.0
0.4
0.3
28.8a
7.0
100.0a
16.6
2.1
100.0a
14.8
1.6
59.5a
54.9
20.2
0.0
0.9
0.0
0.4
0.7
2.3
1.6
0.0
0.1
0.6
2.1
0.0
1.4
26.0a
6.7
0.4
0.3
0.0
0.0
0.7
0.9
0.5
0.0
0.0
0.0
0.0
2.9
9.0
5.1
2.8
0.6
4.6
2.6
0.5
0.1
2.5
57.5c
18.8
7.5
3.9c
3.8
2.1c
0.8
88.8c
30.4
10.1
2.9
50.0c
14.5
3.0
26.1c
5.0
29.7c
7.7
3.8c
1.3
20.1c
5.0
100.0c
34.6
12.4
3.5
1.0
1.1
2.7
1.7
0.0
4.0
2.3
4.1
0.0
1.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.3c
0.9
22.0c
6.9
1.0
0.3
0.3
0.1
0.0
0.0
0.0
0.0
17.1c
7.3
42.4c
12.1
3.0
43.4c
11.3
2.0
100.0c
24.4
28.2
0.0
1.5
1.0
0.4
0.4
0.0
0.0
0.0
0.0
1.1
0.4
0.3
0.0
0.0
0.0
0.0
0.0
0.0
1.4
0.0
0.0
0.0
1.5
1.3
3.0
1.6
0.7
0.3
1.2
0.6
0.0
0.0
1.8
0.0
0.0
0.0
0.0
0.4
0.0
4.4c
2.4
0.9
0.4
0.1
0.0
0.0
0.0
1.5
0.0
0.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
11.0c
4.0
0.0
0.0
0.7
0.0
[13C6]Phe: [ring-13C6]phenylalanine, 1-d3: ( )-[3-OC2H3]secoisolariciresinols, 2-d3: ( )-[3-OC2H3]matairesinols, 6-d3: ( )-[3-OC2H3]pluviatolides, 7-d3: ( )-[3-OC2H3]-5-hydroxypluviatolides. a Ions formed after benzylic
cleavages of A-ring. b Ions formed after benzylic cleavages of B-ring. c Molecular ions. Bold-faced: extra ions due to incorporation of 13C or deuterium atoms.

View Article Online
and [13C6]bursehernin (9-13C12 and 13C6) (Table 3), and [13C12]
and [13C6]yatein (11-13C12 and 13C6) (Table 1)
dibenzylbutyrolactone lignans are physiologically significant
endogenous precursors of yatein (11) in the species.
( )-[3-OC2H3]secoisolariciresinols (1-d3)
[2H3]matairesinol
Thus, our attention was next focused on the physiological
roles of the parallel conversions. First, feeding experiments
with more upstream compounds were conducted. Phenyl-
alanine is the entrance compound of phenylpropanoid bio-
synthesis and upon administration this compound is expected
to move smoothly into a physiologically important pathway.29
Hence we fed [ring-13C6]phenylalanine to A. sylvestris. GC-MS
analysis indicated that 13C was incorporated into matairesinol
(2), 5-methylthujaplicatin (4), 4,5-dimethylthujaplicatin (5),
pluviatolide (6), bursehernin (9), and yatein (11) (Tables 1–3),
but no evidence was obtained for the formation of 13C-labelled
[13C12] or [13C6]-5-hydroxypluviatolide (7-13C12 or 13C6), [13C12] or
[13C6]-4-demethylyatein (8-13C12 or 13C6), or [13C12] or [13C6]-5-
demethylyatein (10-13C12 or 13C6) from [ring-13C6]phenylalanine
(Fig. 5).
(2-d3) (Table 2), [2H3]thujaplicatin (3-d3), [2H3]-5-methyl-
thujaplicatin (4-d3) (Table 2), [2H3]-4,5-dimethylthujaplicatin
(5-d3) (Table 2), [2H3]pluviatolide (6-d3) (Table 2), [2H3]-
bursehernin (9-d3) (Table 3), and [2H3]yatein (11-d3) (Table 1)
( )-[3-OC2H3]matairesinols (2-d3)
[2H3]thujaplicatin (3-d3)
The deuterium incorporation into thujaplicatin (3) was
further confirmed by selected ion monitoring (SIM). The
SIM chromatograms of thujaplicatin (TMS ether) obtained
following administration of (1-d3) and (2-d3) clearly indicate
that the molecular ion of unlabelled (3) at m/z 590 and the
fragment ion at m/z 297 due to the benzylic cleavage of A-ring
of unlabelled (3) are accompanied by extra ions at m/z 593 and
300, respectively, indicating clearly the formation of [2H3]-
thujaplicatin (3-d3) (Figs. 4-B and -C).
On the other hand, no evidence was obtained for the
formation of 13C-labelled [13C12] or [13C6]-5-hydroxypluviatolide
(7-13C12 or 13C6), [13C12] or [13C6]-4-demethylyatein (8-13C12 or
13C6), or [13C12] or [13C6]-5-demethylyatein (10-13C12 or 13C6)
from [ring-13C6]phenylalanine. Thus, as shown in Fig. 5, neither
endogenous unlabelled (7), (8), and (10) which have molecular
ions at m/z 516, 458 and 458 (TMS ether), respectively, nor the
lignans labelled with twelve or six 13C atoms were detected.
Similarly, no deuterium-labelled (7-d3), (8-d3), and (10-d3 or
d6) from any of the deuterium labelled lignans, (1-d3), (2-d3),
(6-d3), and (9-d6) were observed. For example, Fig. 6 demon-
strates that neither unlabelled, endogenous 5-hydroxy-
pluviatolide (7) nor deuterium-labelled (7) was detected
following administration of (6-d3). In addition, no deuterium
incorporation into yatein (11) from ( )-[3-OC2H3]pluviatolides
(6-d3) and ( )-[3,4-(OC2H3)2]bursehernins (9-d6) was observed.
The mass spectra of yatein (11) obtained following administra-
tion of (6-d3) and (9-d6) did not show any extra ions in com-
parison with unlabelled authentic sample (Table 1).
Second, ( )-[3-OC2H3]secoisolariciresinols (1-d3) were
administered, because secoisolariciresinol (1) is known to be
converted to matairesinol (2) by the action of secoisolarici-
resinol dehydrogenase.29–33 This resulted in deuterium incorpor-
ation into matairesinol (2), thujaplicatin (3), 5-methyl-
thujaplicatin (4), 4,5-dimethylthujaplicatin (5), pluviatolide (6),
bursehernin (9), and yatein (11) (Fig. 4 and Tables 1–3),
but again no formation of deuterium-labelled (7-d3), (8-d3), or
(10-d3) was observed.
