in the unknown. These SMY8 cultures showed a distorted
triterpene product ratio relative to the RXY6 ratios (Table
2).15 The results illustrate the pitfalls of using in vivo
Scheme 2. Proposed Biosynthesis of Isomalabaricane
Triterpenoids via Tricycle 3
Table 2. Comparison of in Vivo and in Vitro Product Ratiosa
conditions
3
4
RXY6 (in vitro)
SMY8 (in vivo)
5
90
95
10
a Ratios from GC-FID analyses of crude products.
accumulation to estimate the product profile of a triterpene
synthase.16
We exploited the distorted product profile of SMY8
[SceErg7 Tyr510Phe] to obtain enough 3 for structure
elucidation. A 1-L culture gave after saponification and
partial purification a triterpene alcohol fraction containing
predominantly 3, an analytical sample of which was obtained
by HPLC.17 Compound 3 was identified by NMR spectros-
copy (1H, 13C, DEPT, COSYDEC, HSQC, HMBC, NOE)
as (13RH)-isomalabarica-14(27),17E,21-trien-3â-ol. Details
are given in the Supporting Information.
The novel tricycle 3 is the parent skeleton of isomala-
baricanes, found in nature only in certain Asian sponges.5
These secondary metabolites are tricyclic triterpenoids that
display the distinctive trans-syn-trans ring fusion found in
lanosterol biosynthesis. Sponges synthesize sterols from
lanosterol,18 and the enzyme that constructs the isomalabari-
catrienol skeleton probably evolved from a lanosterol syn-
thase. Sponges could cyclize oxidosqualene to 3 and produce
the isomalabaricane triterpenoids by additional desaturation
and specific oxidation (Scheme 2).19 These sessile marine
animals produce numerous secondary metabolites for a
variety of purposes.20
mutants, nearly all have a trans-anti-trans stereochemistry.
The sole characterized oxidosqualene cyclase that forms
tricycles is thalianol synthase,13 a plant enzyme that generates
a 6-6-5 malabaricatrienol from a trans-anti-trans all-chair
cation. Interestingly, thalianol synthase shares close phylo-
genetic affinity to plant â-amyrin and lupeol synthases which
generate tetracyclic all-chair intermediates. These observa-
tions are consistent with the hypothesis that cyclases rarely
evolve from a B-ring chair to B-ring boat mechanism or vice
versa.21
Some reported cyclase mutants generate 6-6-5 trans-
anti-trans tricycles.22 Malabarica-14(27),17E,21-trien-3â-ol,
a trans-anti-trans isomer of 3, has been produced from 2,3
oxidosqualene by a squalene-hopene cyclase mutant22b and
nonenzymatically under mildly acidic23 or free-radical24
conditions. Other biomimetic reactions also produce 6-6-5
tricycles, all with the trans-anti-trans stereochemistry.25 The
only reported cyclizations to isomalabaricane skeletons
involve reaction of oxidosqualene substrate analogues with
lanosterol synthase.26 However, none of these reactions
produce the novel isomalabaricatrienol 3.
Among the many 6-6-5 tricycles isolated from nature
or from experiments with substrate analogues and cyclase
The product profiles described herein provide some insight
into the cyclization mechanism, although interpretation of
the results is necessarily speculative in the absence of
(15) In vivo SMY8 product ratios varied from 80:20 to 90:10. This
variablity is unsurprising because time of harvest, amount of ergosterol in
the medium, degree of aeration, and other culture conditions could affect
the extent of lanosterol metabolism. By contrast, in vitro RXY6 product
ratios are not affected by such conditions and are reproducible.
(16) This concern applies chiefly to cyclases that generate lanosterol,
parkeol, cycloartenol, and other products that are metabolized by yeast.
(17) SMY8[SceErg7 Tyr510Phe] was cultured in inducing medium
(YPGH, 1 L). Saponification of the cell pellet (10% KOH in 80% EtOH)
followed by hexanes extraction furnished the nonsaponifiable lipids (NSL).
