Biosynthesis of antimalarial lignans from Holostylis reniformis

Article abstract of DOI:10.1016/j.phytochem.2009.02.008

Holostylis reniformis biosynthesizes 8-8′ linked lignans without 9,9′-oxygenation. To elucidate the biosynthetic pathways to these lignans, the reputed precursors [U-¹⁴C]phenylalanine, [9-³H₁]coniferyl alcohol, and [9-

Full text of DOI:10.1016/j.phytochem.2009.02.008

Phytochemistry 70 (2009) 590–596
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Biosynthesis of antimalarial lignans from Holostylis reniformis
*
Gisele B. Messiano, Tito da Silva, Isabele R. Nascimento, Lucia M.X. Lopes
Instituto de Química, São Paulo State University, UNESP, C.P. 355, 14801-970 Araraquara, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Holostylis reniformis biosynthesizes 8-80 linked lignans without 9,90-oxygenation. To elucidate the biosyn-
thetic pathways to these lignans, the reputed precursors [U-14C]phenylalanine, [9-3H1]coniferyl alcohol,
and [9-3H1]isoeugenol were administered to roots of the plant, which led to the incorporation of 3H and
14C into ten 2,70 linked-lignans (aryltetralone lignans) and two 7,70-epoxylignans (furan lignans). These
administration experiments demonstrated that the lignans were propenylphenol-derived and that H.
reniformis can exhibit regioselective control over radical–radical coupling (via isoeugenol radicals). Reg-
iospecific control over propenylphenol-derived lignan biosynthesis was observed, together with diaste-
reoselective control of C2–C70 bond formation for the aryltetralone lignans (70R). These experiments
provide evidence that isoeugenol is a biosynthetic intermediate to the aryltetralone and furan lignans.
Ó 2009 Elsevier Ltd. All rights reserved.
Article history:
Received 16 October 2007
Received in revised form 27 June 2008
Available online 21 March 2009
Keywords:
Holostylis reniformis
Aristolochiaceae
Lignans
Neolignans
Aryltetralone lignans
Epoxylignans
Biosynthesis
Propenylphenols
1. Introduction
Activation of the side-chain alcohol of p-coumaryl and coniferyl
alcohols, e.g. via esterification, to form a more facile leaving
Lignans are a very structurally diverse class of vascular plant
natural products. They are typically dimers and/or higher oligo-
mers (Moss, 2000). Lignans vary substantially in oxidation level,
substitution pattern, and the chemical structure of their basic car-
bon framework. In addition to structural diversity, lignans show
considerable diversity in terms of enantiomeric composition, bio-
synthesis, and phylogenetic distribution (Umezawa, 2003). Based
on structural (chemotaxonomical) differences, lignans can be read-
ily separated into distinct coupling-type subclasses (Gottlieb,
1978; Umezawa, 2003; Davin and Lewis, 2005). An examination
of their corresponding structures and distribution in nature illus-
trates the existence of a large number of distinct phenoxyl radi-
cal–radical coupling modes, including those that are either
stereoselective and/or regiospecific in coupling origin (Davin and
Lewis, 2005). The first demonstration of phenoxyl radical–radical
coupling control was reported during the investigation of (+)-
pinoresinol formation from coniferyl alcohol in Forsythia species
(Paré et al., 1994; Davin et al., 1997). The dirigent protein was pro-
posed to bind and orient coniferyl alcohol-derived radicals in such
a way as to enable 8,80 coupling at the si–si face with subsequent
intramolecular cyclization to afford (+)-pinoresinol (Davin and Le-
wis, 2005).
group via reductive elimination has been demonstrated (Koeduka
et al., 2006; Vassão et al., 2006). This was shown to be the case
using p-coumaryl/coniferyl esters as potential substrates for a
NAD(P)H-dependent reductase to afford chavicol and eugenol.
Lignans present in Larrea tridentata demonstrate an additional
means of control of radical–radical coupling. Instead of being
of monolignol origin, these lignans are derived from propenyl/
allylphenols (presumably from anol) (Davin and Lewis, 2003,
2005). Pathways for the formation of aryltetralin lignans with
oxygenation at C-9,90 have been proposed. The overall pathway
for the formation of podophyllotoxin, for example, has been
shown to involve coniferyl alcohol, (+)-pinoresinol, (+)-mataires-
inol, yatein, and deoxypodophyllotoxin (Seidel et al., 2002; Fuss,
2003).