Third, in order to examine 5-hydroxylation step(s), the
deuterium labelled dibenzylbutyrolactone lignans without the
5-hydroxy or 5-methoxy group were prepared. Administration
of ( )-[3-OC2H3]matairesinols (2-d3) resulted in deuterium
incorporation into thujaplicatin (3) (Fig. 4) as well as pluvia-
tolide (6) and bursehernin (9) (Tables 1–3). In contrast, form-
ation of deuterium-labelled 5-hydroxy- or 5-methoxy-lignans,
(7-d3), (10-d3 or d6), and (8-d3), was not observed when ( )-[3-
OC2H3]matairesinols (2-d3), ( )-[3-OC2H3]pluviatolides (6-d3),
and ( )-[3,4-(OC2H3)2]bursehernins (9-d6) were administered.
These results are in accord with the results of metabolic
profiling, which detected the following lignans in the MeOH
extracts of A. sylvestris young shoots: secoisolariciresinol (1),
matairesinol (2), 5-methylthujaplicatin (4), 4,5-dimethyl-
thujaplicatin (5), pluviatolide (6), bursehernin (9), and yatein
(11). In addition, the presence of endogenous, unlabelled
thujaplicatin (3) was detected by mass chromatography (data
not shown) and selected ion monitoring (Figs. 4-B and 4-C).
However, no evidence indicating the presence of any of
5-hydroxypluviatolide (7), 4-demethylyatein (8), and 5-demeth-
ylyatein (10) was obtained (Fig. 5).
Thus, although 5-hydroxypluviatolide (7), 4-demethylyatein
(8), and 5-demethylyatein (10) were converted to yatein (11)
effectively under the experimental conditions employed, their
conversion to yatein (11) is probably negligible in de novo lignan
biosynthesis in A. sylvestris. This conclusion is based on the
above mentioned facts summarized as follows: First, the
upstream precursors, phenylalanine and secoisolariciresinol (1)
were not transformed to any of (7), (8), and (10). Second, the
lignans, (7), (8), and (10), could not be detected in the MeOH
extracts from the plant. Third, conversion of pluviatolide (6),
bursehernin (9), thujaplicatin (3), and 5-methylthujaplicatin (4)
into (7), (8), and (10) was not observed.
Taken together, these results indicate two independent
branch metabolic pathways (solid arrows in Fig. 2) operating to
affect the fate of matairesinol (2). One pathway leads to yatein
(11) via thujaplicatin (3), 5-methylthujaplicatin (4), and 4,5-di-
methylthujaplicatin (5); and the other gives bursehernin
(9) through pluviatolide (6). The involvement of 4-methyl-
Moreover, administration of ( )-[3-OC2H3]thujaplicatin
(3-d3) and ( )-[3,5-(OC2H3)2]-5-methylthujaplicatins (4-d6) in
the small scale experiments did not lead to the formation of any
of deuterium-labelled (7-d3), (8-d3 or d6), and (10-d3) (data not
shown).
Simultaneous administration of two distinctly labelled lignans.
Simultaneous administration of ( )-[3-OC2H3]thujaplicatins
(3-d3) and ( )-[7,7-2H2, 3-OC2H3]secoisolariciresinols (1-d5)
gave both [2H3]yatein (11-d3) (m/z 403, Table 1) and [2H5]yatein
(11-d5) (m/z 405, Table 1), whereas the administration of
both ( )-[3-OC2H3]pluviatolides (6-d3) and ( )-[7,7-2H2,3-
OC2H3]secoisolariciresinols (1-d5) resulted in [2H5]yatein (11-d5)
(m/z 405, Table 1); the intensity of m/z 403 ion corresponding to
[2H3]yatein (11-d3) formation was insignificant (Table 1).
Discussion
We first examined the formation of the 5-methyl ether of the
A-ring and the methylenedioxy bridge of the B-ring of yatein
(11) (Fig. 1). When the deuterium-labelled dibenzylbutyrol-
actone lignans with 3,3Ј,4,4Ј,5-pentaalkoxy groups {( )-[3-
OC2H3]thujaplicatins (3-d3), ( )-[3,5-(OC2H3)2]-5-methylthu-
japlicatins (4-d6), ( )-[3,5-(OC2H3)2]-4,5-dimethylthujaplicatins
(5-d6), ( )-[3-OC2H3]-5-hydroxypluviatolides (7-d3), ( )-[3,5-
(OC2H3)2]-4-demethylyateins (8-d6), and ( )-[3,4-(OC2H3)2]-5-
demethylyateins (10-d6) (Fig. 2)} were administered individually
to A. sylvestris young shoots, all of them were found to be
transformed to deuterium labelled [2H3] or [2H6]yatein (11-d3 or
11-d6) (Tables 1–3). The results indicate that parallel pathways
on the possible metabolic grid can operate in A. sylvestris,
provided that these dibenzylbutyrolactone lignans are supplied
to an appropriate reaction site of yatein biosynthesis in
A. sylvestris cells. However this does not indicate all of these
thujaplicatin (12) (Fig. 3),
a regioisomer of 5-methyl-
thujaplicatin (4), also seemed possible, but this is unlikely,
because isotope incorporation into this lignan was not observed
from any of labelled precursors tested (data not shown). The
operation of two independent pathways was further reinforced
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 7 4 – 2 4 8 5
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Fig. 3 Structures of benzyl-γ-butyrolactones, benzyl bromides and lignans.
Fig. 4 Selected ion monitoring chromatograms of thujaplicatin (TMS ether). A: unlabelled, synthesized authentic sample; B and C: thujaplicatins
obtained following administration of ( )-[3-OC2H3]secoisolariciresinols (1-d3) and ( )-[3-OC2H3]matairesinols (2-d3), respectively. Shown are the
spectra for the molecular ion m/z 590 and the ion formed after benzylic cleavages of the A-ring of unlabelled thujaplicatin m/z 297. The peaks at
17.14 min (B) and 18.12 min (C) are impurities.
by two sets of simultaneous feeding experiments. When ( )-[3-
OC2H3]thujaplicatins (3-d3) and ( )-[7,7-2H2, 3-OC2H3]secoiso-
lariciresinols (1-d5) were fed at the same time, both [2H3]yatein
(11-d3) and [2H5]yatein (11-d5) were formed, whereas the simul-
taneous administration of both ( )-[3-OC2H3]pluviatolides
(6-d3) and ( )-[7,7-2H2,3-OC2H3]secoisolariciresinols (1-d5)
resulted in [2H5]yatein (11-d5) formation, but [2H3]yatein (11-d3)
formation was insignificant (Table 1). If pluviatolide (6) is con-
verted to yatein (11), both [2H3]yatein (11-d3) and [2H5]yatein
(11-d5) would be formed in both simultaneous feeding
experiments. However, the results indicate that this was not the
case, confirming that two independent pathways from mataires-
inol (2) are operating. Thus, the presence of a metabolic grid is
eliminated. It should be noted that ( )-[3-OC2H3]pluviatolides
(6-d3) were transformed to [2H3]bursehernin (9-d3) in the simul-
taneous feeding, suggesting strongly that the administered ( )-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 7 4 – 2 4 8 5
2480

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Fig. 5 Mass chromatograms of molecular ions and fragment ions formed after benzylic cleavages of A- and B-rings of 5-hydroxypluviatolide (7),
4-demethylyatein (8), and 5-demethylyatein (10) (TMS ether). a: unlabelled, synthesized authentic sample; b: the fraction corresponding to (7), (8),
or (10) following administration of [ring-13C6]phenylalanine. M+: molecular ion of unlabelled, synthesized authentic sample; A+ and B+: fragment
ions formed after benzylic cleavages of A- and B-rings of unlabelled, synthesized authentic sample, respectively.
followed by dual methylation and finally 3Ј,4Ј-methylenedioxy
bridge formation to afford yatein (11). Additionally, the con-
version of matairesinol (2) to bursehernin (9) was also found to
occur, but does not lead to yatein (11).
