Stereochemical course in water addition during LUP1-catalyzed triterpene cyclization

Article abstract of DOI:10.1021/ol062310d

Arabidopsis thaliana LUP1 (At1g78970) catalyzes the cyclization of oxidosqualene into lupeol and 3β,20-dihydroxylupane (lupanediol). The stereochemical course of water addition to the lupanyl cation was studied. The X-ray crystal structure of lupanylepoxi

Full text of DOI:10.1021/ol062310d

ORGANIC
LETTERS
2006
Vol. 8, No. 24
5589-5592
Stereochemical Course in Water
Addition during LUP1-Catalyzed
Triterpene Cyclization
Tetsuo Kushiro,† Masaki Hoshino,† Takehiko Tsutsumi,† Ken-ichi Kawai,‡
Motoo Shiro,§ Masaaki Shibuya,† and Yutaka Ebizuka*,†
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan, Faculty of Pharmaceutical Sciences,
Hoshi UniVersity, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, and Rigaku
Corporation, 3-9-12 Matsubaracho, Akishima, Tokyo 196-8666, Japan
yebiz@mol.f.u-tokyo.ac.jp
Received September 20, 2006
ABSTRACT
Arabidopsis thaliana LUP1 (At1g78970) catalyzes the cyclization of oxidosqualene into lupeol and 3
â
,20-dihydroxylupane (lupanediol). The
stereochemical course of water addition to the lupanyl cation was studied. The X-ray crystal structure of lupanylepoxide 3,5-dinitrobenzoate
established the configuration of epoxide as 20S. LiAlD4 reduction of the epoxide enabled the chemical shift assignment of prochiral methyl
groups at C20 of lupanediol. Correlation of these methyl groups with biosynthetic lupanediol from [1,2-13C2] acetate established the stereochemical
course of water addition.
The enzymatic cyclization of 2,3-oxidosqualene into various
cyclic triterpenes is one of the most fascinating and versatile
chemical transformations found in nature. The responsible
enzyme, oxidosqualene cyclase (OSC), catalyzes the forma-
tion of a multiring system with a number of stereogenic
centers through a series of cation-π cyclizations with
concomitant Wagner-Meerwein rearrangements including
1,2-methyl and hydride shifts in a single step, only part of
which synthetic organic chemistry can mimic.1 OSCs are
widely distributed in nature and play important roles.
Lanosterol synthase in mammals is a typical example, as it
serves as a key enzyme in cholesterol biosynthesis. In the
plant kingdom, nearly 100 of the unique triterpene skeletons
are formed by OSCs. Their further metabolites, comprising
one of the most diverse groups of natural products, exhibit
a range of pharmacological activities.2 Diverse skeletons of
triterpenes suggested the presence of the corresponding
number of OSC genes in plants. However, recent molecular
cloning studies have revealed that many of the plant OSCs
are multifunctional to produce more than one skeletal type
of triterpene product.3,4b,5 Both chimeric and site-directed
mutagenesis studies on OSCs have demonstrated that only
(2) (a) Xu, R.; Fazio, G. C.; Matsuda, S. P. T. Phytochemistry 2004, 65,
261-291. (b) Mahato, S. B.; Sarkar, S. K.; Poddar, G. Phytochemistry 1988,
27, 3037-3067. (c) Dzubak, P.; Hajduch, M.; Vydra, D.; Hustova, A.;
Kvasnica, M.; Biedermann, D.; Markova, L.; Urban, M.; Sarek, J. Nat. Prod.
Rep. 2006, 23, 394-411.
(3) (a) Morita, M.; Shibuya, M.; Kushiro, T.; Masuda, K.; Ebizuka, Y.
Eur. J. Biochem. 2000, 267, 3453-3460. (b) Kushiro, T.; Shibuya, M.;
Masuda, K.; Ebizuka, Y. Tetrahedron Lett. 2000, 41, 7705-7710. (c)
Husselstein-Muller, T.; Schaller, H.; Benveniste, P. Plant Mol. Biol. 2001,
45, 75-92. (d) Kawano, N.; Ichinose, K.; Ebizuka, Y. Biol. Pharm. Bull.
2002, 25, 477-482. (e) Iturbe-Ormaetxe, I.; Haralampidis, K.; Papadopou-
lou, K.; Osbourn, A. E. Plant Mol. Biol. 2003, 51, 731-743.
† The University of Tokyo.
‡ Hoshi University.
§ Rigaku Corporation.
(1) (a) Abe, I.; Rohmer, M.; Prestwich, G. D. Chem. ReV. 1993, 93,
2189-2206. (b) Yoder, R. A.; Johnston, J. N. Chem. ReV. 2005, 105, 4730-
4756.
10.1021/ol062310d CCC: $33.50
© 2006 American Chemical Society
Published on Web 10/28/2006

