STEREOCHEMISTRY OF THE

FORMATION OF ARYLGLYCEROL-8-O4-ARYL ETHERS FROM MONOLIGNOLS

WITH ENZYME REPARATIONS OF
EUCOMMIA ULMOIDES
Alam, M. S.1, Lourith, N.2, Suzuki, T.1, and
Katayama, T.1*
1

Faculty of Agriculture, Kagawa University, Japan

School of Cosmetic Science, Mae Fah Luang University,
Thailand
2

ABSTRACT
Arylglycerol-(8-O-4)-aryl ether linkages in lignins
and 8-O-4' neolignans are the most abundant
intermonomer linkages in natural products except for
glycosidic linkages in carbohydrates. Our group had found
that incubation of cell-free extracts from Eucommia
ulmoides with coniferyl alcohol (CA) in the presence of
hydrogen peroxide gave (+)-erythro- and ()-threoguaiacylglycerol-8-O-4-(coniferyl alcohol) ether (GGCE)
and that the erythro isomer was preferred to the threo one.
In this paper, stereochemistry of the formation of
arylglycerol-8-O-4-aryl ethers from monolignols with two
enzyme preparations of E. ulmoides was investigated.
Incubation of a soluble enzyme preparation of E. ulmoides
with [8-14C]sinapyl alcohol (SA) in the presence of
hydrogen peroxide gave ()-erythro and ()-threo[14C]syringylglycerol-8-O-4-(sinapyl alcohol) ethers
(SGSEs). On the other hand, incubation of an insoluble
enzyme preparation of this plant with [8-14C]SA in the
absence of hydrogen peroxide afforded (+)-erythro- and
()-threo-[8-14C]SGSEs. Both preparations catalyzed the
diastereoselective formation of erythro-SGSEs with
optical activity within 60 min. Interestingly, the soluble
preparation catalyzed the formation of ()-erythro-SGSE,
whereas the insoluble preparation did that of (+)-erythro
one, the opposite enantiomer.
BACKGROUND
Most of the lignans and neolignans in plants have
optical activity. They have important physiological
functions in plant defense and human health.
Arylglycerol- (8-O-4)-aryl ether linkages are present in
lignins and 8-O-4 neolignans. The intermonomer linkages
are the most abundant ones in natural products except for
glycosidic linkages in carbohydrates. An 8-O-4
neolignans have four stereoisomers, erythro and threo
diastereomers (Fig. 1) and their enantiomers. Recently, it
have been reported that Surinamensin (a 9,9-deoxy-8-O4 neolignan) has potential anti-leishmanial activity [1].
*

Author for correspondence. E-mail:

katayama@ag.kagaw-u.ac.jp

Deyama and coworkers [2] first isolated guaiacylglycerol8-O-4-(sinapyl alcohol) ether (GGSE) from Eucommia
ulmoides.
Recently Davin and Lewis discovered a dirigent
protein [3] which catalyzes formation of (+)-pinoresinol
and it was originally obtained from an insoluble residue of
Forsythia plant young shoots [4]. Since biosynthesis that
of 8-O-4 neolignans have not been advanced in contrast
to lignans, our group has investigated the biosynthesis of
8-O-4 neolignans. Katayama and Kado found for the first
time that incubation of cell-free extracts from Eucommia
ulmoides with coniferyl alcohol (CA) in the presence of
H2O2 gave (+)-erythro- and (-)-threo-guaiacylglycerol-8O-4-(coniferyl alcohol) ether (GGCE) and that the
erythro isomer was preferred to the threo one. Very
recently Lourith et al. [5] determined the relative
configuration of GGSE isolated from the plant by Deyama
et al. [2] as erythro form, and found the stereoselective
formation of erythro-GGSE and erythro-syringylglycerol8-O-4-(sinapyl alcohol) ether (SGSE) with optical activity
by feeding experiments.
In this study, to clarify stereochemistry on the
formation of SGSE from sinapyl alcohol (SA) in E.
ulmoides, enzyme reactions with soluble and insoluble
enzyme preparations were carried out.
EXPERIMENTAL
Instrumentation and chromatography materials
All reagents and solvents are reagent grade. Analytical
and preparative thin-layer chromatography (TLC) was
done by using of plates precoated with Mereck silica gel
60 F-254 (0.25 and 0.5 mm thickness, respectively).
Analytical high performance liquid chromatography
(HPLC) was carried out on a Jasco PU-2089 equipped
with a Jasco UV-2075 plus Intelligent UV/Vis detector
and a Shimadzu chromatopac C-R7A plus using a reversed
phase column (TSK-GEL, ODS-80T). Compounds were
separated at a flow rate of 1.0 ml/min using the isocratic
solvent system: MeOH-3% AcOH in H2O (30:70, v/v).
Chiral analysis was performed on a Daicel Chiracel OD
column (250 x 46 mm) eluted with EtOH/n-hexane =
23:77 (v/v) at a flow rate of 0.8 ml/min (for threo-SGSE)
and 1.0ml/min (for erythro SGSE). Radioactivity of the
samples was measured in liquid scintillation cocktail
consisting of scintilationblender-II/toluene/polythelene
glycol mono-p-isooctylphenyl ether (6/54/40; v/v/v)
(nacalai tesque) using a liquid scintillation counter (LSC1000, Aloka). Protein contents of soluble preparations
were determined with Shimadzu spectrophotometer (UV1600) at 595 nm.
Chemical synthesis
Radioactive compounds: Radiolabeled precursors [814
C]SA and [8-14C]CA were synthesized by the literature
methods [6,7].

