Efficient generation of a trisporoid library by combination of synthesis and biotransformation

Article abstract of DOI:10.1021/jo062236c

(Chemical Equation Presented) Trisporic acids and their biosynthetic precursors represent a family of powerful fungal pheromones and morphogenetic factors. A highly flexible synthetic protocol is described that (i) provides rapid access to nonfunctionaliz

Full text of DOI:10.1021/jo062236c

Efficient Generation of a Trisporoid Library by Combination of
Synthesis and Biotransformation
Doreen Schachtschabel and Wilhelm Boland*
Department of Bioorganic Chemistry, Max-Planck-Institute for Chemical Ecology, Hans-Kno¨ll-Strasse 8,
D-07745 Jena, Germany
boland@ice.mpg.de
ReceiVed October 27, 2006
Trisporic acids and their biosynthetic precursors represent a family of powerful fungal pheromones and
morphogenetic factors. A highly flexible synthetic protocol is described that (i) provides rapid access to
nonfunctionalized early trisporoids from â-ionone, (ii) includes a regiospecific oxidative functionalization
of â-ionone leading to 1-acetoxy-â-ionone giving access to functionalized trisporoids, and (iii) utilizes
a biotransformation of early synthetic trisporoids by growing cells of Blakeslea trispora to prepare late
trisporoids including trisporic acids. The same protocol also provides deuterium-labeled trisporoids such
as trisporin B [2H3]-19. Administration of [2H3]-19 to growing cells of the (-)-mating type of B. trispora
resulted in the formation of the labeled trisporols [2H3]-20 and [2H3]-21. Growing cultures containing
both mating types can be used to prepare trisporic acids from early precursors.
Introduction
Scheme 1, both mating types are at least competent to produce
4-dihydrotrisporin (2) but lack the ability to generate the
downstream compounds.7
Instead, the biosynthesis of the late trisporoids was assumed
to require a diffusive exchange of “prohormones” between the
mating partners and results in a “cooperative biosynthesis”8,9
that offers additional regulatory possibilities and molecular
diversification at the level of metabolites.
In fact, the currently known group of trisporoids is structurally
and functionally highly diverse but the specific role of the
individual compounds in the sexual development and function
of the different Zygomycetes is largely unknown. Trisporoids
were grouped within A, B, C, D, and E series according to their
substitution pattern at R4, R5, R6, and R7 and according to further
oxidative functionalization of the ring system (R1, R2, R3) in
trisporols, trisporic acids, (4-dihydro)-methyltrisporates, and (4-
dihydro)-trisporins (Scheme 2).2,7,8,10-12
Carotenoid-derived compounds (Scheme 1) regulate the first
stages of sexual development and stimulate the production of
zygophores in the fungal phylum Zygomycota, for example, in
Blakeslea trispora,1 Phygomyces blakesleeanus,2 Zygorhynchus
moelleri,3 and Mucor mucedo.1,4 Sexual reactions of these fungi
are controlled by a family of pheromones, the trisporic acids,
resulting from oxidative degradation of â-carotene. The com-
pounds are involved in recognition of mating partners, induce
the first steps of sexual differentiation, maintain the development
of sexual structures, and even mediate the recognition between
Zygomycetes and their parasites (e.g., Parasitella parasitica).5
Pioneering analytical work with Blakeslea trispora and Mucor
mucedo revealed a chemical dialogue between the sexual
partners that required an exchange of early trisporoids as
precursors for the bioactive trisporic acids. As outlined in
(6) van den Ende, H. Sexual Interactions in Plants (Experimental Botany);
Academic Press: New York, 1976; Vol. 9, p 64-71.
(7) Bulock, J. D.; Jones, B. E.; Winskill, N. Pure Appl. Chem. 1976, 47,
191-202.
* To whom correspondence should be addressed. Phone: (+49) 3641 571200.
Fax: (+49) 3641 571202.
(1) Austin, D. J.; Bulock, J. D.; Gooday, G. W. Nature 1969, 223, 1178-
1179.
(2) Miller, M. L.; Sutter, R. P. J. Biol. Chem. 1984, 259, 6420-6422.
(3) Schipper, M. A. Trans. Br. Mycol. Soc. 1971, 56, 157-159.
(4) Gooday, G. W. Phytochemistry 1968, 7, 2103-2105.
(5) Schultze, K.; Schimek, C.; Wostemeyer, J.; Burmester, A. Gene 2005,
348, 33-44.
(8) Nieuwenhuis, M.; van den Ende, H. Arch. Microbiol. 1975, 102, 167-
169.
(9) Werkman, B. A. Arch. Microbiol. 1976, 109, 209-213.
(10) Bulock, J. D.; Austin, D. J.; Snatzke, G.; Hruban, L. J. Chem. Soc.
D, Chem. Commun. 1970, 255-256.
10.1021/jo062236c CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/23/2007
1366
J. Org. Chem. 2007, 72, 1366-1372