Experimental
Plant materials
Anthriscus sylvestris Hoffm. (Umbelliferae) plants were
collected in October 2001 at Kyoto University Forest in Ashiu,
Kyoto. The plants were maintained in the experimental garden
of Wood Research Institute, Kyoto University.
Fig. 6 Mass chromatograms of molecular ions and fragment ions
formed after benzylic cleavages of A- and B-rings of 5-hydroxy-
Instrumentation
pluviatolide (7) (ethyl ether). a: unlabelled, synthesized authentic
sample; b: the fraction corresponding to (7) following administration of
( )-[3-OC2H3]pluviatolides (6-d3). M+: molecular ion of unlabelled,
synthesized authentic sample; A+ and B+: fragment ions formed after
benzylic cleavages of A- and B-rings of unlabelled, synthesized
authentic sample.
1H NMR and 13C NMR spectra were taken with a JAM-
LA400MK FT NMR System (JEOL). Chemical shifts and
coupling constants (J) were given in δ and Hz, respectively.
GC-MS was performed on a JMS-DX303HF mass spectro-
meter (JEOL) equipped with a Hewlett-Packard 5890J gas
chromatograph and a JMA-DA5000 mass data system. GC-MS
measurement conditions were as follows: electron-impact
mode, 70 eV; gas-chromatographic column, Shimadzu HiCup
CBP-10 M25–025 (5 m × 0.2 mm); temperature, 40 ЊC at t = 0–2
min, then to 230 ЊC at 30 ЊC minϪ1; carrier gas, He; splitless
injection. Samples dissolved in N,O-bis(trimethylsilyl)-
acetamide were subjected to GC-MS measurement after heat-
ing at 60 ЊC for 45 min.
[3-OC2H3]pluviatolides (6-d3) were transported to the lignan
synthesis site under the conditions employed.
Kawai et al. administrated [7,7-2H2, 3-OC2H3]matairesinol
(2-d5) to Thuja occidentalis shoots, and detected deuterium
incorporation into thujaplicatin (3), which indicates that
the conversion of matairesinol (2) to thujaplicatin (3) is not
specific to A. sylvestris, but also occurs in another species, T.
occidentalis.34
Recently, we have detected pinoresinol/lariciresinol reductase
(PLR) activity in A. sylvestris,26 catalyzing the reduction of
pinoresinol to secoisolariciresinol (1) via lariciresinol (Fig. 2).
This suggests the intermediacy of pinoresinol and laricirsinol in
lignan biosynthesis in this species. Taken together, we propose a
possible biosynthetic pathway of yatein (11) from coniferyl
alcohol, concomitant with a branch leading to bursehernin (9)
from matairesinol (2) (Fig. 2).
The biosynthetic pathway for yatein (11) in Anthriscus
sylvestris (Fig. 2) is in line with the previous reports outlined
in Fig. 1. However, the pathway differs from two other recently
proposed pathways in relation to the biosynthesis of podophyl-
lotoxin derivatives in Linum spp..35,36 One of these proposed
pathways had the intact incorporation of 3,4-methylene-
dioxycinnamic acid into podophyllotoxin,35 and the other
had the benzylic hydroxylation occurring at an earlier stage
(C7Ј of matairesinol was hydroxylated to afford 7Ј-hydroxy-
matairesinol, which was then metabolized to 5-methoxy-
podophyllotoxin36).
Synthesis of lignans and chemicals
Synthesis of benzyl-ꢀ-butyrolactones. ( )-3-(4-Benzyloxy-3-
methyloxybenzyl)-γ-butyrolactones (13a) and ( )-3-(3,4-meth-
ylenedioxybenzyl)-γ-butyrolactones (13c) were prepared
exactly as previously described30,40 (13a): δH (CDCl3) 7.24–7.44
(5 H, m), 6.81 (1 H, d, J 8.0), 6.67 (1 H, d, J 2.0), 6.61 (1 H, dd,
J 8.2 and 1.8), 5.12 (2 H, s), 4.32 (1 H, dd, J 9.1 and 7.0), 4.02
(1 H, dd, J 9.3 and 6.1), 3.87 (3 H, s), 2.75–2.86 (1 H, m), 2.67–
2.72 (2 H, m), 2.59 (1 H, dd, J 17.6 and 8.1), 2.27 (1 H, dd,
J 17.4 and 7.0). (13c): δH(CDCl3) 6.74 (1 H, d, J 7.8), 6.63 (1 H,
s), 6.59 (1 H, d, J 7.8), 5.94 (2 H, s), 4.32 (1 H, dd, J 7.3 and 6.8),
4.01 (1 H, dd, J 9.3 and 6.1), 2.74–2.84 (1 H, m), 2.66–2.72 (2 H,
m), 2.59 (1 H, dd, J 17.6 and 8.0), 2.26 (1 H, dd, J 17.3 and 6.8).
( )-3-(4-tert-Butyldimethylsilyloxy-3-methyloxybenzyl)-γ-
butyrolactones (13b) were prepared by catalytic (Pd–C) hydro-
genation of (13a) followed by tert-butyldimethylsilylation (tert-
butyldimethylsilyl chloride, imidazole, DMF, rt) (13b): δH
(CDCl3) 6.77 (1 H, d, J 8.0), 6.61 (1 H, d, J 2.0), 6.58 (1 H, dd,
J 7.8 and 1.9), 4.31 (1 H, dd, J 9.0 and 7.1), 4.02 (1 H, dd, J 9.1
and 6.2), 3.78 (3 H, s), 2.76–2.86 (1 H, m), 2.66–2.72 (2 H, m),
2.56 (1 H, dd, J 17.4 and 8.2), 2.27 (1 H, dd, J 17.4 and 7.0),
0.98 (9 H, s), 0.13 (6 H, s).
