Recombinant squalene synthase. A mechanism for the rearrangement of presqualene diphosphate to squalene

Article abstract of DOI:10.1021/ja020411a

Squalene synthase (SQase) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP) and the subsequent rearrangement and NADPH-dependent reduction of PSPP to squalene (SQ). These reactions are the fir

Full text of DOI:10.1021/ja020411a

Published on Web 07/04/2002
Recombinant Squalene Synthase. A Mechanism for the
Rearrangement of Presqualene Diphosphate to Squalene
§
‡
Brian S. J. Blagg, Michael B. Jarstfer, Daniel H. Rogers, and C. Dale Poulter*
Contribution from the Department of Chemistry, UniVersity of Utah,
3
15 South 1400 East Room 2020, Salt Lake City, Utah 84112
Received March 21, 2002
Abstract: Squalene synthase (SQase) catalyzes the condensation of two molecules of farnesyl diphosphate
FPP) to form presqualene diphosphate (PSPP) and the subsequent rearrangement and NADPH-dependent
(
reduction of PSPP to squalene (SQ). These reactions are the first committed steps in cholesterol
biosynthesis. When recombinant SQase was incubated with FPP in the presence of dihydroNADPH
3
(NADPH , an unreactive analogue lacking the 5,6-double bond in the nicotinamide ring), three products
were formed: dehydrosqualene (DSQ), a C30 analogue of phytoene; 10(S)-hydroxysqualene (HSQ), a
hydroxy analogue of squalene; and rillingol (ROH), a cyclopropylcarbinyl alcohol formed by addition of
water to the tertiary cyclopropylcarbinyl cation previously proposed as an intermediate in the rearrangement
of PSPP to SQ (Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77). The structure and absolute
stereochemistry of the tertiary cyclopropylcarbinyl alcohol were established by synthesis using two
independent routes. Isolation of ROH from the enzyme-catalyzed reaction provides strong evidence for a
cyclopropylcarbinyl-cyclopropylcarbinyl rearrangement in the biosynthesis of squalene. By comparing the
SQase-catalyzed solvolysis of PSPP in the absence of NADPH to the reaction in the presence of NADPH ,
3 3
it is apparent that the binding of the cofactor analogue substantially enhances the ability of SQase to control
the regio- and stereochemistry of the rearrangements of PSPP.
1
Squalene synthase catalyzes the synthesis of PSPP from FPP
and its subsequent rearrangement to squalene. These reactions
are the first committed steps in sterol biosynthesis. Rilling
discovered that PSPP accumulated in incubations that did not
contain NADPH and was then converted to SQ when NADPH
mimic the enzymatic reaction gave mixed results. Although
several important features predicted by the mechanistic proposals
were duplicated by the model reactions, the regioselectivity of
the c1′-2-3 f 1′-1 rearrangement required to convert the
presqualene skeleton to SQ was poor, giving only trace
2
18-20
was subsequently added. After the structure of PSPP was firmly
established, several groups proposed mechanisms for the
conversion of PSPP to SQ4 based on the rearrangements of
quantities of 1′-1 products.
3
,4
Mechanistic studies with SQase have been hampered by the
unavailability of purified enzyme. A soluble recombinant form
of SQase lacking a putative membrane-spanning R-helix at its
-8
cyclopropylcarbinyl cations.9
-17
Model studies designed to
2
1
C-terminus catalyzed reactions similar to those of the wild-
*
Corresponding author. Phone: (801) 581-6685. Fax: (801) 581-4391.
22-24
type membrane-bound protein.
In an incubation with
E-mail: poulter@chemistry.utah.edu.
§
NADPH, FPP was rapidly converted to PSPP, which was then
rapidly processed to SQ. However, when NADPH was not
added to the buffer, PSPP accumulated and was then slowly
Biological Chemistry Trainee (NIH Grant GM 80357).
‡
Biological Chemistry Trainee (NIH Grant GM 80357); ACS Division
of Organic Chemistry Fellow (sponsored by Boehringer Ingelheim). Current
address: University of North Carolina School of Pharmacy, CB #7360,
Chapel Hill, NC 27599.
(12) Schleyer, P. v. R.; Buss, Y. J. Am. Chem. Soc. 1969, 91, 5880-5882.
(13) Gasic, M.; Whalen, D.; Johnson, B.; Winstein, S. J. Am. Chem. Soc. 1967,
89, 6382-6384.
(
1) All of the abbreviations used except those listed here were defined in the
preceding article (Jarsfter et al., see ref 27): FPPase, farnesyl diphosphate
synthase; NADPH
3
, 5,6-dihydro reduced nicotinamide adenine dinucleotide
(14) Whalen, D.; Gasic, M.; Johnson, B.; Jones, H.; Winstein, S. J. Am. Chem.
Soc. 1967, 89, 6384-6386.
phosphate; NMO, N-methylmorpholine N-oxide; ROH, rillingol; TPAP,
tetrapropylammonium perruthenate.
(15) Wiberg, K. B.; Szeimies, G. J. Am. Chem. Soc. 1970, 91, 571-579.
(16) Baldwin, J. E.; Foglesong, W. D. J. Am. Chem. Soc. 1968, 90, 4303-
4310.
(
(
(
2) Rilling, H. C. J. Biol. Chem. 1966, 241, 3233-3235.
3) Rilling, H. C.; Epstein, W. W. J. Am. Chem. Soc. 1969, 91, 1041-1042.
4) Rilling, H. C.; Poulter, C. D.; Epstein, W. W.; Larsen, B. J. Am. Chem.
Soc. 1971, 93, 1783-1785.
(17) Poulter, C. D.; Winstein, S. J. Am. Chem. Soc. 1969, 91, 3650-3652.
(18) Poulter, C. D.; Muscio, O. J.; Goodfellow, R. J. Biochemistry 1974, 13,
1530-1538.
(19) Poulter, C. D.; Marsh, L. L.; Hughes, J. M.; Argyle, J. C.; Satterwhite, D.
M.; Goodfellow, R. J.; Moesinger, S. G. J. Am. Chem. Soc. 1977, 99, 3816-
3828.
(
5) Altman, L. J.; Kowerski, R. C.; Rilling, H. C. J. Am. Chem. Soc. 1971, 93,
1
782-1783.
(
(
6) Coates, R. M.; Robinson, W. H. J. Am. Chem. Soc. 1971, 93, 1785-1786.
7) van Tamelen, E. E.; Schwartz, M. A. J. Am. Chem. Soc. 1971, 93, 1780-
1
782.
(20) Poulter, C. D.; Hughes, J. M. J. Am. Chem. Soc. 1977, 99, 3824-3829.
(21) Jennings, S. M.; Tsay, T. H.; Fisch, T. M.; Robinson, G. W. Proc. Natl.
Acad. Sci. U.S.A. 1991, 88, 6038-6024.
(
8) Campbell. R. V. M.; Crombie, L.; Pattenden, G. Chem. Commun. 1971,
18-219.
9) Poulter, C. D.; Friedrich, E. C.; Winstein, W. J. Am. Chem. Soc. 1970, 92,
2
(
(22) Sasiak, K.; Rilling, H. C. Arch. Biochem. Biophys. 1988, 260, 622-627.
(23) Zhang, D.; Jennings, S. M.; Robinson, G. W.; Poulter, C. D. Arch. Biochem.
Biophys. 1993, 304, 133-143.
4
274-4281.
(
(
10) Poulter, C. D.; Winstein, S. J. Am. Chem. Soc. 1970, 92, 4282-4288.
11) Ree, B. R.; Martin, J. C. J. Am. Chem. Soc. 1970, 92, 1660-1666.
(24) Radisky, E. S.; Poulter, C. D. Biochemistry 2000, 39, 1748-1760.
8846
9
J. AM. CHEM. SOC. 2002, 124, 8846-8853
10.1021/ja020411a CCC: $22.00 © 2002 American Chemical Society

Recombinant Squalene Synthase
A R T I C L E S
converted to a mixture of triterpenes with 1′-1 and 1′-3 fusions
2
5-27
between the farnesyl residues,
reminiscent of the structures
These reactions were stereo-
seen in earlier model studies.1
8-20
selective, and the absolute stereochemistries of the triterpene
products corresponded to those of the analogous biosynthetic
transformations that give the 1′-1 and 1′-3 isoprenoids found
2
7
in nature. In addition, the proportion of 1′-1 products was
substantially higher in the SQase-catalyzed reaction than in the
model studies.
