Methodology for the preparation of pure recombinant S. cerevisiae lanosterol synthase using a baculovirus expression system. Evidence that oxirane cleavage and A-ring formation are concerted in the biosynthesis of lanosterol from 2,3-oxidosqualene

Article abstract of DOI:10.1021/ja963227w

Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the kinetics of cyclization were determined using Michaelis-Menten analysis for 2,3-oxidosqualene (1) and two analogs in which the C-6 methyl was replaced by H (3) or Cl (4). The measured V(max)/K(M) ratios for 1, 3, and 4 were found to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis studies of each of the six strongly conserved aspartic acid residues (D → N mutation) and each of the nine conserved glutamic acid residues (E → Q) revealed that only one, D456, is essential lor catalytic function of the enzyme. The essential D456 residue is a likely candidate for electrophilic (specifically protic) activation of the oxirane function.

Full text of DOI:10.1021/ja963227w

J. Am. Chem. Soc. 1997, 119, 1277-1288
1277
Methodology for the Preparation of Pure Recombinant S.
cereVisiae Lanosterol Synthase Using a Baculovirus Expression
System. Evidence That Oxirane Cleavage and A-Ring
Formation Are Concerted in the Biosynthesis of Lanosterol
from 2,3-Oxidosqualene
E. J. Corey,* Hengmiao Cheng, C. Hunter Baker, Seiichi P. T. Matsuda,
Ding Li, and Xuelei Song
Contribution from the Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138
ReceiVed September 13, 1996X
Abstract: Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme
from Saccharomyces cereVisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis,
was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the
kinetics of cyclization were determined using Michaelis-Menten analysis for 2,3-oxidosqualene (1) and two analogs
in which the C-6 methyl was replaced by H (3) or Cl (4). The measured Vmax/KM ratios for 1, 3, and 4 were found
to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are
concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic
metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis
studies of each of the six strongly conserved aspartic acid residues (D f N mutation) and each of the nine conserved
glutamic acid residues (E f Q) revealed that only one, D456, is essential for catalytic function of the enzyme. The
essential D456 residue is a likely candidate for electrophilic (specifically protic) activation of the oxirane function.
Introduction
the epoxide function and formation of the A-ring. In addition,
we describe the overproduction of the yeast (S. cereVisiae)
enzyme in a baculovirus system and an effective method for
the purification of this protein.
The most remarkable step in the biosynthesis of steroids (and
one of the most complex in biochemistry) is the conversion of
oxidosqualene (1) to lanosterol (2) catalyzed by lanosterol
synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol
forming), EC 5.4.99.7], which controls precisely the formation
of four rings and six new stereocenters as shown in Scheme 1.1
Although there have been numerous studies of the mechanism
of this complicated process over the past four decades, much
remains to be learned about the enzyme and how it mediates
the individual cyclization and rearrangement steps. Although
it has been often assumed that the cyclization to form the
protosteryl cation is concerted, no compelling evidence for this
proposal had been found. In addition, most of the work over
the past three decades has been done with crude enzyme
preparations. The lipophilic and insoluble nature of this
membrane-associated enzyme necessitates the use of detergents
and glycerol to obtain solutions and greatly complicates
purification. Nonetheless, the application of recombinant DNA
technology has led recently to the cloning of genes and
determination of amino acid sequences for lanosterol synthase
from various organisms.2
Results and Discussion
Overexpression of S. cereWisiae Lanosterol Synthase in E.
coli and S. cereWisiae. In order to obtain large quantities of
lanosterol synthase for detailed study, extensive attempts were
made to overexpress the protein in different systems. Overex-
pression of the enzyme in E. coli was unsuccessful because
active protein could not be obtained, although several different
overexpression vectors and strains were used.3 The exact
reasons for this lack of success are unknown but could result
from protein insolubility, improper folding, or lack of proper
post-translational modification of the protein to form active
enzyme.4
As anticipated, overexpression of the S. cereVisiae lanosterol
synthase in S. cereVisiae, its native organism, provided active
(2) For recent work on the gene cloning and protein sequences for
lanosterol synthase form various organisms, see: (a) Kelly, R.; Miller, S.
M.; Lai, M. H.; Kirsch, D. R. Gene 1990, 87, 177 and Buntel, C. J.; Griffin,
J. H. J. Am. Chem. Soc. 1992, 114, 9711 (Candida albicans). (b) Corey, E.
J.; Matsuda, S. P. T.; Bartel, B. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
11628 (Arabidopsis thaliana). (c) Corey, E. J.; Matsuda, S. P. T.; Bartel,
B. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2211. Shi, Z.; Buntel, C. J.;
Griffin, J. H.; Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7370 (Saccharomyces
cereVisiae). (d) Kasuno, M.; Shibuya, M.; Sankawa, U.; Ebizuka, Y. Biol.
Pharm. Bull. Jpn. 1995, 18, 195. Abe, I.; Prestwich, G. D. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 9274 (Rattus norVegicus). (e) Baker, C. H.; Matsuda,
S. P. T.; Liu, D. R.; Corey, E. J. Biochem. Biophys. Res. Commun. 1995,
213, 154 (Homo sapiens). (f) Corey, E. J.; Matsuda, S. P. T.; Baker, C. H.;
Ting, A.; Cheng, H. Biochem. Biophys. Res. Commun. 1996, 219, 327
(Schizosaccharomyces pombe).
The most recent mechanistic studies of lanosterol biosynthe-
sis, which have been conducted with purified enzyme from
recombinant sources, have revealed that the cyclization involves
discrete carbocations during C-ring formation and specifically
that a five-membered C-ring cationic structure is first formed
and then enlarged by expansion to a six-membered structure as
shown in Scheme 2. Another aspect of the cyclization process
is the subject of this paper, namely, the mode of activation of
X Abstract published in AdVance ACS Abstracts, February 1, 1997.
(1) For a recent review, see Abe, I.; Rohmer, M.; Prestwich, G. D. Chem.
ReV. 1993, 93, 2189.
(3) Vectors used: pKK223-3, pLM1, pKen2, pET-15b. Strains used:
JM101, XA90, BL21-DE(3), BL21-DE(3)pLysS.
S0002-7863(96)03227-1 CCC: $14.00 © 1997 American Chemical Society

1278 J. Am. Chem. Soc., Vol. 119, No. 6, 1997
Corey et al.
Scheme 1. Lanosterol Synthase Catalyzed Cyclization of 2,3-Oxidosqualene to Lanosterol
Scheme 2. Six-Membered C Ring Formation via Ring Expansion
protein. Several promoter systems were tried (GAL1, ADH, and
PGK), but the best expression level was 8-fold higher than wild
type.2c This level of overexpression proved unsatisfactory.2c
The yeast cells (90 g) were lysed by French press and
fractionated by ultracentrifugation. The enzymatically active
microsomes were solubilized in a solution containing 1% Triton
X-100 and 10 mM dithiothreitol (DTT), subjected to DEAE
Sepharose column chromatography and a subsequent hydroxy-
apatite (HAT) column chromatography. At this stage, the purity
was approximately 10%. Further purification by FPLC with a
Mono Q column gave 50 µg of protein of only 90% purity.
Overexpression of S. cereWisiae Lanosterol Synthase in
Baculovirus-Infected Insect Cells and Purification and
Characterization. Because the S. cereVisiae-expressed protein
was difficult to purify and insufficient for extensive studies,
we turned to the baculovirus system. Baculovirus yields are
relatively insensitive to protein toxicity, because polyhedrin-
promoted expression peaks very late in infection when the virus
is already killing the cells, and eukaryotic post-translational
modifications generally occur correctly in the eukaryotic Sf 9
cells. Sf 9 cells were infected with an ERG7-recombinant
Autographa californica nuclear polyhedrosis virus (AcNPV)
constructed with the PharMingen Co. transfection kit. The time
course for expression of the S. cereVisiae lanosterol synthase
in infected Sf 9 cells was studied both on tissue culture plates
and in spinner flasks. Maximum expression of cyclase activity
was seen 3 days post-infection when cell viability (as determined
by Trypan blue staining) was >80%. At 4 days, cell viability
had dropped to 50%. All activity was found in the cell pellet,
rather than in the media.
Figure 1. Protein gel of fractions from different stages of the
purification. Lanes from left to right are as follows: (a) protein
standards, (b) cell homogenate, (c) supernatant after first centrifugation,
(d) redissolved pellet from ammonium sulfate precipitation, (e) protein
solution after DEAE ion exchange chromatography, and (f) pure protein
after HAT chromatography.
Table 1. Lanosterol Synthase Purification
vol. protein conc. total amount
sp. act.
(pmol/µg/h) n-fold
purif.
fraction
(mL)
(mg/mL)
(mg)
Using this recombinant protein source, lanosterol synthase
was purified to greater than 99% purity after only three steps:
(1) ammonium sulfate precipitation, (2) DEAE Sepharose ion
exchange column chromatography, and (3) hydroxyapatite
column chromatography (Figure 1). Starting from 20 g of cells,
2.5 mg of pure protein was obtained (Table 1). The purified
protein showed a single band on SDS-polyacrylamide gel
electrophoresis with a molecular weight of 83 kDa as predicted
by the complementary DNA sequence.
homogenate 63
332
20,900
18,070
4,850
1.26
1.15
8.16
supernatant
(NH4)2SO4
DEAE
63
50
32
40
287
1
7
159
2129
97.8
0.31
0.050
9.61
2.0
182
2448
HAT
enzyme activity. FPLC chromatography was unnecessary, and
large amounts of protein could readily be obtained by conven-
tional column chromatography. Protein solutions were desalted
by dialysis prior to chromatography since ultrafiltration signifi-
cantly reduced enzyme activity. Washing the HAT column with
sodium chloride prior to elution removes all neutral and
positively charged proteins and is essential to purify the protein.
The protein was digested with trypsin, and the resulting
fragments were separated by reversed phase HPLC with a Vydac
Glycerol (20%), Triton X-100 (0.2%), and DTT (3 mM) must
be added to the buffer (sodium phosphate, pH 7.0) to maintain
(4) E. coli lacks the post-translational modification machinery present
in eukaryotes, and might be unable to produce active lanosterol synthase
for this reason.

