Metabolic conversion of 24-methyl-Δ25-cholesterol to 24-methylcholesterol in higher plants

Article abstract of DOI:10.1016/j.bmc.2005.08.061

Feeding of chemically synthesized [27-¹³C]codisterol ([27- ¹³C]2), [27-¹³C]24-epicodisterol ([27-¹³C]3), [23,24-²H₂]codisterol ([23,24-²H ₂]2), and [26,27-²H₆]24-methyldesmosterol ([26,27-²H₆]8) to Oryza sativa cell cultures, followed by MS and NMR analysis of the biosynthesized dihydrobrassicasterol (9)/campesterol (10), revealed that both (24R)- and (24S)-epimers of 24-methyl- Δ²⁵-cholesterol (2/3) were converted to 9 and 10 via the common intermediate 24-methyldesmosterol (8).

Full text of DOI:10.1016/j.bmc.2005.08.061

Bioorganic & Medicinal Chemistry 14 (2006) 732–738
Metabolic conversion of 24-methyl-D25-cholesterol to
24-methylcholesterol in higher plants
Kyoko Takahashi,a,* Kozue Nasu,a Tadahiko Mashino,a Masuo Morisaki,a
Noriyuki Harab and Yoshinori Fujimotob
aKyoritsu University 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 6 July 2005; revised 26 August 2005; accepted 27 August 2005
Available online 6 October 2005
Abstract—Feeding of chemically synthesized [27-13C]codisterol ([27-13C]2), [27-13C]24-epicodisterol ([27-13C]3), [23,24-2H2]codister-
ol ([23,24-2H2]2), and [26,27-2H6]24-methyldesmosterol ([26,27-2H6]8) to Oryza sativa cell cultures, followed by MS and NMR anal-
ysis of the biosynthesized dihydrobrassicasterol (9)/campesterol (10), revealed that both (24R)- and (24S)-epimers of 24-methyl-D25-
cholesterol (2/3) were converted to 9 and 10 via the common intermediate 24-methyldesmosterol (8).
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Oryza sativa, one of our stock plant cell cultures which
contains a 1:1 mixture of dihydrobrassicasterol (9) and
campesterol (10), but is devoid of 2/3.13 Here, we have
investigated on the possibility of 2 and 3 as alternative
biogenetic precursors of dihydrobrassicasterol (9) and/
or campesterol (10) in higher plants. Feeding of the
chemically synthesized [27-13C]codisterol ([27-13C]2),
[27-13C]24-epicodisterol ([27-13C]3), [23,24-2H2]codister-
ol ([23,24-2H2]2), and [26,27-2H6]24-methyldesmosterol
([26,27-2H6]8) to cultured cells of O. sativa, followed
by MS and NMR analysis of the resulting metabolites,
revealed that both 2 and 3 were converted to a mixture
of 9 and 10 via 24-methyldesmosterol (8).
The final step of plant sterol biosynthesis involves
hydrogenation of either D24, D24(28), or D25 olefinic ster-
ols (Scheme 1). It has been established that the tetrasub-
stituted D24 sterol (12) serves for the hydrogenation
substrate to form 24a-ethylsterol, for example, sitosterol
(13), whereas the corresponding methyl analog (8) is re-
duced to 24b-methylsterol, for example, dihydrobrassi-
casterol (9), as well as 24a-methylsterol, for example,
campesterol (10), in higher plants.1–5 The 24-exomethyl-
ene-sterol (4) is a well-known intermediate of ergosterol
biosynthesis in fungi,6,7 while the 24-ethyl-D25-olefinic
compound (5) is hydrogenated to give poriferasterol
(6) in Trebouxia sp.8 and chondrillasterol (7) in Tricho-
santhes kirilowii (Cucurbitaceae).9 However, no hydro-
genation of a major sterol of Ajuga species (Labiatae),
clerosterol (24b-ethyl-D25-cholesterol) appears to
occur.10
2. Results and discussion
The requisite labeled sterols were prepared in a slight
modification of our previous method used for the syn-
thesis of D24- and D25-sterols (Scheme 2).1,14 The known
(24R)-ester 1515 was alkylated with [13C]methyl iodide.