Figure 1. Cyclization of 2,3-oxidosqualene into lupeol and lupanediol by LUP1 and the fate of a carbon atom derived from C2 of mevalonate.
Dots indicate the carbons that originate from C2 of mevalonate. LUP1 catalyzes sequences shown with bold arrows.
a limited number of amino acid residues are responsible for
controlling their product specificity.6,7 The major factors
which determine the product outcome of cyclization are the
folding conformation of the substrate and the position and
mechanism of the termination. Generally, OSCs terminate
the cyclization by elimination of a proton from a cationic
intermediate to yield a product with a double bond. Some
enzymes, however, terminate their reactions by quenching
the final carbocation with a water molecule to release an
alcohol product. The recently reported arabidiol synthase
from Arabidopsis thaliana8 and dammarenediol-II synthase
from Panax ginseng9 are such examples, where water
addition to tri- and tetracyclic cation intermediates terminates
their reactions, respectively. In this regard, LUP1 (At1g78970)
from A. thaliana, one of the first triterpene synthases to be
identified for their enzyme functions, offers a unique
opportunity to explore the mechanism that controls these
processes, as it produces not only lupeol, a deprotonation
product, but also 3â,20-dihydroxylupane (lupanediol), a
water addition product, in nearly equal amount.4 We have
been interested in how the final deprotonation or hydration
of lupanyl cation is controlled by the LUP1 enzyme. To
address this interesting and important issue, we took advan-
tage of the fact that two terminal methyl groups of the lupanyl
cation have different biosynthetic origins, that is either from
C2 or C6 of mevalonate, according to the conventional
mevalonate pathway for isoprenoid biosynthesis (Figure 1).
These two carbon atoms can be easily distinguished by
feeding [1,2-13C2] acetate to the yeast transformant expressing
LUP1 and 13C NMR measurements of the products, as a
methyl group derived from C6 of mevalonate resonates with
an accompanying doublet due to intact incorporation of
acetate whereas a methyl group from C2 of mevalonate
resonates as an enriched singlet. In our previous studies,
LUP1 was demonstrated to eliminate a proton from both
terminal methyl groups in nearly equal ratio, indicating a
lack of regiospecificity in the deprotonation process.6 In the
same feeding experiment, LUP1 was shown to produce
lupanediol with stereospecific addition of water to the lupanyl
cation giving rise to either (A) or (B) in Figure 1.5 Here, we
report the correlation of magnetically and biosynthetically
distinguishable prochiral methyl groups of lupanediol that
establishes the stereochemical course of water addition during
the final termination step of cyclization catalyzed by LUP1.
LUP1 was functionally expressed in Saccharomyces
cereVisiae GIL77, a yeast mutant strain lacking lanosterol
synthase.10 As previously reported, feeding of [1,2-13C2]
acetate to the yeast culture expressing LUP1 yielded 13C-
labeled lupanediol.5 In the 13C NMR spectrum (in CDCl3)
of this specimen (Figure 2A), a methyl signal of natural
abundance resonating at 31.53 ppm was accompanied by a
doublet of J ) 39.6 Hz, and a methyl group resonating at
24.76 ppm appeared as an enriched singlet.5 Accordingly, a
quaternary C20 signal at 73.52 ppm was accompanied by a
doublet of J ) 39.6 Hz.5 To assign the chemical shifts of
the relevant prochiral methyl groups of lupanediol, we relied
on the previous report that m-chloroperbenzoic acid (m-
CPBA) oxidation of natural lupanyl acetate gave only one
enantiomer of the C20-C29 epoxide, although the stereo-
chemistry has not been established yet.11 The stereochemistry
of the epoxide was determined by X-ray crystallographic
analysis of lupanylepoxide 3,5-dinitrobenzoate (3) prepared
(4) (a) Herreira, J. B. R.; Bartel, B.; Wilson, W. K.; Matsuda, S. P. T.
Phytochemistry 1998, 49, 1905-1911. (b) Segura, M. J. R.; Meyer, M.
M.; Matsuda, S. P. T. Org. Lett. 2000, 2, 2257-2259.
(5) Ebizuka, Y.; Katsube, Y.; Tsutsumi, T.; Kushiro, T.; Shibuya, M.
Pure Appl. Chem. 2003, 75, 369-374.
(6) Kushiro, T.; Shibuya, M.; Ebizuka, Y. J. Am. Chem. Soc. 1999, 121,
1208-1216.
(7) Kushiro, T.; Shibuya, M.; Masuda, K.; Ebizuka, Y. J. Am. Chem.
Soc. 2000, 122, 6816-6824.
(8) Xiang, T.; Shibuya, M.; Katsube, Y.; Tsutsumi, T.; Otsuka, M.;
Zhang, H.; Masuda, K.; Ebizuka, Y. Org. Lett. 2006, 8, 2835-2838.
(9) Tansakul, P.; Shibuya, M.; Kushiro, T.; Ebizuka, Y. FEBS Lett. 2006,
580, 5143-5149.
(10) Kushiro, T.; Shibuya, M.; Ebizuka, Y. Eur. J. Biochem. 1998, 256,
238-244.
(11) (a) Corbett, R. E.; Cong, A. N. T.; Wilkins, A. L.; Thomson, R. A.
J. Chem. Soc., Perkin Trans. 1 1985, 2051-2056. (b) Hui, W. H.; Li, M.
M. Phytochemistry 1977, 16, 111-112.
5590
Org. Lett., Vol. 8, No. 24, 2006