Preparation of S. cereVisae Lanosterol Synthase
J. Am. Chem. Soc., Vol. 119, No. 6, 1997 1279
revealed cyclization to a lanosterol analog. For 4, KM was found
to be 2.5 times higher than for 1 (and essentially the same as
for 3) and Vmax was found to be about 2.5 times less than for 1.
Comparison of the Vmax/KM values for 1, 3 and 4 is
instructive: for 1, Vmax/KM ) 138; for 3, Vmax/KM ) 9.44; and
for 4, Vmax/KM ) 21.9. These results show that the rate at which
lanosterol synthase effects cyclization of the substrate correlates
with the nucleophilicity of the 6,7-double bond and strongly
indicates that the 6,7-double bond participates in the closure of
ring A and in the oxirane ring cleavage, i.e., oxirane cleavage
and A ring formation are concerted. Put in another way, an
intermediate tertiary cation (C-2 carbocation) formed by
electrophilically-assisted heterolysis of the oxirane ring is
unlikely to be a discrete intermediate in the conversion of 2,3-
oxidosqualene to lanosterol. If the formation of a localized C-2
carbocation were involved in the rate-limiting step of the
cyclization, essentially identical Vmax/KM values would be
expected for the enzymic cyclization of 1, 3, and 4. The
observed order of Vmax/KM values, 1 (138) > 4 (21.9) > 3 (9.14),
clearly correlates with the carbocation stabilizing ability of the
C-6 group, CH3 > Cl > H.
The proposal that cleavage of the electrophilically activated
oxirane function of 1 and A-ring closure are concerted implies
that the tight control by lanosterol synthase on the conformation
of 2,3-oxidosqualene7 enforces a suitable prefolded, three-
dimensional geometry on 1, including proximity of C-2 and
C-7 which are joined during A-ring formation. The concerted
A-ring closure/oxirane cleavage model has received strong
support from the study of the spiro epoxide 5 in which the
isopropyl subunit of 2,3-oxidosqualene is replaced by cyclo-
propyl. It is well-known that oxaspiropentanes such as 5
undergo extremely facile acid-catalyzed conversion to cyclobu-
tanones, doubtless by pinacolic rearrangement of an intermediate
stabilized cyclopropylcarbinyl cation.8 Consequently, it seemed
very possible that the spiro epoxide 5 would be converted by
lanosterol synthase to a monocyclic cyclobutanone without
involvement of any of the olefinic units and simply as a
consequence of electrophilic activation of the oxirane by the
enzyme. In fact, analog 5 was resistant to change by lanosterol
synthase at pH 7 under conditions for the enzymic conversion
of 1 to lanosterol. This rather striking result seems inexplicable
in terms of a two-stage mechanism involving (1) electrophili-
cally induced oxirane cleavage and (2) subsequent closure of
the A ring. On the other hand, it does seem reconcilable with
the concerted oxirane cleavage-A-ring closure pathway since
that process would require a less potent activating electrophile
and a substrate folding which brings C-2 and C-7 into
proximity. The geometry of the oxaspiropentane subunit of 5
is such that one of the cyclopropyl methylenes would be
involved in strong steric repulsion with the methylene group
attached to C-7 in the appropriate conformation for the
concerted oxirane cleavage-A-ring closure pathway.
Figure 2. Michaelis-Menten plot of lanosterol synthase with 2,3-
oxidosqualene.
narrowbore column (C18, 300 Å, 2.1 mm × 150 mm). Two of
the resulting oligopeptides were subjected to Edman degradation
on a Hewlett Packard G1005 protein sequencer. The resulting
sequences NTVCGVDLYYPHSTTLNIANSLVVF and LFEGID-
VLLNLQNIGSFEYG are identical to the sequences predicted
from the DNA sequence of S. cereVisiae lanosterol synthase.
In 150 mM sodium phosphate buffer (pH 6.3) containing 20%
glycerol, 0.2% Triton X-100, and 3 mM DTT, the pure enzyme
has KM of 18 µM and Vmax of 2448 pmol/µg/h for 2,3-
oxidosqualene (Figure 2). The maximum activity of the
overexpressed enzyme occurs at pH 6.3, which is the same as
for the wild type yeast enzyme.
Epoxide Activation and A-Ring Cyclization Are Con-
certed. Granted that the closure of the A ring in lanosterol
synthesis is a cationic process, an important question is whether
epoxide opening occurs prior to or concurrently with cyclization
by C-C bond formation. In order to investigate this question,
a number of mechanistically relevant analogs of 2,3-oxi-
dosqualene were synthesized and studied with regard to the
catalytic action of lanosterol synthase. We discuss first the
investigation of two analogs in which the 6-methyl group of
2,3-oxidosqualene (which becomes the A/B angular methyl of
lanosterol) is replaced by hydrogen or chlorine, i.e., 6-desmethyl-
2,3-oxidosqualene (3) and 6-chloro-6-desmethyl-2,3-oxidosqualene
(4). These compounds and their 3-tritiated radioactive coun-
3
terparts (3H-3 and H-4) were synthesized as outlined in the
section on synthesis which appears below. The 6,7-Z isomer
of 3 and the 6,7-E isomer of 4 were also available from
synthesis. As expected, these double bond isomers with
unnatural olefinic geometry were inert to the action of lanosterol
synthase. They served neither as substrates for the enzyme nor
inhibitors of the cyclization of 2,3-oxidosqualene to lanosterol.
In contrast 6-desmethyl-2,3-oxidosqualene (3) was converted
by lanosterol synthase to a product of the same chromatographic
mobility as lanosterol (Rf 0.5 on silica gel plates with 1:1 ether-
Epoxides 6, 7, and 8 were also tested as substrate analogs of
2,3-oxidosqualene with lanosterol synthase and were found
neither to be substrates nor time-dependent inhibitors, again
consistent with the idea of a concerted A-ring closure process
with only modest electrophilic activation provided by the
enzyme. Substrates 6 and 7 clearly would require powerful
1
hexane) and which was shown by H NMR comparison with
lanosterol5 to be 10-desmethyllanosterol. It was also observed
that the rate of cyclization of 3 was less than that of 2,3-
oxidosqualene and that 3 caused a marked time-dependent
inactivation of lanosterol synthase. Careful Michaelis-Menten
kinetic studies with 3H-3 following the reaction with pure
lanosterol synthase from zero to 2.5% conversion revealed that
the KM value for 3 is about 2.5 times higher than for 1 and that
Vmax is about 6 times less.6 Similar studies and kinetic analysis
with 6-desmethyl-6-chloro-2,3-oxidosqualene (4) and 3H-4
(6) Measurements of kinetics were carried out under the same standard
conditions for 1, 3, and 4 (with respect to medium, concentrations,
temperature etc.) to allow comparison. Obviously, KM values are not absolute
since detergent, which can associate with the substrate, is present.
(7) (a) Corey, E. J.; Virgil, S. C.; Cheng, H.; Baker, C. H.; Matsuda, S.
P. T.; Singh, V.; Sarshar, S. J. Am. Chem. Soc. 1995, 117, 11819. (b) Corey,
E. J.; Virgil, S. C.; Liu, D. R.; Sarshar, S. J. Am. Chem. Soc. 1992, 114,
1524.
(5) Emmons, G. T.; Wilson, W. K.; Schroepfer, G. J. Magn. Reson.
Chem. 1989, 1012.
(8) Trost, B. M. Acc. Chem. Res. 1974, 7, 85.

1280 J. Am. Chem. Soc., Vol. 119, No. 6, 1997
Corey et al.
Figure 3. Alignment of lanosterol synthase amino acid sequences from Saccharomyces cereVisiae (Sc), Candida albicans (Ca), Schizosaccharomyces
pombe (Sp), Homo sapiens (Hs), and Rattus norVegieus (Rn). Residues conserved in at least three sequences are shaded.
electrophilic activation because of the lack of charge stabilizing
methyl groups at the oxirane C-terminus. Epoxide 8 (a nor
oxidosqualene) not only has a spatially different location of the
oxirane oxygen but also poor stereoelectronic geometry for
concerted oxirane cleavage and ring closure. Interesting, but
of less mechanistic relevance, is the complete resistance of vinyl
ether 9 and aldehyde 10 to lanosterol synthase induced cycliza-
tion.
Finally, Z-4,5-dehydro-2,3-oxidosqualene (11) was synthe-
sized (Vide infra) and tested as a substrate for lanosterol
synthase. This epoxide clearly bound very well to the enzyme
since it is a strong competitive inhibitor (IC50 8.3 µM) for the
cyclization of 2,3-oxidosqualene (KM 18 µM). However,
epoxide 11 was totally resistant to either cyclization or epoxide
cleavage by lanosterol synthase. Given the fact that epoxide
11 is very sensitive to protic activation (for example, it is very
unstable on silica gel), as might be expected for an allylic
epoxide, the ineffectiveness of lanosterol synthase as a catalyst
for oxirane cleavage in 11 is impressive. There are two factors
which may give rise to this inertness of 11. First, the
electrophilic activation by the enzyme may be weak and may
be effective only for concerted nucleophilic attack by the 6,7-
double bond. Second, the three-dimensional arrangement
required for concerted oxirane cleavage-A-ring cyclization of
11 is somewhat different than that for 1 and involves signifi-
cantly greater steric repulsions of the C-1, C-1′, and C-6
methyl groups and poorer orbital overlap between C-2 and
C-7. The experimental results with substrate 11 appear to favor
the concerted over the nonconcerted ring A closure pathway.
D456 Is The Prime Candidate As Electrophilic Activator
of 2,3-Oxidosqualene in Lanosterol Biosynthesis. It is
generally accepted that the cyclization of 2,3-oxidosqualene to
lanosterol proceeds by way of cationic intermediates1 and that
the process is initiated by electrophilic activation of the oxirane
function. One possibility for such electrophilic activation is
Lewis acid catalysis by a cationic metal center bound to the
enzyme. In order to test for the type of metal ion activation
samples of purified enzyme in purified buffer solution (which
had been treated to remove transition metals and zinc) was
passed through a hydroxyapatite column (which itself removes
these metal ions), collected, and analyzed by atomic absorption
(carried out by Drs. Robert Shapiro and Bert L. Vallee of the
Harvard Medical School). These analyses were negative for
Mn, Cu, and Fe cations and essentially negative for Zn2+ (since
the amount found was only 0.13 Zn2+ per molecule of enzyme).
As a result of these findings we believe that oxirane activation
is unlikely to be caused by a Lewis acidic metal ion and is
probably effected by an acidic group (i.e., protic acid) on the
enzyme. This conclusion is also consistent with the observations
that metal ion chelators such as EDTA and 1,10-phenanthroline

Preparation of S. cereVisae Lanosterol Synthase
Scheme 3. Oxidosqualene Analogs
J. Am. Chem. Soc., Vol. 119, No. 6, 1997 1281
Scheme 4. Possible Role of D456 in Catalysis by Lanosterol
Scheme 5. Synthesis of 3a
Synthase
do not inhibit lanosterol synthase and that H2S and trisnor-
squalenethiol are also without inhibitory effect.
Information regarding the most likely acidic amino acid unit
for protic activation of the oxirane function of lanosterol
synthase was obtained from site-directed mutagenesis experi-
ments involving the aspartic acid (D) and glutamic acid (E)
residues of the enzyme which are conserved in five different
species, identified in Figure 3. The species, Saccharomyces
cereVisiae,2c Candida albicans,2a Schizosaccharomyces pombe,2f
Homo sapiens,2e and Rattus norVegicus,2d arose over a large
evolutionary time scale (ca. 880 million years). Each of the
six conserved Asp residues, D140, D286, D370, D456, D580,
and D629, was mutated to asparagine, and each of the nine
conserved Glu residues, E216, E264, E460, E483, E487, E511,
E520, E526, and E634, was mutated to glutamine. Site-directed
mutagenesis experiments were carried out using the ERG7 gene
by the method of Kunkel.9 The steps used were as follows:
(1) annealing of the appropriate oligonucleotide to the uracil
containing single-stranded DNA template, (2) primer extension
using T4 DNA polymerase and nick sealing using T4 DNA
ligase, (3) transformation into E. coli DH5R and selection of
a (a) BnBr, NaH, 95%. (b) NMO, OsO4, acetone/H2O, 83%. (c) 2,2-
dimethoxypropane, p-toluenesulfonic acid, 97%. (d) H2, 10% Pd/C,
98%. (e) MsCl, Et3N, CH2Cl2; (f) NaI, acetone, 91% over two steps.
(g) PPh3, CH3CN, 99%. (h) n-BuLi, THF, -78 °C, then R20CH2CHO.
(i) s-BuLi, THF, -78 °C. (j) 2-phenyl-1-propanol, -78 °C to 23 °C,
55% overall. (k) TsOH, MeOH, 60%. (l) MsCl, Pyr, CH2Cl2. (m)
K2CO3, MeOH, 80% overall.
mutants using restriction fragment analysis, (4) transformation
into the mutant yeast strain SMY8 (MATa erg7::HIS3 hem1::
TRP1 ura3-52 trp1- 63 leu2-3,112 his3-D200 ade2 Gal+),2f
and (5) testing for ergosterol production by the complementation
method. SMY8 transformants were selected after growth in a
synthetic complete medium lacking leucine and supplemented
with ergosterol. The transformant with the desired mutant was
then restreaked on a YPG plate without supplementary ergos-
terol. Of the nine conserved glutamates none is essential since
all of the individual E f Q mutants were viable in an ergosterol-
free medium. Of the six conserved aspartates only one, D456,