In conclusion, the biosynthetic pathway from matairesinol
(2) to yatein (11) has been presented for the first time based
on concrete experimental evidence. The pathway involves
5-hydroxylation of matairesinol (2) to give thujaplicatin (3),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 7 4 – 2 4 8 5
2481

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Scheme 1 Synthetic routes for deuterium-labelled benzyl bromide derivatives (14a–14h). Reagents and conditions: i, NaBH4, MeOH, rt, 10 min;
ii, PBr3, Et2O, rt, 1 h; iii, c. HCl, AcOH, reflux, 1 h; iv, I2, KI, NaOH (aq), rt, 2 h; v, NaOH (aq), CuSO4, 125 ЊC, 6 h; vi, BnBr, K2CO3, DMF, 70 ЊC,
4 h; vii, C2H3O2H, Na, 128 ЊC, 3 h; viii, C2H3I, K2CO3, DMF, rt, 2 h; ix, CH(OMe)3, p-TsOH, MeOH, 0 ЊC, 10 min; x, O2, BuLi, Et2O, Ϫ35 ЊC,
40 min; xi, 1 M HCl, acetone, 0 ЊC, 5 min; xii, KMnO4, acetone, rt, 1 h; xiii, CH3CHN2, Et2O, 0 ЊC, 3 min; xiv, LiAl2H4, Et2O, Ϫ35 ЊC, 40 min.
Synthesis of benzyl bromides. Unlabelled and deuterium-
labelled benzyl bromides (14a–14h) were synthesized from
the vanillin derivatives [vanillin (14j), syringaldehyde (14r), 3,4-
dimethoxy-5-hydroxybenzaldehyde (14y) (Aldrich), and [3-
OC2H3]benzylvanillin (14i)30], respectively, by the methods
described previously,30,40–43 which are outlined in Scheme 1.
4,5-Dibenzyloxy-3-methoxybenzyl bromide (14a): δH(CDCl3)
7.24–7.50 (10 H, m), 6.68 (1 H, d, J 2.0), 6.63 (1 H, d, J 2.0),
5.10 (2 H, s), 5.03 (2 H, s), 4.44 (2 H, s), 3.85 (3 H, s).
4-Benzyloxy-3,5-dimethoxybenzyl bromide (14b): δH(CDCl3)
7.23–7.52 (5 H, m), 6.60 (2 H, s), 4.99 (2 H, s), 4.42 (2 H, s), 3.83
(6 H, s).
were prepared by lithium hexamethyldisilylamide-catalyzed
condensation of appropriate combinations of γ-butyrolactones
(13a–13c) and benzyl bromides (14a–14h) followed by
deprotection as previously reported,30,40–43 which is outlined in
Scheme 2.
( )-[3-OC2H3]Matairesinols (2-d3): δH(CDCl3) 6.80 (1 H, d,
J 8.6), 6.78 (1 H, d, J 7.8), 6.56–6.62 (2 H, m), 6.50 (1 H, d,
J 8.0), 6.34 (1 H, s), 4.13 (1 H, dd, J 8.6 and 7.6), 3.90 (1 H, dd,
J 8.8 and 8.8), 3.80 (3 H, s), 2.94 (1 H, dd, J 13.7 and 4.9), 2.87
(1 H, dd, J 13.7 and 7.3), 2.40–2.64 (4 H, m); m/z (EI) 361.1578
5-Benzyloxy-3,4-dimethoxybenzyl bromide (14c): δH(CDCl3)
7.17–7.42 (5 H, m), 6.60 (1 H, d, J 1.9), 6.56 (1 H, d, J 2.0), 5.02
(2 H, s), 4.35 (2 H, s), 3.80 (3 H, s), 3.80 (3 H, s).
[3-OC2H3]-4-Benzyloxy-3-methoxybenzyl bromide (14d):
δH(CDCl3) 7.24–7.46 (5 H, m), 6.92 (1 H, d, J 1.9), 6.87 (1 H,
dd, J 8.3 and 2.0), 6.81 (1 H, d, J 8.0), 5.15 (2 H, s), 4.48 (2 H, s).
[3-OC2H3]-4,5-Dibenzyloxy-3-methoxybenzyl bromide (14e):
δH(CDCl3) 7.24–7.47 (10 H, m), 6.68 (1 H, d, J 2.0), 6.63 (1 H,
d, J 2.0), 5.09 (2 H, s), 5.02 (2 H, s), 4.44 (2 H, s).
[3,5-(OC2H3)2]-4-Benzyloxy-3,5-dimethoxybenzyl bromide
(14f ): δH(CDCl3) 7.24–7.50 (5 H, m), 6.59 (2 H, s), 4.99 (2 H, s),
4.45 (2 H, s).
[3,4-(OC2H3)2]-5-Benzyloxy-3,4-dimethoxybenzyl bromide
(14g): δH(CDCl3) 7.16–7.47 (5 H, m), 6.59 (1 H, s), 6.55 (1 H, s),
5.06 (2 H, s), 4.36 (2 H, s).
[α,α-2H2, 3-OC2H3]-4-Benzyloxy-3-methoxybenzyl bromide
(14h): δH(CDCl3) 7.21–7.40 (5 H, m), 6.87 (1 H, d, J 2.2), 6.82
(1 H, dd, J 8.2 and 2.1), 6.74 (1 H, d, J 8.1), 5.09 (2 H, s).
Synthesis of dibenzylbutyrolactone lignans. ( )-Matairesinols
(2),37 ( )-pluviatolides (6),38 and ( )-bursehernins (9)39 were
prepared previously.
The dibenzylbutyrolactone lignans, ( )-[3-OC2H3]mataires-
inols (2-d3), ( )-[7,7-2H2, 3-OC2H3]matairesinols (2-d5), ( )-thu-
japlicatins (3), ( )-[3-OC2H3]thujaplicatins (3-d3), ( )-5-methyl-
thujaplicatins (4), ( )-[3,5-(OC2H3)2]-5-methylthujaplicatins
(4-d6), ( )-[3-OC2H3]pluviatolides (6-d3), ( )-5-hydroxy-
pluviatolides (7), ( )-[3-OC2H3]-5-hydroxypluviatolides (7-d3),
( )-4-demethylyateins (8), ( )-[3,5-(OC2H3)2]-4-demethylyat-
eins (8-d6), ( )-5-demethylyateins (10), ( )-[3,4-(OC2H3)2]-5-
demethylyateins (10-d6), and ( )-4-methylthujaplicatins (12)
Scheme 2 Synthetic routes for unlabelled and deuterium-labelled
lignans. Reagents and conditions: i, Lithium hexamethyldisilylamide,
HMPA, THF, Ϫ35 ЊC, 45 min; ii, 10% Pd-C, H2, THF, MeOH, rt, 1 h;
iii, LiAlH4, Et2O, Ϫ35 ЊC, 40 min; iv, CH3I, K2CO3, DMF, rt, 2 h; v, n-
Bu4NF, THF, 0 ЊC, 20 min; vi, C2H3I, K2CO3, DMF, rt, 2 h; vii,
CH3CHN2, Et2O, rt, 10 h.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 7 4 – 2 4 8 5
2482

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(Mϩ, 42.8%. C20H192H3O6 requires361.1604), 360(1.1), 359(1.1),
358 (0.5), 149 (10.5), 140 (100), 137 (42.1), 131 (12.4), 122 (12.5).