SQase binds its substrates in an ordered manner.24 Two
molecules of FPP add to the enzyme, followed by NADPH, to
generate an E‚FPP‚FPP‚NADPH complex. Under normal
conditions, PSPP is synthesized from FPP and then converted
directly to SQ without dissociating from the enzyme. NADPH
plays an important role in catalysis beyond serving as a
reductant. Without the cofactor, the rate of PSPP synthesis
decreases substantially.27 Although SQase can trigger the
reaction that normally leads to SQ from PSPP, the enzyme can
no longer control the regiochemistry of the ensuing rearrange-
ment, and substantial amounts of 1′-3 products are formed
along with 1′-1 structures.27 Also, there is an accompanying
reduction in stereocontrol. We now report studies in which
SQase was incubated with FPP and 5,6-dihydroNADPH
Figure 1. HPLC trace of products from incubation of SQase with FPP
and NADPH3. Analysis on a normal-phase Ranin microsorb MV silica gel
column with isocratic elution using 1:19 (v/v) MTBE/hexanes at 1 mL/
min.
(
NADPH3), an unreactive analogue of the normal cofactor.
When the NADPH binding site was occupied by NADPH3, no
′-3 products were seen. In addition, a new cyclopropylcarbinyl
1
Figure 2. Products from incubation of SQase with FPP and NADPH3.
triterpene alcohol with a c1′-1-2 fusion of farnesyl units was
formed along with the 1′-1 compounds. This new triterpene
alcohol provides direct evidence for a c1′-2-3 f c1′-1-2
cyclopropylcarbinyl-cyclopropylcarbinyl cationic rearrange-
ment within the active site of SQase during the biosynthesis of
tion buffer.27 The third compound, rillingol (ROH, 17%), which
was not previously observed, eluted between HSQ and the 1′-3
triterpene hydroxybotryococcene (HBO) (see Figure 1). The
new product gave a molecular ion at m/z 426.3865, correspond-
ing to a triterpene alcohol that is isomeric with HSQ and HBO.
1
′-1 isoprenoids.
Results
Studies with NADPH3. We used NADPH3 to determine the
1
The H NMR spectrum of the alcohol had a three-proton
resonance at 1.14 ppm for a tertiary methyl group, resonances
for four cyclopropyl protons, and a vinyl proton that appeared
as a doublet at 4.6 ppm. The chemical shifts and coupling
patterns for the cyclopropyl and vinyl protons were reminiscent
of those reported for rothrockene, an irregular monoterpene with
effect of occupying the NADPH-binding site on the product
distribution from the SQase-catalyzed rearrangement of PSPP.
NADPH3 was prepared by a partial catalytic hydrogenation of
+
30
NADP according to the procedure of Dave et al. A steady-
state kinetic analysis indicated that the analogue was a competi-
2
8
a c1′-1-2 structure. The mechanism originally proposed by
4
Rilling et al. for the conversion of PSPP to SQ involved
tive inhibitor against NADPH, KI ) 45 µM. A comparison of
rearrangement of a c1′-2-3 presqualene cation to its c1′-1-2
isomer. The structure proposed for ROH (see Figure 2) is the
product expected from capture of the c1′-1-2 cation at the
carbinyl carbon by water. ROH has the same carbon skeleton
as “compound X”, originally proposed by Rilling as the structure
NADPH
KI with KM
(∼100 µM) suggests that the affinity of the
analogue for the NADPH site is similar to that of the normal
cofactor.24
Incubation of recombinant SQase with saturating levels of
FPP and NADPH3 resulted in the rapid formation of PSPP,
2
for PSPP, and has previously been synthesized as a racemic
-1
kcat ≈ 0.2 s , followed by a much slower consumption of PSPP,
2
9
mixture of diastereomers.
-
1
kcat ≈ 0.002 s . Similar results were found for incubations in
the absence of NADPH.27 After 1.5 h, the incubation mixture
was extracted with methyl tert-butyl ether (MTBE). Analysis
of the mixture by HPLC revealed three products. Two of the
compounds, DSQ (23%) and HSQ (55%), were seen previously
in incubations where NADPH3 was not included in the incuba-
Absolute Stereochemistry of Rillingol. On the basis of the
absolute stereochemistry of PSPP and the suprafacial stereo-
selectivity for 1,2-bond migrations during cyclopropylcarbinyl-
cyclopropylcarbinyl rearrangements, we predicted that the chiral
cyclopropane carbons in ROH should have a 1S,2R configu-
3
0
ration. This prediction was verified, and the absolute stereo-
chemistry of the carbinyl center was established by the two
independent syntheses of ROH shown in Schemes 1 and 2. The
synthesis presented in Scheme 1 established the absolute
stereochemistry of the cyclopropane ring, while that in Scheme
2 established the absolute stereochemistry of the carbinyl carbon.
Together, they unambigiously determine the structure of ROH
produced by the SQase-catalyzed rearrangement of PSPP.
(
25) Zhang, D.; Poulter, C. D. J. Am. Chem. Soc. 1995, 117, 1641-1642.
26) Jarstfer, M. B.; Blagg, B. S. J.; Rogers, D. H.; Poulter, C. D. J. Am. Chem.
Soc. 1996, 118, 13089-13090.
(
(
(
(
(
27) Jarstfer, M. B.; Zhang, D.; Poulter, C. D. J. Am. Chem. Soc. 2002, 124,
8
834-8845 (preceding article in this issue).
28) Epstein, W. W.; Gaudioso, L. A.; Brewster, G. B. J. Org. Chem. 1984, 49,
748-2754.
29) Corey, E. J.; Ortiz de Montellano, P. R. Tetrahedron Lett. 1968, 49, 5113-
115.
30) Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77.
2
5
J. AM. CHEM. SOC.
9
VOL. 124, NO. 30, 2002 8847

A R T I C L E S
Blagg et al.
Scheme 1. Synthesis of Rillingol: Absolute Stereochemistry of
the Cyclopropane Ring
diastereomeric secondary cyclopropylcarbinols 10. The mixture
of diastereomers was oxidized with TPAP/NMO, and the
resulting ketone (11) was treated with methylmagnesium
bromide to furnish ROH as a 1:6 mixture of diastereomers.
Although the diastereomers did not resolve during flash chro-
2
9
matography, we were able to separate and purify the com-
pounds by HPLC. When the mixture of diastereomers was co-
injected with ROH from the enzymatic reaction, the alcohol
produced by SQase coeluted with the minor diastereomer
1
obtained by synthesis. In addition, the H NMR and mass spectra
of the minor diastereomer and the enzyme-derived material were
identical.
The absolute stereochemistry of the cyclopropane ring in 6
was established as shown in Scheme 1. Cyclopropyl diacetate
7
was obtained from 6 by ozonolysis, reduction of the ozonides
with LiAlH4, and treatment of the resulting alcohols with acetic
2
3
anhydride. A comparison of the optical rotation of 7, [R]
D
D
3
6
23
-
-
14.1°, with the literature value for (R,R)-diacetate, [R]
17.7°, served to establish its stereochemistry and the 2R,3S
stereochemistry of primary alcohol 6.
(b) The Carbinyl Carbon. The absolute stereochemistry of
the carbinyl carbon in ROH was established by a second
synthesis (see Scheme 2). (R)-Nerolidol (12) was prepared from
37
farnesol in a 19:1 er by the procedure of Yasuda et al. Vinyl
2
4
Scheme 2. Synthesis of Rillingol: Absolute Stereochemistry of
the Carbinyl Carbon
iodide 12 was prepared from geranyl bromide by the procedure
38
of Zheng et al. and coupled to 13 with palladium(II) acetate
and Ag2CO3. The trans disubstituted double bond in alcohol
4 was then regioselectively cyclopropanated using Sm/HgCl2/
3
9
1
40
CH2I2 to give ROH. The cyclopropanation reaction produced
a single diastereomer in 29% yield after purification by HPLC.