1282 J. Am. Chem. Soc., Vol. 119, No. 6, 1997
Corey et al.
3
Scheme 6. Synthesis of H-Labeled 3a
a (a) NaIO4, THF/H2O, 79%. (b) NaBT4, MeOH, 90%. (c) Dess-Martin reagent, CH2Cl2, NaHCO3, 81%. (d) Isopropyldiphenylsulfonium
tetrafluoroborate, LDA, DME, -78 °C, 99%.
3
is essential since only the D456N mutant was not viable in the
absence of ergosterol.
hydes upon treatment with Dess-Martin reagent. When H-
labeled aldehydes 18 and 6-Z-18 were treated with the ylide
generated from diphenylisopropylsulfonium tetrafluoroborate,
3H-labeled 3 was obtained together with 3H-labeled 6-Z-3. The
validity of the kinetic parameters measured using the mixture
of 3H-labeled 3 and 6-Z-3 was confirmed by control experiments
which showed that addition of 6-Z-3 to a concentration five
times higher than that of 3 did not change the measured kinetics,
as expected since 6-Z-3 is not a substrate.
Incorporation of the mutagenic oligonucleotide was confirmed
by restriction fragment analysis since the oligonucleotides were
designed to introduce a new restriction site within the gene. To
further confirm incorporation of the desired mutations DNA
sequencing of the mutated erg7 gene was performed on the
following mutants: D140N, D370N, D456N, D580N, E460Q,
and E634Q. DNA sequence analysis showed both that the
desired mutations had been incorporated and that no other
mutations had been introduced during the mutagenesis proce-
dure.
The essentiality of D456, its occurrence in a highly conserved
region, and the dispensability of all 14 of the other conserved
aspartates and glutamates all point to D456 as a likely candidate
for protic activation of 2,3-oxidosqualene to initiate cyclization.
A matter of concern in this regard is the question of whether
D456 is sufficiently acidic to catalyze fast oxirane cleavage,
since experiments by Ms. Donnette Daley in this laboratory have
revealed that 2,3-oxidosqualene (and similar oxiranes) are stable
in glacial acetic acid at 23 °C for many hours and that stronger
acids such as trichloroacetic acid are required for catalyzed
oxirane cleavage. However, considerable enhancement in the
acidity of D456 by the presence of neighboring positive charge10
is clearly a reasonable possibility. For instance, a proximate
protonated histidine hydrogen bonded to the D456 carboxyl endo
lone pair as shown in Scheme 4 should enhance the acidity of
that carboxyl by several orders of magnitude.11
Synthesis of 2,3-Oxidosqualene Analogs. 5-Methyl-4-
hexen-1-ol (12), prepared in 86% yield by reaction of γ-buty-
rolactol with isopropylidenetriphenylphosphorane in THF, was
converted to acetonide 13 as shown in Scheme 5. Conversion
of the alcohol 13 to the corresponding iodide and subsequent
treatment with triphenylphosphine yielded the triphenylphos-
phonium salt 14. Coupling of 14 with the aldehyde 15 using
the modified Schlosser-Wittig reaction conditions gave 6-E-
16 and 6-Z-16 in 1:1 ratio (reaction not optimized). The mixture
of Z and E isomers of 16 was treated with TsOH in MeOH to
remove the protecting group and give diols 17 and 6-Z-17, which
were separated by preparative TLC using 3:1 hexane-EtOAc.
The 6-E olefin 17 was converted to 6-desmethyl-2,3-oxi-
dosqualene (3) via the monomesylate in the standard way. 6-Z-
17 was converted 6-Z-3 by the same method. Wittig reaction
of aldehyde 15 with the ylide from 14 under salt-free conditions
(THF, KN(TMS)2 as base) produced 6-Z-16 cleanly which was
further transformed into pure 6-Z-3. The stereochemistry of 3
at the 6,7-olefinic linkage was clearly shown by the observation
of a 15 Hz coupling between the protons at C(6) and C(7) as
contrasted with J ) ca. 6 Hz for the corresponding protons of
6-Z-3. These assignments are consistent with the findings that
3 is a substrate for lanosterol synthase, whereas 6-Z-3 is not.
3H-labeled 6-desmethyl-2,3-oxidosqualene was prepared as
shown in Scheme 6. A mixture of Z and E isomers of 17 was
cleaved with NaIO4 to give the corresponding aldehydes 18 and
6-Z-18. The aldehydes were first reduced with NaBT4 to yield
3H-labeled alcohols, which were converted to 3H-labeled alde-
The chloro analog 4 was synthesized by N-chlorosuccinimide
chlorination12 as shown in Scheme 7. The resulting mixture of
6-Z and 6-E isomers of 19 as well as nonchlorinated 6-Z-16
was deprotected to the corresponding diol mixture and separated
first by chromatography on silica gel containing 10% AgNO3
to give the Z and E isomers of diol 20 and then by preparative
silica gel plate chromatography to give pure 20 and the Z isomer.
Conversion of 6-Z olefin 20 to 4 and of 6-E-20 to 6-E-4 were
carried out in the standard way via the mono mesylate. 3H-
labeled 4 was synthesized by a process analogous to that used
3
for the synthesis of H-3 as outlined in Scheme 6.
The stereochemical assignment to 4 and 6-Z-4 is supported
by the following data: (1) the observed and calculated13 δ values
for the olefinic proton at C(7) in 4 are 5.48 and 5.49; (2) the
observed and calculated13 δ values for the olefinic proton at
C(7) in 6-Z-4 are 5.60 and 5.62; and (3) epoxide 4 is a substrate
for lanosterol synthase, whereas 6-Z-4 is not.
From trisnorsqualene aldehyde, compounds 5-10 were
prepared as shown in Scheme 8. Spiro epoxide 5 was prepared
by reaction of trisnorsqualene aldehyde with the ylide generated
from cyclopropyldiphenylsulfonium tetrafluoroborate.14 1,1′-
Bisnorsqualene epoxide (6) was prepared by coupling trisnor-
squalene aldehyde with trimethylsulfonium methylide. Treat-
ment of trisnorsqualene aldehyde with MeLi gave the corre-
sponding alcohol which by oxidation with PCC generated the
methyl ketone 21. Upon treatment with the ylide generated from
trimethylsulfonium methylide, 21 was converted to 7. After
oxidation of trisnorsqualene aldehyde with NaClO2 to the
corresponding acid, and methylation with diazomethane, the
resulting methyl ester was treated with LDA and NCS to give
the corresponding R-chloromethyl ester 22. When 22 was
treated with MeLi, epoxide 8 was generated. The vinyl ether
9 was prepared by coupling trisnorsqualene aldehyde with
methoxymethylene triphenylphosphorane. Upon acidic hy-
drolysis, 9 was converted to aldehyde 10.
(9) Kunkel, T. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 488.
(10) Westheimer, F. H. Tetrahedron 1995, 51, 3.
(11) (a) Gandour, R. D. Bioorganic Chem. 1981, 10, 169. (b) This
proposal contrasts with the position taken by Abe and Prestwich (Abe, I.;
Prestwich, G. D. Lipids 1995, 30, 231) that two adjacent asp residues in rat
lanosterol synthase, corresponding to D456 and C457 in the yeast enzyme,
“may act as point charge to stabilize incipient cationic charge”. See, also:
Abe, I.; Prestwich, G. D. J. Biol. Chem. 1994, 269, 802.
(12) Corey, E. J.; Shulman, J. I.; Yamamoto, H. Tetrahedron Lett. 1970,
447.
(13) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric
Identification of Organic Compounds, 4th ed.; John Wiley and Sons: New
York, 1981, p 228.
(14) Trost, B. M.; Bogdanowicz, M. J. J. Am. Chem. Soc. 1973, 95, 5311.

Preparation of S. cereVisae Lanosterol Synthase
Scheme 7. Synthesis of 4a
J. Am. Chem. Soc., Vol. 119, No. 6, 1997 1283
a (a) n-BuLi, THF, -78 °C. (b) NCS, THF, -78 °C. (c) s-BuLi, THF, -78 °C, then 15, 36% overall. (d) TsOH, MeOH, 28%. (e) MsCl, Pyr,
CH2Cl2. (f) K2CO3, MeOH, 74% overall.
Scheme 8. Synthesis of 5-10a
Conclusions
In summary, new data and insights have been presented on
the initiation step in sterol biosynthesis from 2,3-oxidosqualene.
Specifically, the possibility has been advanced that electrophilic
oxirane cleavage and cyclization to form the A ring are
concerted15 and that oxirane activation involves a particularly
acidic, highly conserved aspartic acid residue, D456. An X-ray
crystal structure of lanosterol synthase would be of great value
in connection with these hypothesis. However, given the
hydrophobic character, water insolubility, and instability of the
enzyme, the prospects for such a determination in the foreseeable
future seem questionable.
Experimental Section
Proton and carbon nuclear magnetic resonance (1H and 13C NMR)
spectra were recorded on an AM-500 (500 MHz), AM-400 (400 MHz),
or AM-300 (300 MHz) spectrometers using chloroform-d as a solvent.
Chemical shifts are reported as δ, parts per million downfield shift
from tetramethylsilane (δ 0.0) using the residual solvent signal as an
internal standard; δ 7.24 (1H), δ 77.0 triplet (13C). All coupling
constants are in units of Hertz. Infrared spectra (IR) were recorded on
a Nicolet 5 ZDX FTIR spectrometer with an internal polystyrene sample
as a reference. Mass spectral analyses were performed with JEOL
model AX-505 or SX-102 spectrometers. Reactions were monitored
by thin layer chromatography (TLC) using Merck 60 F254 precoated
silica gel plates (0.25 mm thickness). After ultraviolet illumination at
254 nm, the plates were visualized by immersion in the indicated
solution and warming on a hot plate. Baker silica gel and reagent grade
solvents were used for flash chromatography. Preparative thin layer
chromatography was performed using Merck 60 F254 precoated silica
gel plates (0.50 mm thickness, 20 × 20 cm). AgNO3 impregnated silica
gel plates were prepared by the addition of 5% (w/w silica gel) AgNO3
to the water used in typical chromatotron plate preparation. All
substrate analogs were purified chromatographically to homogeneity
by TLC analysis and purity was confirmed as >97% by 1H NMR
analysis. All solvents are reagent grade unless otherwise stated, and
anhydrous solvents were dried immediately prior to use.
a (a) Cyclopropyldiphenylsulfonium tetrafluoroborate, KOH, DMSO,
23 °C, 62%. (b) Trimethylsulfonium iodide, n-BuLi, 20%. (c) MeLi,
THF, -78 °C, 21%. (d) PCC on alumina, hexane, 23 °C, 84%. (e)
Trimethylsulfonium iodide, n-BuLi, THF, 3%. (f) NaClO2, NaH2PO4,
H2O, t-BuOH, 2-methyl-2-butene. (g) CH2N2, Et2O, 0 °C, 8% overall.
(h) LDA, THF, -78 °C; then NCS, -78 °C, 43%. (i) MeLi, THF,
39%. (j) Methoxymethyltriphenylphosphonium chloride, LDA, THF,
0 °C, 34%. (k) HCl, H2O, THF, 23 °C, 4%.
Scheme 9. Synthesis of 11a
Spodoptera frugiperda (Sf 9) insect cells, BaculoGold Transfection
Kit, and pVL1393 were purchased from PharMingen. IPL-41 powdered
media and fetal bovine serum were purchased from GIBCO. Grace’s
liquid insect media was obtained from Invitrogen or PharMingen,
Pluronic F-68 from JRH Biosciences, restriction enzymes and T4 DNA
polymerase from New England Biolabs or GIBCO, and T4 DNA ligase
from GIBCO. QIAEX gel extraction kit was purchased from QIAGEN.
CsCl was purchased from Boehringer Mannheim Biochemicals. DEAE
Sepharose ion exchange resin, dialysis membrane tubing, and Triton
X-100 were purchased from Sigma. Hydroxyapatite and BioRad protein
content assay kit were purchased from BioRad. Standard protein
molecular weight markers were purchased from Promega. Protogel
for SDS-PAGE was purchased from Kimberly Research. Aquasol from
Du Pont was used for liquid scintillation counting.
a (a) Triethylphosphonopropionate, NaH, THF, -78 °C, 77%. (b)
DIBAL, CH2Cl2, -78 °C, 90%. (c) MsCl, Et3N, CH2Cl2, -40 °C. (d)
LiBr, THF; (e) Ph3P, CH3CN, 97% overall. (f) t-BuOK, THF, -78
°C. (g) Me2C(-O-)-CHCHO, -78 °C to 20 °C, 66%.
To prepare 4,5-dehydro-2,3-oxidosqualene (11), triethyl
phosphonopropionate was condensed with 15 using NaH as a
base as shown in Scheme 9. A mixture of E and Z (ratio 4:1)
of R,â-unsaturated esters 23 was obtained. After the Z isomer
of 23 was removed by flash chromatography, the trans isomer
23 was treated with DIBAL to give the corresponding alcohol,
which was converted to the triphenylphosphonium salt 24 by
reaction with MsCl and subsequent reaction with LiBr and PPh3.
Coupling of 24 with the epoxide of â,â-dimethylacrolein using
t-BuOK as a base produced 4,5-(Z)-dehydro-2,3-oxidosqualene
(11).
(15) Kinetic studies on the trichloroacetic acid-catalyzed cyclization of
2,3-oxido-2,6-dimethylhept-6-ene and related substrates in CHCl3 carried
out by Ms. Donnette Daley in these laboratories have demonstrated
conclusively that such chemical cyclizations also involve concerted oxirane
cleavage and cyclization (manuscript in preparation for this Journal). For
early studies on such cyclizations, see: Goldsmith, D. J. J. Am. Chem. Soc.
1962, 84, 3913.