The C-25 methylated ester was converted to [27-13C]2
in four steps, that is, reduction with LiAlH4, catalytic
hydrogenation, dehydration, and final regeneration of
D5-3b-ol. The corresponding 24-epimer, [27-13C]3, was
prepared starting with the (24S)-ester 1615 in the same
manner. For the preparation of [23,24-2H2]2, the known
(22R)-acetylenic alcohol 1715,16 was first converted to
the 23,24-di-deuterated analog of 15 in two steps (reduc-
As for 24-methyl-D25 sterols, codisterol [(24S)-24-meth-
ylcholesta-5,25-dien-3b-ol] (2) and 24-epicodisterol (3)
were only rarely found in higher plants and marine
sponges,11 and information on their metabolism is lack-
ing (dashed arrow in Scheme 1).12 The same is true of
Keywords: Steroids and sterols; Biosynthesis; Campesterol; Dihydro-
brassicasterol; Codisterol; Oryza sativa.
2
tion with LiAl2H4 followed by quenching with H2O
16
*
Corresponding author. Tel.: +81 3 5400 2664; fax: +81 3 5400 2691
(K.T); e-mail: takahashi-ky@kyoritsu-ph.ac.jp
and orthoester Claisen rearrangement). The deuterated
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.061

K. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 732–738
733
28
St =
SAM
27
25
26
24
+
2H
2H
St
St
St
HO
1
codisterol (24S, 2)
epicodisterol (24R, 3)
St
dihydrobrassicasterol (9)
2H
SAM
St
St
St
St
St
St
8
4
poriferasterol (∆5, 6)
campesterol (10)
5
chondrillasterol (∆7, 7)
SAM
2H
2H
St
St
St
ergosterol (14)
11
sitosterol (13)
12
Scheme 1. Biosynthetic pathway of phytosterol (24-alkylsterol). St, D5-, D7-, or D5,7-3b-hydroxysterol skeleton; 2H, hydrogenation; SAM,
S-adenosylmethionine.
X
Y
Y
X
X
Y
X
Y
13CH3
13CH3
24
13CH3
a
c
b
CO2Et
CO2Et
CH2OH
CH2OH
OMe
15: X=H, Y=Me
16: X=Me, Y=H
Y
X
13CH3
13CH3
d
13CH3
e
or
St
St
[27-13C]-3
[27-13C]-2
HO
22R
D
OH
D
D
D
D
c
g
b
f
24
D
D
CH2OH
CO2Et
D
CH2OH
d, e
OMe
17
D
COOMe
St
D
CD3
CD3
CD3
OH
CD3
h
i, j
[23,24-2H2]-2
St
[26,27-2H6]-8
TBSO
18
Scheme 2. Preparation of labeled substrates. St denotes 3b-hydroxy-D5-sterol skeleton. Reagents: (a) LDA, 13CH3I, (b) LiAlH4, (c) H2-Pd/C,
(d) m-NO2-PhSeCN, Bu3P, then H2O2, (e) aq dioxane, TsOH, (f) LiAlD4/THF, then D2O, (g) CH3CH2C(OEt)3, EtCO2H, (h) CD3MgBr, (i) POCl3,
(j) HCl.
ester was subjected to the procedure described above to
give [23,24-2H2]2. [26,27-2H6]8 was prepared starting
from the methyl ester 1817 according to the procedure
used for the synthesis of [26,27-13C2]24-methyldesmos-
terol1 with C2H3MgI as a source of the 26,27-C2H3
groups.
NMR and MS spectroscopy, which established no con-
figurational interconversion on C-24 and C-25 during
incubation.
The 13C NMR spectra of 24-methylsterols (9/10) biosyn-
thesized from [27-13C]2 and [27-13C]3 are illustrated in
Figure 1. The signals19 of the pro-R-Me (d 17.6) of 9
and the pro-S-Me (d 18.3) of 10 were enriched compared
to those of non-labeled samples, whereas the signal
intensities for the pro-S-Me (d 20.5) of 9 and the pro-
R-Me (d 20.2) of 10 were the same as those of endoge-
nous samples. These data indicated that both codisterol
2 and 24-epicodisterol 3 were metabolized to 24-meth-
Feeding experiments of the labeled substrates to O. sati-
va cell cultures were carried out as described previously.1
The biosynthesized 24-methylcholesterols (9/10) ob-
tained as a mixture (24S/24R = ca. 1:1) after final HPLC
fractionation were subjected to spectroscopic analysis as
such.18 The recovered substrates were also analyzed by

734
K. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 732–738
A
B
pro-R (9)
pro-S (10)
pro- R (9)
pro-S (10)
pro-S (9)
pro-R
(10)
pro-R
(10)
pro-S (9)
ppm
16
ppm
22
20
18
22
20
18
16
Figure 1. Partial 13C NMR spectra (125 MHz, CDCl3) of 24-methyl-
cholesterols 9/10 derived from [27-13C]codisterol (A) and from
[27-13C]24-epicodisterol (B).
ylsterols (9/10) in a stereospecific manner.20 It should be
noted that both [27-13C]2 (24b) and [27-13C]3 (24a) were
converted to give a mixture of 9 (24b) and 10 (24a).