Lignans (1–19) and neolignans (20 and 21) have been iso-
lated from the roots of H. reniformis (da Silva and Lopes,
2004, 2006; Andrade-Neto et al., 2007) (Fig. 1). Of these, lignans
6, 12, 13, and 15 have attracted much interest due to their
antiplasmodial activity (da Silva et al., 2004; Andrade-Neto
et al., 2007). All of these lignans and neolignans are apparently
derived from propenylphenols and lack the oxygenated func-
tionalities at C-9,C-90 which are characteristic of many other
phenylpropanoids.
The biosyntheses of chavicol and eugenol have been shown to
occur via the phenylpropanoid pathway to p-coumaryl alcohol.
The aim of this work was to contribute to the elucidation of the
biosynthetic pathways of lignans without oxygenation at C-9,C-90
especially those from H. reniformis, by in vivo conversion of phenyl-
propanoid intermediates into aryltetralone lignans.
* Corresponding author. Tel.: +55 16 3301 6663; fax: +55 16 3301 6692.
E-mail address: lopesxl@iq.unesp.br (L.M.X. Lopes).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.02.008

G.B. Messiano et al. / Phytochemistry 70 (2009) 590–596
591
O
O
O
R1O
RO
H3CO
RO
R1O
R2
RO
OR3
OCH3
OCH3
OR2
OR3
OR4
R2
R
R1
R3 R4
CH3 CH3 OH CH3 CH3
R
H
R
H
R1 R2
CH2 CH2
CH3 CH3 CH3
CH2
R3
1
2
3
4
5
6
7
8
9
14
15
10
11
12
13
H
CH3
CH3 CH3
CH2
CH3 CH3
CH3
H
H
H
H
H
H
H
H
H
CH3
CH3 CH3
CH3
CH2
H
CH
3
CH3 CH3
H
CH3 CH3 CH3 CH3
CH2
H
CH3
CH2
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
OH
H3CO
H3CO
H3CO
H3CO
O
OCH3
OCH3
O
O
OCH3
OCH3
OCH3
18
OCH3
16
17
O
O
O
O
H3CO
HO
H
O
O
O
O
O
H3CO
H3CO
O
O
O
OCH3
OCH3
19
20
21
Fig. 1. Chemical structures for compounds 1–21.
The best result of recovered activity was observed for acetone
extract using [9-3H1]isoeugenol 23 (75.4%) followed by
[9-3H1]coniferyl alcohol 22 (11.0%). While practically all
[9-3H1]coniferyl alcohol 22 was incorporated into lignans in the ex-
2. Results and discussion
Preliminary in vivo experiments using the potential radiola-
beled precursor [9-3H1]isoeugenol 23 were carried out at two dif-
ferent durations (4 and 12 h) to identify satisfactory conditions
for uptake and metabolism into lignans. After uptake, 9, the most
abundant aryltetralone lignan in acetone extracts (Fig. 2), was iso-
lated by semi-preparative HPLC. Incorporation of the radiolabel
into lignan 9 increased slightly with an increase in the duration
of metabolism. Therefore, 12 h was chosen for the experiments
using the three potential precursors [U-14C]phenylalanine,
[9-3H1]coniferyl alcohol 22, and [9-3H1]isoeugenol 23 and for the
‘‘blank”/control experiment (Table 1). To verify if the radioactivity
shown by the respective extracts obtained after uptake was due to
the incorporation of labeled precursor into lignans, the extracts
were subjected to semi-preparative HPLC, and the radioactivity
of the isolated lignans was individually measured (Tables 2–4).
Radiolabel was present in 10 aryltetralone lignans (2, 4–10, 12,
and 13) and two furan lignans (18 and 19), which were identified
by comparison of their Rt, ESI-MS, and 1H NMR spectra with those
of the lignans from the blank/control experiment as well as those
of authentic samples previously isolated from this plant (da Silva
and Lopes, 2004, 2006; Andrade-Neto et al., 2007).
9
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.0
12
8
4+7
13
0.5
0.0
2
6
5
10
19
18
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Minutes
Time (min)
Fig. 2. Representative chromatogram of the acetone extract from the roots of H.
reniformis. Peaks corresponding to the radiolabeled lignans are shown (C18,
150 ꢀ 3.9 mm, UV detection at 254 nm, and flow rate: 0.8 ml minꢁ1).

