Stereochemical fate of C-26 and C-27 during the conversion of isofucosterol to sitosterol and of 24-methylenecholesterol to campesterol and dihydrobrassicasterol in Oryza sativa cell cultures

Article abstract of DOI:10.1016/S0031-9422(00)00122-9

Administration of pro-R-methyl-¹³C-labeled isofucosterol to cultured cells of Oryza sativa revealed that the pro-R and pro-S methyls at C-25 become the pro-R and pro-S methyls at C-25 of sitosterol, respectively. Similar administration experime

Full text of DOI:10.1016/S0031-9422(00)00122-9

Phytochemistry 54 (2000) 381±385
www.elsevier.com/locate/phytochem
Stereochemical fate of C-26 and C-27 during the conversion of
isofucosterol to sitosterol and of 24-methylenecholesterol to
campesterol and dihydrobrassicasterol in Oryza sativa cell cultures
Kozue Nasua, Kyoko Takahashia, Masuo Morisakia, Yoshinori Fujimotob,*
aKyoritsu College of Pharmacy, Shibakoen, Minato-ku, Tokyo 105-8512, Japan
bDepartment of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan
Received 9 December 1999; received in revised form 1 March 2000
Abstract
Administration of pro-R-methyl-13C-labeled isofucosterol to cultured cells of Oryza sativa revealed that the pro-R and pro-S
methyls at C-25 become the pro-R and pro-S methyls at C-25 of sitosterol, respectively. Similar administration experiments
using pro-S-methyl-13C-labeled 24-methylenecholesterol established that the pro-R and pro-S methyls at C-25 of 24-
methylenecholesterol become the pro-R and pro-S methyls of campesterol, and the pro-S and pro-R methyls of
dihydrobrassicasterol, respectively. These results are compatible with our recently proposed `syn-SE2' mechanism' for double
bond isomerization of D24(28) into D24(25). 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Oryza sativa; Sterol; Biosynthesis; Gramineace; Isofucosterol; Sitosterol; 24-Ethyldesmosterol; 24-Methyldesmosterol
1. Introduction
origins of C-26 and C-27 were deduced mainly on the
basis of administration experiments using
[1,2-13C2]acetate. A contrasting ®nding that the pro-R
methyl on C-25 of 4 derives from C-2 of mevalonate
was reported with Pinus pinea (Nicotra et al., 1981).
We recently established that the (E )-methyl of 6
becomes the pro-S methyl of 1 (Fujimoto et al.,
1998a), and that the corresponding methyl of 7
becomes the pro-S methyl of 2 and the pro-R methyl
of 3 (Fujimoto et al., 1997b) in Oryza sativa cell cul-
tures. Thus, reduction of 6 and 7 should proceed with
the anti-addition of hydrogen atoms. On the basis of
these ®ndings with O. sativa cell cultures, combined
with the observations of the origin of C-26 and C-27
of 4 and 5 reported for other plants (Seo et al., 1990,
1992; Fujimoto et al., 1998b), the double bond isomer-
ization is suggested to follow a mechanism in which
the pro-S methyls of 4 and 5 become the (E )-methyls
of 6 and 7. The purpose of the present investigation is
to substantiate this hypothesis. Because sterols 6 and 7
are not present in isolable amount in O. sativa cell cul-
In the biosynthesis of the side chain of sitosterol (1),
campesterol (2) and dihydrobrassicasterol (3), common
24-alkylsterols in higher plants, D24(28)-ole®nic sterols,
e.g., isofucosterol (4) and 24-methylenecholesterol (5)
isomerize to D24(25)-ole®nic sterols, e.g., 24-ethyldes-
mosterol (6) and 24-methyldesmosterol (7), which are
then reduced to furnish 1±3 (Scheme 1) (Nes and
McKean, 1977). Concerning the two methyl groups
(C-26 and C-27) on the prochiral C-25 center of the
phytosterols, it has been reported that the pro-S
methyl of 1, 2, 4 and 5 and the pro-R methyl of 3 orig-
inate from C-2 of mevalonate, while the other methyls
are derived from C-6 of mevalonate (Seo et al., 1978,
1984, 1986, 1990, 1992; Nes et al., 1992; Guo et al.,
1996; Fujimoto et al., 1997a, 1998a). The metabolic
* Corresponding author. Tel.: +81-3-5734-2241; fax: +81-3-5734-
2241.