These results ruled out a direct reductive conversion of
2 and 3 to 9/10 and suggested that the D25-sterols first
isomerize into the common intermediate D24-sterol (8)
which should be a substrate for hydrogenation. Howev-
er, there remains a possibility that the direct hydrogena-
tion of D25 is cooperative as a minor pathway since
dihydrobrassicasterol (9) was more 13C-enriched than
campesterol (10) when [27-13C]codisterol was fed, and
vice versa when [27-13C]epicodisterol was fed (Fig. 1).
Figure 2. Partial mass spectra of 24-methylcholesterols 9/10. A, non-
labeled; B, biosynthesized from [23,24-2H2]codisterol; C, biosynthe-
sized from [26,27-2H6]24-methyldesmosterol. The spectra were scanned
at a relatively early part of the GC peak wherein the deuterium-labeled
compound was more enriched than the center of the peak due to the
isotope effect.
T. kirilowii, in which the D25 precursor (5) is directly
hydrogenated without isomerization to D24-olefin
(12).8,9
24-Methyl-D25-sterols (2 and 3) would be formed by
deprotonation of a C-25 cationic species (1, Scheme 1)
generated by the methyl transfer from SAM onto D24-
sterol. However, isomerization of the 24-double bond
of 24-methyldesmosterol (8) into the 25-double bond
might be an alternative way to yield 2/3. To explore this
possibility, [26,27-2H6]8 was fed to O. sativa cell cul-
tures. The mass spectrum of the biosynthesized 24-meth-
ylcholesterol 9/10 (Fig. 2C) exhibited a molecular ion
peak at m/z 406 due to 2H6-24-methylcholesterol in
addition to a molecular ion peak at m/z 400 for the
This possibility can be elucidated by tracing the meta-
bolic fate of the 24-hydrogen atom of 2 and/or 3. For
this purpose, a feeding experiment of [23,24-2H2]2 was
carried out. The resulting 24-methylcholesterol (9/10)
was analyzed by GC–MS. As can be seen in Figure
2B, the biosynthesized 9/10 clearly showed a molecular
ion peak at m/z 401 for a mono-deuterated species,
along with a molecular ion peak at m/z 400 for the
endogenous 9/10, whereas a molecular ion peak (m/z
402) for di-deuterated species was negligible. Analysis
of dehydration (M+ÀH2O) ions supported the above re-
sults. It is thus likely that 24-2H of [23,24-2H2]2 was
eliminated and 23-2H retained during the bioconversion.
The location of the retained deuterium atom in the bio-
synthesized 24-methylcholesterols (9/10) was unambigu-
C-22 (9/10)
2
ously determined by H-decoupled 13C NMR analysis.
C-24 (9)
C-24 (10)
C-23 (9)
C-23 (10)
The spectrum (Fig. 3) showed weak but significant sig-
nals due to deuterium isotope-shifts in addition to the
signals of the endogenous 24-methylcholesterols. The
assignments of the signals are summarized in Table 1
which clearly indicated that the C-24 deuterium atom
of [23,24-2H2]2 was eliminated during the bioconversion
into 9/10, whereas the C-23 deuterium atom of the sub-
strate was retained at the same position. This implies
that the direct reductive conversion of 2/3 to 9/10 is
not operative in O. sativa cell cultures and the double-
bond isomerization from D25 to D24 is an obligatory step
for 2 and 3 to be reduced. This is in marked contrast to
the 24b-ethylsterol biosynthesis in Trebouxia sp. and
c
d
a
b
e
ppm
38
36
34
32
30
Figure 3. Partial {1H}{2H}-13C NMR spectrum (125 MHz, CDCl3) of
24-methylcholesterols 9/10 derived from [23,24-2H2]2. For the assign-
ments of peaks a–e, see Table 1.

K. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 732–738
735
Table 1. Assignments of pertinent 13C signals of 24-methylcholesterol
9/10 biosynthesized from [23,24-2H2]2
reduction of 8 to 9/10, has been characterized in Arabid-
opsis thaliana.22 Database search showed that there is no
homologous gene in A. thaliana. This is the case also in
O. sativa, which has only one ortholog of Arabidopsis
Dwarf1.23 It is therefore suggested that the isomerization
from D25 to D24 and the subsequent reduction would be
catalyzed by DWARF1 enzyme of a poor substrate
specificity. The metabolism of (24R)- and (24S)-24-eth-
yl-D25-cholesterols in higher plants is under investiga-
tion in our laboratory.
Carbon
d
Upfield shift
Non-labeled
2H-labeled
C-24 of 9
C-24 of 10
C-23 of 9
39.02
38.79
30.52
30.22
33.67
38.92 (c)
38.70 (d)
30.08 (a)
29.78 (b)
33.57 (e)
0.10
0.09
0.44
0.47
0.15
C-23 of 10
C-22 of 9/10
Note. (a)–(e) refer to indicated points in Fig. 1.
4. Experimental
4.1. Preparation of the labeled substrates
4.1.1. General. 1H NMR (500 MHz) spectra were
recorded on a JEOL JNM-DX 500 spectrometer in
CDCl3 solution and chemical shifts (d) are reported in
ppm downfield from tetramethylsilane (used as an inter-
nal reference). 13C NMR (125 MHz) spectra are
obtained on the same spectrometer and chemical shifts
were referenced to the signal (d 77.0) of CDCl3. 13C-
{1H}{2H} NMR (125 MHz) spectra are recorded on a
Bruker DRX500 spectrometer. GC–MS analyses
were performed on a JEOL AUTOMASS SUN200
instrument with HP-1 capillary column (0.32 mm ·
30 m) using He gas (1 mL/min), with an oven tempera-
ture from 250 to 260 ꢁC (1 ꢁC/min). HRMS were record-
ed with a JEOL JMS-700 spectrometer. HPLC was
performed on a Shimadzu LC-6A apparatus equipped
with a UV detector using a reversed-phase column,
PREP-ODS(H) (25 cm · 20 mm i.d.). TLC analyses
were performed on commercial glass plates bearing a
0.25 mm layer of Merck DC-Fertigplatten Kieselgel 60
F245. Column chromatography was done with Merck
Kieselgel 60 (70–230 mesh). Tetrahydrofuran (THF)
was distilled before use from benzophenone ketyl under
N2 atmosphere. Diethyl ether was distilled from LiAlH4
before use. [13C]Methyl iodide (99% 13C), lithium alumi-
num deuteride (98% 2H), deuterium oxide (99% 2H), and
2H3-methylmagnesium bromide (98% 2H) were pur-
chased from Merck.
Scheme 3. Proposed mechanism of the metabolic conversion of
codisterol 2 and 24-epicodisterol 3 to 24-methylcholesterol 9/10 in O.
sativa. Dots represent correlations of C-26 and C-27.
endogenous 24-methylcholesterol, whereas ions at m/z
405, 404, and 403 were negligible. The results revealed
that [26,27-2H6]8 was converted to 9/10 without any loss
of deuterium atom, thus ruling out the possibility of the
double-bond isomerization from D24 to D.25
We previously established in the 13C-labeling study with
cell cultures of O. sativa and Catharanthus roseus that
the pro-(S)-Me of 24-exomethylenesterol (4) becomes
stereospecifically the (E)-Me of 24-methyldesmosterol
821 and reduction of 8 with anti-addition mechanism
yields 9 and 10.2 The 13C-labeling pattern (Fig. 1) of
24-methylcholesterols (9 and 10) derived from
[27-13C]2 and 3 in the present study is identical with
those of 9 and 10 derived from [(E)-Me-13C]8 previous-
ly.2,21 This implies that the conversion of 2 and 3 to 8
takes place in such a stereospecific manner that the
13C-labeled carbons (C-27) of 2 and 3 become the (E)-
Me of 8 (Scheme 3).