1284 J. Am. Chem. Soc., Vol. 119, No. 6, 1997
Corey et al.
Construction of Recombinant Baculovirus Transfer Vector
pVL1393-ERG7. A 2.3 kb DNA fragment encoding lanosterol
synthase from S. cereVisiae was excised from pSM61.5 by digestion
with EcoRV and NotI, isolated by agarose gel electrophoresis, and
purified using the QIAEX gel extraction protocol (QIAGEN). The
fragment was then ligated into the baculovirus transfer vector pVL1393,
which had previously been digested with SmaI and NotI and treated
with calf intestinal phosphatase. The ligation reaction mixture was
used to transform competent E. coli DH5R. Restriction analysis of
the resulting clones confirmed construction of the transfer vector with
correct orientation. One clone was grown up on a large-scale, and the
recombinant plasmid (pVL1393-ERG7) was purified by CsCl gradient.
The transfer vector and PharMingen’s linearized BaculoGold viral DNA
were used to transfect Sf 9 cells using PharMingen’s calcium phosphate
transfection procedure.16 After the transfected cells were incubated
for 4 days at 27 °C, the media containing the recombinant virus
(AcMNPV-ERG7) was collected. A portion of the recombinant viral
stock was used to infect a fresh plate of Sf 9 cells in order to amplify
the virus.
ether to 2 cm and then fully developed in hexane-diethyl ether (1:1).
The TLC plate was visualized by staining with p-anisaldehyde. Rf
values for 2,3-oxidosqualene and lanosterol were 0.91 and 0.54,
respectively.
Quantitative assays were performed in duplex with the following
modifications. The substrate, 2,3-oxidosqualene, was radiolabeled with
tritium (100 mCi/mmol). The assays for homogenate, supernatant, and
ammonium sulfate precipitated fractions were terminated at 30 min,
and the assays for DEAE and HAT fractions were terminated at 2.5 h.
After chromatography, the areas corresponding to oxidosqualene and
lanosterol were added to scintillation cocktail and counted.
To measure the Michaelis-Menten constants and reaction rates of
2,3-oxidosqualene, 6-desmethyloxidosqualene, and 6-chlorooxidosqualene,
the following procedures were used. Substrate or modified substrate
at different concentrations in 20% Triton X-100 in water was added to
lanosterol synthase in 100 mM sodium phosphate buffer (pH 7.0)
containing 20% glycerol, 0.2% Triton X-100, and 3 mM DTT. After
vortexing, the reaction mixture was incubated at room temperature.
Aliquots were withdrawn for analysis at different times before the
reaction reached 5% conversion. Km and Vmax were obtained by fitting
the initial rates vs. substrate concentrations to the Michaelis-Menten
equation. Those parameters can also be obtained from the double
reciprocal plot of initial rates vs. substrate concentrations.
Protein Content Determination. Protein content of homogenate,
supernatant, and ammonium sulfate precipitated fractions were deter-
mined based on the pellet weight. The protein amount in DEAE column
chromatography fractions were determined by BioRad protein content
determination methods. The protein concentration in HAT column
chromatography fractions were determined by comparing with known
amounts of protein standards on SDS-PAGE.
Expression of Lanosterol Synthase in Sf 9 Cells Using AcMNPV-
ERG7. A 10 L bioreactor of Sf 9 cells growing in serum-free Sf900 II
media with 0.05% Pluronic F-68 was infected with AcMNPV-ERG7
at a multiplicity of infection of ca. 1. At 48 h post infection, the cells
were harvested yielding a wet cell paste (120 g), which was frozen at
-78 °C.
Protein Purification. Sf 9 cells (20.9 g) infected with the ERG7-
recombinant baculovirus were suspended in 100 mM sodium phosphate
buffer (pH 7.0, 63 mL). The resulting suspension was sonicated using
four 30 s blasts (power lever 4) at 4 °C. The cell lysate was centrifuged
(16 000 × g, 30 min), and to the resulting supernatant was added
saturated ammonium sulfate solution (30 mL). This suspension was
stirred at 4 °C for 1 h, after which the precipitated proteins were pelleted
by centrifugation (16 000 × g, 30 min). The pellet was resuspended
in 20 mM sodium phosphate buffer (pH 7.0, 50 mL) containing 20%
glycerol, 1% Triton X-100, and 3 mM DTT. This solution was dialyzed
against 20 mM sodium phosphate buffer (pH 7.0, 1 L) containing 20%
glycerol, 0.2% Triton X-100, and 3 mM DTT at 4 °C overnight and
then applied to a DEAE Sepharose CL-6B column (50 mL bed volume)
at 4 °C preequilibrated with 20 mM sodium phosphate buffer (pH 7.0)
containing 20% glycerol, 0.2% Triton X-100, and 3 mM DTT. The
column was eluted with a linear gradient from 20 mM sodium phosphate
(pH 7.0) to 200 mM sodium phosphate (pH 7.0) (250 mL × 250 mL)
both containing 20% glycerol, 0.2% Triton X-100, and 3 mM DTT.
Fractions of 15 mL each were collected. Enzyme activity was eluted
off the column between 85 and 100 mM sodium phosphate concentra-
tion.
The most active fractions were combined, dialyzed against 7.5 mM
sodium phosphate buffer (pH 7.0, 1 L) containing 20% glycerol, 0.2%
Triton X-100, and 3 mM DTT at 4 °C overnight, and then applied to
a hydroxyapatite (HAT) column (25 mL bed volume) preequilibrated
with 7.5 mM sodium phosphate buffer (pH 7.0) containing 20%
glycerol, 0.2% Triton X-100, and 3 mM DTT at 4 °C. The column
was washed with seven column volumes of 1 M sodium chloride
solution, followed by one column volume of 7.5 mM sodium phosphate
buffer (pH 7.0) containing 20% glycerol, 0.2% Triton X-100, and 3
mM DTT. The enzyme was eluted with a linear gradient from 7.5
mM sodium phosphate (pH 7.0) to 200 mM sodium phosphate (pH 7)
(180 mL × 180 mL) containing 20% glycerol, 0.2% Triton X-100,
and 3 mM DTT. Fractions of 10 mL each were collected. Enzyme
activity was eluted between 110 and 150 mM sodium phosphate
concentration. Aliquots from fractions containing the most cyclase
activity were analyzed by SDS-PAGE for purity.17
Expression Fold Determination. Wild type S. cereVisiae was
hydrated by soaking with water and lysed by suspending in five times
weight of 100 mM sodium phosphate buffer (pH 7.0) and vortexing
against one volume of glass beads. The homogenate was then incubated
with tritium labeled substrate for 3.5 h and assayed as described above.
The specific activity of the homogenate solutions from wild type yeast
(2.18 pmol/min/mg) and Sf 9 cells (21.0 pmol/min/mg) were used to
calculate the overexpression fold, which is 10-fold.
Mutagenesis. Mutants were generated according to the method of
Kunkel.9 Uracil-containing single-stranded DNA was prepared using
helper phage M13KO7 from E. coli RZ1032 transformed with the vector
pSM61.21, a LEU2-based pRS30518 derivative containing the S.
cereVisiae lanosterol synthase gene driven by the GAL promoter.
Oligonucleotides (approximately 40 bases long) containing the desired
mutation as well as silent mutations which created or deleted a
restriction site were used as primers. Oligonucleotides were synthesized
by an automated DNA synthesizer. Mutant clones were screened by
restriction fragment analysis. Some of the mutants, D140N, D370N,
D456N, D580N, E460Q, and E634Q, were sequenced by the dideoxy-
nucleotide method using the USB Sequenase Kit (version 2.0) to further
confirm incorporation of the mutagenic oligonucleotide.19 E. coli strain
DH5R was used for all DNA manipulations. E. coli were transformed,
selected, and propagated according to published procedures.19 The
plasmid pSM61.21 was linearized with BstEII and transformed into S.
cereVisiae strain SMY8 (MATa erg7::HIS3 hem1::TRP1 ura3-52 trp1-
63 leu2-3,112 his3-D200 ade2 Gal+)2f in order to integrate into the
LEU2 locus in the genome by homologous recombination. SMY8
transformants were selected at 30 °C on synthetic complete medium
lacking leucine, supplemented with hemin chloride (13 mg/L), ergos-
terol (20mg/L), and Tween 80 (0.5%). Complementation analysis was
performed on 1% yeast extract, 2% peptone, 2% galactose, and 2%
agar, supplemented with heme chloride (13 mg/L), and Tween 80
(0.5%).20
Enzyme Activity Assay. To 47.5 µL of the enzyme solution was
added 2.5 µL solution containing racemic 2,3-oxidosqualene (20 mg/
mL) and Triton X-100 (200 mg/mL) to yield a final concentration of
1 mg/mL substrate and 1% Triton X-100. The resulting solution was
incubated at 18 °C for 8 to 12 h. The reaction mixture (5 mL) was
spotted on a TLC plate, and water was removed by placing the TLC
plate in Vacuo for 15 min. The TLC plate was then developed in diethyl
5-Methyl-4-hexen-1-ol (12). To a stirred solution of γ-butyrolactone
(11.0 g, 128 mmol) in CH2Cl2 (50 mL) at -78 °C was added gradually
1 M diisobutylaluminum hydride in CH2Cl2 (150 mL). After the
(18) Sikorski, R. S.; Hieter, P. Genetics 1989, 122, 19.
(19) Current Protocols in Molecular Biology; Ausubel, F. M., Brent,
R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K.,
Ed.; Wiley-Interscience: New York, 1995.
(16) Burand, J. P.; Summers, M. D.; Smith, G. E. Virology 1980, 101,
286.
(20) Guide to Yeast Genetics and Molecular Biology; Guthrie, C., Fink,
G. R., Ed.; Academic: New York, 1991.
(17) Laemmli, U. K. Nature 1970, 227, 680.