Preparation of SGSE: To a stirred solution of sinapyl

alcohol (43.0mg, 0.205mmol) in 1,4-dioxane (3ml), a
solution of FeCl36H2O (25.8mg, 0.095mmol) in H2O
(0.3ml) was added dropwise at room temperature over a
period of 5 min. After a drop of the aqueous solution was
added, the light yellow color of the original reaction
solution changed to light green, then the original color of
the solution returned. The dropwise addition of the reagent
was continued. The reaction was quenched by the addition
of a small amount of granulated NaCl. The reaction
mixture was then extracted three times with EtOAc. The
EtOAc solutions were combined, washed with saturated
brine, dried over anhydrous Na2SO4, and then evaporated
to dryness in vacuo. The residue was purified by
preparative TLC (5% MeOH in CH2Cl2) to give SGSE
(25.8 mg, 28.8%) as a mixture of the erythro and threo
isomers. The diastereomeric ratio of this SGSE was
quantified by reversed-phase HPLC and then
diastereomeric separation was carefully carried out by
preparative TLC [benzene/acetone 2:1 (X5)] to give threoSGSE (Rf 0.27, 2.60mg) and erythro-SGSE (Rf 0.25, 2.63
mg). The diastereomeric identification was achieved by
comparison of 1H NMR spectra with those of the erythro
and threo SGSEs synthesized previously [8]. ErythroSGSE: 1H NMR (acetone-d6 ): 3.46 (1H, dd, J = 7.08,
3.44, H-9a), 3.38-3.42 (1H, o, 9-OH), 3.84-3.88 (2H, o, 9OH & H-9b), 3.81 (6H, s, A-OCH3), 3.90 (6H, s, B-OCH3)
4.24 (2H, dt, J = 5.32, 1.54, H-9), 4.17 (1H, m, H-8), 4.38
(1H, d, J = 4.14, 7-OH), 4.99 (1H, t, J = 4.27, H-7), 6.39
(1H, td, J = 15.85, 5.13, H-8), 6.57 (1H, td, J = 15.86,
1.55, H-7), 6.72 (2H, s, H-2 & 6), 6.82 (2H, s, H-2 & 6),
7.07 (1H, s, 4-OH). EIMS m/z (%): 436 (3.9) [M]+, 418
(11.1) [M-H2O]+, 210 (100), 93 (3.7), 77(9). Threo-SGSE:
1
H NMR (acetone-d6): 3.33 (1H, m, 9-OH), 3.52 (1H, dd,
J = 7.92, 4.76, H-9a), 3.66 (1H, m, H-9b), 3.81 (6H, s, AOCH3), 3.88-3.90 (1H, o, 9-OH), 3.92 (6H, s, B-CH3)
3.94-3.96 (1H, o, H-8), 4.23 (2H, dt, J = 4,76, 1.50, H-9),
4.38 (1H, d, J= 2.93 7-OH), 4.97 (1H, dd, J = 7.44, 2.81,
H-7), 6.39 (1H, td, J = 15.86, 5.12, H-8), 6.56 (1H, dt, J =
15.85, 1.59, H-7), 6.77 (2H, s, H-2 & 6), 6.82 (2H, s, H2 & 6), 7.10 (1H, s, 4-OH). EIMS m/z (%): 436 (3.9)
[M]+, 418 (12) [M-H2O]+, 210 (100), 93 (4), 77(6).