592
G.B. Messiano et al. / Phytochemistry 70 (2009) 590–596
Table 1
Activities of precursors and extracts from H. reniformis.
Radiolabeled precursor
[U-14C]Phenylalanine
Administered activity (Bq)
Extract code
Extract mass (mg)
Extract activity (Bq)
Yielda (%)
1.42 ꢀ 106
9.63 ꢀ 106
1.28 ꢀ 105
II-Hexane
II-Acetone
III-Hexane
III-Acetone
IV-Hexane
IV-Acetone
I-Hexane
36.8
546.7
20.9
399.6
39.8
429.0
32.7
6.25 ꢀ 102
1.72 ꢀ 104
1.20 ꢀ 104
1.06 ꢀ 106
4.75 ꢀ 103
9.65 ꢀ 104
0.04
1.21
0.12
11.01
3.71
75.39
[9-3H1]Coniferyl alcohol 22
[9-3H1]Isoeugenol 23
–
I-Acetone
929.6
a
Percentage calculated in relation to total administered radioactivity (Bq).
Table 2
Lignans from in vivo [U-14C]phenylalanine administration experiment.
tract, the incorporation of [9-3H1]isoeugenol 23 into lignans repre-
sented only 8.7% of extract activity. Although the recovered activity
for [U-14C]phenylalanine was low (1.2%) in the extract, the frac-
tions that contained lignans contributed to 77.7% of extract activ-
ity. The yield of 77.7% was calculated considering the total lignan
activity (2.71 ꢀ 103 Bq, Table 2) in 110.0 mg, and the extract activ-
ity (1.72ꢀ104 Bq, Table 1) in 546.7 mg. Nonetheless, since phenyl-
alanine and coniferyl alcohol 22 may be biosynthetic precursors of
many different metabolites in H. reniformis, their activity decrease
in the extracts could be related to other biosynthetic pathways.
Thus, administration of [U-14C]phenylalanine to the roots of H.
reniformis resulted in its conversion into the [14C]aryltetralone (2,
5, 6, 8–10, 12, and 13) and [14C]furan (18) lignans, whereas admin-
istration of [9-3H1]coniferyl alcohol 22 and [9-3H1]isoeugenol 23
led to [9,90-3H1]lignans (2, 4–10, 12, 13, 18, and 19). These results
suggest the following biosynthetic sequence: phenylalanine,
coniferyl alcohol (and coniferyl ester), and isoeugenol for lignan
formation in H. reniformis (Fig. 3), which is in accordance with that
established for eugenol, isoeugenol, and anol (Reichling and
Martin, 1990; Kota et al., 2004; Koeduka et al., 2006).
Fractiona
Mass (mg)
Compound
Activity (Bq)
Yieldb (%)
II-3
II-4
II-5
II-6
II-7
II-8
II-9
II-10
II-11
Total
9.3
17.5
4.9
7.1
3.5
8.2
9.6
6.1
4.3
70.5
2
9
1.70 ꢀ 103
0.59
0.02
0.11
0.05
0.03
0.05
0.05
0.03
0.01
0.94
65
13
315
140
80
147
154
85
c
8
6
12
5
10 + 18
25
2.71 ꢀ 103
a
Fractions obtained from the acetone extract (110.0 mg) by HPLC.
Percentage calculated in relation to total administered radioactivity (Bq).
Mixture of lignans.
b
c
Table 3
Lignans from in vivo [9-3H1]coniferyl alcohol 22 administration experiment.