( )-[7,7-2H2, 3-OC2H3]Matairesinols (2-d5): δH (CDCl3)
6.70–6.79 (2 H, m), 6.50–6.57 (2 H, m), 6.44 (1 H, dd, J 6.4
and 2.0), 6.34 (1 H, d, J 2.0), 5.45 (2 H, d, J 8.6), 4.08 (1 H, dd,
J 9.2 and 7.2), 3.82 (1 H, dd, J 8.3 and 8.0), 3.74 (3 H, s),
2.58–2.35 (4 H, m); m/z (EI) 363.1734 (Mϩ, 48.2%. C20H17-
2H5O6 requires 363.1730), 362 (3.5), 361 (1.5), 360 (0.6), 359
(0.2), 358 (0.0), 149 (11.5), 142 (100), 137 (47.5), 131 (10.9),
122 (7.8).
( )-Thujaplicatins (3): δH(CDCl3) 6.72 (1 H, d, J 8.1), 6.44
(1 H, dd, J 7.9 and 1.8), 6.38 (1 H, d, J 1.7), 6.30 (1 H, d, J 2.0),
6.17 (1 H, d, J 2.0), 4.09 (1 H, dd, J 9.1 and 6.9), 3.82 (1 H, dd,
J 9.1 and 6.9), 3.76 (3 H, s), 3.73 (3 H, s), 2.85 (1 H, dd, J 13.9
and 4.9), 2.75 (1 H, dd, J 14.0 and 6.9), 2.36–2.58 (4 H, m); m/z
(EI) 374.1380 (Mϩ, 73.9%. C20H22O7 requires 374.1366), 237
(15.0), 223 (9.7), 163 (11.9), 153 (100), 137 (92.8), 131 (24.7),
122 (20.3).
( )-[3,5-(OC2H3)2]-4-Demethylyateins (8-d6): δH (CDCl3) 6.69
(1 H, d, J 7.8), 6.46 (1 H, d, J 7.6), 6.45 (1 H, s), 6.34 (2 H, s),
5.92–5.93 (2 H, m), 4.13 (1 H, dd, J 9.5 and 7.6), 3.86 (1 H, dd,
J 9.0 and 7.6), 2.89 (2 H, d, J 6.1), 2.43–2.65 (4 H, m); m/z (EI)
392.1735 (Mϩ, 57.9%. C21H162H6O7 requires 392.1742), 391
(0.0), 390 (2.4), 389 (1.0), 388 (0.5), 387 (0.7), 386 (0.8), 256
(15.9), 173 (100), 161 (6.6), 149 (18.8), 135 (32.0), 131 (9.8).
( )-5-Demethylyateins (10): δH (CDCl3) 6.62 (1 H, d, J 8.3),
6.37–6.42 (2 H, m), 6.32 (1 H, d, J 2.0), 6.21 (1 H, d, J 1.7),
5.83–5.87 (2 H, m), 4.07 (1 H, dd, J 9.3 and 6.8), about 3.79
(1 H), 3.79 (3 H, s), 3.79 (3 H, s), 2.88 (1 H, dd, J 13.9 and 4.9),
2.73 (1 H, dd, J 13.9 and 7.1), 2.35–2.57 (4 H, m); m/z (EI)
386.1382 (Mϩ, 90.3%. C21H22O7 requires 386.1365), 250 (24.8),
237 (12.9), 205 (14.6), 167 (100), 161 (16.9), 135 (70.8), 131
(18.3).
( )-[3,4-(OC2H3)2]-5-Demethylyateins (10-d6): δH (CDCl3)
6.68 (1 H, d, J 8.3), 6.43–6.49 (2 H, m), 6.38 (1 H, d, J 2.0), 6.26
(1 H, d, J 1.7), 5.87–5.93 (2 H, m), 4.13 (1 H, dd, J 9.3 and 6.6),
3.85 (1 H, dd, J 9.3 and 7.0), 2.94 (1 H, dd, J 14.0 and 4.8), 2.80
(1 H, dd, J 13.9 and 7.1), 2.46–2.65 (4 H, m); m/z (EI) 392.1740
(Mϩ, 75.1%. C21H162H6O7 requires 392.1742), 391 (0.0), 390
(2.3), 389 (1.4), 388 (1.1), 387 (0.0), 386 (0.0), 256 (30.2), 243
(15.3), 211 (14.6), 173 (100), 161 (12.6), 135 (56.0), 131 (17.5).
( )-4-Methylthujaplicatins (12): δH(CDCl3) 6.79 (1 H, d,
J 8.0), 6.50 (1 H, dd, J 8.1 and 1.9), 6.47 (1 H, d, J 2.0), 6.37
(1 H, d, J 2.0), 6.27 (1 H, d, J 1.9), 4.14 (1 H, dd, J 9.0 and 6.8),
about 3.87 (1 H), 3.86 (3 H, s), 3.83 (3 H, s), 3.79 (3 H, s), 2.92
(1 H, dd, J 13.9 and 5.1), 2.83 (1 H, dd, J 13.9 and 6.8),
2.44–2.64 (4 H, m); m/z (EI) 388.1503 (Mϩ, 92.3%. C21H24O7
requires 388.1522), 167 (100), 149 (39.7), 137 (55.8), 131 (27.2),
122 (20.3).
( )-4,5-Dimethylthujaplicatins (5): δH (CDCl3) 6.74 (1 H, d,
J 8.0), 6.45 (1 H, dd, J 8.1 and 1.7), 6.37 (1 H, d, J 1.7), 6.26
(2 H, s), 4.12 (1 H, dd, J 9.0 and 7.3), 3.83 (1 H, dd, J 8.8 and
8.0), 3.75 (3 H, s), 3.74 (3 H, s), 3.74 (6 H, s), 2.88 (1 H, dd,
J 13.9 and 5.4), 2.83 (1 H, dd, J 14.2 and 6.6), 2.36–2.62 (4 H,
m); m/z (EI) 402.1696 (Mϩ, 100%. C22H26O7 requires 402.1679),
251 (13.4), 181 (92.9), 137 (45.0), 131 (11.3), 122 (11.5).
( )-[3,5-(OC2H3)2]-4,5-Dimethylthujaplicatins (5-d6): δH
(CDCl3) 6.80 (1 H, d, J 8.0), 6.52 (1 H, dd, J 8.0 and 2.0), 6.43
(1 H, d, J 2.0), 6.32 (2 H, s), 4.18 (1 H, dd, J 9.2 and 7.2), 3.88
(1 H, dd, J 9.0 and 7.6), 3.81 (3 H, s), 3.80 (3 H, s), 2.94 (1 H,
dd, J 13.9 and 5.4), 2.88 (1 H, dd, J 14.2 and 6.6), 2.42–2.67
(4 H, m); m/z (EI) 408.2037 (Mϩ, 75.1%. C22H202H6O7 requires
408.2055), 407 (0.0), 406 (0.0), 405 (0.0), 404 (0.0), 403 (0.8),
402 (0.0), 257 (8.0), 187 (100), 137 (35.5), 131 (10.2), 122 (6.0).