1
13
The H and C NMR spectra of the tertiary cyclopropylcarbinyl
alcohol obtained by this route were identical to those for the
minor product obtained in Scheme 1 and ROH from the
enzymatic reaction. Hence, ROH is the 10R,11R,12S stereoi-
somer.
Absolute Stereochemistry of HSQ. The absolute configu-
ration and optical purity of HSQ isolated from an incubation
when NADPH3 was included in the buffer were determined as
27
described in the preceding paper. The Mosher’s ester derivative
of HSQ obtained by treatment of the alcohol with (S)-Mosher’s
chloride gave a single peak that had the same retention time as
an authentic sample of the Moshers ester from (R)-HSQ on a
Chiracel OD column. The ester was degraded with ozone, the
ozonide was reduced to the corresponding diol with NaBH4,
and the hydroxyl groups were esterified with (S)-Mosher’s
chloride. The tris-Mosher ester gave a single peak that comi-
grated with a sample of tris ester prepared from (R)-1,2,4-
butanetriol on normal-phase HPLC. Thus, HSQ isolated from
the incubation of SQase with FPP and NADPH3 is the R
enantiomer. We estimate that at least 1% of the S enantiomer
would have been detected in the HPLC traces of the Mosher’s
esters of HSQ and (R)-1,2,4-butanetriol.
(
a) The Cyclopropane Ring. In the synthesis described in
Scheme 1, farnesal was converted to trans-allylic alcohol 5 in
high yield by a Horner-Wadsworth-Emmons condensation,
followed by reduction of the resulting ethyl ester (4). Alcohol
was then complexed with (D)-dioxaboralane and added to
Furukawa’s reagent to give disubstituted cyclopropylcarbinol
. The enantioselectivity of the cyclopropanation was determined
by converting 6 to the corresponding Mosher’s ester.
NMR spectrum of the ester had two peaks for the trifluoromethyl
resonance in a 9:1 ratio. The primary cyclopropylcarbinol was
oxidized with TPAP/NMO34 to cyclopropyl aldehyde 8, which
was treated with homogeranylmagnesium iodide35 to give
31
5
32
6
3
3
19
A
F
Stereochemistry of the Conversion of FPP to DSQ. The
stereochemistry of the double bonds in the central conjugated
(
36) Inamasu, S.; Horiike, M.; Inouye, Y. Bull. Chem. Soc. Jpn. 1969, 42, 1393-
1398.
(
(
31) Charette, A. B.; Juteau, H. J. Am. Chem. Soc. 1994, 116, 2651-2652.
32) Furukawa, J.; Kawabata, N.; Nishimura, J. Tetrahedron Lett. 1966, 22,
(37) Yasuda, A.; Yamamoto, H.; Nozaki, H. J. Chem. Soc. Bull. Jpn. 1979, 52,
1757-1759.
3
353-3354.
(38) Zheng, Y. F.; Oehlschlager, A. C.; Hartman, P. G. J. Org. Chem. 1994,
59, 3-5809.
(
(
(
33) Mosher, D. J. Am. Chem. Soc. 1973, 95, 512-519.
34) Griffith, W. P.; Ley, S. V. Aldrichimica Acta 1990, 23, 13-19.
35) Leopold, E. J. Org. Synth. 1985, 64, 164-170.
(39) Jeffrey, T. Chem. Commun. 1991, 324-325.
(40) Molander, G. A.; Harring, L. S. J. Org. Chem. 1989, 54, 5-3532.
8848 J. AM. CHEM. SOC.
9
VOL. 124, NO. 30, 2002

Recombinant Squalene Synthase
A R T I C L E S
triene of DSQ was determined to be E,Z,E by UV spectroscopy
and described in the preceding paper.27 The stereochemistry for
removal of a proton from C(1) of each of the molecules of FPP
incorporated into DSQ was determined by mass spectrometry
Scheme 3. Mechanisms for the Conversion of Presqualene
Diphosphate to Squalene
2
27
for samples of DSQ obtained from (R)- and (S)-[1- H]FPP.
2
DSQ produced from (R)-[1- H]FPP had a molecular ion at m/z
4
10, indicating the retention of two deuterium atoms, whereas
2
the material from (S)-[1- H]FPP had a molecular ion at m/z
08, indicating that both deuterium atoms had been lost. These
4
results are similar to those reported when FPP was incubated
without NADPH.
SQ produced from the incubation of SQase with (R)-[1- H]-
2
FPP and NADPH had a molecular ion at m/z 412, while material
2
from an incubation with (S)-[1- H]FPP gave SQ with a
molecular ion at m/z 411. These results are consistent with earlier
stereochemical studies of squalene biosynthesis by Cornforth
4
1
and Popjak, where a si-hydrogen is lost from C(1) of one of
the molecules of FPP during the formation of PSPP.
Effects of Solvent and Divalent Metal Ions. In the preceding
paper we found that methanol competed with water as a
2
7
nucleophile in the substitution reaction that produced HSQ.
Under similar conditions in buffer containing 20% methanol
and NADPH3 in addition to the normal components, only 2%
of the HSQ methyl ether was formed. This observation is
consistent with our proposal that the products are formed from
the SQase‚PSPP‚NADPH3 complex, and apparently binding of
NADPH3 either excludes some of the methanol from the active
site or interferes with addition of methanol to the cationic
intermediates. We also reported that the mixture of products
formed upon the SQase-catalyzed solvolysis reaction was
sensitive to the divalent metal.27 In contrast, we found no change
in the distribution of products when Mg2+ was replaced by Mn2+
for incubations in the presence of NADPH3.
Discussion
+
cation might be the transition state that links c1′-2-3 and
The steps leading from PSPP to SQ have been a topic of
speculation ever since PSPP was discovered as an intermediate
in the 1′-1 coupling of FPP. Salient features of the mechanisms
that have been proposed are summarized in Scheme 3. All
involve formation of a primary c1′-2-3 cyclopropylcarbinyl
+
8
c1′-1-2 . The 11R and 12S stereocenters in ROH are exactly
what one expects for the c1′-2-3 f c1′-1-2 rearrangement
that converts (1R,2R,3R)-PSPP to ROH. The 10R stereocenter
in ROH is generated by addition of water to the re face of the
tertiary cation. One would not anticipate a stereoelectronic
preference for this reaction, and presumably the direction of
addition to the trigonal center in c1′-1-2 is imposed by
structural features in the catalytic site of the enzyme. Finally,
formation of (12R)-HSQ from c1′-1-2 is the predicted result
from addition of water to C(1′) of the tertiary cyclopropylcarbi-
nyl cation with inversion. With no compelling evidence requiring
′-1 as a precursor of c1′-1-2 or 1′-1 products, we view
+
+
8
cation from PSPP, followed by rearrangements that eventually
lead to the 1′-1 system.4-8,42,43
+
We now have data that allow us to distinguish among these
proposals. Isolation of ROH from incubations of FPP with
SQase in the presence of NADPH3 provides direct evidence
for a cyclopropylcarbinyl-cyclopropylcarbinyl rearrangement
+
+
+
of c1′-2-3 to c1′-1-2 . These rearrangements are known
+
1
to be highly stereoselective9
-12
and can be envisioned as the
its inclusion in the mechanism for formation of SQ from PSPP
net result of two consecutive suprafacial [1,2] rearrangements
where the C(2)-C(1′) bond in the primary c1′-1-2 cation
rearranges to form the c1′-1-3 cyclobutyl isomer, followed
as an unnecessary embellishment.