E-mail address: fujimoto@cms.titech.ac.jp (Y. Fujimoto).
0031-9422/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00122-9

382
K. Nasu et al. / Phytochemistry 54 (2000) 381±385
Scheme 1. Biosynthetic pathway of the side chain of common plant sterols 1±3. Dots designate the carbons derived from C-2 of mevalonate.
tures, no direct correlation between 4 and 6, and 5 and
7 is possible. On the other hand, an analysis of the
metabolic correlation of C-26 and C-27 between 4 and
1, and between 5 and 2/3, should be informative par-
ticularly since the stereochemical course of 6 to 1, and
7 to 2/3 has been ®rmly established (Fujimoto et al.,
1997b, 1998a).
methyl-13C-labeled 24-ketone 8 (83% of the 13C label
resided at the pro-R methyl and 17% at the pro-S
methyl) was reacted with triphenylethylidenephosphor-
ane (Dusza, 1960) to a€ord the ethylidene 9 which
upon desilylation gave desired 4a [78% of the 13C
label resided at the pro-R methyl ꢀd 21.08) and 22% at
the pro-S methyl ꢀd 21.00), containing ca. 15% of
fucosterol] (Seo et al., 1990). The small amount of
fucosterol contamination was discounted, as it is not
converted into sitosterol in O. sativa cell cultures
(Okuzumi and Fujimoto, unpublished data). The
known pro-S-methyl-13C-labeled 24-ketone 10 (85% of
the 13C label resided at the pro-S -methyl and 15% at
the pro-R -methyl) was reacted with triphenylmethyle-
nephosphorane (Bergmann and Dusza, 1957) to give
exomethylene derivative 11 which upon desilylation
under acidic conditions furnished 5a [80% of the 13C
label resided at the pro-S methyl ꢀd 21.81) and 20% at
the pro-R methyl ꢀd 21.99)] (Fujimoto et al., 1997a).
The pro-R-methyl-13C-labeled isofucosterol 4a was
administered to O. sativa cell cultures as described pre-
viously (Yamada et al., 1997), and the resulting sterol
2. Results and discussion
We considered that the metabolic correlation of
interest might be obtained conveniently by administer-
ing stereospeci®cally 13C-labeled substrates combined
with 13C-NMR spectroscopic analyses. The requisite
stereochemically de®ned 13C-labeled sterols, pro-R-
methyl-13C-labeled isofucosterol (4a) and pro-S-
methyl-13C-labeled 24-methylenecholesterol (5a), were
synthesized according to Scheme 2 from stereospeci®-
cally 13C-labeled 24-oxocholesterol derivatives for
which we had previously described a convenient syn-
thetic method (Fujimoto et al., 1990). Thus, the pro-R-
Scheme 2. Synthesis of pro-R-methyl-13C-labeled isofucosterol (4a) and pro-S-methyl-13C-labeled 24-methylenecholesterol (5a).

K. Nasu et al. / Phytochemistry 54 (2000) 381±385
383
fraction obtained was separated by reversed-phase
HPLC to furnish sitosterol (1). The 13C-NMR spec-
trum of 1 (Fig. 1) showed an enriched signal ꢀd 19.04)
due to the pro-R methyl at C-25, accompanied by a
weakly enriched signal ꢀd 19.82) due to the pro-S
methyl at C-25 (Horibe et al., 1989). The intensities
(4:1) of the two peaks were approximately equal to
those of the substrate. It is therefore established that
the pro-R methyl at C-25 of 4 becomes the pro-R
methyl of 1, while the pro-S methyl of 4 becomes the
pro-S methyl of 1. This ®nding is consistent with the
aforementioned biosynthetic origin of the pro-S (de-
rived from C-2 of mevalonate) and pro-R methyl
groups at C-25 of 4 in Catharanthus roseus (Fujimoto
et al., 1998b) and Physalis perviana (Seo et al., 1990).