( )-[3,4-(OC2H3)2]Bursehernins (9-d6): δH(CDCl3) 6.76 (1 H,
d, J 8.1), 6.62–6.78 (3 H, m), 6.44 (1 H, d, J 8.1), 6.42 (1 H, s),
5.91–5.95 (2 H, m), 4.10 (1 H, dd, J 9.2 and 6.7), 3.84 (1 H, dd,
J 9.0 and 7.1), 2.95 (1 H, dd, J 14.0 and 5.1), 2.87 (1 H, dd,
J 14.0 and 7.0), 2.40–2.70 (4 H, m); m/z (EI) 376.1779 (Mϩ,
47.1%. C21H162H6O6 requires 376.1793), 375 (0.0), 374 (1.3), 373
(0.4), 372 (0.7), 371 (0.0), 370 (0.0), 240 (13.2), 214 (5.4), 161
(5.6), 157 (100), 135 (22.4).
( )-Yateins (11): δH(CDCl3) 6.68 (1 H, d, J 8.0), 6.45 (1 H, d,
J 8.1), 6.44 (1 H, s), 6.34 (2 H, s), 5.90–5.94 (2 H, m), 4.16 (1 H,
dd, J 9.1 and 7.2), 3.86 (1 H, dd, J 9.0 and 5.6), 3.81 (3 H, s),
3.81 (6H, s), 2.92 (1 H, dd, J 14.2 and 5.4), 2.87 (1 H, dd, J 13.9
and 6.4), 2.40–2.66 (4 H, m); m/z (EI) 400.1509 (Mϩ, 76.4%.
C22H24O7 requires 400.1522), 251 (11.2), 181 (100), 167 (10.5),
149 (12.4), 135 (28.6).
( )-4,5-Diethyl-5-hydroxypluviatolides (4,5-DiEt-7): δH
(CDCl3) 6.62 (1 H, d, J 8.5), 6.36–6.42 (2 H, m), 6.28 (1 H, d,
J 2.0), 6.27 (1 H, d, J 2.0), 5.85–5.88 (2 H, m), 4.08 (1 H, dd,
J 9.2 and 7.2), 3.96 (2 H, q, J 7.1), 3.95 (2 H, q, J 7.1), 3.80 (1 H,
dd, J 9.2 and 7.3), 3.74 (3 H, s), 2.87 (1 H, dd, J 13.9 and 5.1),
2.79 (1 H, dd, J 14.0 and 7.0), 2.46–2.58 (4 H, m), 1.35 (3 H, t,
J 7.0), 1.27 (3 H, t, J 7.1); m/z (EI) 428.1833 (Mϩ, 100%.
C24H28O7 requires 428.1835), 399 (12.0), 210 (33.9), 181 (38.6),
153 (30.6), 135 (59.4).
( )-[3-OC2H3]Thujaplicatins (3-d3): δH(CDCl3) 6.78 (1 H, d,
J 7.8), 6.50 (1 H, dd, J 8.0 and 2.0), 6.44 (1 H, d, J 1.7), 6.36
(1 H, d, J 2.0), 6.23 (1 H, d, J 1.7), 4.15 (1 H, dd, J 9.2 and 7.0),
3.88 (1 H, dd, J 9.2 and 7.2), 3.82 (3 H, s), 2.92 (1 H, dd, J 14.0
and 5.0), 2.81 (1 H, dd, J 14.0 and 6.9), 2.42–2.64 (4 H, m); m/z
(EI) 377.1557 (Mϩ, 76.8%. C20H192H3O7 requires 377.1554), 376
(4.1), 375 (4.1), 374 (1.4), 240 (14.2), 226 (10.6), 163 (14.1), 156
(100), 137 (98.5), 131 (25.8), 122 (20.5).
( )-5-Methylthujaplicatins (4): δH(CDCl3) 6.75 (1 H, d,
J 7.8), 6.45 (1 H, dd, J 7.9 and 1.8), 6.34 (1 H, d, J 1.7), 6.25
(2 H, s), 4.11 (1 H, dd, J 9.0 and 7.3), 3.82 (1 H, dd, J 9.2 and
7.4), 3.76 (6 H, s), 3.73 (3 H, s), 2.86 (1 H, dd, J 13.8 and 5.4),
2.80 (1 H, dd, J 13.9 and 6.6), 2.33–2.60 (4 H, m); m/z (EI)
388.1512 (Mϩ, 73.5%. C21H24O7 requires 388.1522), 173 (11.7),
167 (100), 149 (13.7), 137 (46.1), 131 (9.9), 122 (11.9).
( )-[3,5-(OC2H3)2]-5-Methylthujaplicatins (4-d6): δH(CDCl3)
6.79 (1 H, d, J 8.0), 6.51 (1 H, dd, J 8.1 and 1.9), 6.40 (1 H, d,
J 1.9), 6.30 (2 H, s), 4.17 (1 H, dd, J 9.1 and 7.2), 3.88 (1 H, dd,
J 9.1 and 7.4), 3.79 (3 H, s), 2.92 (1 H, dd, J 14.2 and 5.4), 2.87
(1 H, dd, J 14.2 and 6.8), 2.40–2.65 (4 H, m); m/z (EI) 394.1897
(Mϩ, 57.9%. C21H182H6O7 requires 394.1898), 393 (0.0), 392
(2.3), 391 (0.8), 390 (0.6), 389 (0.4), 388 (0.0), 173 (100), 149
(4.0), 137 (35.1), 131 (10.6), 122 (6.8).
( )-[3-OC2H3]Pluviatolides (6-d3): δH (CDCl3) 6.76 (1 H, d,
J 8.1), 6.52–6.65 (3 H, m), 6.34–6.42 (2 H, m), 5.83–5.87 (2 H,
m), 4.03 (1 H, dd, J 9.0 and 7.1), 3.77 (1 H, dd, J 10.4 and 7.1),
2.88 (1 H, dd, J 14.1 and 5.1), 2.80 (1 H, dd, J 14.2 and 6.8),
2.23–2.56 (4 H, m); m/z (EI) 359.1429 (Mϩ, 42.2%. C20H172H3O6
requires 359.1448), 358 (2.6), 357 (1.5), 356 (0.6), 223 (11.4),
162 (10.1), 140 (100), 135 (38.2), 131 (9.5), 122 (9.6).
( )-5-Hydroxypluviatolides (7): δH (CDCl3) 6.68 (1 H, d,
J 8.3), 6.45 (1 H, m), 6.44 (1 H, s), 6.37 (1 H, d, J 1.7), 6.26 (1 H,
d, J 1.4), 5.83–5.87 (2 H, m), 4.11 (1 H, dd, J 9.3 and 7.3), 3.84
(1 H, dd, J 9.0 and 6.8), 3.81 (3 H, s), 2.91 (1 H, dd, J 13.9 and
4.9), 2.80 (1 H, dd, J 13.9 and 6.8), 2.40–2.63 (4 H, m); m/z (EI)
372.1205 (Mϩ, 89.5%. C20H20O7 requires 372.1209), 236 (31.4),
191 (16.3), 161 (16.7), 153 (100), 149 (15.8), 135 (86.1), 131
(34.0), 122 (13.0).