Efficient Generation of a Trisporoid Library
and C15 of the most important A, B, and C series.7,10,14
However, the very low concentrations of the hormones ham-
pered their direct structural characterization and made the
chemical synthesis imperative for physiological studies. Several
routes to the late trisporoids such as methyl trisporates,
trisporols, and free trisporic acids have been developed and
reported.7,15-23 The disadvantage of previous routes is the large
number of steps required for the synthesis of the final targets
and the lack of flexibility to produce early and late trisporoids
along the same protocol using common intermediates. Espe-
cially, the lack of efficient routes to trisporoids with position-
specific isotopic labeling hampered systematic in vivo studies
and kinetic analyses from the early trisporoids to the late
bioactive signals in the competent mating types.
SCHEME 1. Postulated Biosynthetic Pathway for Trisporic
Acid Series B6
Here, we present a novel and highly flexible concept that is
based on three key elements: (i) rapid access to the nonfunc-
tionalized C18-apocarotenoid skeleton from commercial â-ion-
one, (ii) regiospecific oxidative functionalization of the cyclo-
hexene moiety of the apocarotenoids at C4, and (iii) biotransfor-
mation of early synthetic trisporoids by growing cultures of the
zygomycete Blakeslea trispora. The approach has no need for
complex functionalized or chiral precursors because the growing
cells of the fungus are expected to oxidize the correct methyl
group at C1 of the cyclohexene. The method also provides a
rapid access to deuterium-labeled compounds as valuable tools
for the analysis of trisporic acid biosynthesis in mucoraceous
fungi.
Results
Synthesis of Early Trisporoids. The concept for the
synthesis of trisporoids was designed to provide the different
groups of trisporoids shown in Scheme 1, namely, the non-
functionalized early trisporoids (e.g., the â-C18-ketone), the
group of C4- and/or C15-modified intermediates, and finally
the group of late trisporoids that are functionalized at the methyl
group at C1. A particular simple and highly flexible access to
the apocarotenoid C18-skeleton is possible from the vinyl
bromide 9 that allows the completion of differently function-
alized side chains in a single operation with limited or no
protecting group strategies (Scheme 3 and Scheme 4). Moreover,
â-ionone (8) is an ideal target for an isotope exchange reaction
yielding [2H3]-â-ionone (18)24 which can be used for the
synthesis of labeled trisporoids25,26 along the same protocol.
The central building block 9 (E/Z ) 1:1) is obtained by Wittig
olefination of â-ionone (8) with bromomethylidenetriphenyl-
SCHEME 2. Functionalization Patterns of Trisporoidsa
(14) Reschke, T. Tetrahedron Lett. 1969, 3435-3439.
(15) Edwards, J. A.; Schwarz, V.; Fajkos, J.; Maddox, M. L.; Fried, J.
H. J. Chem. Soc. D, Chem. Commun. 1971, 292-293.
(16) Isoe, S.; Hayase, Y.; Sakan, T. Tetrahedron Lett. 1971, 3691-3694.
(17) Secrist, J. A.; Hickey, C. J.; Norris, R. E. J. Org. Chem. 1977, 42,
525-527.
(18) Prisbylla, M. P.; Takabe, K.; White, J. D. J. Am. Chem. Soc. 1979,
101, 762-763.
a Characters A-E represent the different series.
(19) Trost, B. M.; Ornstein, P. L. Tetrahedron Lett. 1983, 24, 2833-
2836.
Until now, systematic studies correlating structural features
with biological activities have been missing and only certain
aspects have been investigated.13 Besides the configurational
isomers, natural trisporoids possess chiral centers at C1, C4,
(20) Takabe, K.; White, J. D. Tetrahedron Lett. 1983, 24, 3709-3712.
(21) White, J. D.; Takabe, K.; Prisbylla, M. P. J. Org. Chem. 1985, 50,
5233-5244.
(22) Takahashi, S.; Oritani, T.; Yamashita, K. Agric. Biol. Chem. 1987,
51, 2291-2293.
(23) Reeder, M. R.; Meyers, A. I. Tetrahedron Lett. 1999, 40, 3115-
(11) Sutter, R. P. Science 1970, 168, 1590-1592.
(12) Sutter, R. P.; Dadok, J.; Bothnerby, A. A.; Smith, R. R.; Mishra, P.
K. Biochemistry 1989, 28, 4060-4066.
(13) Schachtschabel, D.; Schimek, C.; Wostemeyer, J.; Boland, W.
Phytochemistry 2005, 66, 1358-1365.
3118.
(24) Bergen, H. R.; Furr, H. C.; Olson, J. A. J. Labelled Compd.
Radiopharm. 1988, 25, 11-21.
(25) Ramamurthy, V.; Liu, R. S. H. Tetrahedron 1975, 31, 201-206.
(26) Camps, J.; Font, J.; Demarch, P. Tetrahedron 1981, 37, 2493-2499.
J. Org. Chem, Vol. 72, No. 4, 2007 1367