+
+
The active site of SQase is designed to selectively promote
the c1′-2-3 f c1′-1-2 rearrangement over other possible
reactions of c1′-2-3 . In model studies, rearrangement of the
by rearrangement of the C(1)-C(3′) bond to give the tertiary
+
+
c1′-1-2 cation. Cyclopropylcarbinyl-cyclopropylcarbinyl
primary c1′-2-3 cation to its allylic 1′-3 isomer was highly
rearrangements can proceed through cyclobutyl cations as
discrete intermediates. However, the putative cyclobutyl species
in the c1′-2-3 to c1′-1-2 rearrangement could not be
trapped during model studies, suggesting that the c1′-1-3
-4
favored where kc1′-1-2/k1′-3 ≈ 4 × 10 . When SQase catalyzed
the “solvolysis” of PSPP in the absence of NADPH, kc1′-1-2/
k1′-3 ) 5.2, and the ratio increased to >200 when NADPH3
was bound in the active site along with PSPP. These rate ratios
correspond to differences in transition-state energies leading
from c1′-2-3 to c1′-1-2 or 1′-3 , where ∆G c1′-2-3 -
∆G 1′-3 ≈ 4.7 kcal/mol in the model system, -1.0 kcal/mol
+
+
(
41) Cornforth, J. W.; Cornforth, R. H.; Donninger, C.; Popjak, G.; Ryback,
G.; Schroepfer, G. J. Proc. R. Soc. B 1966, 163, 436-464.
42) Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77.
+
+
+
q
(
(
q
43) Poulter, C. D. J. Agric Food Chem. 1974, 22, 167-173.
J. AM. CHEM. SOC.
9
VOL. 124, NO. 30, 2002 8849

A R T I C L E S
Blagg et al.
Scheme 4. Stereochemistry for Formation of
Hydroxybotryococcene, Hydroxysqualene, and Dehydrosqualene
from Presqualene Diphosphate
On the basis of the stereochemical analysis presented above,
it is straightforward to envision how botryococcene synthase,
which generates a 1′-3 structure from PSPP, might have
evolved from squalene synthase by mutations that repositioned
NADPH slightly so that the hydrogen atom transferred from
the cofactor is moved from near C(1′), where a stereoelectronic
+
barrier prevents addition to c1′-2-3 with retention of con-
figuration, to near C(3′), where addition to the si face of the
double bond is allowed.
The stereochemical analysis of the reactions catalyzed by
SQase also has important mechanistic implications for the c1′-
+
+
2
-3 f 1′-3 rearrangement. In an unconstrained model
+
+
system, the transition state for c1′-2-3 f 1′-3 was ∼4.7
+
+
kcal/mol below that for c1′-2-3 f c1′-1-2 . To achieve
the observed level of regioselectivity seen during the biosyn-
+
thesis of SQ, the transition state for the c1′-2-3 f c1′-1-
+
2
rearrangement must be selectively stabilized by >7.8 kcal/
+
mol in the SQase‚NADPH3‚c1′-2-3 ‚PPi complex. Assuming
that PSPP is bound in the conformation shown in Scheme 4,
the negatively charged pyrophosphate anion is located near the
bond between C(2) and C(3) in the cyclopropane ring after the
C(1)-oxygen bond in bound PSPP is ruptured. We propose
that regioselectivity in the rearrangement is achieved through
electrostatic interactions between positively and negatively
+
+
charged partners in the c1′-2-3 ‚PPi and c1′-1-2 ‚PPi
intimate ion pairs. These interactions should facilitate c1′-2-
for the enzyme catalyzed “solvolysis” in the absence of NADPH,
+
+
3
f c1′-1-2 relative to the other reactions seen in model
and > -3.2 kcal/mol when NADPH3 is bound! Thus, SQase
studies or in the enzyme-catalyzed reaction in the absence of
NADPH.
+
+
selectively catalyzes the c1′-2-3 f c1′-1-2 rearrangement
by lowering the barrier of the cyclopropylcarbinyl-cyclopro-
pylcarbinyl rearrangement by over 7.9 kcal/mol relative to the
The stereochemistry for elimination of a proton from C(1)
+
in c1′-1-2 to form the 1′-1 double bond in (Z)-DSQ is
+
+
barrier for the accompanying c1′-2-3 f c1′-3 rearrange-
ment. Thus, we propose the pathway in Scheme 3 delineated
by heavy arrows.
identical to that seen during biosynthesis of (Z)-phytoene from
44
prephytoene diphosphate. Interestingly, the other C(1) proton
44
is lost during biosynthesis of (E)-phytoene. All of these results
The stereochemistries for formation of DSQ, HSQ, HBO,
and ROH are summarized in Scheme 4. A detailed analysis of
these reactions provides important insights about the conforma-
tion of PSPP in the active site of the enzyme and the mechanism
for formation of the products. During synthesis of squalene,
the C(1) methylene in PSPP is inverted during the c1′-2-3 to
can be easily explained if one assumes that the elimination of
the proton removed from C(1′) in the corresponding c1′-1-2
cation proceeds through a least motion transition state to give
the respective Z and E double bonds.
Several aspects of the reactions catalyzed by SQase and
PHase suggest that the two proteins share a common ancestor.
The cyclopropane rings in PSPP and PPPP have identical
1
′-1 rearrangement.30,41 This requires that the C(1) oxygen and
the C(2)-C(1′) bonds in PSPP be antiperiplanar in the E‚PSPP‚
NADPH complex in order to accommodate the mandatory
suprafacial [1,2] bond migration of the C(1′)-C(2) bond from
1
R,2R,3R absolute stereochemistries, and the two enzymes
catalyze identical c1′-2-3 f 1′-1 cyclopropylcarbinyl-
cyclopropylcarbinyl rearrangements. When deprived of NADPH,
DSQ, a C30 analogue of phytoene, is one of the major products
synthesized by SQase. In addition to these chemical similarities,
multiple alignments of amino acid sequences for squalene and
phytoene synthases from widely diverged organisms indicate
that the enzymes are genetically related. Table 1 lists conserved
amino acids in three different regions that are found in both
enzymes. When the conserved regions are mapped onto the
+
+
C(2) to C(1) during rearrangement of c1′-2-3 to c1′-1-2 .
Both SQ and HSQ are formed with inversion at C(1′), again in
accord with the stereoelectronic requirements for addition of
nucleophiles to the cyclopropane carbons in cyclopropylcarbinyl
cations. Thus, the nucleophile (NADPH or water) must be
+
+
located at the backside of C(1′) in c1′-1-2 after c1′-2-3
+
f c1′-3 . Interestingly, this particular topology would require
+
the nucleophile to add to C(1′) in c1′-2-3 with retention of
45
recently published X-ray structure of human SQase, all three
configuration, a stereoelectronically unfavorable orientation.
cluster around the putative active site of the enzyme, as
2
7
Finally, the Z double bond between C(1′) and C(2′) HBO
illustrated in Figure 3. In addition, SQase consists of R-helicies
requires that the dihedral angle between the C(1′)-H and
C(2′)-H bonds in the E‚PSPP complex be greater than 90°
and probably near 180°, assuming that SQ and HBO are both
46
arranged in an “isoprenoid fold” similar to the one first
47
reported for farnesyl diphosphate synthase (FPPase), suggest-
ing a familial link between the cyclopropanation enzymes and
+
derived from c1′-2-3 , as suggested by the regioselective
formation of HSQ and HBO, to the exclusion of their respective
allylic isomers isoHBO and isoHSQ, when SQase is incubated
with FPP in the absence of NADPH.27
(44) Gregonis, D. E.; Rilling, H. C. Biochemistry 1974, 13, 1538-1542.
(
45) Pandit, J.; Danley, D. E.; Schulte, G. K.; Mazzalupo, S.; Pauly, T. A.;
Hayward, C. M.; Hamanaka, E. S.; Thompson, J. F.; Harwood, H. J., Jr. J.
Biol. Chem. 2000, 275, 30610.
8850 J. AM. CHEM. SOC.
9
VOL. 124, NO. 30, 2002

Recombinant Squalene Synthase
A R T I C L E S
a
b
c
Table 1. Conserved Regions in Squalene and Phyotene Synthases
a
Uppercase designates conserved amino acids with no more than one mismatch in the sequence alignments. Lowercase designates conserved amino acids
with no more than three mismatches in the sequence alignments. Amino acids in blue denote aspartate/glutamate-rich putative diphosphate binding motifs.
b
c
Based on 19 sequences. Based on 27 sequences. Includes one sequence for a dehydrosqualene synthase.