( )-[3-OC2H3]-5-Hydroxypluviatolides (7-d3): δH (CDCl3)
6.68 (1 H, d, J 8.3), 6.44–6.48 (1 H, m), 6.45 (1 H, s), 6.37 (1 H,
d, J 1.7), 6.27 (1 H, d, J 1.7), 5.91–5.94 (2 H, m), 4.11 (1 H, dd,
J 9.3 and 6.8), 3.85 (1 H, dd, J 9.2 and 7.0), 2.92 (1 H, dd, J 14.0
and 5.0), 2.81 (1 H, dd, J 13.9 and 6.8), 2.42–2.64 (4 H, m); m/z
(EI) 375.1375 (Mϩ, 89.1%. C20H172H3O7 requires 375.1397), 374
(5.7), 373 (7.3), 372 (2.5), 239 (28.8), 194 (13.9), 161 (19.0), 156
(100), 149 (34.0), 135 (73.1), 173 (23.1), 122 (11.3).
( )-4-Demethylyateins (8): δH (CDCl3) 6.69 (1 H, d, J 7.6),
6.43–6.48 (2 H, m), 6.35 (2 H, s), 5.92–5.93 (2 H, m), 4.15 (1 H,
dd, J 9.3 and 7.3), about 3.87 (1 H), 3.85 (6 H, s), 2.89 (2 H, d,
J 6.1), 2.42–2.64 (4 H, m); m/z (EI) 386.1362 (Mϩ, 55.3%.
C21H22O7 requires 386.1366), 250 (13.8), 167 (100), 161 (8.8),
135 (32.7), 131 (8.9).
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( )-4-Ethyl-4-demethylyateins (4-Et-8): δH(CDCl3) 6.62 (1 H,
d, J 8.3), 6.36–6.42 (2 H, m), 6.29 (2 H, s), 5.85–5.87 (2 H, m),
4.09 (1 H, dd, J 9.1 and 6.9), 3.95 (2 H, q, J 7.1), 3.80 (1 H, dd,
J 9.3 and 7.6), 3.74 (6 H, s), 2.88 (1 H, dd, J 13.9 and 5.2), 2.80
(1 H, dd, J 13.9 and 6.8), 2.35–2.60 (4 H, m), 1.27 (3 H, t, J 7.1);
m/z (EI) 414.1660 (Mϩ, 100%. C23H26O7 requires 414.1678), 385
(16.8), 278 (7.1), 217 (6.2), 195 (34.8), 167 (86.5), 135 (66.0), 131
(10.5).
Feeding experiments
Administration of lignans to Anthriscus sylvestris: deuterium
labelled lignans (0.4 mg each), ( )-[3-OC2H3]matairesinols (2-
d3), ( )-[3-OC2H3]thujaplicatins (3-d3), ( )-[3,5-(OC2H3)2]-5-
methylthujaplicatins (4-d6), ( )-[3,5-(OC2H3)2]-4,5-dimethyl-
thujaplicatins (5-d6), ( )-[3-OC2H3]pluviatolides (6-d3), ( )-[3-
OC2H3]-5-hydroxypluviatolides (7-d3), ( )-[3,5-(OC2H3)2]-4-
demethylyateins (8-d6), ( )-[3,4-(OC2H3)2]bursehernins (9-d6),
and ( )-[3,4-(OC2H3)2]-5-demethylyateins (10-d6), were dissol-
ved individually in MeOH or DMSO (62 µl) and dispersed in
distilled water (438 µl). Young shoots (about 3 cm long) of A.
sylvestris were cut by means of scissors, and the cut end of each
shoot was placed directly in each solution of the deuterium
labelled compounds. Following uptake, each shoot was
metabolized for 3 d and freeze-dried. The resulting dried
material was hand-disintegrated using scissors and extracted
with hot MeOH. The MeOH extract was treated with β-glucos-
idase and was submitted to trimethylsilylation followed by GC-
MS analysis. The peaks on GC corresponding to the lignans (1)
(tR = 10.6 min), (2) (tR = 15.3 min), (3) (tR = 17.5 min), (4) (tR =
18.4 min), (5) (tR = 18.9 min), (6) (tR = 14.6 min), (7) (tR = 16.6
min), (8) (tR = 17.4 min), (9) (tR = 15.2 min), (10) (tR = 16.1 min),
(11) (tR = 18.3 min), and (12) (tR = 16.9 min) were analyzed for
13C or deuterium incorporation.
In separate experiments, individual administration of ( )-[3-
OC2H3]secoisolariciresinols (1-d3), ( )-[3-OC2H3]matairesinols
(2-d3), ( )-[3-OC2H3]pluviatolides (6-d3), and ( )-[3,4-
(OC2H3)2]bursehernins (9-d6) was conducted exactly as above
but scaled up proportionately ten times using ten shoots and
the same concentration of the solutions. Following uptake, the
shoots were metabolized for 7 d and then freeze-dried and
extracted with hot MeOH. The MeOH extracts were treated
with β-glucosidase and the reaction products were extracted
with AcOEt as above.
The AcOEt extracts obtained after the administration of ( )-
[3-OC2H3]pluviatolides (6-d3) and ( )-[3,4-(OC2H3)2]burse-
hernins (9-d6) were treated with an excess of diazoethane
followed by TLC separation (solvents: appropriate mixtures
of AcOEt–CHCl3) to give the following two fractions: Fr. 1
corresponding to 4-demethylyatein (8) and 5-demethylyatein
(10), and Fr. 2 corresponding to 5-hydroxypluviatolide (7) and
yatein (11). The whole of each fraction was then dissolved in
14 µl of BSA for 45 min at 60 ЊC44 and analyzed by GC-MS. At
the retention times corresponding to the authentic ethylated
lignans ( )-4,5-diethyl-5-hydroxypluviatolides (4,5-DiEt-7) (tR
= 18.6 min), ( )-4-ethyl-4-demethylyateins (4-Et-8) (tR = 18.5
min), ( )-[3,4-(OC2H3)2]-5-ethyl-5-demethylyateins (5-Et-10-d6)
(tR = 18.3 min), and ( )-yateins (11) (tR = 18.3 min), mass spec-
tra were analyzed for deuterium incorporation.
The AcOEt extracts so obtained following administration of
( )-[3-OC2H3]secoisolariciresinols (1-d3) and ( )-[3-OC2H3]-
matairesinols (2-d3) were submitted individually to TLC separ-
ation (solvents: appropriate mixtures of AcOEt–CHCl3) to give
the following three fractions: Fr. 1 corresponding to pluviato-
lide (6), 4-demethylyatein (8), bursehernin (9), 5-demethylyatein
(10), and yatein (11); Fr. 2 corresponding to matairesinol (2),
5-methylthujaplicatin (4), 4,5-dimethylthujaplicatin (5),
5-hydroxypluviatolide (7), and 4-methylthujaplicatin (12); and
Fr. 3 corresponding to secoisolariciresinol (1) and thujaplicatin
(3). The whole of each fraction was then dissolved in 14 µl of
BSA for 45 min at 60 ЊC44 and subjected to GC-MS analysis to
determine deuterium incorporation into lignans.