Fractiona
Mass (mg)
Compound
Activity (Bq)
Yieldb (%)
Rodríguez-Evora and Schepp (2005) observed that isoeugenol
radical cations undergo rapid deprotonation, as well as rapid
reprotonation under moderately acidic conditions in a non-protic,
polar solvent. They also suggested that in a biological system, iso-
eugenol radical cations need not undergo a dimerization event as
soon as they are produced by enzymatic oxidation; instead, the
radical cations can be ‘‘stored” in their less-reactive 2-methoxy-
4-vinylphenoxyl radical form, and then regenerated by enzymatic
protonation when dimerization is ready to proceed. It seems that
all of the lignans from H. reniformis have a C8–C80 bond as a result
of isoeugenol radical dimerization. All of the aryltetralone lignans
have a 70R configuration. Thus, H. reniformis lignans should result
from regiospecific 8-80 coupling, and the corresponding
diastereoisomeric hydroxylated intermediates can still be diaste-
reospecifically metabolized to produce either aryltetralol or furan
lignans. Furthermore, considering the principle of the lower molec-
ular movement to reach the transition state to form these lignans,
from the 10 possible isomers (where the reactive species approach
each other from their re–re, re–si, si–re or si–si faces), only seven
(re–re, si–re and re–si faces) could lead to the desirable 70R config-
uration. In addition, the participation of dirigent protein in the for-
mation of intermediate ‘‘diarylbutanes”, which in turn could give
rise to aryltetralol lignans, such as 16, by attack of radical or anion
hydroxyl on the exo faces of quinone methide, or attack on the endo
faces of quinone methide to yield furan lignans, should be consid-
ered. Thus, the number of the possible isomers could be reduced to
three (24–26, Fig. 3). For tetralol lignan formation, an anti-peripla-
nar addition at the double bonds in the quinone methides would
result in asymmetric induction in the formation of up to two addi-
tional chiral centers (C-70 and C-7). Therefore, independent of
the mechanism, whether it involves radical or carbocation
intermediates (Fig. 4), it is important that the hydroxyl group be
III-2
III-3
III-4
III-5
III-6
III-7
III-8
III-9
III-10
III-11
Total
4.3
19.7
4.4
2.7
10.1
5.5
12.2
6.6
5.4
2.0
72.9
2 + 4 + 7
9
2.80 ꢀ 105
3.05 ꢀ 103
1.05 ꢀ 103
1.02 ꢀ 103
1.67 ꢀ 103
1.04 ꢀ 103
2.00 ꢀ 103
4.83 ꢀ 102
1.65 ꢀ 103
1.66 ꢀ 102
2.92 ꢀ 105
10.57
0.12
0.04
0.04
0.06
0.04
0.08
0.02
0.06
0.01
11.04
13
c
8
6
12
5
10 + 18
19
a
Fractions obtained from the acetone extract (110.0 mg) by HPLC.
Percentage calculated in relation to total administered radioactivity (Bq).
Mixture of lignans.
b
c
Table 4
Lignans from in vivo [9-3H1]isoeugenol 23 administration experiment.
Fractiona
Mass (mg)
Compound
Activity (Bq)
Yieldb (%)
IV-3
IV-4
IV-5
IV-6
IV-7
IV-8
IV-9
IV-10
IV-11
IV-12
IV-13
Total
5.4
2 + 4 + 7
2
9
1.98 ꢀ 103
78.20
20.0
12.49
7.32
11.17
11.70
9.67
6.04
0.24
0.06
0.04
0.02
0.03
0.04
0.03
0.01
0.02
0.03
6.56
10.4
15.2
2.8
2.7
7.0
1.4
7.5
3.9
2.3
13
c
8
6
12
5
10 + 18
19
4.67
8.00
5.8
64.4
9.50
2.15 ꢀ 103
a
Fractions obtained from the acetone extract (110.0 mg) by HPLC.
Percentage calculated in relation to total administered radioactivity (Bq).
Mixture of lignans.
b
c

G.B. Messiano et al. / Phytochemistry 70 (2009) 590–596
593
COOH
NH2
CHTOH
CH2T
HO
HO
OCH3
OCH3
22
23
Regiospecific
coupling
in vivo
R
H3CO
O
H3CO
O
H3CO
S
R
S
S
CH2T
H
H
H
re
CH2T
CH2T
O
H
H
si
H
H
H-OH
H
CH2T
H
CH2T
H
re
CH2T
R
O
O
O
H
H
si
H3CO
H3CO
H3CO
isomer 24
isomer 25
isomer 26
trans-cis
trans-trans
cis-trans
Intramolecular cyclization (a)
TH2C
Intramolecular cyclization (a)
TH2C
CH2T
CH2T
O
O
HO
H3CO
OH
HO
H3CO
OH
OCH3
OCH3
19*
18*
Fig. 3. Proposed biochemical pathways to lignans from [U-14C]phenylalanine, [9-3H1]coniferyl alcohol, and [9-3H1]isoeugenol in H. reniformis roots. (*Radiolabeled lignans.
aPossible mechanisms, which depend on the hydroxyl species attack and protonation positions as shown from intermediate 26, for the conversion of quinone methides into
epoxy lignans.)
anti-periplanar to the new C2–C70 bond, so that it could lead to
intramolecular stereoselective cyclization. Hence, it seems that
the preferential configurations for the OH attack are the re faces.