1
′-1 products with a high degree of regioselectivity by relying
on electrostatic interactions between PPi and the cyclopropyl-
carbinyl cations.
Experimental Section49
+
1
,4,5,6-TetrahydroNADPH (NADPH
3
). NADP (NH
4
salt, 100 mg,
, and 13 mg
O. The solution
was added, and the progress of the
0
(
.12 mmol), 100 mg (5% Pd, 0.05 mmol) of Pd on BaCO
0.11 mmol) of NH SO were dissolved in 15 mL of H
with outgassed with N before H
3
4
4
2
2
2
reaction was followed by UV until the ratio of A260/A290 reached ∼1.2.
The reaction mixture was passed through a 0.45 µm filter and
concentrated by lyophilization. The residue was resuspended in 50 mM
NH
exchange column using 100 mL of 100 mM NH
gradient to 300 mM NH HCO , and 300 mM NH
NADPH eluted. The purest fractions, judged by A260/A290 ) 1.2, were
pooled and lyophilized to give a white solid (75 mg, 0.073 mmol based
4
HCO
3
and chromatographed on a 100 mL DEA-sephacyl ion-
HCO , a 300 mL linear
HCO until the
4
3
4
3
4
3
3
on A290, 61%). NADPH
3
was stored at -80 °C as the ammonium salt:
UV λmax ) 263 (ꢀ ) 16 500 M- cm ) and 291 nm (ꢀ ) 13 750 M
1
-1
-1
-
1
1
cm ); H NMR (D
2
O) δ 8.44 (s, 1H), 8.21 and 8.18 (s, 1H), 7.22 (s,
1
)
4
(
H,), 6.17 (d, 1H, J ) 5 Hz), 6.16 (d, 1H, J ) 5 Hz), 4.93 (dt, 1H, J
6.6, 5 Hz), 4.56 (t, 1H, J ) 5 Hz), 4.34 (m, 1H), 4.23-4.12 (m,
H), 3.97-3.75 (m, 4H), 3.01 (ddd, 2H, J ) 6.5, 6.5, 4.2 Hz), 1.96
dd, 2H, J ) 6.5, 6.5 Hz), 1.75-1.52 (m, 2H); HRMS (-) FAB calcd
for C21 17 (M - 1) 746.0991, found 746.0973.
SQase Assay. SQase (SA ) 2.0 µmol min mg ) was assayed by
Figure 3. Ribbon diagram of human SQase showing the “isoprenoid fold”
of the protein. The first (red), second (green), and third (gold) conserved
regions common to SQase and PHase cluster around the putative active
site, and the aspartate/glutamate-rich regions are shown in blue.
31 3 7
H P N O
-
1
-1
25
3
the chain elongation enzymes in the isoprenoid pathway. Also,
the aspartate/glutamate-rich motifs in regions 1 and 2 map onto
the highly conserved aspartate-rich sequences that bind the
diphosphate residues or the substrates for FPPase. Finally, the
recently published sequence for chrysanthemyl diphosphate
synthase, an enzyme that catalyzes the cyclopropanation of two
molecules of dimethylallyl diphosphate, is strikingly similar to
the procedure of Zhang and Poulter. In a typical assay, [1- H]FPP
3
(100 µM, 7.5 µCi H/µM) was incubated with 200 ng of SQase in 200
µL of 50 mM MOPS buffer, pH 7.2, containing 20 mM MgCl , 1 mM
DTT, 1 mg/mL BSA, and 2% (v/v) Tween 80. Reactions were initiated
2
by the addition of SQase. A 4 × 4 matrix of varied concentrations of
3
NADPH and NADPH at fixed concentrations of FPP and enzyme was
used to determine mode of inhibition by NADPH
constant.
Analysis of Products from Incubations of FPP with SQase in
the Presence of NADPH . (a) Analytical Scale. Using conditions
similar to those described for incubation of SQase with FPP in the
absence of NADPH, FPP (300 µM) was incubated with 1 mg (22
µM) of SQase in 50 mM MOPS buffer, pH 7.2, containing 700 µM
NADPH and 10 mM MgCl . The extracts were dried, concentrated,
and analyzed by normal-phase HPLC.
b) Preparative Scale. Typically, 5 mg (11.4 µmol) of FPP and 12
mg (0.3 µmol) of SQase were incubated at 30 °C in 12 mL of 50 mM
MOPS buffer, pH 7.2, containing 10 mM MgCl and 700 µM NADPH .
3
The reaction was worked-up as previously described to give the
following products in order of elution:
3
and the inhibition
4
8
FPPase.
In conclusion, the biosynthesis of 1′-1 structures from c1′-
-3 cyclopropylcarbinyl diphosphates in the sterol and caro-
tenoid pathways proceeds via c1′-2-3 and c1′-1-2 car-
3
2
+
+
27
bocationic intermediates. The key step in these reactions is a
+
+
c1′-2-3 f c1′-1-2 cyclopropylcarbinyl-cyclopropyl-
carbinyl rearrangement. Without catalysis, the rearrangement
is a minor reaction channel for the c1′-2-3 cation, where the
3
2
(
+
+
preferred reaction is a c1′-2-3 f c1′-3 rearrangement.
However, within the active site of the enzymes, the transition
state for the cyclopropylcarbinyl-cyclopropylcarbinyl rear-
rangement is stabilized by more than 7.9 kcal/mol relative to
the competing 1′-3 rearrangement. We suggest that squalene
synthase and phytoene are able to synthesize their respective
2
27
Dehydrosqualene (DSQ, 1), 4.0 min, 23%.
0-Hydroxysqualene (HSQ, 2),27 28.5 min, 55%.
Rillingol (ROH, 3), 24 min, 22%; H NMR (500 MHz, CDCl
1
1
3
) δ
5
.15 (t, 1H, J ) 6.3 Hz), 5.11-5.07 (m, 3H), 4.61 (d, 1H, J ) 9.3
(
(
(
46) Lesburg, C. A.; Zhai, G.; Cane, D. E.; Christianson, D. W. Science 1997,
77, 1820.
47) Tarshis, L. C.; Yan, M.; Poulter, C. D.; Sacchettini, J. C. Biochemistry
Hz), 2.15-1.95 (m, 16H), 1.73 (s, 3H), 1.68 (s, 6H), 1.62 (s, 3H),
1.59 (s, 3H), 1.47-1.41 (m, 1H), 1.13 (bs, 1H), 1.12 (s, 3H), 0.85-
2
1
994, 33, 10871-10877.
0
.79 (m, 2H), 0.42-0.39 (m, 1H); LRMS (EI, 70 eV) m/z 426 (M, 3),
(49) General experimental conditions are described in ref 27.
J. AM. CHEM. SOC. VOL. 124, NO. 30, 2002 8851
48) Rivera, S.; Swedlund, B. D.; King, G. J.; Bell, R. N.; Hussey, C. E., Jr;
Shattuck-Eidens, D. M.; Wrobel, W. M.; Peiser, G. D.; Poulter, C. D. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 4373-4378.
9

A R T I C L E S
Blagg et al.
4
(
08 (M - H
EI, 70 eV) for C30
Ethyl (E,E,E)-5,9,13-Trimethyl-2,4,8,12-tetradecatetraenoate (4).
NaH (2.15 g, 53.5 mmol, 60% in mineral oil) was washed twice with
0 mL of pentane and suspended in 180 mL of dry toluene. The flask
2
O, 19), 271 (33), 149 (42), 81 (55), 69 (C
5
H
9
100); HRMS
mL of brine was added. Ethyl acetate (10 mL) was added, and the
material was filtered through 6 g of Celite. The eluent was concentrated
and dissolved in 10 mL of CH Cl , and 2 mL (14.3 mmol) of Et N
2 2 3
was added at room temperature. After the mixture was stirred for 5
min, DMAP (10 mg, 0.1 equiv) was added, followed by 1 mL (10.6
mmol) of acetic anhydride. The solution was stirred for 4.5 h at room
temperature before 1 mL of brine was added. The mixture was diluted
with ether (5 mL), and the organic layer was separated, dried, and
concentrated. The residue was purified by flash chromatography (1:9
H50O calcd 426.3861, found 426.3865.