4.1.2. Synthesis of [27-13C]codisterol ([27-13C]2). n-Butyl
lithium (1.54 M solution in toluene, 11.3 mL,
17.0 mmol) was added to a solution of freshly distilled
diisopropylamine (2.5 mL, 17.8 mmol) in THF
(169 mL) at 0 ꢁC under N2 atmosphere and the solution
was stirred for 0.5 h. The resultant LDA solution was
added to a solution of the (24R)-ester 1515 (3.87 g,
8.47 mmol) in THF (170 mL) at À78 ꢁC and the mixture
was stirred for 1 h. [13C]Methyl iodide (1.14 mL,
17.0 mmol) was added to the enolate solution and the
mixture was stirred at À78 ꢁC for 2 h. The reaction mix-
ture was diluted with satd NH4Cl and extracted with
ethyl acetate. The organic layer was washed with satd
NaHCO3 and brine, dried over MgSO4, and concentrat-
ed under reduced pressure. The crude product was chro-
matographed on silica gel (hexane/ethyl acetate = 30:1)
to give ethyl [27-13C]-(22E,24S)-6b-methoxy-3a,5-cycl-
oergost-22-en-26-oate (3.82 g, 89%) as a colorless oil.
1H NMR d: 0.42 (dd, J = 8.5, 5.4 Hz, H-4a), 0.64 (t,
3. Conclusion
We have investigated the metabolism of codisterol (2)
and epicodisterol (3) in cell cultures of a higher plant
for the first time and found that the C-24 epimers of
24-methyl-D25-cholesterol were indifferently converted
to both (24S)- and (24R)-24-methylcholesterols (9/10).
Detailed investigation of the conversion mechanism
established that neither 2 nor 3 is a substrate for the
reduction, but both 2 and 3 first isomerize to the com-
mon intermediate 24-methyldesmosterol (8), which is
then reduced to give 9 and 10. The gene, Dwarf1, which
encodes a multifunctional enzyme catalyzing the conver-
sion of 24-exomethylene-sterol (4) to 8 as well as the

736
K. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 732–738
J = 4.0 Hz, H-4b), 0.72 (s, H3-18), 1.00 (d, J = 8.5 Hz,
H3-21), 1.01 (d, J = 8.5 Hz, H3-28), 1.02 (s, H3-19),
1.08 (dd, J = 107, 6.4 Hz, H3-27), 1.26 (t, J = 7.8 Hz,
OCH2CH3), 2.76 (s, H-6), 3.23 (s, OCH3), 4.12 (q,
J = 7.8 Hz, OCH2CH3), 5.11 (m, H-22), 5.23 (m,
H-23). 13C NMR d: 13.71 and 14.65 (enriched
25R- and 25S-13CH3).
11.83 (C-18), 18.52 (C-21), 18.66 (enriched C-27),
19.39 (C-28), 20.12 (C-19), 21.07 (C-11), 24.27 (C-15),
28.15 (C-2), 31.15 (C-16), 31.65 (C-23), 31.89 (C-7),
31.89 (C-8), 33.66 (C-22), 35.65 (C-20), 36.49 (C-10),
37.24 (C-4), 39.76 (C-1), 41.55 (C-12), 42.3 (C-13),
42.3 (C-24), 50.12 (C-9), 55.97 (C-17), 56.75 (C-14),
71.78 (C-3), 109.32 (C-26), 121.69 (C-6), 140.74 (C-5),
150.05 (C-25). HREIMS found m/z: 399.3584 (M+),
calcd for C2713CH46O: 399.3582. The labeled codisterol
was found be a mixture of 92% of [27-13C]codisterol
and 8% of [27-13C]epicodisterol on the basis of the signal
intensity of the enriched carbons at d: 18.66 and 19.14.
A solution of the methylated ester (3.80 g, 8.06 mmol) in
THF (80 mL) was stirred at room temperature for 0.5 h
after addition of LiAlH4 (758 mg, 19.9 mmol). Extrac-
tive (ethyl acetate) work-up gave the crude product,
which was chromatographed on silica gel (hexane/ethyl
acetate = 15:1) to afford the C-26 alcohol (3.01 g, 86%)
as a colorless oil. A mixture of the alcohol (1.00 g,
2.32 mmol) and 5% Pd/C (200 mg) in ethyl acetate
(58 mL) was stirred at room temperature for 17 h under
hydrogen atmosphere. The catalyst was filtered off
through a pad of Celite and the filtrate was concentrated
under reduced pressure. The crude product was chro-
matographed on silica gel (hexane/ethyl acetate = 15:1)
to give [27-13C]-(24S)-6b-methoxy-3a,5-cycloergostan-
26-ol (900 mg, 90%). 1H NMR d: 0.43 (dd, J = 8.5,
5.3 Hz, H-4a), 0.65 (t, J = 4.0 Hz, 4-Hb), 0.72 (s, H3-
18), 0.88 (d, J = 8.5 Hz, H3-28), 0.91 (d, J = 7 Hz, H3-
21), 0.92 (dd, J = 125, 6.4 Hz, H3-27), 1.02 (s, H3-19),
2.77 (s, H-6), 3.32 (s, OCH3), 3.45 and 3.63 (m,
H2-26). 13C NMR d: 11.05 and 13.94 (enriched
25R- and 25S-13CH3).