For furan lignans, such as 18 and 19, it is important that the inter-
mediate can adopt a conformation in which intramolecular cycliza-
tion takes place without great changes in the conformation.
Therefore, the biosynthesis of all of these lignans should involve
enzymes and/as proteins that are capable of stereospecifically
interacting with units of trans–trans (24) and trans–cis (26) isomers
with an 8S configuration. The former give rise to 70R,8S,80S (2–9, 16,
and19)and 70R,8R,80S(1)lignans, fromwhichthecorrespondingaryl-
tetralonelignans (7 and 9) could give lignans with a 70R,8R,80S (14and
15, respectively) configuration via enolization; while 70R,8S,80R lign-
ans (10–13 and 18) could be obtained from 26 (Figs. 3 and 4).
As previously stated, administration of the hypothetical precur-
sors to the plant roots resulted in the incorporation of 3H and 14C
into 2,70-lignans (2, 4–10, 12, and 13, aryltetralone lignans) and
7,70-epoxylignans (18 and 19, furan lignans). Although 9 was the
lignan isolated in highest concentration from the acetone extracts,
the highest yields (calculated in relation to administered activity of
precursor) were observed for fractions containing 2 either alone or
in mixture with 4 and 7 (Tables 2–4). Quantification of these com-
pounds was not quite accurate because their retention times were
very similar and thus their peaks overlapped with that of 2. In fact,
considering the amounts of extracts subjected to semi-preparative
HPLC, the production of 2 + 4 + 7 (ꢂ14%) was substantially higher in
the acetone extract from [9-3H1]isoeugenol than in the ‘‘blank” ace-
tone extract (ꢂ8%). Considering these results and the B ring stereo-
chemistry of these lignans, the most feasible biosynthetic pathway
for them seems to involve intermediate 24. These administration
experiments thus support the pathways proposed by Cho et al.
(2003) and Vassão et al. (2007). Instead of originating from mono-
lignol coupling, these lignans should be derived from propenylphe-
nols, which agrees with the Gottlieb hypothesis (Gottlieb, 1978)
that allyl- and/or propenylphenols are biosynthetic precursors of
lignans with absence of oxygen at C-9,C-90 (called neolignans at
that time). These results also agree with in vivo feeding experiments
using L. tridentata (Davin and Lewis, 2003). However, the aryltetra-
lol lignan intermediate that gives aryltetralone differs from the re-
sults obtained in biosynthetic studies on podophyllotoxin, which
involve the aryltetralin deoxypodophyllotoxin. If a similar pathway
was followed by lignans in H. reniformis, then an anti-periplanar hy-
dride attack on the re face, followed by intramolecular cyclization,
should be proposed. However, in this case the loss of the stability
of the intermediate carbocation (29) by the unshared electron pair
of the oxygen would be lost. Besides, the absence of aryltetralin
lignans is not in favor of this pathway in this plant.
3. Conclusions
Holostylis reniformis exhibits control over radical–radical cou-
pling (isoeugenol radical). This control should be regioselective
since the plant contains an abundant group of lignans (aryltetr-
alone) that have been shown to be exclusively derived from 8-80
coupling. Regiospecific control over propenylphenol-derived lig-
nan biosynthesis was observed, together with the diastereoselec-
tive control of C2–C70 bond for aryltetralone lignans (70R). These
controls suggest that an anti-periplanar OH group could promote
stereoselectivity for aryltetralol formation, and a syn-periplanar
orientation could lead exclusively to formation of furan lignans.