( )-[3,4-(OC2H3)2]-5-Ethyl-5-demethylyateins (5-Et-10-d6):
δH(CDCl3) 6.63 (1 H, d, J 8.3), 6.37–6.43 (2 H, m), 6.28 (1 H, d,
J 2.0), 6.26 (1 H, d, J 2.0), 5.85–5.88 (2 H, m), 4.09 (1 H, dd,
J 9.1 and 7.2), 3.97 (2 H, q, J 7.0), 3.80 (1 H, dd, J 9.0 and 7.6),
2.85 (1 H, dd, J 13.9 and 5.1), 2.80 (1 H, dd, J 13.9 and 6.3),
2.36–2.65 (4 H, m), 1.36 (3 H, t, J 7.1); m/z (EI) 420.2047 (Mϩ,
100%. C23H202H6O7 requires 420.2055), 419 (0.0), 418 (0.0), 417
(0.0), 416 (1.0), 415 (1.0), 414 (0.0), 284 (12.3), 201 (92.0), 173
(30.9), 135 (46.9) 131 (12.4).
Synthesis of dibenzylbutane lignans. ( )-[3-OC2H3]Secoiso-
lariciresinols (1-d3) and ( )-[7,7-2H2, 3-OC2H3]secoisolaricires-
inols (1-d5) were prepared by reduction of ( )-[3-OC2H3]-
matairesinols (2-d3) and ( )-[7,7-2H2, 3-OC2H3]matairesinols
(2-d5), respectively, as previously.44 (1-d3): δH (CDCl3) 6.79 (2 H,
d, J 7.8), 6.55–6.65 (4 H, m), about 3.82 (2 H), 3.80 (3 H, s), 3.54
(2 H, dd, J 11.3 and 4.3), 2.73 (2 H, dd, J 13.7 and 8.1), 2.63 (2
H, dd, J 13.7 and 6.2), 1.85 (2 H, m); m/z (EI) 365.1935 (Mϩ,
18.0%. C20H232H3O6 requires 365.1917), 364 (2.9), 353 (3.3), 362
(2.7), 347 (11.8), 192 (8.6), 189 (9.2), 140 (100), 137 (98.7), 122
(19.4). (1-d5): δH (CDCl3) 6.73 (2 H, d, J 8.0), 6.50–6.58 (4 H,
m), about 3.75 (2 H), 3.74 (3 H, s), 3.48 (2 H, dd, J 11.5 and
4.4), 2.67 (1 H, dd, J 13.8 and 8.2), 2.57 (1 H, dd, J 13.8 and
6.7), 1.78 (2 H, m); m/z (EI) 367.2031 (Mϩ, 27.1%. C20H212H5O6
requires 367.2043), 366 (3.6), 365 (6.1), 364 (3.1), 363 (8.7), 362
(1.3), 349 (14.8), 194 (7.6), 189 (6.8), 142 (99.2), 137 (100), 122
(11.2).
Chemicals
-[ring-13C6]phenylalanine (99 atom % 13C),C2H3I (99.5 atom%
2
2H), and LiAl2H4 (98 atom% H) were purchased from ICON,
Aldrich, and Aldrich, respectively. All chemicals used were
reagent grade unless otherwise stated.
Lignans in Anthriscus sylvestris
Young shoots of Anthriscus sylvestris were harvested and
washed sequentially with tap and distilled water. The plant
materials thus obtained (2.4 g) were frozen (liquid N2), freeze-
dried, disintegrated with scissors, and then extracted with hot
MeOH (16 ml). The MeOH extract was treated with β-glucos-
idase (from almonds, SIGMA G-0395, 68 units in 5.0 ml of
NaOAc buffer at pH 5.0) for 24 h at 37 ЊC. The reaction mixture
was extracted with AcOEt (3.0 ml × 3), and the solvent was
evaporated off. The AcOEt extracts (16.3 mg) thus obtained
were submitted to TLC separation (solvents: appropriate
mixtures of AcOEt–CHCl3) to afford 3 fractions. An aliquot of
each fraction was subjected to GC-MS analysis after heating
with 14 µ l of N,O-bis(trimethylsilyl)acetamide (BSA) for 45
min at 60 ЊC44 so that lignans present in the fractions were
identified. In a separate experiment, freeze-dried, aerial parts
(including leaves, petioles and racemes) of A. sylvestris (39.8 g)
were pulverized and extracted with hot MeOH as above but
scaled up proportionately. The MeOH extract (17.3 g) thus
obtained was treated with β-glucosidase and the reaction
products were extracted with AcOEt. The AcOEt extract (1.2 g)
was subjected to repeated silica gel column and thin layer
chromatography to afford pure yatein (11) (90.2 mg) and
deoxypodophyllotoxin (17.9 mg).
Administration of -[ring-13C6]phenylalanine: -[ring-13C6]-
phenylalanine (1.0 mg) was dissolved in 0.1 M KPB buffer (pH
7.0, 217.0 µl), and administered to ten young shoots. Following
uptake and metabolism for 7 d, the plant materials were sub-
mitted to GC-MS analysis after TLC purification exactly as
above. Simultaneous administration of two distinct lignans: A
solution of a mixture of ( )-[3-OC2H3]pluviatolides (6-d3) (2.0
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mg) and ( )-[7,7-2H2, 3-OC2H3]secoisolariciresinols (1-d5) (2.0
mg) in a mixture of DMSO (620 µl) and distilled water (4380 µl)
was administered to ten young shoots of A. sylvestris. Follow-
ing uptake and metabolism for 7 d, the plant materials were
treated with β-glucosidase and were submitted to GC-MS
analysis after TLC purification exactly as above. Simultaneous
administration of ( )-[3-OC2H3]thujaplicatins (3-d3) and ( )-
[7,7-2H2, 3-OC2H3]secoisolariciresinols (1-d5) were conducted
exactly as above.
19 W.-J. Hu, S. A. Harding, J. Lung, J. L. Popko, J. Ralph, D. D. Stokke,
C. J. Tsai and V. L. Chiang, Nat. Biotechnol., 1999, 17, 812.
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26 S. Suzuki, N. Sakakibara, T. Umezawa and M. Shimada, J. Wood
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Acknowledgements
27 Y.-H. Leem, M.-J. Lim, D.-H. Shin, H.-B. Chang, S.-W. Hong,
E.-Y. Moon, D.-K. Lee, S.-J. Yoon and W.-S. Woo, Arch Pharm Res.,
1999, 22, 208.
This research was partly supported by Grants-in-Aid for
Scientific Research (Nos. 1066016666, 12660150) and the
Encouragement for Young Scientists (No. 4542) from the
Ministry of Education, Culture, Sports, Science and Tech-
nology of Japan; and by a grant from the New Energy and
Industrial Technology Development Organization. The authors
thank Jacqueline Leshkevich for valuable comments on English
writing.
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33 T. Okunishi, N. Sakakibara, S. Suzuki, T. Umezawa and
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