4.1.3. Synthesis of [27-13C]24-epicodisterol ([27-13C]3).
[27-13C]24-Epicodisterol ([27-13C]3) was synthesized
from the (24S)-ester 1615 in the same procedure as de-
scribed for the synthesis of [27-13C]2. [27-13C]3: mp
142–143 ꢁC; 1H NMR d: 0.67 (s, H3-18), 0.92 (d,
J = 6.5 Hz, H3-21), 0.99 (d, J = 7.4 Hz, H3-28), 1.01 (s,
H3-19), 1.63 (d, J = 130 Hz, H3-27), 3.48 (m, H-3),
4.65 (m, H2-26), 5.36 (m, H-6). 13C NMR d: 11.83 (C-
18), 18.79 (C-21), 19.14 (enriched C-27), 19.39 (C-28),
19.56 (C-19), 20.12 (C-11), 24.27 (C-15), 28.17 (C-2),
31.35 (C-16), 31.66 (C-23), 31.90 (C-7), 31.90 (C-8),
33.67 (C-22), 35.84 (C-20), 36.49 (C-10), 37.24 (C-4),
39.75 (C-1), 41.46 (C-12), 42.30 (C-13), 42.30 (C-24),
50.12 (C-9), 55.93 (C-14), 56.76 (C-17), 71.78 (C-3),
108.92 (C-26), 121.68 (C-6), 140.75 (C-5), 150.86 (C-
25). HREIMS found m/z: 399.3579 (M+), calcd for
C2713CH46O: 399.3582. The labeled epicodisterol was
found be a mixture of 91% of [27-13C]epicodisterol
and 9% of [27-13C]codisterol on the basis of the signal
intensity of the enriched carbons at d: 19.14 and 18.66.
A mixture of the saturated alcohol (768 mg, 1.78 mmol),
2-nitrophenylselenocyanate (2.0 g, 10.8 mmol), and tri-
n-butylphosphine (2.2 mL, 10.8 mmol) in THF (25 mL)
was stirred at room temperature for 30 min under N2
atmosphere. The reaction mixture was stirred, after
addition of 30% H2O2 solution (35 mL), for another
1 h and extracted with ethyl acetate. The organic layer
was washed with sat NaHCO3 and brine, dried over
MgSO4, and concentrated under reduced pressure. The
crude product was chromatographed on silica gel (hex-
ane/ethyl acetate = 30:1) to afford [27-13C]-(24S)-6b-
methoxy-3a,5-cycloergost-25-ene (570 mg, 78%) as a
4.1.4. Synthesis of [23,24-2H2]codisterol ([23,24-2H2]2).
LiAl2H4 (2.10 g, 49.9 mmol) was added to a solution of
the (22R)-acetylenic alcohol 1715,16 (1.09 g, 2.83 mmol)
in THF (28 mL) and the mixture was refluxed under
N2 atmosphere for 18 h. The mixture was cooled with
ice water and 2H2O (6.0 mL) was carefully added in por-
tions. The mixture was extracted with ethyl acetate and
the organic layer was washed with satd NaHCO3 and
brine, dried over MgSO4, and concentrated under re-
duced pressure. The crude product was chromato-
graphed on silica gel (hexane/ethyl acetate = 25:1) to
1
colorless oil. H NMR d: 0.43 (dd, J = 8.5, 5.3 Hz, H-
4a), 0.65 (t, J = 4 Hz, H-4b), 0.71 (s, H3-18), 0.90 (d,
J = 6.5 Hz, H3-21), 0.99 (d, J = 6.5 Hz, H3-28), 1.02 (s,
H3-19), 1.63 (d, J = 125 Hz, H3-27), 2.77 (s, H-6), 3.32
(s, OCH3), 4.66 (m, H2-26). 13C NMR d: 18.68 (enriched
27-13C).
give
[23,24-2H2]-(23E)-6b-methoxy-26,27-dinor-3a,5-
cyclocholest-23-en-22-ol (347 mg, 32%) as a pale yellow
oil. 1H NMR d: 0.43 (dd, J = 8.5, 5.3 Hz, H-4a), 0.65 (t,
J = 4.0 Hz, H-4b), 0.72 (s, H3-18), 0.90 (d, J = 5.5 Hz,
H3-21), 1.02 (s, H3-19), 1.71 (s, H3-25), 2.77 (s, H-6),
3.33 (s, OCH3), 4.19 (m, H-22). EIMS m/z: 388 (M+),
373 ([M-CH3]+).