Preparation of S. cereVisae Lanosterol Synthase
J. Am. Chem. Soc., Vol. 119, No. 6, 1997 1285
reaction mixture was stirred at -78 °C for 1 h, it was quenched with
MeOH (10 mL) and saturated Rochelle’s salt (10 mL). The mixture
was warmed to 23 °C and stirred for 2 h. The layers were separated,
and the aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The
combined extracts were dried (MgSO4) and concentrated in Vacuo to
yield the desired product as a colorless oil. The crude product was
used directly for the next reaction without purification.
To a suspension of isopropyltriphenylphosphonium iodide (20.6 g,
47.7 mmol) in THF (50 mL) at -78 °C was added t-BuOK (5.9 g,
52.7 mmol) in THF (40 mL). The mixture was stirred at 0 °C for 30
min, γ-butyrolactol (4.0 g, 45.5 mmol) in THF (20 mL) was added
dropwise, and the resulting mixture was stirred overnight. The reaction
mixture was treated with water and extracted with ether (3 × 50 mL).
The combined organic layers were dried (MgSO4) and concentrated in
Vacuo. The crude product was purified by flash column chromatog-
raphy using 3:7 ether-pentane (4.50 g, 86%). 1H NMR (400 MHz,
CDCl3) δ 5.11 (m, 1 H), 3.61 (t, J ) 6.6, 2 H), 2.03 (m, 2 H), 1.67 (s,
3 H), 1.59 (s, 3 H), 1.56 (m, 3 H); 13C NMR (100 MHz, CDCl3) δ
132.0, 123.8, 62.5, 32.6, 25.6, 24.3, 17.5; IR (neat) 3334 (br), 2965,
2928, 2877, 2871, 2863, 1450, 1436, 1059, 1041 cm-1; HRMS (CI)
114.1043 (114.1045 calcd for C7H14O‚NH4+).
2870, 1371, 1236, 1217, 1200, 1117, 1063, 1034, 1008 cm-1; HRMS
(CI) 189.1483 (189.1491 calcd for C10H21O3).
Triphenylphosphonium Salt 14. The alcohol acetonide 13 (300
mg, 1.60 mmol) and triethylamine (640 mg, 6.32 mmol) were dissolved
in dichloromethane (40 mL) at 0 °C, and methanesufonyl chloride (724
mg, 6.32 mmol) was added with stirring. The reaction mixture was
stirred at 0 °C for 1 h, allowed to warm to room temperature, and
poured into a solution of 5% aqueous sodium bicarbonate solution.
The methanesulfonate ester was isolated by extraction with ether and
removal of solvent. The mesylate was then heated at reflux with sodium
iodide in acetone under argon for 3 h. The mixture was cooled to
room temperature, and the solvent was removed in Vacuo. After
workup, the crude product was purified by flash chromatography to
generate the corresponding iodide (430 mg, 91%). 1H NMR (400 MHz,
CDCl3) δ 3.67 (m, 1 H), 3.31-3.18 (m, 2 H), 2.09 (m, 1 H), 1.89 (m,
1 H), 1.56 (m, 2 H), 1.32 (s, 3 H), 1.27 (s, 3 H), 1.25 (s, 3 H), 1.10 (s,
3 H); 13C NMR (100 MHz, CDCl3) δ 106.7, 82.3, 80.1, 30.9, 30.2,
28.5, 26.8, 25.9, 22.8, 6.6; IR (neat) 2981, 2936, 2866, 1377, 1370,
1271, 1236, 1217, 1168, 1002 cm-1; HRMS (CI): 316.0767 (316.0774
calcd for C10H19O2I + NH4).
The above prepared iodide (210 mg, 0.70 mmol) and triphenylphos-
phine (600 mg, 2.29 mmol) were dissolved in acetonitrile (20 mL),
and the resulting solution was heated at reflux under argon overnight.
The solvent was then removed in Vacuo. The crude product was
purified by flash column chromatography using 2% MeOH in CHCl3
to give 14 (390 mg, 99%). 1H NMR (400 MHz, CDCl3) δ 7.85-7.67
(m, 15 H), 4.05(m, 1 H), 3.59 (m, 1 H), 3.73 (m, 1 H), 2.00 (m, 1 H),
1.86 (m, 2 H), 1.68 (m, 1 H), 1.35 (s, 3 H), 1.29 (s, 3 H), 1.25 (s, 3 H),
1.04 (s, 3 H); 13C NMR (CDCl3) δ 135.0, 133.7, 130.4, 118.5, 117.6,
106.8, 82.6, 80.6, 29.4, 29.2, 28.5, 27.1, 26.0, 23.1, 20.5; IR (neat)
2979, 2934, 2927, 2865, 2361, 2342, 2333, 1438, 1113 cm-1; HRMS
(FAB) 433.2298 (433.2296 calcd for C28H34O2P).
Triol Monoacetonide 13. To a solution of 12 (1.55 g, 13.6 mmol)
in THF (30 mL) at 0 °C was added sodium hydride (359 mg, 15.0
mmol), followed by benzyl bromide (1.78 mL, 15.0 mmol, dropwise).
The resulting solution was stirred at 23 °C overnight, filtered, and
concentrated in Vacuo. The crude product was purified by flash column
chromatography using 5% ethyl acetate in hexane to give the benzyl
ether of 12 (2.6 g, 95%). 1H NMR (400 MHz, CDCl3) δ 7.42-7.26
(m, 5 H), 5.13 (m, 1 H), 4.51 (s, 2 H), 3.49 (t, 2 H), 2.09 (m, 2 H),
1.71 (s, 3 H), 1.69 (m, 2 H), 1.64 (s, 3 H); 13C NMR (100 MHz, CDCl3)
δ 138.8, 132.2, 129.0, 128.7, 128.3, 127.6, 127.4, 123.9, 72.8, 69.9,
33.5, 25.7, 24.5, 17.6; IR (neat) 2965, 2927, 2855, 1454, 1363, 1102,
1076 cm-1; HRMS (CI): 222.1867 (222.1858 calcd for C14H20O +
Acetonide 16. To the triphenylphosphonium salt 14 (150 mg, 0.268
mmol) in THF (2 mL) at -78 °C was added n-BuLi (161 µL, 1.67 M
solution in hexane), and the resulting solution was stirred at room
temperature for 30 min. The reaction mixture was then cooled to -78
°C, and the aldehyde R20CH2CHO (84.6 mg, 0.268 mmol) in THF (2
mL) was added. The aldehyde R20CH2CHO was prepared by peracid
epoxidation of squalene, cleavage to a diol mixture by aqueous acid,
chromatography, diol cleavage with periodate, and vacuum distillation.
After 5 min stirring at -78 °C, s-BuLi (227 µL, 1.3 M solution in
cyclohexane) was added, and the reaction mixture was stirred at -78
°C for 3 h. 2-Phenylpropanol (40.0 mg, 0.295 mmol) was then added,
and the solution was warmed to room temperature. After workup, the
product was purified by flash chromatography using 4% diethyl ether
in hexane (69.1 mg, 55%). The product consisted of a 1:1 mixture of
+
NH4
)
N-Methylmorpholine oxide (630 mg, 5.4 mmol), the benzyl ether
of 12 (1 g, 4.9 mmol), and OsO4 (8 mg) were dissolved in 5:2 acetone-
water solution with stirring, and the resulting solution was stirred at
23 °C overnight, after which it was quenched with sodium hydrogen
sulfite (50 mg). After another 20 min of stirring, the solution was
extracted with ethyl acetate (3 × 50 mL). The combined organic layers
were dried (MgSO4) and concentrated in Vacuo. The crude diol was
purified by flash column chromatography using first 20% ethyl acetate
in hexane then ethyl acetate (970 mg, 83%). 1H NMR (400 MHz,
CDCl3) δ 7.36-7.25 (m, 5 H), 4.51 (s, 2 H), 3.51 (t, J ) 5.9, 2 H),
3.35 (m, 1 H), 3.32 (m, 1 H), 2.63 (s, 1 H), 1.86-1.33 (m, 4 H), 1.17
(s, 3 H), 1.12 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 138.0, 128.3,
127.7, 127.6, 78.1, 72.9, 70.3, 28.8, 26.9, 26.3, 23.2; IR (neat) 3423
(br), 2970, 2933, 2862, 1364, 1090, 1077 cm-1; HRMS (CI):256.1909
(256.1913 calcd for C14H22O3 + NH4).
To a solution of the above prepared benzyl ether diol (640 mg, 2.69
mmol) in 2,2-dimethoxypropane (30 mL) was added p-toluenesulfonic
acid (20 mg). The resulting solution was stirred for 30 min, saturated
sodium bicarbonate solution (20 mL) was added, and the organic layer
was separated. The aqueous layer was extracted with ethyl acetate (3
× 25 mL). The combined organic layers were washed with brine, dried
(MgSO4), and concentrated in Vacuo to give the acetonide (730 mg,
97%). 1H NMR (CDCl3) δ 7.37-7.25 (m, 5 H), 4.51 (s, 2 H), 3.68
(m, 1 H), 3.57-3.48 (m, 2 H; m), 1.82 (m, 1 H), 1.70 (m, 1 H), 1.56
(m, 2 H), 1.31 (s, 3 H), 1.27 (s, 3H), 1.24 (s, 3 H), 1.09 (s, 3 H); 13C
NMR (100 MHz, CDCl3) δ 138.5, 128.3, 127.6, 127.5, 106.4, 83.1,
80.1, 72.8, 69.9, 28.5, 27.1, 26.8, 26.0, 22.8; IR (neat) 2981, 2935,
2860, 1370, 1217, 1197, 1115, 1008 cm-1; HRMS (CI) 279.1965
(279.1960 calcd for C17H27O3).
1
Z and E isomers. Rf 0.77 (silica gel, 1:1 diethyl ether-hexane); H
NMR (400 MHz, CDCl3) δ 1.06 (s, 3 H), 1.21 (s, 3 H), 1.31 (s, 3 H),
1.40 (s, 3 H), 1.38 (m, 1 H), 1.49 (m, 1 H), 1.58 (s, 12 H), 1.66 (s, 3
H), 1.95-2.19 (m, 18 H), 3.66 (m, 1 H), 5.09 (m, 4 H), 5.36 (m, vinyl
H for cis double bond), 5.41 (m, vinyl H for trans double bond); IR
(neat) 2980, 2929, 2854, 1376, 1217 cm-1; MS (CI) m/z 488 (M +
NH4+); HRMS: 470.4129 (470.4124 calcd for C32H54O2).
Diol 17. To a solution of the acetonide 16 (58.5 mg) in THF (0.4
mL) and MeOH (3 mL) was added p-toluenesulfonic acid (5.00 mg),
and the resulting solution was stirred at room temperature for 24 h.
The solvent was then removed in Vacuo, and the product was purified
by flash chromatography using 20% diethyl ether in hexane (32.0 mg,
60%). The isomers 17 and 6-Z-17 were then separated by preparative
TLC using 3:1 hexane-EtOAc.
Data for 17: R
f 0.62 (silica gel, diethyl ether); 1H NMR (400 MHz,
CDCl3) δ 1.13 (s, 3 H), 1.18 (s, 3 H), 1.38 (m, 1 H), 1.50 (m, 1 H),
1.57 (s, 12 H), 1.66 (s, 3 H), 1.95-2.21 (m, 20 H), 3.36 (dd, J ) 10.5
Hz, J ) 2 Hz, 1 H), 5.09 (m, 4 H), 5.43 (m, 2 H, vinyl H); 13C NMR
(100 MHz, CDCl3) δ 16.01, 16.05, 17.69, 23.21, 25.71, 26.46, 26.65,
26.76, 28.24, 29.78, 31.17, 31.44, 39.71, 39.74, 73.02, 78.09, 124.24,
124.40, 124.53, 129.65, 131.10, 131.29, 134.74, 135.17; IR (neat) 2966,
2927, 2855, 1448, 1380, 1007 cm-1; MS (EI) m/z 430 (M+); HRMS:
430.3821 (430.3811 calcd for C29H50O2).
A mixture of the above prepared benzyl ether acetonide (650 mg,
2.34 mmol) and 10% palladium-carbon (0.5 g) in ethanol (30 mL)
was hydrogenated at room temperature under atmospheric pressure.
After the reaction was complete, the mixture was filtered, and the filtrate
was concentrated in Vacuo to afford alcohol acetonide 13 (430 mg,
98%). 1H NMR (500 MHz, CDCl3) δ 3.71-3.68 (m, 3 H), 2.20 (s, 1
H), 1.74 (m, 2 H), 1.57 (m, 2 H), 1.43 (s, 3 H), 1.34 (s, 3 H), 1.25 (s,
3 H), 1.10 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 106.7, 83.5, 80.5,
62.7, 30.6, 28.5, 26.9, 26.3, 25.9, 22.8; IR (neat) 3417 (br), 2982, 2937,
1
Data for 6-Z-17: Rf 0.67 (silica gel, diethyl ether); H NMR (400
MHz, CDCl3) δ 1.13 (s, 3 H), 1.18 (s, 3 H), 1.38 (m, 1 H), 1.50 (m,
1 H), 1.58 (s, 12 H), 1.66 (s, 3 H), 1.93-2.23 (m, 20 H), 3.37 (d, J )