1
was cooled to 0 °C, and 9.39 g (53.5 mmol) of triethyl phosphonoacetate
was added dropwise by syringe. The mixture was stirred for 50 min at
0
°C before 9.8 g (44.6 mmol) of farnesal was added dropwise. The
reaction was stirred for an additional 2.5 h at 0 °C and was quenched
by the addition of 20 mL of saturated NH Cl solution. The organic
layer was separated, and the aqueous layer was washed 3 times with
5 mL of ether. The combined organic layers were dried and
concentrated. The residue was chromatographed on silica (1:19 (v/v)
to 100 (v/v) ethyl acetate/hexanes) to give 62 mg (29%) of a colorless
4
oil: [R]23
-14.1° (c 2.1, EtOH); H NMR δ 3.93 (d, 4H, J ) 7 Hz),
1
D
13
2
2.06 (s, 6H), 1.08-1.18 (m, 2H), 0.61 (dd, 2H, J ) 7, 7 Hz); C NMR
δ 171.0, 67.4, 21.0, 16.1, 8.9; MS (CI) m/z 187 (M + 1, 8.6), 163 (3),
+
1
ethyl acetate/hexane) to yield 10.61 g (82%) of a colorless oil:
H
127 (100), 67 (31); HRMS calcd for C
9 15 4
H O 187.0969, found
NMR δ 7.61 (dd, 1H, J ) 15, 12 Hz), 5.99 (d, 1H, J ) 12 Hz), 5.78
187.0939.
(
(
1
1
1
d, 1H, J ) 15 Hz), 5.05-5.13 (m, 2H), 4.20 (q, 2H, J ) 7 Hz), 2.17
m, 4H), 1.94-2.12 (m, 4H), 1.9 (s, 3H), 1.68 (s, 3H), 1.61 (s, 6H),
(2R,3S)-Cyclopropyl Aldehyde 8. A solution of 1.28 g (4.88 mmol)
of cyclopropyl trienol 6, 30 mL of CH Cl , and 3 mL of CH CN over
2
2
3
.30 (t, 3H, J ) 7 Hz); 13C NMR δ 167.1, 149.8, 141.0, 135.8, 131.4,
1.2 g of crushed 4 Å activated molecular sieves was cooled to 0 °C
before 1.05 g (9.0 mmol) of NMO and 85 mg (0.24 mmol, 5 mol %)
of TPAP were added. The suspension was stirred at 0 °C for 10 min,
warmed to room temperature, and stirred for an additional 2 h. The
material was filtered through 50 g of silica, dried, and concentrated.
24.2, 123.3, 123.2, 118.8, 60.1, 40.2, 39.7, 26.7, 26.2, 25.7, 17.7, 17.4,
+
6.0, 14.34; MS m/z 290 (M , 1.3), 245 (3.9) 154 (65), 137 (13), 69
(
100); HRMS calcd for C19
E,E,E)-5,9,13-Trimethyltetradeca-2,4,8,11-tetraenol (5). A solu-
tion of 8.3 g (28.6 mmol) of 4 and 200 mL of CH Cl was cooled to
78 °C before 68.6 mL (68.6 mmol, 1 M in hexanes) of DIBALH
was added dropwise. The mixture was stirred at -78 °C for 1.5 h before
addition of 5 mL of saturated NH Cl solution. The layers were
30 2
H O 290.2245, found 290.2254.
(
The residue was purified by flash chromatography (toluene) to give
2
2
1.12 g (89%) of a colorless oil: [R]23
-61.1° (neat); H NMR δ 9.19
1
-
D
(d, 1H, J ) 5 Hz), 5.05-5.15 (m, 2H), 4.67 (d, 1H, J ) 9 Hz), 2.16-
2.36 (m, 1H), 1.93-2.12 (m, 8H), 1.78-1.86 (m, 1H), 1.74 (s, 3H),
1.70 (s, 3H), 1.62 (s, 3H), 1.60 (s, 3H), 1.54 (dt, 1H, J ) 5, 9 Hz),
1.10 (ddd, 1H, J ) 5, 7, 8 Hz); 13C NMR δ 200.3, 138.7, 135.8, 131.9,
124.2, 123.8, 123.5, 40.0, 39.7, 32.2, 27.0, 26.6, 26.0, 22.5, 18.0, 17.0,
4
separated, the aqueous layer was extracted with ether, and the combined
organic layers were washed with brine, dried, and concentrated.
Purification of the residue by flash chromatography (1:9 (v/v) ethyl
acetate/hexane) yielded 7.02 g (99%) of a colorless oil: H NMR δ
6
1
-1
16.3, 16.2; FTIR (film, cm ) 2918 (s), 2855 (s), 2724 (m) 1709 (s);
.48 (dd, 1H, J ) 15, 11 Hz), 6.86 (d, 1H, J ) 11 Hz), 5.74 (dt, 1H,
28
MS m/z 260 (5), 136 (14), 123 (11), 69 (100); HRMS calcd for C18H O
260.2133, found 260.2153.
J ) 15, 6 Hz), 5.06-5.16 (m, 2H), 4.15-4.23 (t, 2H, J ) 6 Hz), 1.94-
2
.18 (m, 8H), 1.78 (s, 3H), 1.69 (s, 3H), 1.61 (s, 6H), 1.56 (br s, 1H);
C NMR δ 140.0, 135.5, 131.4, 129.5, 128.5, 124.5, 124.0, 123.9,
Homogeranyl Iodide (9). A solution of 2.85 g (16.9 mmol) of
homogeraniol and 100 mL of CH Cl was cooled to -42 °C before
2 2
13
35
6
3
4
2
4.0, 40.1, 40.0, 27.0, 26.7, 26.0, 17.9, 16.9, 16.2; FTIR (film, cm-1
356 (br), 2919 (s), 2825 (m), 1742 (m), 1654 (w); MS m/z 248 (M ,
.9), 232 (1.2), 137 (24.3), 69 (100.0); HRMS calcd for C17
48.2133, found 248.2120.
)
+
3.28 g (25.5 mmol) of diisopropylethylamine was added, followed by
100 mg of DMAP. The solution was stirred for 5 min before 1.63 mL
(20.4 mmol) of methyl sulfonyl chloride was added. The solution was
stirred for 2 h at -42 °C and at 0 °C for an additional 2.5 h. Five
milliliters of water was added, and the organic layer was separated,
dried, and concentrated. The yellow oil was added to a solution of 19
g (119 mmol) of NaI in 150 mL of acetone. After the mixture was
stirred at reflux for 36 h, 20 mL of water was added. The layers were
separated, and the aqueous layer was washed with ether. The combined
organic layers were dried and concentrated. The residue was purified
by flash chromatography (hexanes) to give 3.65 g (78%) of a colorless
oil: 1H NMR δ 5.06-5.16 (m, 2H), 3.11 (t, 2H, J ) 7 Hz), 2.59 (q,
28
H O
(
2R,3S)-Cyclopropylcarbinol 6. A solution of 1.60 g (6.4 mmol)
of 5 and 2.05 g (7.6 mmol) of d-dioxaborolane in 10 mL of CH Cl
was stirred for 10 min. In parallel, 8.32 mL (8.32 mmol, 1 M in hexanes)
of ZnEt and 1.4 mL (16.0 mmol) of CH were added to 20 mL of
CH Cl at -42 °C, and the mixture was stirred for 5 min. The contents
of the first flask were slowly transferred via cannula into the zinc
solution at -42 °C. The mixture was slowly warmed to room
temperature and stirred for 8 h before addition of 5 mL of saturated
2
2
2
2 2
I
2
2
1
3
NH
4
Cl solution. The layers were separated, the aqueous layer was
2H, J ) 7 Hz), 1.96-2.17 (m, 4H), 1.69 (s, 3H), 1.61 (s, 3H);
C
extracted with three 25 mL portions of ether, and the combined organic
layers were washed with brine, dried, and concentrated. The residue
was purified by flash chromatography (1:9 (v/v) ethyl acetate/hexanes)
NMR δ 138.0, 131.5, 124.0, 123.0, 39.6, 32.4, 26.5, 25.8, 17.8, 16.3,
+
6.1; MS m/z 278 (M , 9.7), 149 (22), 69 (100); HRMS calcd for C11
19
H I
278.0526, found 278.0491.