Soluble enzyme assay

Each assay mixture (5.75ml X 2) consisted of the
soluble enzyme (5.0 ml) from defoliated young shoots of
E. ulmoides, 0.43 mM of H2O2 (10 mM, 0.25 ml), and 2.6
mM of [8-14C]SA (5.0 MBq/mmol, 30 mM, in 0.5ml of KPi buffer). After incubation at 300C for various time
intervals, glacial AcOH (0.5 ml) was added. The assay
mixture was then extracted with EtOAc (30ml) containing
the mixture of unlabeled erythro- and threo-SGSEs as cold
carriers (100g). The aqueous layer was further extracted
twice with EtOAc (30ml). The EtOAc solutions were
combined, washed twice with saturatwd brine, dried over
anhydrous Na2SO4, and evaporated under reduced pressure.
[14C]SGSE (a mixture of erythro and threo forms) was
isolated by preparative TLC (7% MeOH in CH2Cl2). The
erythro and threo diastereomers were quantified by
reverse-phase column HPLC and LSC, and isolated by the
HPLC. Each diastereomer was then subjected to chiral
column HPLC and LSC to quantify the enantiomers.
Insoluble enzyme assay
(a) Each assay consisted of the insoluble residue (3.5 g)
suspended in K-Pi buffer (50 mM, pH 7, 12 ml) and the
solution of 1.7 mM [8-14C]SA (30 mM, in 0.7ml of K-Pi
buffer). After incubation at 300C for various time intervals,
glacial AcOH (0.5 ml) was added.
(b) Each assay consisted of the insoluble enzyme
preparation (3.5 g) suspended in K-Pi buffer (50mM, pH 7,
12ml). The reaction was initiated by addition of solutions
of 15mM [8-14C]coniferyl alcohol (4.53MBq/mmol) and
15mM [8-14C]sinapyl alcohol (5.0MBq/mmol) in K-Pi
buffer (30mM, pH 7, 0.7ml). After incubation at 300C for
various time intervals, glacial AcOH (0.5 ml) was added.
Those assay mixtures were then extracted with EtOAc
containing the mixture of unlabeled erythro- and threoSGSEs as cold carriers (100g) and stereoisomers of the
resulting [14C]SGSE were analyzed similar to the soluble
enzyme assay.

Enzyme preparations
A soluble enzyme preparation [cell-free extracts with
potassium phosphate (K-Pi) buffer] was prepared by the
method of Katayama and Kado [6]. Protein content in the
enzyme samples was determined by the Bio-Rad micro
assay procedure using bovine serum albumin as standard.
An insoluble enzyme preparation (cell wall residue
removing soluble and ionically bound enzymes) was
prepared by the method of Davin et al. [4].

HO

Plant materials
Eucommia ulmoides plants were obtained from Sanyo
Nouen Inc. were maintained at Faculty of Agriculture,
Kagawa University.

CH2OH

CH3O

HO

OCH3

7
1
6

2
A

CH3O

4'

OH

erythro SGSE
OCH3

HO

OCH3

threo SGSE

3
4

CH2OH

CH3O

CH3O

OH

OCH3
OH

Fig. 1: The configuration of erythro and threosyringylglycerol-8-O-4-(sinapyl alcohol) ethers

(SGSE)
RESULTS AND DISCUSSION
Soluble enzyme assay
Incubation of a soluble enzyme preparation with [814
C]SA in the presence of H2O2 gave [14C]SGSE with
[14C]syringaresinol (SYR). During the incubation at
various time intervals, maximum amount of SGSE

(41.7mol/mg protein) accumulated at 30 min. The

SGSEs were diastereoselectively formed in erythro isomer
from 2 to 60 min incubations with 63.2 % d.e. at 30 min,
except for a preferential formation of threo-SGSE (31.6%
d.e) at 120 min (Table 1). Furthermore, soluble
preparation catalyzed the formation of erythro SGSE was
more favored than the threo SGSE. Both products, (-)erythro- and (-)-threo-SGSEs have optical activity with
47.4% e.e. and 21.6% e.e., respectively, at 60 min.
Table 1. Formation of SGSE, its percent
diastereomeric excess and part of enantiomeric excess,
following incubation of [8-14C] sinapyl alcohol with a
soluble enzyme preparation in the presence of H2O2 at
various time intervals
SGSE
Time
(min)
Erythro
Threo