1286 J. Am. Chem. Soc., Vol. 119, No. 6, 1997
Corey et al.
10.4 Hz, 1 H), 5.09 (m, 4 H), 5.37 (m, 2 H); 13C NMR (100 MHz,
CDCl3) δ 16.00, 16.06, 17.68, 23.23, 24.44, 25.68, 25.88, 26.51, 26.68,
26.79, 28.27, 31.59, 39.62, 39.74, 73.03, 78.21, 124.27, 124.42, 124.71,
129.05, 130.54, 131.25, 134.68, 135.16; IR (neat) 2966, 2927, 2855,
1448, 1380, 1007 cm-1; MS (EI) m/z 430 (M+); HRMS: 430.3821
(430.3811 calcd for C29H50O2).
6-Desmethyltrisnorsqualene Aldehyde (18). Diol 17 (19.0 mg,
0.0442 mmol) was dissolved in THF (0.27 mL), water was added to
saturation (slight cloudiness) followed by NaIO4 (28.4 mg, 0.133 mmol),
and the resulting solution was stirred at room temperature for 1 h. After
workup, the product was purified by flash chromatography using 3%
diethyl ether in hexane (13.0 mg, 79%). Rf 0.8 (silica gel, 1:1 diethyl
ether-hexane); 1H NMR (400 MHz, CDCl3) δ 1.58 (s, 12 H), 1.66 (s,
3 H), 1.97-2.65 (m, 20 H), 5.07 (m, 4 H), 5.31 (m, vinyl H for cis
double bond), 5.39 (m, vinyl H for trans double bond); IR (neat) 2961,
2927, 2856, 1729, 1446 cm-1; MS (EI) m/z 370 (M+); HRMS:
370.3247 (370.3236 calcd for C26H42O).
worked up by extraction. The product was purified by flash chroma-
tography using 3% diethyl ether in hexane (6.9 mg, 99%). The specific
activity of the product was 68 µCi/µmol; trans/cis ratio was 53:47.
Acetonide of 6-Chloro-6-desmethyl-2,3-dihydroxysqualene 19.
To the triphenyl phosphonium salt 14 (208 mg, 0.371 mmol) in THF
(1 mL) at -78 °C was added n-BuLi (222 µL, 1.67 M solution in
hexane), and the resulting solution was stirred at room temperature for
30 min. The reaction mixture was cooled to -78 °C, and N-
chlorosuccinimide (59.2 mg, 0.443 mmol) in THF (1 mL) was added.
After stirring for 20 min at -78 °C, s-BuLi (616 µL, 1.3 M solution
in cyclohexane) was added until the solution became clear, and the
reaction mixture was stirred at -78 °C for 90 min. To this mixture
was added the aldehyde R20CH2CHO (95.0 mg, 0.300 mmol) in THF
(1 mL) with stirring which was continued at -78 °C for 5 min. The
reaction mixture was then warmed to room temperature, and after
extractive workup, the product was purified by flash chromatography
using 4% diethyl ether in hexane (54.0 mg, 36%). The product was a
mixture of 6-E and 6-Z chloroolefins and 6-E-6-desmethyloxidosqualene
3H-Labeled 6-Desmethyltrisnorsqualene Aldehyde (3-3H-18). Al-
dehyde 18 (10.7 mg, 0.0288 mmol) was dissolved in MeOH (1 mL),
and the resulting solution was cooled to 0 °C. Under stirring conditions
was added [3H]-NaBH4 (25 mCi, 359.8 mCi/mmol) in MeOH (1 mL),
and the stirring was continued for 2 h. Acetone (100 mL) was added
to quench the unreacted [3H]-NaBH4. The solvent was removed in
Vacuo, and the product was purified by flash chromatography using
20% diethyl ether/hexane to give the 3H-labeled alcohol (9.6 mg, 0.0257
mmol, 90%). A solution of the resulting alcohol in CH2Cl2 (1 mL)
was treated with NaHCO3 (23.5 mg, 0.280 mmol) and Dess-Martin
reagent (18.7 mg, 0.0431 mmol). After stirring for 1 h at 23 °C,
saturated Na2S2O3 solution (1 mL), saturated NaHCO3 solution (1 mL),
and CH2Cl2 (1 mL) were added. The stirring was continued until two
phases became clear. The reaction was then worked up, and the product
was purified by flash chromatography using 3% diethyl ether in hexane
1
(1:1:2) as determined by 400 MHz H NMR analysis.
Conversion of Acetonide 19 to the Corresponding Diol (20). To
a solution of the acetonide 19 (50.0 mg, 0.100 mmol) in THF (0.2
mL) and MeOH (2 mL) was added p-toluenesulfonic acid (5.00 mg,
0.0263 mmol), and the resulting solution was stirred at room temper-
ature for 48 h. The solvent was then removed in Vacuo, and the crude
product was subjected to flash chromatography using 20% diethyl ether
in hexane to give a mixture of chlorinated and nonchlorinated
compounds. The nonchlorinated component was then removed by flash
chromatography on silica gel containing 10% silver nitrate using diethyl
ether as an eluant. The compounds eluted first were the 2,3-dihydroxy-
6-Z and E-chloro olefins (1:1) (13.0 mg, 28%). The Z and E isomers
were then separated by use of a preparative TLC silica gel plate using
50% diethyl ether-hexane.
3
(7.8 mg, 0.0211 mmol, 81%). This material was used to make H-
Data for 2,3-dihydroxy-6-chloro-6-desmethylsqualene (20). Rf
0.83 (silica gel, diethyl ether); 1H NMR (400 MHz, CDCl3) δ 1.15 (s,
3 H), 1.20 (s, 3 H), 1.45 (m, 1 H), 1.58 (s, 12 H), 1.66 (s, 3 H), 1.76
(m, 1 H), 1.95 (m, 14 H), 2.25 (m, 2 H), 2.41 (m, 1 H), 2.52 (m, 1 H),
3.33 (d, J ) 10.7 Hz, 1 H), 5.09 (m, 4 H), 5.49 (t, J ) 6.8 Hz, 1 H );
labeled 6-desmethyloxidosqualene (8.6 mg, 68 µCi/µmol, 99%) fol-
lowing method (b) which is described below.
6-Desmethyl-2,3-oxidosqualene (3). Diol 17 (7.0 mg, 0.0163
mmol) was dissolved in CH2Cl2 (80 µL) and pyridine (13.2 µL, 0.163
mmol) with stirring at 23 °C, MsCl (1.51 µL) was added, and stirring
continued at room temperature for 12 h. MeOH (1 mL) and K2CO3
(35 mg) were then added, and after stirring for another 15 min, the
solvent was removed in Vacuo, and the product was purified by flash
chromatography using 3% diether-hexane to give 3 (5.5 mg, 80%). Rf
0.86 (silica gel, 1:1 diethyl ether-hexane); 1H NMR (400 MHz, CDCl3)
δ 1.24, (s, 3 H), 1.28 (s, 3 H), 1.51-1.66 (m, 2 H), 1.58 (s, 12 H),
1.66 (s, 3 H), 1.93-2.13 (m, 18 H), 2.72 (m, 1 H), 5.09 (m, 4 H), 5.43
(m, 2 H); 13C NMR (100 MHz, CDCl3) δ 16.01, 16.05, 17.67, 18.79,
24.89, 25.68, 26.68, 26.79, 28.26, 28.93, 29.50, 31.22, 39.71, 39.74,
58.27, 64.05, 124.27, 124.42, 124.50, 129.00, 130.97, 131.25, 134.92,
135.15; IR (neat) 2966, 2927, 2855, 1448, 1380, 1007 cm-1 IR (neat)
2961, 2927, 2855, 1448, 1378 cm-1; MS (EI) m/z 412 (M+); HRMS:
412.3710 (412.3705 calcd for C29H48O).
6-Z-3. Diol 6-Z-17 (2.8 mg, 0.00651 mmol) was dissolved in CH2-
Cl2 (80 µL) and pyridine (5.26 µL, 0.0651 mmol) with stirring at room
temperature, MsCl (0.655 µL in 10 µL CH2Cl2, 0.00847 mmol) was
added, and the stirring was continued at room temperature for 12 h.
MeOH (0.5 mL) and K2CO3 (15 mg) were then added. After stirring
for another 15 min, the solvent was removed in Vacuo, and the product
was purified by flash chromatography using 3% diether-hexane to give
6-Z-3 (0.50 mg, 19%). Rf 0.86 (silica gel, 1:1 diethyl ether-hexane);
1H NMR (400 MHz, CDCl3) δ 1.25 (s, 3 H), 1.29 (s, 3 H), 1.51-1.66
(m, 2 H), 1.58 (s, 12 H), 1.66 (s, 3 H), 1.95-2.24 (m, 18 H), 2.72 (m,
1 H), 5.09 (m, 4 H), 5.38 (m, 2 H); IR (neat) 2961, 2927, 2855, 1448,
1378 cm-1; MS (EI) m/z 412 (M+); HRMS: 412.3710 (412.3705 calcd
for C29H48O).
IR (neat) 3406 (br), 2966, 2925, 2854, 1446, 1382, 1158, 1079 cm-1
;
MS (FAB) m/z 487 (M + Na+); HRMS: 487.3310 (487.3319 calcd
for C29H49ClO2).
1
Data for 6-E-20. Rf 0.91 (silica gel, diethyl ether); H NMR (400
MHz, CDCl3) δ 1.16 (s, 3 H), 1.21 (s, 3 H), 1.48 (m, 1 H), 1.58 (s, 12
H), 1.66 (s, 3 H), 1.79 (m, 1 H), 2.05 (m, 14 H), 2.16 (m, 2 H), 2.51
(m, 2 H), 3.33 (d, J ) 10.7 Hz, 1 H), 5.09 (m, 4 H), 5.60 (t, J ) 7.5
Hz, 1 H); 13C NMR (100 MHz, CDCl3) δ 15.96, 16.00, 16.06, 17.62,
23.50, 25.57, 26.67, 26.84, 26.95, 27.34, 28.25, 28.35, 29.47, 30.80,
39.34, 39.80, 57.52, 73.09, 124.29, 124.45, 124.59, 125.55, 128.53,
133.50, 133.98, 134.97, 135.34; IR (neat) 3406 (br), 2966, 2925, 2854,
1446, 1382, 1158, 1079 cm-1; MS (FAB) m/z 487 (M + Na+);
HRMS: 487.3310 (487.3319 calcd for C29H49ClO2).
6-Chloro-6-desmethyltrisnorsqualene Aldehyde (21). A solution
of a 1:1 mixture of 20 and 6-E-20 (13 mg, 0.0267mmol) in THF (0.30
mL) was treated with water to slight turbidity, then NaIO4
(25 mg,
0.117 mmol) was added, and the resulting solution was stirred at room
temperature for 1 h. After workup, the product was purified by flash
chromatography using 3% diethyl ether in hexane (6.9 mg, 61%). Rf
0.76 (silica gel, 1:1 diethyl ether-hexane); 1H NMR (400 MHz, CDCl3)
δ 1.57 (s, 12 H), 1.65 (s, 3 H), 1.93-2.24 (m, 16 H), 2.61-2.70 (m,
4 H), 5.09 (m, 4 H), 5.49 (t, J ) 6.9 Hz, vinyl H for cis double bond),
5.59 (t, J ) 7.6 Hz, vinyl H for trans double bond), 9.76, 9.79 (s, s, 1
H); IR (neat) 2964, 2918, 2852, 1728, 1438 cm-1
; MS (EI) m/z 404
(M+); HRMS: 404.2831 (404.2846 calcd for C26H41ClO).
3H-Labeled 6-Chloro-6-desmethyltrisnorsqualene Aldehyde (3H-
21). Aldehyde 21 (6.9 mg, 0.0171 mmol) was dissolved in MeOH
(0.5 mL), and the resulting solution was cooled to 0 °C. A solution of
[3H]-NaBH4 (25 mCi, 359.8 mCi/mmol) in MeOH (0.5 mL) was added
with stirring, and the stirring was continued for 2 h. Acetone (100
mL) was added to consume the unreacted [3H]-NaBH4. The solvent
was removed in Vacuo, and the product was purified by flash
chromatography using 20% diethyl ether-hexane to give the 3H-labeled
alcohol (5.5 mg, 0.0135 mmol, 79%). The alcohol was then dissolved
in CH2Cl2 (0.5 mL) and treated with NaHCO3 (11.3 mg, 0.135 mmol)
3H-Labeled 6-Desmethyl-2,3-oxidosqualene (3-3H-3). To a solu-
tion of diphenylisopropylsulfonium boron tetrafluoride (121 mg, 0.383
mmol) in DME (2 mL) at -78 °C was added LDA solution (0.100
mL, 0.38 M solution in DME), and the resulting solution was stirred
at -78 °C for 30 min. Aliquots of this solution were then added to a
solution of 6-desmethyl trisnorsqualene aldehyde (3-3H-18) (6.1 mg,
0.0165 mmol) in DME (1 mL) at -78 °C until the solution turned
yellow. The reaction mixture was stirred at -78 °C for 1 h and then

