Mechanism of the second methylation in sitosterol side-chain biosynthesis in higher plants: Metabolic fate of 28-hydrogens of 24- methylenecholesterol in Morus alba cell cultures

Article abstract of DOI:10.1016/S0040-4039(00)00428-7

Biosynthesis of the side-chain of sitosterol in higher plants involves two methylation steps by attack of S-adenosylmethionine. The stereochemical features of the second methylation, namely, of the conversion from 24- methylenecholesterol to isofucosterol in higher plants has been investigated. Feeding studies of synthesized [28E-²H]- and [28Z-²H]-24- methylenecholesterols to cultured cells of Morus alba followed by ²H NMR analysis of the resulting isofucosterol established that the second methylation proceeded in such a manner that addition of the methyl group and proton loss occur on opposite faces of the original Δ(24(28))-double bond. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00428-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 3623–3626
Mechanism of the second methylation in sitosterol side-chain
biosynthesis in higher plants: metabolic fate of 28-hydrogens of
24-methylenecholesterol in Morus alba cell cultures
Tatsuya Okuzumi,a Yuko Kaji,a Hiroki Hamada b and Yoshinori Fujimoto a,∗
aDepartment of Chemistry and Materials Science, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japan
bDepartment of Applied Science, Okayama Science University, Ridai-cho, Okayama 700-0005, Japan
Received 7 February 2000; revised 6 March 2000; accepted 10 March 2000
Abstract
Biosynthesis of the side-chain of sitosterol in higher plants involves two methylation steps by attack of S-
adenosylmethionine. The stereochemical features of the second methylation, namely, of the conversion from 24-
methylenecholesterol to isofucosterol in higher plants has been investigated. Feeding studies of synthesized [28E-
2H]- and [28Z-2H]-24-methylenecholesterols to cultured cells of Morus alba followed by 2H NMR analysis of the
resulting isofucosterol established that the second methylation proceeded in such a manner that addition of the
methyl group and proton loss occur on opposite faces of the original ∆24(28)-double bond. © 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: steroids and sterols; biosynthesis; NMR; stereochemistry.
In the biosynthesis of the side-chain of sitosterol (4), one of the most popular sterols in higher
plants, 24-methylenecholesterol (1) which is produced by the first methylation of 24(25)-olefinic sterol,
is subjected to a second methylation leading to isofucosterol (2). Isofucosterol is isomerized to 24-
ethyldesmosterol (3) which is then reduced to yield 4 (Scheme 1).1 Concerning the stereochemical
features of this transformation, we have recently reported that the reduction of 3 to 4 takes place with
an anti-addition of hydrogen atoms from the 24-Si and 25-Re face in cell cultures of Oryza sativa.2 It
has also been demonstrated that isomerization of 2 to 3 proceeds in a syn-SE20 fashion in which the
hydrogen newly introduced at C-28 of 2 comes from the 28-Si face in the same culture.3 However, the
mechanism of the second methylation in higher plants remains to be established, although Djerassi’s
group have reported the mechanism of the second methylation in a marine sponge which contains 2 and
its ∆24(28)E-isomer, fucosterol (vide infra).4
Although the direction of transfer of the methyl group from S-adenosylmethionine (SAM) in higher
plants has not been fully established, the methyl transfer is suggested to occur from the 28-Si face of
∗
Corresponding author. Tel/fax: +81-3-5734-2241; e-mail: fujimoto@cms.titech.ac.jp (Y. Fujimoto)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00428-7
tetl 16708