Complete
(mol / mg protein)

Control

Table 2. Formation of SGSE, its percent

diastereomeric excess and part of percent enantiomeric
excess, following incubation of [8-14C] sinapyl alcohol
with an insoluble enzyme preparation in the absence of
H2O2 at various time intervals
SGSE
Time
(min)
Erythro
Threo

Complete
(nmol / mg residue)

Control
Boiled

10

30

19.6

7.41

3.84

4.87

5.08

2.60

60
3.28
[(+) 24.8a]
2.61
[(-) 14.6 a]

120

60

0.82

2.35

0.97

1.30

Percent
diastereo60.8
18.7
19.3
11.4
-3.83
meric
excess
a
Percent enantiomeric excess was analyzed at 60 min.

28.8

-H2O2 Boiled
2

10

30

1.23

23.0

34.0

0.77

8.68

7.66

60

120

8.76
8.53
[(-) 47.4a]
5.07
16.4
[(-) 21.6a]

Percent
diastereo23.0
45.0
63.2
26.7
-31.6
meric
excess
a
Percent enantiomeric excess was analyzed at 60 min

60

60

2.48

2.19

3.21

0.94

-12.8

39.9

Insoluble enzyme assay (a)

Incubation of an insoluble enzyme preparation with [814
C]SA also gave [14C]SGSE with [14C]SYR. Throughout
the time course, maximum amount of SGSE (24.4
nmol/mg residue) accumulated at 2 min. The SGSEs were
diastereoselectively formed in erythro isomer from 2 to 60
min incubations with 60.8% d.e. at 2 min (Table 2), except
for a preferential formation of threo-SGSE (18.7% d.e. at
10 min). Both products, (+)-erythro- and (-)-threo-SGSEs
have optical activity with 24.8% e.e. and 14.6% e.e.,
respectively, at 60 min. Interestingly, the soluble
preparation produced (-)-erythro- and (-)-threo-SGSEs,
whereas the insoluble preparation produced (+)-erythroand (-)-threo-SGSEs. Both preparations catalyzed the
diastereoselective formation of erythro- and threo-SGSEs
with optical activity. On the other hand, the soluble
preparation catalyzed the formation of ()-erythro-SGSE,
whereas the insoluble preparation did that of (+)-erythro
one, the opposite enantiomer.
Insoluble enzyme assay (b)
Insoluble enzyme preparations from defoliated young
shoots of E. ulmoides were incubated with a mixture of [814
C]CA and [8-14C]SA for various time intervals. After
affluent of incubation they produced a mixture of SGCE,
GGCE, SGSE, and GGSE respectively. A preferential
formation of erythro-[14C]SGSE was detected after 10 min
and lasted for the period of observation (Table 3). The
formation rate increased from 10 to 30min. The highest %
d.e. (82.8) was found at 60min, when the enantiomeric

Table 3. Formation of SGSE, its percent

diastereomeric excess and part of percent enantiomeric
excess, following incubation with a mixture of [8-14C]
sinapyl alcohol and [8-14C] coniferyl alcohol with an
insoluble enzyme preparation in the absence of H2O2 at
various time intervals
SGSE
Time
(min)
Erythro
Threo

Complete
(nmol / mg residue)

Control
Boiled

10

30

nd

0.85

4.85

nd

0.22

1.45

60
5.10
[(+) 21.4a]
0.48
[(-) 4.3 a]