2
3
to give 1.28 g (77%) of a colorless oil: [R]
D
-35.6° (c 2.5, EtOH);
(10R/S,11R,12S)-Cyclopropylcarbinol 10. A suspension of 2.5 g
(9.0 mmol) of homogeranyl iodide (9), 4 mL of ether, and 400 mg
(16.4 mmol) of magnesium turnings was heated at reflux for 6 h, during
which time 25 mg (1.0 mmol) of magnesium was added every hour.
The Grignard reagent was added by cannula to a solution of 782 mg
(3.0 mmol) of cyclopropyl aldehyde 8 in 5 mL of ether at -42 °C.
The solution was stirred at -42 °C for 1 h and allowed to warm to
room temperature over the next 1.5 h before addition of 2 mL of
1
H NMR δ 5.06-5.14 (m, 2H), 4.63 (d, 1H, J ) 9 Hz), 3.44-3.60
m, 2H), 1.94-2.14 (m, 8H), 1.74 (s, 3H), 1.69 (s, 3H), 1.60 (s, 6H),
(
1
0
.45 (t, 1H, J ) 6 Hz), 1.33-1.44 (m, 1H), 1.02-1.14 (m, 1H), 0.63-
.71 (dt, 1H, J ) 6, 5 Hz), 0.51-0.58 (dt, 1H, J ) 6, 5 Hz); 13C NMR
δ 135.2, 135.0, 131.5, 126.7, 124.5, 124.2, 67.0, 40.0, 39.7, 27.0, 26.8,
1
2
2
6.0, 23.2, 17.9, 16.8, 16.3, 16.3, 12.1; FTIR (film, cm- ) 3336 (br),
+
924 (s), 2855 (m), 1652 (m), 1559 (w); MS m/z 262 (M , 28), 244
(1.5), 177 (53), 107 (89), 69 (100); HRMS calcd for C18H30O 262.2289,
4
saturated NH Cl solution. The layers were separated, and the aqueous
found 262.2296.
layer was washed with ether. The combined organic layers were dried
and concentrated. The residue was purified by flash chromatography
(1:9 (v/v) ethyl acetate/hexanes)
(R,R)-Cyclopropyl Diacetate 7. A solution of 295 mg (1.13 mmol)
of 6 in 10 mL of CH Cl was cooled to -78 °C, and ozone was added
2
2
until a blue color persisted for 2 min. Excess ozone was removed by
bubbling nitrogen gas through the solution. The solvent was removed
f
Diastereomer 1 was isolated as a colorless oil, 430 mg (35%), R )
0.34: [R]23
-29.3° (c 5.8, EtOH); H NMR δ 5.04-5.18 (m, 4H),
1
D
in vacuo. Ether (15 mL) and 0.5 g (13.1 mmol) of LiAlH
and the suspension was stirred at room temperature for 13 h before 1
4
were added,
4.60 (d, 1H, J ) 9 Hz), 3.03 (q, 1H, J ) 8 Hz), 1.92-2.18 (m, 16H),
1.74 (s, 3H), 1.68 (s, 6H), 1.60 (s, 12H), 1.50 (br s, 1H), 1.36-1.49
8
852 J. AM. CHEM. SOC. VOL. 124, NO. 30, 2002
9

Recombinant Squalene Synthase
A R T I C L E S
(
m, 1H), 0.80-0.94 (m, 1H), 0.65 (dt, 5 Hz, J ) 9 Hz), 0.52 (dt, 1H,
Hz, J ) 9 Hz); C NMR δ 135.6, 135.0, 134.5, 131.4, 131.3, 126.8,
2H), 0.38-0.44 (m, 1H); 13C NMR δ 135.7, 135.2, 134.3, 131.7, 131.6,
127.8, 124.7, 124.6, 124.5, 124.4, 71.4, 43.4, 40.0, 39.8, 30.6, 27.0,
26.9, 26.8, 26.4, 26.0, 23.0, 18.0, 16.8, 16.3, 16.3, 13.9, 10.1; FTIR
1
3
5
1
2
2
24.4, 124.3, 124.1, 124.1,75.4, 39.7, 39.5, 37.0, 27.3, 26.8, 26.6, 26.5,
-
1
-1
5.7, 24.2, 17.7, 16.6, 16.0, 15.7, 11.7; FTIR (film, cm ) 3385 (br),
(film, cm ) 3474 (br), 2922 (s), 2854 (s); MS m/z 426 (0.5), 408 (1.7),
+
968 (s), 2854 (s), 1668 (w); MS m/z 412 (M , 3.0), 394 (2.3), 257
339 (1.6), 137 (13.6), 109 (15.5), 69 (100); HRMS calcd for C30
50
H O
(4.3), 137 (17.4), 69 (100); HRMS calcd for C29H48O 412.3693, found
426.3849, found 426.3865.
4
12.3681.
Diastereomer 2 was isolated as a colorless oil, 620 mg (50%), R
Alcohol 14. A mixture of 400 mg (1.4 mmol) of iodotriene 12, 932
mg (4.2 mmol) of (R)-nerolidol (13), 16 mg (0.072 mmol, 0.05 equiv)
of Pd(II) acetate, 390 mg (1.4 mmol) of Ag CO , and 5 mL of DMF
f
)
2
3
1
0
4
1
.25: [R]
D
-29.4° (c 6.8, EtOH); H NMR δ 5.04-5.18 (m, 4H),
2
3
.60 (d, 1H, J ) 9 Hz), 3.05 (q, 1H, J ) 7 Hz), 1.93-2.18 (m, 16H),
.72 (s, 3H), 1.69 (s, 6H), 1.61 (s, 12H), 1.49 (br s, 1H), 1.33-1.43
was heated at 65 °C for 2 h. The material was cooled to room
temperature and concentrated in vacuo. The residue was diluted with
15 mL of ether and filtered through 2.0 g of Celite. The eluent was
concentrated, and the residue was purified by flash chromatography
(1:9 ethyl acetate/hexanes) to give 440 mg (76%) of a colorless oil:
(m, 1H), 0.8-0.91 (m, 1H), 0.73 (dt, 1H, J ) 8, 5 Hz), 0.52 (dt, 1H,
1
3
J ) 8, 5 Hz); C NMR δ 135.6, 135.0, 134.5, 131.3, 131.2, 126.6,
1
2
3
24.3, 124.2, 124.0, 124.0, 75.5, 39.7, 39.5, 37.4, 27.3, 26.8, 26.7, 26.6,
5.7, 24.3, 17.7, 16.5, 16.0, 16.0, 16.0, 16.0, 11.6; FTIR (film, cm
-
1
23
1
)
[R] -8.0° (c 1.7, EtOH); H NMR δ 6.43 (dd, 1H, J ) 15, 11 Hz),
D
+
362 (br), 2922 (s), 2856 (s), 1668 (w); MS m/z 412 (M , 6.8), 394
5.82 (d, 1H, J ) 11 Hz), 5.63 (d, 1H, J ) 15 Hz), 5.02-5.18 (m, 4H),
(3.8), 257 (7.5), 137 (17.4), 69 (100); HRMS calcd for C29
H
48
O
1.92-2.15 (m, 16H), 1.76 (s, 3H), 1.66 (s, 6H), 1.58 (s, 12H), 1.29 (s,
4
12.3693, found 412.3666.