A solution of the 25-ene (570 mg, 1.38 mmol) and p-tol-
uensulfonic acid monohydrate (223 mg, 0.322 mmol) in
dioxane (27.5 mL) and water (27.5 mL) was stirred at
75 ꢁC for 2.5 h and then cooled to room temperature.
The mixture was extracted with ether and the organic
layer was washed with satd NaHCO3 and brine, dried
over MgSO4, and concentrated under reduced pressure.
The crude product was chromatographed on silica gel
(hexane/ethyl acetate = 10:1) and crystallization from
methanol to give [27-13C]2 (422 mg, 77%) as colorless
A solution of the allylic alcohol (326 mg, 0.89 mmol),
triethyl orthopropionate (1.90 mL, 4.45 mmol), and pro-
pionic acid (40 lL, 0.53 mmol) in xylene (20 mL) was
stirred at 140 ꢁC for 3 h under N2. Concentration of
the mixture under reduced pressure gave ethyl
[23,24-2H2]-(22E,24S)-6b-methoxy-3a,5-cycloergost-22-
en-26-oate (371 mg, 93%) as a pale yellow oil. 1H NMR
d: 0.43 (dd, J = 8.5, 5.4 Hz, H-4a), 0.65 (t, J = 4.0 Hz, H-
4b), 0.73 (s, H3-18), 0.95 (d, J = 7.5 Hz, H3-21), 0.96 (d,
H3-28), 1.02 (s, H3-19), 1.25 (t, J = 7.8 Hz, OCH2CH3),
1
needles, mp 143–144 ꢁC. H NMR d: 0.67 (s, H3-18),
0.91 (d, J = 6.5 Hz, H3-21), 0.99 (d, J = 7.4 Hz, H3-28),
1.01 (s, H3-19), 1.65 (d, J = 111 Hz, H3-27), 3.51 (m,
H-3), 4.65 (m, H2-26), 5.35 (m, H-6). 13C NMR d:

K. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 732–738
737
1.26 (s, H3-27), 2.77 (s, H-6), 3.26 (s, OCH3), 4.12 (q,
J = 7.8 Hz, OCH2CH3), 5.25 (m, H-22, H-23).
C-27); HREIMS found m/z: 404.3917 (M+), calcd for
C28H402H6O: 404.3919.
The unsaturated ester was converted to [23,24-2H2]2 as
described for the synthesis of [27-13C]2. [23,24-2H2]2:
white needles, mp 143–144 ꢁC. 1H NMR d: 0.67 (s,
H3-18), 0.91 (d, J = 7.4 Hz, H3-21), 0.99 (s, H3-28),
1.01 (s, H3-19), 1.63 (s, H3-27), 3.51 (m, H-3), 4.65 (m,
H-26), 5.35 (m, H-6). 13C NMR d: 11.83 (C-18), 18.63
(C-27), 18.71 (C-21), 19.39 (C-19), 19.98 (C-28), 21.07
(C-11), 24.27 (C-15), 28.15 (C-2), 28.15 (C-16), 30.61
(t, JCD = 19 Hz, C-23), 31.89 (C-7), 31.89 (C-8), 33.54
(C-22), 35.63 (C-20), 36.5 (C-10), 37.24 (C-1), 39.76
(C-12), 41.01 (t, JCD = 19 Hz, C-24), 42.3 (C-4), 42.3
(C-13), 50.12 (C-9), 55.98 (C-17), 56.75 (C-14), 71.8
(C-3), 109.29 (C-26), 121.71 (C-6), 140.75 (C-5), 150.19
(C-25). 13C-{1H}{2H} NMR d: 30.53 (C-23), 40.99 (C-
24). HREIMS found m/z: 400.3670 (M+), calcd for
C28H442H2O: 400.3672.