Removal of ergosterol by silica gel column chromatography gave a triterpene
alcohol fraction containing mainly 3, with some lanosterol, 4,4-dimethyl-
cholest-8-en-3â-ol (T-MAS), traces of sterol intermediates and possibly
unidentified triterpenes.
(21) Xiong, Q.; Rocco, F.; Wilson, W. K.; Xu, R.; Ceruti, M.; Matsuda,
S. P. T. J. Org. Chem. 2005, 70, 5362-5375.
(22) (a) Hoshino, T.; Sato, T. Chem. Commun. 2002, 291-301. (b)
Hoshino, T.; Shimizu, K.; Sato, T. Angew. Chem., Int. Ed. 2004, 43, 6700-
6703.
(23) van Tamelen, E. E.; Willet, J.; Schwartz, M.; Nadeau, R. J. Am.
Chem. Soc. 1966, 88, 5937-5938.
(24) Justicia, J.; Rosales, A.; Bun˜uel, E.; Oller-Lo´pez, J. L.; Valdivia,
M.; Ha¨ıdour, A.; Oltra, J. E.; Barrero, A. F.; Ca´rdenas, D. J.; Cuerva, J. M.
Chem. Eur. J. 2004, 10, 1778-1788.
(25) Bartlett, P. A. In Asymmetric Synthesis; Morrison, J. D., Ed.;
Academic Press: New York, 1984; Vol. 3, pp 341-409. The trans-syn-
trans stereochemistry has been detected in minor products of biomimetic
reactions: Nishizawa, M.; Yadav, A.; Iwamoto, Y.; Imagawa, H. Tetra-
hedron 2004, 60, 9223-9234 and ref therein; however these reactions did
not generate 6-6-5 tricycles. A trans-syn-cis tetracycle was made
biomimetically with a specially designed substrate: Corey, E. J.; Wood,
H. B., Jr. J. Am. Chem. Soc. 1996, 118, 11982-11983.
(26) (a) Van Tamelen, E. E.; Sharpless, K. B.; Hanzlik, R.; Clayton, R.
B.; Burlingame, A. L.; Wszolek, P. C. J. Am. Chem. Soc. 1967, 89, 7150-
7151. (b) Krief, A.; Schauder, J. R.; Guittet, E.; Herve du Penhoat, C.;
Lallemandt, J. Y. J. Am. Chem. Soc. 1987, 109, 7910-7911. (c) Guittet,
E.; Herve du Penhoat, C.; Lallemand, J. Y.; Schauder, J. R.; Krief, A. J.
Am. Chem. Soc. 1987, 109, 7911-7913. (d) Krief, A.; Pasau, P.; Guittet,
E.; Shan, Y. Y.; Herin, M. Bioorg. Med. Chem. Lett. 1993, 3, 365-368.
(18) (a) Kerr, R. G.; Stolilov, I. L.; Thompson, J. E.; Djerassi, C.
Tetrahedron 1989, 45, 1893-1904. (b) Silva, C. J.; Wu¨nsche, L.; Djerassi,
C. Comp. Biochem. Physiol. 1991, 99B, 763-773.
(19) The ∆13(14) isomer of 3 could alternatively be a precursor of
isomalabaricanes: Xu, R.; Fazio, G. C.; Matsuda, S. P. T. Phytochemistry
2004, 65, 261-290. However, 3 should be easier to generate enzymatically
since a C14 methyl proton is more readily abstracted than the more hindered
C13 methine proton. Also, abstraction of the C13 proton would probably
require rotation of the side chain to align the cationic 2p orbital with the
C-H bond; see discussion of horizontal and vertical cations in: Matsuda,
S. P. T.; Wilson, W. K.; Xiong, Q. Org. Biomol. Chem. 2006, in press,
DOI: 10.1039.
(20) (a) Keyzers, R. A.; Davies-Coleman, M. T. Chem. Soc. ReV. 2005,
34, 355-365. (b) Kubanek, J.; Whalen, K. E.; Engel, S.; Kelly, S. R.;
Henkel, T. P.; Fenical, W.; Pawlik, J. R. Oecologia 2002, 131, 125-136.
Org. Lett., Vol. 8, No. 3, 2006
441