Similar administration experiments using pro-S-
methyl-13C-labeled
24-methylenecholesterol
(5a)
Fig. 2. The 13C-NMR spectrum (125 MHz, in CDCl3) of a mixture
of campesterol and dihydrobrassicasterol derived from pro-S-
methyl-13C-labeled 24-methylenecholesterol. The pro-R and pro-S
methyls at C-25 of 2 resonate at d 20.19 and 18.26, respectively, and
the corresponding signals of 3 resonate at d 17.60 and 20.50.
a€orded, after HPLC separation, campesterol (2) and
dihydrobrassicasterol (3) as an inseparable mixture.
The 13C-NMR spectrum (Fig. 2) of the mixture
showed enriched signals at d 18.26 (the pro-S methyl
of 2) and 17.60 (the pro-R methyl of 3), accompanied
by weakly enriched signals at d 20.19 (the pro-R
methyl of 2) and 20.50 (the pro-S methyl of 3)
(Colombo et al., 1990). These data clearly indicate that
the pro-S methyl of 5 is converted to the pro-S methyl
of 2 and the pro-R methyl of 3. The fact that the ratio
of the two major enriched signals in Fig. 2 was closely
similar to that obtained upon incubation of (E )-
methyl-13C-labeled 24-methyldesmosterol (Fujimoto et
al., 1997b) supports the view that conversion proceeds
via 24-methyldesmosterol as an obligatory intermedi-
ate, ruling out a mechanism of direct reduction of the
D24(28)-double bond leading to 2 and 3.
In the present paper, it has been unequivocally
established that the pro-S methyl (one derived from C-
2 of mevalonate) of 4 is converted to the pro-S methyl
of 1 in O. sativa cell cultures. Further, it has been elu-
cidated that the pro-S methyl (one derived from C-2
of mevalonate) of 5 becomes the pro-S methyl of 2
and the pro-R-methyl of 3. These ®ndings, combined
with our earlier observations (Fujimoto et al., 1997a,
1998b) in the conversion from 6 to 1 and 7 to 2 and 3,
allow us to depict the metabolic correlation of C-26
and C-27 in O. sativa cell cultures as illustrated in
Scheme 1.
We recently demonstrated that C-28 hydrogen of 4
become the pro-R hydrogen at C-28 of 1, proposing a
`syn-SE2' mechanism' (Fig. 3, R = Me) for the double
bond isomerization of 4 to 6 (Okuzumi et al., 1999). A
similar mechanism is suggested to be operating in the
conversion of 5 to 7 (Fig. 3, R = H). In conclusion,
the present study has provided further experimental
basis for our proposal that isomerization of the D24(28)
-
ole®nic sterols (4 and 5) to the D24(25)-ole®nic sterols (6
and 7) follow a `syn-SE2' mechanism' and the sub-
Fig. 1. 13C-NMR spectrum (125 MHz, in CDCl3) of sitosterol de-
rived from pro-R-methyl-13C-labeled isofucosterol (4a). The pro-R
and pro-S methyls at C-25 of 1 resonate at d 19.04 and 19.82, re-
spectively.
Fig. 3. Postulated stereostructure (syn-SE2' mechanism) for the
double bond isomerization from D24(28) to D24(25)
.

384
K. Nasu et al. / Phytochemistry 54 (2000) 381±385
sequent reduction proceeds via an `anti-addition mech-
anism'. These mechanisms appear to be general for
phytosterol biosynthesis in higher plants, although
such a thorough study including a D24(25)-ole®nic sterol
is limited.
enriched signal), 24.29 (C-15), 27.89 (C-23), 28.22 (C-
2
16), 28.59 ꢀd, JC±C = 35 Hz, C-25), 31.66 (C-2), 31.91
(C-7 and C-8), 35.95 (C-22), 36.16 (C-20), 36.50 (C-
10), 37.24 (C-1), 39.77 (C-12), 42.30 (C-4), 42.33 (C-
13), 50.12 (C-9), 55.99 (C-17), 56.75 (C-14), 71.80 (C-
3), 116.45 (C-28), 121.71 (C-6), 140.75 (C-5), 145.88
(C-24).