Preparation of S. cereVisae Lanosterol Synthase
J. Am. Chem. Soc., Vol. 119, No. 6, 1997 1287
and Dess-Martin reagent (8.8 mg, 0.0203 mmol). After stirring for 1
h, saturated Na2S2O3 solution (0.5 mL), saturated NaHCO3 solution
(0.5 mL), and CH2Cl2 (1 mL) were added. The stirring was continued
until two phases were clear. The reaction was then worked up, and
the product was purified by flash chromatography using 3% diethyl
ether in hexane (4.3 mg, 0.0106 mmol, 78%). This material was then
used to make 3H-labeled 6-chloro-6-desmethyl-2,3-oxidosqualene (4.7
mg, 64 mCi/mmol, 99%) following the method (b) described below.
6-Desmethyl-6-chloro-2,3-oxidosqualene (4). Method (a). Diol
20 (3.1 mg, 0.00667 mmol) was dissolved in CH2Cl2 (50 µL) and
pyridine (5.59 µL, 0.0691 mmol), and MsCl (0.695 µL in 220 µL CH2-
Cl2, 0.00898 mmol) was added with stirring at room temperature. After
12 h, MeOH (1 mL) and K2CO3 (25 mg) were added, and stirring was
continued for 15 min. The solvent was removed in Vacuo, and the
product was purified by flash chromatography using 3% diether/hexane
to give 4 (2.2 mg, 74%), further purified by normal phase HPLC using
0.1% isopropyl alcohol in hexane. Rf 0.5 (silica gel, 1:4 diethyl ether-
concentrated in Vacuo. The final product (62% yield) was purified on
a silica gel column (1.1 cm × 19 cm) pretreated with triethylamine.
1H NMR (CDCl3) δ 5.16-5.07 (5H; m; H’s of CdCH-), 3.45 (1H; t,
J ) 5.9; H’s of epoxide ring), 2.16-1.96 (18H; m; Hs of CH2-CdCH),
1.68 (3H; s; CH3), 1.62 (3H; s; CH3), 1.60 (12H; s; H’s of CH3), 1.10-
0.83 (4H; m; H’s of cyclopropyl ring); IR (neat) 2966, 2916, 2871,
2852, 1580, 1475, 1439, 1024 cm-1; HRMS 424.3684 (424.3705 calcd
for C30H48O).
1,1′-Bisnorsqualeneoxide 6. A suspension of dry trimethylsulfo-
nium iodide (106 mg, 0.26 mmol) in anhydrous THF (1 mL) was cooled
to 0 °C and treated with 319 µL of 1.626 M n-BuLi in hexane to form
the ylide over 15 min at 0 °C with stirring. This solution was added
to 100 mg of trisnorsqualene aldehyde in 1 mL of THF via cannula.
The reaction mixture was stirred for 30 min at 0 °C and then allowed
to warm to 25 °C. After 1 h all of the starting material had been
consumed. The product was separated by extractive isolation and
purified on a silica gel column using 20:1 hexane-Et2O as the eluant.
1H NMR (300 MHz, CDCl3) δ 5.09 (m, 5H), 2.90 (m, 1H), 2.74 (dd,
J ) 4.9, 4.0 Hz, 1H), 2.47 (dd, J ) 2.7, 4.9 Hz, 1H), 2.06 (m, 20H),
1.68 (s, 3H), 1.60 (m, 15H); 13C NMR (400 MHz, CDCl3) δ 135.20,
135.00, 134.97, 133.88, 131.32, 125.02, 124.46, 124.32, 52.17, 47.26,
39.80, 39.69, 35.87, 31.07, 28.32, 26.82, 26.72, 26.66, 25.76, 17.74,
16.11, 16.07, 16.00.
1
hexane); H NMR (500 MHz, CDCl3) δ 1.25 (s, 3 H), 1.28, (s, 3 H),
1.58 (s, 12 H), 1.66 (s, 3 H), 1.69 (m, 1 H), 1.81 (m, 1 H), 1.97 (m, 14
H), 2.25 (m, 2 H),, 2.41 (m, 1 H), 2.47 (m, 1 H), 2.69 (dd, J ) 6.3 Hz,
J ) 5.9 Hz, 1 H), 5.10 (m, 4 H), 5.48 (t, J ) 6.8 Hz, 1 H); IR (neat)
2948, 2926, 2857, 1444 cm-1; MS (EI) m/z 446 (M+); HRMS:
446.3303 (446.3315 calcd for C29H47ClO).
Synthesis of 6-E-4. 6-E-20 (6.0 mg, 0.0123 mmol) was converted
by method (a) to 6-E-4 (3.7 mg, 67%). Rf 0.5 (silica gel, 1:4 diethyl
ether-hexane); 1H NMR (400 MHz, CDCl3) δ 1.27 (s, 3 H), 1.29, (s,
3 H), 1.58 (s, 12 H), 1.66 (s, 3 H), 1.72 (m, 1 H), 1.82 (m, 1 H), 1.93
(m, 14 H), 2.13 (m, 2 H), 2.48 (m, 2 H), 2.72 (dd, J ) 6.9 Hz, J ) 5.8
Hz, 1 H), 5.09 (m, 4 H), 5.60 (t, J ) 7.5 Hz, 1 H); 13C NMR (100
MHz, CDCl3) δ 15.94, 16.00, 16.05, 17.67, 18.79, 24.86, 25.68, 26.68,
26.79, 26.88, 27.18, 28.17, 28.27, 30.76, 39.18, 39.74, 58.53, 63.22,
124.14, 124.26, 124.41, 125.45, 128.44, 131.25, 132.60, 133.77, 134.92,
135.28; IR (neat) 2948, 2926, 2857, 1444 cm-1; MS (EI) m/z 446 (M+);
HRMS: 446.3303 (446.3315 calcd for C29H47ClO).
Synthesis of 3H-Labeled 4. Method (b). To a solution of
diphenylisopropylsulfonium boron tetrafluoride (121 mg, 0.383 mmol)
in DME (2 mL) at -78 °C was added LDA solution (0.100 mL, 0.38
M in DME), and the resulting solution was stirred at -78 °C for 30
min. Aliquots of this solution were then added to a solution of
6-desmethyl-6-chlorotrinorsqualene aldehyde 3-3H-21 consisting of a
1:1 mixture of Z/E isomers (4.3 mg, 0.0104 mmol) in DME (1 mL)
until the solution turned yellow. The reaction mixture was stirred at
-78 °C for 1 h and then worked up. The product was purified by
flash chromatography using 3% diethyl ether in hexane (4.7 mg, 99%).
The exact ratio of Z/E was 55:45, and the specific activity was 64 µCi/
µmol.
Methyl Ketone 21. A solution of azeotropically dried trisnor-
squalene aldehyde (353.8 mg, 0.92 mmol) in THF (5 mL) was cooled
to -78 °C and treated with excess MeMgCl in THF (1.5 mL of a 2.9
M solution). After 4 h the reaction mixture was quenched by the
addition of brine, and the product was isolated by extractive workup
and purified by silica gel chromatography on a flash column (2.7 cm
× 24 cm) using 25% diethyl ether in hexane as the eluant to give the
methyl carbinol (76.6 mg, 0.191 mmol). 1H NMR (300 MHz, CDCl3)
δ 5.18-5.08 (m, 5H), 3.78 (t × q, J ) 6.15 Hz, 1H), 2.14-1.97 (m,
20H), 1.68 (s, 3 H), 1.60 (s, 15H), 1.14 (d, J ) 20.1 Hz, 3H). HRMS
(EI+) m/z [M+] calculated for C28OH48 400.3705, observed 400.3694.
This carbinol (76.6 mg, 0.191 mmol) was dissolved in hexane (2 mL)
and treated with pyridinium chlorochromate on alumina (1.2 g). After
stirring for 24 h at 23 °C, the reaction mixture was filtered through
silica gel, and the crude product was purified on a silica gel flash column
using 5% diethyl ether in hexane as eluant to give pure methyl ketone
21 (64 mg, 0.161 mmol) in 84% yield. 1H NMR (300 MHz, CDCl3)
δ 5.13 (m, 5H), 2.51 (t, J ) 7.26 Hz, 2H), 2.23 (t, J ) 7.93, 2H), 2.13
(s, 3H), 2.05-1.97 (m, 16H), 1.67 (s, 3H), 1.59 (s, 15H). HRMS (EI+)
m/z [M+] calculated for C28OH46 398.3549, observed 398.3535.
Epoxide 7. The above methyl ketone 21 in THF (2 mL) was added
to a cold (-78 °C) solution of dimethylsulfonium methylide. The
reaction mixture was allowed to warm to room temperature and kept
for 16 h. After extractive isolation the desired epoxide was purified
on a silica gel flash column (1.5 cm × 28 cm) using 4% diethyl ether
in hexane as the eluant to give epoxide 7 (2 mg, 0.0048 mmol). 1H
NMR (300 MHz, CDCl3) δ 5.14-5.09 (m, 5H), 2.62 (d × d, J ) 7.54,
9.69 Hz, 2H), 2.04-1.98 (m, 20H), 1.68 (s, 3H), 1.60 (m, 9H), 1.55
(s, 6H), 0.94 (t, J ) 7.35 Hz, 3H). HRMS (EI+) m/z [M+] calculated
for C29H48O 412.3705, observed 412.3695.
Trisnorsqualene Methyl Ester. A solution of trisnorsqualene
aldehyde (469 mg, 1.2 mmol) in tert-butyl alcohol (30 mL) and
2-methyl-2-butene (10 mL) was cooled to 0 °C and treated with NaClO2
(1.09 g, 12 mmol) in 20% NaH2PO4 (10 mL) aqueous solution. After
1 h the reaction mixture was treated with a saturated aqueous solution
of Na2S2O3. After isolation by extractive workup, a solution of the
resulting trisnorsqualene carboxylic acid in diethyl ether (20 mL) was
cooled to 0 °C. To the stirred solution was added excess CH2N2 in
diethyl ether. After 3 min, acetic acid (2 N) was added to consume
the excess CH2N2. The product was isolated by extractive workup and
purified on a silica gel column (2.9 cm × 23 cm) using 5% diethyl
ether in hexane as the eluant to give the methyl ester. 1H NMR (300
MHz, CDCl3) δ 5.14-5.07 (m, 5H), 3.66 (s, 3H), 2.31-2.26 (m, 2H),
2.43-2.38 (m, 2H), 2.06-1.98 (m, 18H), 1.68 (s, 3H), 1.60 (s, 15H).
HRMS (EI+) m/z [M+] calculated for C28H46O2 414.3498, observed
414.3496.
Biosynthetic Product from 6-Desmethyl-2,3-oxidosqualene and
Lanosterol Synthase. Compound 3 (6.9 mg) was dissolved in dH2O/
Triton X-100 (8:2, 200 µL), and the resulting solution was added to a
solution of lanosterol synthase (1.2 µM, 6 mL) in buffer containing
sodium phosphate (200 mM, pH 6.4), glycerol (20%), Triton X-100
(0.2%), and DTT (3 mM). After incubation at 20 °C for 48 h, the
unconverted starting material and the product were extracted with
CHCl3, and the product was purified by flash chromatography using
5% diethyl ether in hexane and then by preparative TLC using 25%
diethyl ether in hexane (0.15 mg, 2.2%). 1H NMR (500 MHz, CDCl3)
δ 0.687 (s, 3 H), 0.742 (s, 3 H), 0.864 (s, 3 H), 0.899 (d, J ) 6.9 Hz,
3 H), 0.998 (s, 3 H), 1.142-2.003 (m, 24 H), 1.584 (s, 3 H), 1.662 (s,
3 H), 3.218 dd, J ) 11.8 Hz, J ) 4.5 Hz, 1 H), 5.082 (t, J ) 6.1 Hz,
1
1 H). Comparison with the assigned methyl peaks in the H NMR
spectrum of lanosterol5 indicates the absence of the A/B ring angular
methyl group.
Spiro Epoxide 5. Trisnorsqualene aldehyde (65.6 mg, 0.171 mmol)
and cyclopropyldiphenylsulfonium tetrafluoroborate (53.7 mg, 0.171
mmol) were introduced into a 25-mL round-bottomed flask fitted with
a stir bar and septum. DMSO (2 mL) was added to the flask, and the
stirred solution at room temperature was treated with freshly crushed
KOH (19.2 mg, 0.342 mmol). After 2 h at room temperature, the
reaction was complete as indicated by silica gel TLC using triethyl-
amine-treated plates. The reaction mixture was extracted four times
with hexane. The combined organic layers were washed twice with
saturated aqueous NaHCO3 solution, dried over Na2SO4, filtered, and
r-Chloro Methyl Ester 22. A solution of azeotropically dried
trisnorsqualene methyl ester (39.6 mg, 0.095 mmol) in THF (1 mL) at
-78 °C was added to a solution of LDA gradually. After 15 min the