4.2. Feeding experiments
Cell cultures of O. sativa were maintained as described
previously.1
To cultured cells of O. sativa (2 weeks, four 500 mL-
flasks, each containing 350 mL N6 medium (SIGMA,
Chu (N6) basal salt mix) supplemented with sucrose
30 g/L, proline 2.8 g/L, casein hydrolysate 300 mg/L,
and 2,4-D 2 mg/L) was added evenly a solution of each
substrate (50 mg) in acetone (1 mL) and Tween 80
(1.5 mL) through a membrane filter. Incubation was
continued for another 2 weeks and the cells were collect-
ed by filtration. The CHC13-MeOH extract was chro-
matographed on silica gel to give the sterol fraction as
described previously.1 24-Methylsterol was separated
by HPLC [methanol, 9.9 mL/min, detection with UV
(210 nm), typical retention time, 45 min]. Sterol analysis
of the 24-methylsterols was performed with Shim pack
CLC-ODS column (15 cm · 6 mm i.d.) using methanol
(1.0 mL/min) as solvent. The separated 24-methylcholes-
terols (typical amount, 8 mg) were analyzed by 13C
NMR (Fig. 1), 13C-{1H}{2H} NMR (Fig. 3) and
GC–MS (Fig. 2).
4.1.5. Synthesis of [26,27-2H6]24-methyldesmosterol
([26,27-2H6]8). C2H3MgI (1.0 M solution in diethyl
ether, 3.3 mL, 3.3 mmol) was added to a solution of
the TBS ether 1817 (420 mg, 0.79 mmol) in THF
(2.6 mL) at room temperature under N2 atmosphere
and the mixture was stirred for 2 h. Extractive (ethyl
acetate) work-up gave the crude product, which was
chromatographed on silica gel (hexane/ethyl ace-
tate = 15:1), to afford [26,27-2H6]3b-t-butyldimethylsily-
loxyergost-5-en-25-ol (402 mg, 62%) as a colorless oil.
1H NMR d: 0.057 (s, H6-Me2Si), 0.67 (s, H3-18), 0.89
(s, H9-t-Bu), 0.92 (d, J = 6.5 Hz, H3-21), 1.00 (s, H3-
19), 3.48 (m, H-3), 5.31 (br s, H-6).
References and notes
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POCl3 (0.56 mL, 6.0 mmol) was added to a solution of the
C-25 alcohol (300 mg, 0.56 mmol) in pyridine (8.0 mL)
and the mixture was stirred at room temperature for
3 h. The mixture was diluted with water and extracted
with ethyl acetate. The organic layer was washed with satd
NaHCO3 and brine, dried over MgSO4, and concentrated
under reduced pressure to give a ca. 1:1 mixture of D24-
and D25-olefins (365 mg). A solution of the olefinic mix-
ture in THF (10 mL) and conc HCl (1 mL) was stirred
at room temperature for 3 h. The reaction mixture was
diluted with satd NaHCO3 and extracted with ethyl ace-
tate. The organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The
crude product was separated by reversed-phase HPLC
[methanol, 9.9 mL/min, UV detection (215 nm), typical
retention times for the D24- and D25-olefins, 35.0 and
38.0 min, respectively] and crystallization of the desired
D25-olefin from methanol furnished [26,27-2H6]8
(148 mg, 60% from the C-25 alcohol) as white needles,
mp 138 ꢁC. 1H NMR d: 0.68 (s, H3-18), 0.96 (d,
J = 6.6 Hz, H3-21), 1.01 (s, H3-19), 1.61 (s, H3-28), 3.54
(m, H-3), 5.35 (br s, H-6). 13C NMR d: 11.85 (C-18),
18.45 (C-28), 18.79 (C-21), 19.39 (C-19), 21.08 (C-11),
24.32 (C-15), 28.18 (C-16), 31.05 (C-23), 31.68 (C-2),
31.91 (C-7), 31.91 (C-8), 34.33 (C-20), 35.96 (C-22),
36.51 (C-10), 37.25 (C-1), 39.75 (C-12), 42.31 (C-4),
42.34 (C-13), 50.13 (C-9), 55.83 (C-17), 56.74 (C-14),
71.81 (C-3), 121.71 (C-6), 123.13 (C-24), 128.42 (C-25),
140.75 (C-5). 13C-{1H}{2H} NMR d: 19.06, 19.62 (C-26,
11. Rubinstein, I.; Goad, L. J. Phytochemistry 1974, 13, 481–
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ergosterol in Prototheca wickerhamii was recently report-
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925–936.

738
K. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 732–738
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icasterol (12%), and cholesterol (2%).
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labeled.
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20. Feeding of [27-13C]codisterol to the cultured cells of
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and 10 (unpublished results).
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23. GenBank Accession No. S71189.