3. Experimental
3.3. Synthesis of pro-S-methyl-13C-labelled 24-
methylenecholesterol (5a)
3.1. General
Cell cultures of O. sativa were maintained as
1
described previously (Yamada et al., 1997). H-NMR
To a suspension of triphenylmethylphosphonium
bromide (235 mg, 0.66 mmol) in dry THF (1.6 ml)
placed in a pressure ¯ask was added n-BuLi (1.50 M
hexane soln., 436 ml) at room temperature under N2
and the mixture was stirred for 30 min. To this ylide
solution, the ketone 10 (170 mg, 0.33 mmol) was
added and the mixture was heated at 1008C overnight.
After cooling, it was diluted with ether and dil. HCl.
The organic layer was washed with sat. NaHCO3 and
brine, dried over MgSO4, and concentrated. The crude
product was chromatographed over silica gel with hex-
ane±AcOEt (15:1) to give the TBS ether 11 (107 mg),
and then with hexane±AcOEt (3:1) to give 5a (21 mg).
The former compound was desilylated as described for
9. The resulting 5a was mixed with the above 5a and
recrystallized from MeOH to yield pure 5a (77 mg,
58% from 10a) as white crystals, mp 138±1408C. EI-
MS m/z: 399 (M+), 384, 381, 366, 314, 299, 281, 271,
(500 MHz) spectra were obtained on a JEOL Alpha
500 spectrometer in CDCl3 solutions and chemical
shifts ꢀd† are reported in ppm down®eld from tetra-
methylsilane (used as internal reference). 13C-NMR
(125 MHz) spectra were recorded on the same spec-
trometer and chemical shifts are referenced to the sig-
nal ꢀd 77.0) of CDCl3. HPLC separations were
performed on a Shimadzu LC-6A with a SPD-6A UV
detector, equipped with Shim-pack CLC-ODS column
(6 mm i.d. Â 15 cm).
3.2. Synthesis of pro-R-methyl-13C-labeled isofucosterol
(4a)
To a suspension of triphenylethylphosphonium bro-
mide (464 mg, 1.2 mmol) in dry ether (5 ml) placed in
a pressure ¯ask was added n-BuLi (1.50 M hexane
soln., 653 ml) at room temperature under N2 and the
mixture was stirred for 30 min. To this ylide solution,
the ketone 8 (260 mg, 0.49 mmol) was added and the
mixture was heated at 1008C for 2 h. After cooling,
the mixture was diluted with ether and dil. HCl. The
organic layer was washed with sat. NaHCO3 and
brine, dried over MgSO4, and concentrated. The crude
product was subjected to silica gel chromatography
with hexane±AcOEt (100:1) as eluent to give the TBS
ether 9 (67 mg), and then with hexane±AcOEt (10:1)
to recover 8 (169 mg). The former was dissolved in
THF (2 ml) with the resulting solution stirred for 2.5 h
after addition of conc. HCl (200 ml). Extractive
workup gave a product, which was recrystallized from
MeOH to a€ord 4a (32 mg, 11%) as white crystals,
mp 113±1158C. EI-MS m/z: 413 (M+), 398, 380, 314,
1
253, 229. H-NMR d: 0.69 (s, 18-H3), 0.95 (d, J = 6.5
Hz, 21-H3), 1.01 (s, 19-H3), 1.02 (t, J = 6.5 Hz, JC±H
2
1
= 6.5 Hz, pro-R-Me), 1.03 (dd, J = 6.5 Hz, JC±H
=
126 Hz, pro-S-Me), 3.49 (m, 3-H), 4.66 (brs, 28-Ha),
4.71 (brs, 28-Ha), 5.35 (1H, m, 6-H). 13C-NMR d:
11.85 (C-18), 18.70 (C-21), 19.39 (C-19), 21.08 (C-11),
21.86 (pro-S-Me, enriched signal), 21.99 (pro-R-Me,
enriched signal), 24.27 (C-15), 28.21 (C-16), 30.97 (C-
2
23), 31.66 (C-2), 31.91 (C-7 and C-8), 33.79 (d, JC±C
= 35 Hz, C-25), 34.69 (C-22), 35.74 (C-20), 36.50 (C-
10), 37.25 (C-1), 39.77 (C-12), 42.30 (C-4), 42.35 (C-
13), 50.12 (C-9), 55.99 (C-17), 56.77 (C-14), 71.80 (C-
3), 105.94 (C-28), 121.68 (C-6), 140.77 (C-5), 156.9 (C-
24).