3624
Scheme 1. Two possible steric courses of the second methylation in sitosterol side-chain biosynthesis. The present study has
established that the Ha hydrogen is retained at the C-28 positions of compound 2, 3 and 4. The carbons derived from C-2 of
mevalonate are indicated by black circles
∆
24(28)-double bond of 1 from the analogy of the first methylation5 as well as from stereochemical
consideration of clerosterol [(24S)-stigmasta-5,25-dien-3β-ol] biosynthesis.6 For simplicity, this text
including Scheme 1 is written on the assumption that this is correct. Thus, the purpose of this paper
is to elucidate whether Ha (designated as a ‘cis-process’, since addition of the methyl group and proton
loss occur on the same face of the ∆24(28)-double bond) or Hb (designated as a ‘trans-process’, since the
two events occur on opposite faces) is eliminated. To differentiate the two mechanisms, [28E-2H]- (1a)
and [28Z-2H]- (1b) 24-methylenecholesterols were synthesized and their feeding studies were carried
out using cell cultures of Morus alba,7 which was selected from several plant cell cultures since our
preliminary studies indicated a labeled-1 was most efficiently incorporated into 2.
Scheme 2. Synthesis of [28E-2H]- (1a) and [28Z-2H]- (1b) 24-methylenecholesterols. Reagents: (i) Ph3P, CBr4, py, 80%; (ii)
n-BuLi, then D2O, 99%; (iii) i-Pr2CuMgCl, then HCl, 66%; (iv) n-BuLi, then H2O, 95%; (v) i-Pr2CuMgCl, then DCl, D2O,
73%
28E-2H compound 1a was synthesized from the known aldehyde 5 via deuterated acetylene in three
steps (dibromoolefination,8 elimination–deuteration, and carbocupration–deprotection) according to the
published method (Scheme 2).9 The synthesis of 28Z-2H compound 1b was also achieved in a similar
three-step sequence, but introducing the deuterium atom by quenching the carbocupration reaction with
1
D2O/DCl. The H NMR spectra (Fig. 1) of 1a (δ 4.70 for 28Z-H), 1b (δ 4.64 for 28E-H), and non-

3625
labeled 1 (δ 4.71 and 4.65 for 28Z-H and 28E-H, respectively) clearly indicated that the deuterium label
was regiospecifically introduced. The (E)-geometry for the ∆24(28) of 1a was confirmed by NOE studies
in which irradiation of the C-26 or C-27 methyl signal caused enhancement of the signal intensity of
28-H, whereas analogous NOE studies of 1b did not show such signal enhancement.
Fig. 1. Partial 1H NMR spectra (300 MHz, in CDCl3) of 1a (A), 1b (B), and non-labeled 1 (C)
Compounds 1a and 1b (50 mg each) were individually fed to cell cultures of M. alba (three 500
ml flasks containing 250 ml of Murashige–Skoog medium) which had been preincubated for 2 weeks,
and the cultures were incubated for another 2 weeks. The sterol fraction was obtained by silica gel
chromatography of the CHCl3–MeOH extract of the cells. This was further separated by RP-HPLC to
2
give a mixture of isofucosterol (2) and cholesterol.† The H NMR spectrum (Fig. 2A) of the mixture
Fig. 2. 2H NMR spectra (61 MHz, in CHCl3) of 2 derived from 1a (A), and from 1b (B), and 1H NMR spectrum (300 MHz, in
CHCl3) of authentic 2 (C). A weak signal around δ 4.70 is apparently due to 28E-2H of the slightly contaminated substrate 1a
†
The cells grown without feeding external sterol contain sitosterol (81%), campesterol (4%), isofucosterol (8%) and
cholesterol (7%).

3626
derived from 1a exhibited a deuterium signal at δ 5.13 due to 28-H of 2. Complementary results were
obtained from the incubation of 1b, as shown in Fig. 1B in which no signal was observed at δ 5.13. The
results unambiguously established that 28E-hydrogen (Ha) of 24-methylenecholesterol is retained while
28Z-hydrogen (Hb) is eliminated during the conversion of 1 to 2 in the cultured cells of M. alba. Thus, it
can be concluded that the second methylation of 1 proceeded in the ‘trans-process’.
Furthermore, sitosterol 4 derived from 1a, but not derived from 1b, exhibited a signal at δ 1.23 which
2
corresponds to a 28-hydrogen in the H NMR spectrum (the pro-R and pro-S hydrogens of 4 resonate
at δ 1.24 and 1.28, respectively3). This is compatible with the above findings that the Ha hydrogen is
retained in the second methylation process. The observed 2H signal in 4 is most likely to be assigned to
28-pro-R hydrogen in view of our finding that isomerization of 2 to 3 occurs in the SN20 mechanism.3
It is interesting to compare the present results with those reported in the sponge, Xestospongia
testudinaria.4 In the case of the sponge, it was reported that the label of 28E-3H-1 was incorporated
into isofucosterol, but not fucosterol, whereas the label of 28Z-3H-1 was incorporated into isofucosterol
as well as fucosterol. The authors deduced from these results that a ‘trans-process’ operates in a sponge
and the tritium isotope effect seemed to be responsible for the anomalous behavior. In contrast to the
complicated observations, our results in cell cultures of M. alba are very simple: feeding of 1a and 1b
furnished isofucosterol, but not fucosterol, as evidenced by 1H NMR analysis, and the elimination of Hb
takes place in a highly stereospecific manner even when Hb is replaced with a deuterium atom.
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