594
G.B. Messiano et al. / Phytochemistry 70 (2009) 590–596
24
25
26
1) H2O (Mechanism b or c)
2) Oxidation
H2O
(b)
(c)
HO-
HO-
O
O
H3CO
HO+
H3CO
H
H3CO
CH2T
CH2T
CH2T
H
H3CO
HO
CH2T
CH2T
O
H
H
HO
H
H
CH2T
H
H
HO+
CH2T
CH2T
OCH3
O
OCH3
OH
OH
O
H3CO
H3CO
28
27
Dienone-phenol
rearrangement
10*, 12* and 13*
H3CO
HO
H3CO
HO
HO
OH
H
H
H
CH2T
+
CH2T
H3CO
HO
CH2T
CH2T
H
CH2T
H
H
CH2T
O
2* and 4-9*
OCH3
HO
H
29
OCH3
H3CO
OH
OH
H3CO
HO
CH2T
Oxidation
Intramolecular
cyclization
CH2T
OCH3
OH
Fig. 4. Proposed biochemical pathways to aryltetralol and aryltetralone lignans from 24–26 intermediates in H. reniformis roots. (*Radiolabeled lignans. b,cPossible
mechanisms, which depend on the hydroxyl species attack and protonation positions on 27 and 28, for the conversion of quinone methides into aryltetralol lignans.)
specimen (ESA 88282) was deposited at the herbarium of the Esco-
la Superior de Agricultura, Luiz de Queiroz (ESALQ), Piracicaba, SP,
Brazil. The cultivated plants were collected in February, 2006.
4. Experimental
4.1. General experimental procedures
One-dimensional (1H, 13C, and gNOESY) and two-dimensional
(1H-1H gCOSY, gHMQC, gHMBC, and gNOESY) NMR experiments
were recorded on a Varian INOVA 500 spectrometer (11.7 T) at
500 MHz (1H) and 126 MHz (13C), with the solvents used as inter-
nal standards. Mass spectra (LC–ESI-MS) were obtained on a Fisons
Platform II, and flow injection into the electrospray source was
used for LC–ESI-MS. LC analyses were carried out using C18
(MeOH–H2O 7:3). HPLC analyses were carried out using a Shima-
dzu liquid chromatograph 10 Avp equipped with a UV–vis detec-
tor. Columns were RP18 (Shimadzu, C18, 150 ꢀ 3.9 mm, and flow
rate: 0.8 ml minꢁ1 for analytical analysis and 250 ꢀ 20 mm, and
flow rate: 8 ml minꢁ1 for semi-preparative analysis), and chro-
matograms were acquired at 254 and 280 nm. Optical rotations
were measured on a Perkin Elmer 341-LC polarimeter. Thin-layer
chromatography (TLC): silica gel 60 PF254. Radioactive samples
were analyzed in 2 ml biodegradable counting scintillant (Amer-
4.3. Materials
[U-14C]L-Phenylalanine and NaB3H4 were purchased from Per-
kin Elmer Life. All of the other reagents were purchased from Al-
drich Chemical Co., Sigma Chemical Co., or Baker Analysed, and
used without purification, except where noted otherwise. All of
the yields refer to chromatographically and spectroscopically (1H
and 13C NMR) homogenous material unless otherwise stated.
Radioactivity decontaminant was purchased from SigmaÒ/Sigma-
cleanÒ. All solvents, either HPLC- or reagent-grade, were purchased
from Mallinckrodt Baker.
4.4. Chemical syntheses
All reactions were monitored by TLC analyses using UV and 10%
H2SO4 – heat as developing agents. All reactions were carried out
under an argon atmosphere with dry freshly distilled solvents un-
der anhydrous conditions unless otherwise noted.
sham Biosciences) and measured using
21000TR liquid scintillation counter.
a Packard Tri-carb
4.4.1. [9-3H1]Coniferyl alcohol (22)
4.2. Plant material
[9-3H1]Coniferyl alcohol (22) was prepared based on a proce-
dure for obtaining coniferyl alcohol 22 previously described by
Nascimento et al. (2000). To coniferyl aldehyde (256.0 mg,
1.44 mmol) dissolved in MeOH (10 ml) in an ice-bath was added
Seedlings of H. reniformis were grown in the garden at the
Chemistry Institute, UNESP, SP, Brazil, using a commercial garden-
ing soil mixture. The seeds were from plants collected in Ituiutaba,
MG, Brazil, and were identified as H. reniformis Duch. by Dr. Con-
dorcet Aranha and by Dr. Lindolpho Capellari Júnior. A voucher
NaB3H4 (4.0 mg, 0.11 mmol, a = 7.55 GBq mmolꢁ1
)
along with
MeOH (5 ml). The resulting reaction mixture was stirred for

G.B. Messiano et al. / Phytochemistry 70 (2009) 590–596
595
30 min under an argon atmosphere, after which NaBH4 (238.0 mg,
6.29 mmol) was added and the mixture was stirred for an addi-
tional 30 min. Subsequently, MeOH (5 ml), H2O (10 ml), and a satd.