Figure 2. Partial 13C NMR spectra of lupanediol from (A) feeding [1,2-13C2] acetate to LUP1 expressing yeast culture, (B) deuterium
labeled by LiAlD4 reduction of (3), and (C) an authentic nonlabeled sample. Asterisk indicates the signal of two terminal methyl groups
of lupanediol.
from lupenyl 3,5-dinitrobenzoate. The colorless plates suit-
able for X-ray analysis (monoclinic, P21, a ) 7.942 (5), b
) 10.334 (6), c ) 19.76 (1) Å, â ) 92.16 (5)°, Z ) 2)
were prepared by crystallization from acetone.12 The structure
was solved by direct methods using SIR9713 with an R (Rw)
value of 0.069 (0.112) and established as shown in Figure
3. The bond lengths and angles are not significantly different
group resonating at a higher field (1H, 1.121 ppm; 13C: 24.76
1
ppm) was confirmed by H NMR, 13C NMR, and HMQC
analysis. A deuterium shift (0.3 ppm) and direct 13C-2H
coupling (1JC-D, J ) 19 Hz) were observed on the 13C NMR
spectrum (Figure 2B). Therefore, it is now firmly established
that the methyl group resonating at a higher field (24.76 ppm)
is pro-R, whereas the one in the lower field (31.53 ppm) is
pro-S. By comparing this spectrum with that of lupanediol
produced from [1,2-13C2] acetate by LUP1 expressing yeast,
it is now clear that the pro-R methyl group is derived from
C2 of mevalonate, and the pro-S methyl group is derived
from C6 of mevalonate (Figure 2).
Taken together, these results indicated that water addition
to the lupanyl cation takes place stereospecifically from only
one face of the plane of the isopropyl cation moiety during
lupanediol formation as shown in Figure 4 to produce species
Figure 3. X-ray crystal structure of lupanylepoxide 3,5-dinitro-
benzoate (3) (ORTEP drawing).
from the expected ones.14 This structure confirmed that the
configuration of the epoxide is 20S, as the absolute stereo-
chemistry at C3 is retained from (3S)-2,3-oxidosqualene.
LiAlD4 reduction of this epoxide gave deuterated lu-
panediol. The incorporation of deuterium into the methyl
Figure 4. (a) Stereochemical course of final water addition to
lupanyl cation. (b) Nonspecific deprotonation during lupeol forma-
tion. Carbons originating from C2 of mevalonate are shown with
dots.
(12) Diffraction intensities were collected from a crystal of dimensions
0.25 × 0.15 × 0.03 mm on a Rigaku RAXIS RAPID imaging plate area
detector with graphite monochromated Mo KR radiation. A total of 19 530
reflections, 4983 unique reflections, within a 2θ range of 60.1° were used
in the solution and refinement of the structure. The data were collected for
Lorenz and polarization effects.
(13) Altomare, A.; Buria, M.; Camalli, M.; Cascarano, G.; Giacovazzo,
C.; Guagliardi, A.; Moliterni, A.; Polidori, G.; Spagna, R. J. Appl. Cryst.
1999, 32, 115-119.
(14) Lists of atomic parameters, bond lengths, and bond angles will be
deposited to the Cambridge Crystallographic Data Centre.
(A) in Figure 1. Simultaneous production of the water
addition product and the deprotonation product by LUP1
implies that a water molecule in the active site may be
quenching the cation to produce lupanediol and at the same
time serving as a base to eliminate a proton to yield lupeol,
although participation of any basic residue of the enzyme
Org. Lett., Vol. 8, No. 24, 2006
5591