11R,12S)-Cyclopropyl Ketone 11. A suspension of 913 mg (2.22
mmol) of a mixture of secondary alcohol diastereomers (10), 20 mL
of CH Cl , and 1 g of finely ground 4 Å molecular sieves was cooled
to 0 °C before 400 mg (3.42 mmol) of NMO and 40 mg (0.11 mmol,
.05 equiv) of TPAP were added. The mixture was warmed to room
3H); 13C NMR δ 138.6, 137.8, 135.5, 135.3, 131.4, 131.3, 124.3, 124.3,
(
124.2, 124.1, 123.9, 123.8, 73.4, 42.6, 40.0, 39.8, 28.4, 28.3, 26.8, 26.7,
26.5, 25.8, 23.0, 22.8, 17.8, 16.8, 16.1, 16.1; FTIR (film, cm-1) 3727
+
2
2
(br), 2917 (s), 2850 (s), 1653 (w); MS m/z 412 (M
, 4.0), 395 (81.7),
257 (27.4), 149 (48.7), 69 (100); HRMS calcd for C H O 412.3705,
29
48
0
found 412.3736.
10R,11R,12S)-Rillingol (ROH). A suspension of 482 mg (3.2
temperature and stirred for 3.5 h, poured over a plug of silica, and
eluted with ethyl acetate. The eluent was concentrated to give 870 mg
(
0
2
mmol) of Sm and 80 mg (0.30 mmol) of HgCl in 5 mL of THF was
2
3
1
(96%) of a colorless oil: [R]
D
-141.2° (c 5.2, EtOH); H NMR δ
stirred at room temperature for 15 min before 100 mg (0.25 mmol) of
alcohol 14 was added. The suspension was cooled to 0 °C, and 540
5
.03-5.14 (m, 4H), 4.62 (d, 1H, J ) 9 Hz), 2.54-2.61 (m, 2H), 2.29
(q, 2H, J ) 7 Hz), 1.92-2.12 (m, 13H), 1.87 (ddd, 1H, J ) 4, 5, 8
mg of CH
to room temperature, and stirred for an additional 5 h. Saturated K
CO solution (2 mL) and 10 mL of ether were added. The layers were
2 2
I was added. The mixture was stirred at 0 °C for 2 h, warmed
Hz), 1.70 (s, 3H), 1.68 (s, 6H), 1.59 (br s, 12H), 1.45 (ddd, 1H, J ) 4,
2
-
1
3
5
1
3
1
, 8 Hz), 0.89 (ddd, 1H, J ) 4, 6, 8 Hz); C NMR δ 209.3, 137.5,
36.2, 135.2, 131.4, 131.3, 124.9, 124.3, 124.2, 123.9, 122.7, 43.8,
9.7, 39.7, 39.4, 29.9, 26.8, 26.7, 26.4, 25.7, 24.9, 22.7, 18.4, 17.7,
3
separated, and the aqueous layer was washed three times with 8 mL of
ether. The combined organic layers were washed with brine, dried, and
concentrated. The residue was purified by flash chromatography (1:49
-
1
6.7, 16.0, 16.0; FTIR (film, cm ) 2918 (s), 2854 (s), 1695 (s); MS
+
m/z 410 (M , 1.6), 341 (3.4), 193 (8.0), 149 (8.7), 137 (14.5), 123
(v/v) ethyl acetate/hexanes) to give 61 mg of a mixture that contained
(
47.3), 81 (45.7), 69 (100); HRMS calcd for C29
10.3533.
10R/S,11R,12S)-Rillingol (ROH). A solution of 100 mg (0.24
mmol) of cyclopropyl ketone 11 in 4 mL of ether was cooled to -42
C before 400 µL (1.2 mmol, 5 equiv) of methylmagnesium bromide
3.0 M in diethyl ether) was added dropwise. The mixture was stirred
at -42 °C for 2 h before 1 mL of saturated NH Cl solution was added.
H46O 410.3537, found
starting material. Additional purification by HPLC (18:82 (v/v) MTBE/
4
23
hexanes) gave 29 mg (29%) of a colorless oil: [R]
D
-9.7° (c 1.2,
(
1
EtOH); H NMR (500 MHz) δ 5.06-5.18 (m, 4H), 4.61 (d, 1H, J )
9
1
Hz), 1.94-2.16 (m, 16H), 1.73 (s, 3H), 1.68 (s, 6H), 1.62 (s, 3H),
°
(
.60 (s, 6H), 1.59 (s, 3H), 1.41-1.48 (ddd, 1H, J ) 13, 9, 5 Hz), 1.16
13
(br s, 1H), 1.12 (s, 3H), 0.78-0.86 (m, 2H), 0.38-0.44 (m, 1H);
C
4
NMR δ 135.7, 135.2, 134.3, 131.7, 131.6, 127.8, 124.7, 124.6, 124.5,
The layers were separated, and the organic layer was washed with brine,
dried, and concentrated. The residue was purified by flash chromatog-
raphy (1:9 ethyl acetate/hexanes) to give 102 mg (99%) of a mixture
of diastereomers (6:1 ratio). The diastereomers were separated by HPLC
on silica using a linear gradient (1:5 to 1:1.5, tBME in hexanes).
1
1
24.4, 71.4, 43.4, 40.0, 39.8, 30.6, 27.0, 26.9, 26.8, 26.4, 26.0, 23.0,
8.0, 16.8, 16.3, 16.3, 13.9, 10.1; FTIR (film, cm ) 3474 (br), 2922
-1
(
(
s), 2854 (s); MS m/z 426 (1.2), 408 (2.4), 339 (4.3), 137 (25.7), 109
26.6), 69 (100); HRMS calcd for C30 50O 426.3849, found 426.3888.
H
23
Acknowledgment. This work was supported by NIH Grant
GM25521 from the National Institutes of Health.
The major diastereomer was isolated as a colorless oil: [R]
D
-8°
c 0.8, EtOH); H NMR (500 MHz) δ 5.06-5.17 (m, 4H), 4.61 (d,
H, J ) 9 Hz), 1.94-2.16 (m, 16H), 1.73 (s, 3H), 1.68 (s, 6H), 1.60
s, 9H), 1.59 (s, 3H), 1.50-1.57 (m, 1H), 1.17 (s, 3H), 1.04 (br s, 1H),
.81 (ddd, 1H, J ) 9, 6, 5 Hz), 0.74 (ddd, 1H, J ) 9, 4, 6 Hz), 0.38
1
(
1
(
0
Supporting Information Available: Three tables showing a
list of proteins used in multiple sequence alignments, pairwise
percent identities among squalene synthases, and pairwise
identities among phytoene synthases and a dehydrosqualene
synthase; four figures showing amino acid sequence alignments
for 19 squalene synthases, a phylogenetic analysis of squalene
synthases based on amino acid sequences, amino acid sequence
alignments for 26 phytoene synthases and a dehydrosqualene
synthase, and a phylogenetic analysis of phytoene synthases and
a dehydrosqualene synthase based on amino acid sequences
1
3
(ddd, 1H, J ) 9, 5, 4 Hz); C NMR δ 135.4, 135.0, 134.0, 131.5,
1
2
9
0
31.3, 127.6, 124.5, 124.4, 124.3, 124.2, 71.5, 43.1, 39.8, 39.6, 30.3,
7.1, 26.8, 26.7, 26.7, 25.8, 22.7, 17.7, 16.6, 16.1, 16.0, 16.0, 13.7,
.5; FTIR (film, cm ) 3474 (br), 2922 (s), 2854 (s); MS m/z 426 (M ,
-1
+
.3), 408 (1.3), 339 (1.4), 175 (4.7), 137 (16.4), 109 (17.9), 69 (100);
HRMS calcd for C30
H50O 426.3849, found 426.3879.
23
The minor diastereomer was a colorless oil, [R]
D
-9° (c 0.3, EtOH),
1
whose H NMR spectrum was identical to that of ROH isolated from
the incubation of FPP with SQase: H NMR (500 MHz) δ 5.06-5.18
1
(PDF). This material is available free of charge via the Internet
(m, 4H), 4.61 (d, 1H, J ) 9 Hz), 1.94-2.16 (m, 16H), 1.73 (s, 3H),
at http://pubs.acs.org.
1
1
.68 (s, 6H), 1.62 (s, 3H), 1.60 (s, 6H), 1.59 (s, 3H), 1.41-1.48 (ddd,
H, J ) 13, 9, 5 Hz), 1.16 (br s, 1H), 1.14 (s, 3H), 0.78-0.86 (m,
JA020411A
J. AM. CHEM. SOC.
9
VOL. 124, NO. 30, 2002 8853