soln. of NH4Cl (3 ml) were added and the soln. was extracted with
EtOAc (3 ꢀ 10 ml). The combined organic phase was washed with
H2O (5 ml), dried (dry Na2SO4), and concentrated to yield 22
(255.1 mg, 98.0%, a = 1.83 ꢀ 107 Bq mmolꢁ1).
(12.3 mg), 6 (4.7 mg), 12 (14.0 mg), 18 (5.7 mg), and 19 (3.2 mg),
respectively. This feeding procedure was also carried out using
[9-3H1]isoeugenol, for 4 h and 12 h after uptake, and this was fol-
lowed by isolation and determination of the activity of 9 (a =
4.50 ꢀ 102 Bq mmolꢁ1 and 4.83 ꢀ 102 Bq mmolꢁ1, respectively).
Lignans 2, 4, 6–9, 12, 13, 18, and 19 obtained from I-acetone ex-
tract were identified by comparison of their
aD, Rt (Fig. 2), and
spectroscopic data (LC–ESI-MS, 1H and 13C NMR) with those of
authentic samples (da Silva and Lopes, 2004, 2006; Andrade-Neto
et al., 2007), whereas labeled lignans (2, 4–10, 12, 13, 18, and 19)
were identified by comparison of their Rt, 1H NMR, and LC–ESI-
MS data with those of unlabeled lignans from I-acetone extract
and authentic samples.
4.4.2. [9-3H1]Isoeugenol (23)
[9-3H1]Isoeugenol (23) was prepared based on a procedure for
obtaining of isoeugenol previously described by Nascimento et al.
(2000). To a stirred solution of [9-3H1]coniferyl alcohol (22,
126.0 mg, 0.70 mmol, a = 1.83 ꢀ 107 Bq mmolꢁ1
) and coniferyl
alcohol (252.0 mg, 1.40 mmol) in dry CH2Cl2 (6 ml) were added
MsCl (650
l
l, 8.4 mmol) and Et3N (1.70 ml, 12.6 mmol). After
Acknowledgements
30 min of stirring, ice water was poured into the soln., which
was then extracted with CH2Cl2 (3 ꢀ 5 ml). The combined organic
phase was washed twice with 10% HCl (5 ml), satd. NaHCO3 soln.
(5 ml) and brine (5 ml), and then dried (dry Na2SO4), and the sol-
vent was evaporated. The resulting oil was subjected to flash chro-
matography (silica gel 60, CH2Cl2) to yield [9-3H1]coniferyl alcohol
mesylate (201.9 mg, 28.6%, 0.60 mmol). The mesylate (192.8 mg,
0.57 mmol) was dissolved in dry THF (6 ml) and, in one portion
by means of syringe injection, a 1 M LiEt3BH solution in THF
(2.9 ml) was added to the stirring reaction mixture, which was
then maintained in reflux for 9 h. Following reduction, excess hy-
dride was quenched by the dropwise addition of MeOH and H2O.
The organoboranes were oxidized by adding 3 N NaOH (0.5 ml),
followed by slow, dropwise addition of 30% H2O2 (0.5 ml). The
reaction mixture was next poured into H2O (7 ml), extracted with
CH2Cl2 (3 ꢀ 7 ml), and then washed with H2O to remove dissolved
THF, dried (dry Na2SO4), and concentrated to give 23 (93.8 mg,
99.0%, a = 2.27 ꢀ 106 Bq mmolꢁ1).
The authors thank the Fundação de Amparo à Pesquisa do Esta-
do de São Paulo (FAPESP) for financial support and the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for
fellowships to G.B.M. and T. da S. We also thank Dr. Jorge M. David
(Bahia Federal University, Brazil) for performing LC–ESI-MS. We
are very grateful to Dr. Norman G. Lewis and Laurence B. Davin
(Washington State University, USA) for introducing us to this re-
search field.
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tive vials was repeated for four times following uptake. Water was
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