during the deprotonation step is equally possible. Detailed
kinetic studies on the formation of both lupeol and lupanediol
are necessary to clarify the detailed mechanism regarding
proton abstraction by the water molecule. Lack of depro-
tonation specificity during lupeol formation6 might suggest
a possible rotation of the isopropyl moiety at the nascent
lupanyl cation intermediate. However, the observed precise
control of the direction of water access with regard to the
face of the isopropyl moiety of the lupanyl cation excludes
the rotation of the C19-C20 bond before water addition.
Nonspecific deprotonation could be achieved by a water
molecule approaching both terminal methyl groups from one
face of the isopropyl moiety. Therefore, LUP1 seems to
control the position of the water molecule with respect to
the plane of the isopropyl cation moiety but not accurately
enough to let it eliminate a proton from only one methyl
group. This was in striking contrast to monofunctional lupeol
synthases of OEW from olive (Olea europaea) and TRW
from dandelion (Taraxacum officinale), as they eliminate a
proton specifically from the methyl group derived from C6
of mevalonate.15 These lupeol synthases are monofunction-
ally producing lupeol as a sole product and no lupanediol or
other triterpenes. These high-fidelity enzymes might have
evolved to maintain control of deprotonation specificity so
as not to produce other byproducts. On the other hand, low-
fidelity multiproduct LUP1 might have a different evolution-
ary path and has lost control in the deprotonation process.
In fact, high-fidelity lupeol synthases of the OEW and TRW
type form a branch in a phylogenetic tree of plant OSCs,16
whereas LUP1 does not join this branch and situates more
closely to a branch of â-amyrin synthases. One might
speculate that LUP1 has evolved from â-amyrin synthase
and has lost an ability to control the reaction path after the
lupanyl cation stage, which accounts for the observed
multiproduct nature to produce multiple lupane triterpenes.
Production of both the deprotonated olefinic product and
the hydrated alcohol product by LUP1 is reminiscent of
squalene hopene cyclase (SHC) from bacteria, which pro-
duces hopene and hopanol in a 5:1 ratio.17 It has been shown
that in a SHC-catalyzed reaction deprotonation takes place,
in contrast to a LUP1 reaction, specifically from the (Z)-
methyl group of the terminal isopropenyl moiety during
hopene formation.18 Water addition to the hopanyl cation is
also stereospecific during hopanol formation,18 although the
stereochemistry of this water addition has not been estab-
lished yet. The present study demonstrated, for the first time,
the stereochemical course of the termination of triterpene
cyclization with regard to water addition to produce prochiral
methyl groups. These results provided a valuable insight into
the active site structure of OSCs. In combination with future
structural studies, we would be able to draw a precise picture
of this fascinating series of enzymatic cation-π cyclization
reactions and the observed product diversity.
Acknowledgment. We thank Dr. K. Masuda of Showa
Pharmaceutical University for the generous gift of lupenyl
acetate and Dr. Y. Fujimoto of Tokyo Institute of Technology
for helpful discussions. A part of this research was financially
supported by a Grant-in-Aid for Scientific Research (S)
(No.15101007) to Y.E. from Japan Society for the Promotion
of Science, Japan.
Supporting Information Available: Details of the syn-
thetic procedure, 1H and 13C NMR data, and 1H and 13C NMR
spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL062310D
(15) Kushiro, T.; Shibuya, M.; Ebizuka, Y. Tetrahedron Lett. 1999, 40,
5553-5556.
(16) Shibuya, M.; Zhang, H.; Endo, A.; Shishikura, K.; Kushiro, T.;
Ebizuka, Y. Eur. J. Biochem. 1999, 266, 302-307.
(17) Pale-Grosdemange, C.; Feil, C.; Rohmer, M.; Poralla, K. Angew.
Chem., Int. Ed. 1998, 37, 2237-2240.
(18) Hoshino, T.; Nakano, S.; Kondo, T.; Sato, T.; Miyoshi, A. Org.
Biomol. Chem. 2004, 2, 1456-1470.
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