1288 J. Am. Chem. Soc., Vol. 119, No. 6, 1997
Corey et al.
resulting solution of enolate was added to a suspension of N-
chlorosuccinimide in THF (1 mL) at -78 °C. After stirring for 30
min the reaction mixture was quenched by the addition of brine. The
product was isolated by extractive workup and purified on a silica gel
column (1.7 cm × 28 cm) using 2% diethyl ether in hexane as the
eluant to give the R-chloro methyl ester 22 in 43% yield. 1H NMR
(300 MHz, CDCl3) δ 5.26 (t, J ) 5.88 Hz, 1H), 5.14-5.07 (m, 4H),
4.39-4.33 (m, 1H), 3.66 (s, 3H), 2.75-2.49 (m, 2H), 2.09-1.93 (m,
16H), 1.68 (s, 3H), 1.66 (s, 3H), 1.60 (s, 12H). HRMS (EI+) m/z [M+]
calculated for C28O2H45Cl 448.3108, observed 448.3091.
(0.65 mL, 1.0 M solution in CH2Cl2), and the resulting solution was
stirred at -78 °C for 30 min. Saturated NH4Cl solution (5 mL) was
then added. After workup, the product was essentially pure (105 mg,
90%). 1H NMR (400 MHz, CDCl3) δ 1.58 (s, 12 H), 1.65 (s, 3 H),
1.66 (d, J ) 1.0 Hz, 3 H), 1.93-2.14 (m, 16 H), 3.97 (s, 2 H), 5.10
(m, 4 H), 5.37 (m, 1 H); 13C NMR (100 MHz, CDCl3) δ 135.2, 134.7,
131.3, 126.2, 124.6, 124.4, 124.2, 69.1, 39.7, 39.3, 36.1, 35.8, 28.2,
26.8, 26.3, 25.7, 17.7, 16.1, 16.0, 13.7; IR (neat) 3324 (br), 2964, 2918,
2854, 1382, 1010 cm-1; MS (EI) m/z 358 (M+); HRMS: 358.3245
(358.3236 calcd for C25H42O).
Oxide 8. A solution of the azeotropically dried R-chloro methyl
ester 22 (18.3 mg, 0.041 mmol) in THF (1 mL) was cooled to -78 °C
and treated with an excess of MeLi (80 mL, 1.53 M in diethyl ether).
After stirring for 5 min, the reaction mixture was warmed to 0 °C.
After stirring for 2 h, the reaction mixture was quenched by the addition
of saturated aqueous NaHCO3 solution. The product was isolated by
extractive workup and purified on a silica gel flash column (1.7 cm ×
28 cm) using 3% diethyl ether in hexane as the eluant to give oxide 8
(6.6 mg, 0.016 mmol). 1H NMR (300 MHz, CDCl3) δ 5.23-5.09 (m,
5H), 2.79 (t, J ) 6.24 Hz, 1H), 2.18-1.99 (m, 18H), 1.60 (m, 9H),
1.68 (m, 6H), 1.57 (s, 3H), 1.32 (s, 3H), 1.28 (s, 3H). HRMS (EI+)
m/z [M+] calculated for C29H48O 412.3705, observed 412.3690.
Enol Ether 9. A stirred solution of methoxymethyltriphenylphos-
phonium chloride (86 mg, 0.25 mmol) in toluene (1 mL) at 0 °C was
treated with LDA over 5 min to form the red Wittig reagent. To a
solution of azeotropically dried trisnorsqualene aldehyde (89 mg, 0.23
mmol) at 0 °C was added the preformed Wittig reagent. The reaction
mixture immediately turned yellow and was quenched after stirring
for 1 h at 0 °C by the addition of saturated aqueous NH4Cl solution.
The product was isolated by extractive workup and chromatographed
on silica gel to give a mixture of diastereomers (1:1.3) enol ether 9
(32 mg) in 34% yield. 1H NMR (300 MHz, CDCl3) (Mixture of
diastereomers, 1:1.3 E/Z) δ 6.29 (MeO-CHdC, E, d, J ) 12.5 Hz,
1H), 5.85 (MeO-CHdC, Z, d, J ) 6.2 Hz, 1H), 5.14-5.10 (m, 5H),
4.71 (MeO-CHdCH, E, m, 1H), 4.31 (MeO-CHdCH, Z, m 1H), 3.57
(s, 3H), 3.49 (s, 3H), 2.07-2.00 (m, 20H), 1.68 (s, 3H), 1.60 (s, 15
H). HRMS (EI) calculated 412.3705, observed 412.3693.
Bisnorsqualene Aldehyde (10). A stirred solution of enol ether 9
(12 mg, 29 mmol) in THF (1 mL) was treated with 12% HCl (0.2
mL). After 4 h at 23 °C the reaction mixture was quenched by the
addition of ice-cold saturated aqueous NaHCO3 solution. The product
was isolated by extractive workup and chromatographed on silica gel
to give the aldehyde 10. 1H NMR (300 MHz, CDCl3) δ 9.76 (s, 1H),
5.14-5.09 (m, 5H), 2.39 (t, J ) 7.4 Hz, 2H), 2.06-2.01 (m, 20H),
1.60 (s, 12H), 1.55 (s, 6H). HRMS (EI) calculated 398.3549, observed
398.3533.
Ester 23. To a solution of triethyl phosphonopropionate (151 mg,
0.633 mmol) in THF (2 mL) at -78 °C was added NaH (25.3 mg,
0.633 mmol), and the resulting solution was stirred for 30 min. Then
the aldehyde (100 mg, 0.316 mmol) was added, and the reaction mixture
was stirred for 1 h. After workup, the trans isomer was purified by
flash chromatography using 1% diethyl ether in hexane (97.0 mg, 77%).
1H NMR (400 MHz, CDCl3) δ 1.27 (t, J ) 7.0 Hz, 3 H), 1.58 (s, 12
H), 1.66 (s, 3 H), 1.81 (s, 3 H), 1.92-2.27 (m, 16 H), 4.15 (q, J ) 7.0
Hz, 2 H), 5.10 (m, 4 H), 6.72 (t, J ) 7.3 Hz, 1 H); 13C NMR (100
MHz, CDCl3) δ 168.2, 141.9, 135.2, 134.9, 134.0, 131.2, 127.7, 125.1,
124.2, 124.2, 124.1, 60.3, 39.7, 38.3, 28.3, 28.2, 27.4, 26.8, 26.7, 25.7,
17.7, 16.0, 14.3, 12.4; IR (neat) 2964, 2926, 2856, 1711, 1444, 1269,
1121, 1079 cm-1; MS (EI) m/z 400 (M+); HRMS: 400.3333 (400.3341
calcd for C27H44O2).
To a solution of the alcohol prepared above (89.0 mg, 0.249 mmol)
in CH2Cl2 (2 mL) at -40 °C was added Et3N (70.0 µL, 0.497 mmol)
and MsCl (28.9 µL, 0.375 mmol). The reaction mixture was stirred at
-40 °C for 30 min. Then LiBr (216 mg, 2.49 mmol) in THF (4 mL)
was added, and the resulting solution was warmed to room temperature.
After extractive workup, the corresponding bromide was dissolved in
CH3CN (2.5 mL), followed by addition of triphenylphosphine (326 mg,
1.25 mmol). The resulting solution was stirred at 40 °C for 12 h. Then
solvent was removed in Vacuo, and product was purified by flash
chromatography eluting with first CHCl3 then 4% MeOH in CHCl3 to
give triphenylphosphonium salt 24 (165 mg, 97%). 1H NMR (400
MHz, CDCl3) δ 1.44 (s, 3 H), 1.45 (s, 3 H), 1.54 (s, 9 H), 1.62 (s, 3
H), 1.72-2.03 (m, 16 H), 4.53 (d, J ) 14.6 Hz, 2 H), 4.91 (m, 1 H),
5.04 (m, 3 H), 5.22 (m, 1 H), 7.62-7.80 (m, 15 H); 13C NMR (100
MHz, CDCl3) δ 136.4, 136.2, 134.9, 134.8, 134.1, 134.0, 133.9, 130.2,
130.1, 124.6, 124.2, 124.1, 123.9, 122.0, 121.9, 118.8, 117.9, 39.6,
38.5, 28.1, 28.0, 26.6, 26.5, 25.5, 18.4, 17.5, 15.9, 15.8, 15.7; IR (neat)
2965, 2922, 2871, 1438, 1112 cm-1; MS (FAB+) m/z 603 (M+);
HRMS: 603.4100 (603.4120 calcd for C43H56P+).
4,5-Dehydrooxidosqualene (11). To a solution of triphenylphos-
phonium salt 24 (39.0 mg, 0.0571 mmol) in THF (2 mL) at -78 °C
was added t-BuOK (7.59 mg, 0.676 mmol) in THF (0.31 mL), and the
reaction mixture was warmed to room temperature and stirred for 30
min. Then it was cooled to -78 °C, and to it was added the
epoxyaldehyde in ether until the red color disappeared. After the
resulting solution was stirred at -78 °C for 5 min and then room
temperature for 1 h, it was worked up. The product was purified by
flash chromatography with Et3N treated silica gel using 3% diethyl
ether in hexane (16.0 mg, 66%). 1H NMR (500 MHz, CDCl3) δ 1.18
(s, 3 H), 1.20 (s, 3 H), 1.56 (s, 3 H), 1.57 (s, 3 H), 1.60 (s, 3 H), 1.61
(s, 3 H), 1.69 (s, 3 H), 1.98-2.21 (m, 16 H), 3.63 (d, J ) 7.4 Hz, 1H),
5.23-5.30 (m, 4 H), 5.33 (dd, J ) 11.8 Hz, J ) 7.4 Hz, 1 H), 5.49 (t,
J ) 7.0 Hz, 1 H), 6.06 (d, J ) 11.8 Hz, 1 H); 13C NMR (100 MHz,
benzene) δ 138.0, 135.3, 134.0, 135.0, 132.6, 131.1, 125.5, 125.3, 124.9,
124.8, 124.7, 61.2, 40.2, 39.7, 28.7, 27.2, 27.1, 27.0, 25.8, 24.4, 19.7,
17.7, 16.5, 16.2, 16.1; IR (neat) 2961, 2923, 2854, 1448, 1377 cm-1
;
MS (EI) m/z 424 (M+); HRMS: 424.3695 (424.3705 calcd for
C30H48O).
Acknowledgment. This research was generously supported
by the National Institutes of Health. We are grateful also to
Dr. S. Edward Lee and Mr. Steven J. Hawrylik of Pfizer Inc.
Central Research for scaling up the baculovirus expression of
lanosterol synthase and for supplying large quantities of crude
protein. Thanks are also due to Professor Tom Maniatis and
his group for helpful advice in the initial stage of our work
with the baculovirus system.
Triphenylphosphonium Salt 24. To a solution of ester 23 (130
mg, 0.325 mmol) in CH2Cl2 (5 mL) at -78 °C was added DIBAL
JA963227W