120

60

3.34

nd

0.33

nd

Percent
diastereo45.0
54.0
82.8
82.0
meric
excess
a
Percent enantiomeric excess was analyzed at 60 min

composition was examined by chiral HPLC. (-)-Erythroand (-)-threo-[14C]SGSE were formed with 21.4% e.e. and
4.3% e.e., respectively, (Table 3). No [14C]SGSE
formation was detectable in denatured (10min heating in
boiling water) preparations (Table 3). In addition to SGSE,
other
8-O-4-neolignans,
GGCE,
GGSE,
and
syringylglycerol-8-O-4-(coniferyl alcohol) ether (SGCE),
were also produced in the order of each quantity (data not
shown). The predominant diastereomer of SGSE and
GGCE was the erythro isomer and the diastereomeric
excess of SGSE was higher than that of GGCE. On the
other hand, the predominant diastereomer of GGSE and
SGCE was the threo isomer and the diastereomeric excess
of GGSE was higher than that of SGCE.
Discussion
The soluble preparation catalyzed the formation of ()erythro-SGSE, whereas the insoluble preparation did that
of (+)-erythro one, the opposite enantiomer. These results

were analogous with those of feeding experiments by

Lourith et. al [9]. The diastereoselective formation of
erythro-SGSE in the enzymatic reactions was consistent
with that in the feeding experiments (full data not shown).
Erythro-SGSE formed by the insoluble enzyme preparation
[(-) 21.4] had the same optical activity as that of free erythroSGSE derived from stems in the feeding experiments [(-) 9.1].
Furthermore, the optical activity of (-)-threo-SGSE in the
both soluble [(-) 14.6] and insoluble assays [(-) 4.3] were in
accord with that of free threo-SGSE derived from leaves in
the feeding experiments [(-) 7.4]. Opposite optical activities
of stem-derived erythro-SGSE between the organic (SGSE)
and aqueous (SGSE glucosides) layers were observed. One
reason may be that the ()-erythro-SGSE, in preference to

the (+)-enantiomer, was transformed into its glucosides.

The selectivity of enantiomer glucosylation in leaves also
remain obscure, although there is a possibility that
selective formation of ()-erythro-SGSE was followed by
its selective transformation to ()-erythro-SGSE glucoside.
The observation of soluble and insoluble enzyme
preparations to preferences for different enantiomers of the
erythro isomer suggests that different enzymes regulate
the 8-O-4 coupling of sinapyl alcohol in E. ulmoides.
Recently, two classes of pinoresinol-lariciresinol
reductases have been identified in Western Red cedar
(Thuja pilcata). Each class is specific for one enantiomer
of the substrate [10]. This finding also supports the
suggestion.
CONCLUSIONS
Incubation of a soluble enzyme preparation of E.
ulmoides with [8-14C]sinapyl alcohol (SA) in the presence
of hydrogen peroxide gave ()-erythro and ()-threo[14C]syringylglycerol-8-O-4-(sinapyl alcohol) ethers
(SGSEs). On the other hand, incubation of an insoluble
enzyme preparation of this plant with [8-14C]SA in the
absence of hydrogen peroxide afforded (+)-erythro- and (
)-threo-[8-14C]SGSEs. Both preparations catalyzed the
diastereoselective formation of erythro-SGSEs with
optical activity within 60 min. Interestingly, the soluble
preparation catalyzed the formation of ()-erythro-SGSE,
whereas the insoluble preparation did that of (+)-erythro
one, the opposite enantiomer.
REFERENCES
1. Barata L.E.S., Santos L.S, Ferri P.H., Phillipson J.D.,
Paine A., Croft S.L. (2000) Phytochemistry 55: 589595
2. Deyama T., Ikawa T., Nishibe S. (1985) Chem. Pharm.
Bull. 33: 3651-3656.
3. Davin L.B., Wang H.B., Crowell A.L., Bedger D.L.,
Martin D.M., Sarkanen S., Lewis N.G. (1997) Science
275: 362-366
4. Davin L.B., Bedger D.L., Katayama T., Lewis N.G.
(1992) Phytochemistry 31: 3869-3874
5. Lourith N., Katayama T., Suzuki T. (2005) J. Wood
Sci. 51: 370-378
6. Katayama T., Kado Y. (1998) J. Wood Sci. 44: 244246

7.

Nakazawa Y., Odagiri N., Imai R., Yoshii T.,

Tagashira E., nakata C., Nakamura T., Asaumi M.,
Onizuka S., Yahara M., Nohara T. (1997) Natural
medicines 51: 392-398
8. Tanahashi M., Takeuchi H., Higuchi T. (1976) Wood
Res. 61: 44-53
9. Lourith N., Katayama T., Suzuki T. (2005) J. Wood
Sci. 51: 379-386
10. Fujita M., Gang D.R., Davin L.B., Lewis N.G. (1999)
J. Biol. Chem. 274:618-627