3.4. Precursor administration experiments
1
299, 281, 271, 255, 229. H-NMR d: 0.68 (s, 18-H3),
0.95 (d, J = 6.5 Hz, 21-H3), 0.97 (dd, J = 6.5 Hz,
To cultured cells of O. sativa (2 weeks, four 500 ml-
¯asks, each containing 250 ml of N6 medium (Chu et
al., 1975) supplemented with sucrose 30 g/l, proline 2.8
g/l, casein hydrolysate 300 mg/l, 2,4-D 2 mg/l), a sol-
ution of 4a (30 mg) in acetone (1 ml) and Tween 80 (1
ml) was added evenly through a membrane ®lter. Incu-
bation was continued for another 2 weeks and the cells
were collected by ®ltration. The sterol fraction was
extracted and separated from the wet cells as described
previously (Yamada et al., 1997). HPLC separation
2
1JC±H = 125 Hz, pro-R-Me), 0.98 (t, J = 6.5 Hz, JC±
= 6.5 Hz, pro-S-Me), 1.01 (s, 19-H3), 1.59 (3H, d, J
H
= 7.0 Hz, 29-H3), 2.82 (m, 25-H), 3.53 (m, 3-H), 5.11
(q, J = 7.0 Hz, 28-H), 5.18 (0.15H, q, J = 7.0 Hz, H-
28 of fucosterol), 5.35 (m, 6-H). 13C-NMR d: 11.85 (C-
18), 12.74 (C-29), 18.80 (C-21), 19.39 (C-19), 21.00
(pro-S-Me, enriched signal), 21.08 (pro-R-Me, enriched
signal), 21.08 (C-11), 22.22 (pro-R-Me of fucosterol,

K. Nasu et al. / Phytochemistry 54 (2000) 381±385
385
H., Morisaki, M., 1998b. Metabolic origin of C-26 and C-27 of
isofucosterol in tissue cultures of Catharanthus roseus. Nat. Prod.
Lett. 11, 207±210.
(conditions: solvent, MeOH; ¯ow rate 1.0 ml/min;
retention time 11.9 min) of the sterol fraction gave
pure 1 (4 mg). Administration of compound 5a (50
mg) was similarly carried out to a€ord a mixture of 2
and 3 (27 mg) after HPLC separation (conditions: sol-
vent, MeOH; ¯ow rate 1.0 ml/min; retention time 11.9
min for 2/3).
Guo, D-.A., Jia, Z., Nes, D., 1996. Stereochemistry of hydrogen mi-
gration from C-24 to C-25 during phytosterol biomethylation. J.
Am. Chem. Soc. 118, 8507±8508.
Horibe, I., Nakai, H., Sato, T., Seo, S., Takeda, K., 1989.
Stereoselective synthesis of C-24 and C-25 stereoisomeric pairs of
24-ethyl-26-hydroxy- and 24-ethyl-[26-2H]sterols and their D22-de-
rivatives: reassignment of 13C NMR. signals of the pro-R and the
pro-S methyl groups at C-25 of 24-ethylsterols. J. Chem. Soc.,
Perkin Trans. 1, 1957±1967.
Acknowledgements
Nes, W.R., McKean, M., 1997. Biochemistry of Steroids and Other
Isopentenoids. University Press, Baltimore, pp. 326±410.
Nes, W.D., Norton, R.A., Benson, M., 1992. Carbon-13 NMR stu-
We thank Dr Seiichi Toki, National Institute of
Agrobiological Resources, for providing tissue cultures
of O. sativa. Thanks are also due to Ms Miho Aka-
bane and Ms Hana Arita for excellent technical help.
dies on sitosterol biosynthesized from
Phytochemistry 31, 805±811.
[
13C]mevalonate.
Nicotra, F., Ronchetti, F., Russo, G., Lugaro, G., Casellato, M.,
1981. Stereochemical fate of the isopropylidene methyl groups of
lanosterol during the biosynthesis of isofucosterol in Pinus pines.
J. Chem. Soc., Perkin Trans. 1, 498±502.
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