Schachtschabel and Boland
SCHEME 3. Synthesis of the â-C18-Ketone (1)a
14 and 16 or to produce trisporoids with aliphatic side chains
of different length for structure-function studies.13 For trisporin
A (13) and 15, an almost quantitative conversion (99% GC-
MS) was observed. The trisporoids 14 and 16 resulted from
alkylation of 11 or 12 with an organozincate derived from (4-
iodobutan-2-yloxy)(tert-butyl)dimethylsilane. The latter is ob-
tained from methyl-3-hydroxymethylbutanoate by protection
with tert-butyldimethylsilylchloride,40 reduction of the methyl
ester moiety with DIBALH,41 and iodination of the resulting
alcohol with I2/PPh3 to (4-iodobutan-2-yloxy)(tert-butyl)di-
methylsilane.42 The sequence has the advantage that the race-
mate and both enantiomers of methyl-3-hydroxybutanoate are
available, thus allowing also the synthesis of both enantiomers
of 14 and 16. In all alkylation reactions, the original ratio of
the E/Z-isomers (ca. 1:1) of 11 and 12 was nearly maintained
in the products.
a Reagents and conditions: (a) (BrCH2PPh3)+Br-, LiHMDS, THF,
-78 °C for 1 h, room temperature for 12 h; (b) Pd(Ac)2, PPh3, Et3N, THF,
60 °C, 120 h.
Deuterium-labeled trisporoids were obtained from â-ionone
(8) or 1-acetoxy-â-ionone 10 as outlined in Scheme 5. Exchange
of the acidic protons in â-ionone (8) by deuterium atoms was
achieved in CH3OD in the presence of N-ethyl-diisopropylamine
phosphorane using LiHMDS as a soft base for deprotonation
of the phosphonium salt. The vinyl bromide 9 is an excellent
component for transition-metal-catalyzed alkylations, and in the
case of the â-C18-ketone (1), this is easily achieved by a Heck-
type alkylation of 9 with but-3-en-2-one.27 The corresponding
iodide proved to be too unstable and decomposed during
workup.
2
and resulted in [2H3]-17 or [2H3]-18 (>97% H per position
according to NMR).
The subsequent Wittig reaction with bromomethylidenetri-
phenylphosphorane and the final alkylation with but-3-en-2-
one under Heck conditions generated [2H3]-19 and proceeded
without exchange of deuterium atoms.43 Owing to the flexibility
of the synthetic protocol, almost all early and late (vide infra)
trisporoids can be prepared in a few steps and high overall
yields.
The alkylation sequence could be successfully extended to
the acetoxy-â-ionone 10 which is readily available from
â-ionone (8) by allylic oxidation with benzoquinone and Pd-
(O2CCF3)2/o-methoxyacetophenone.28 Alternative methods proved
to be less satisfactory.29-33 The key intermediate 11 is obtained
by Wittig olefination in 82% yield as described for the synthesis
of 9 followed by deacetylation with methanolic KOH.34
The third building block, namely, the ketone 12, was obtained
Biotransformation of Early Trisporoids by Growing
Cultures of Blakeslea trispora. The last important aspect of
the envisaged uniform concept from early to late trisporoids
was the successful administration and transformation of the
synthetic precursors by growing cells of Blakeslea trispora. In
a representative feeding experiment, [2H3]-trisporin B (20) was
added to growing cells of the (-)-mating type of B. trispora
precultivated on a SUP medium for 3 days (Scheme 6).44 After
48 h, the mycelia was removed and the culture broth was
adjusted to pH 8. The produced trisporols [2H3]-21 and [2H3]-
22 were extracted with chloroform/isopropanol (10% yield,
equal amounts of [2H3]-21 and [2H3]-22). The presence of the
deuterium atoms corroborated the origin of the [2H3]-trisporols
from 20 and confirmed the feasibility of the synthetic concept.
The formation of 21 and 22 by cells representing the (-)-
mating type of B. trispora is not consistent with the originally
proposed cooperative biosynthetic pathway (Scheme 1) but
experimentally confirms the revised proposed interaction
scheme.7,45
35
best by oxidation of 11 with MnO2. Direct oxidation of the
vinyl bromide 9 with CrO3‚pyridine was possible, but the yield
was low (35%).36 Using either 11 or 12 for the Pd(0)-catalyzed
cross-coupling with organozincates, the two series of C4-
hydroxylated (2, 15, 16) or C4-keto (4, 13, 14) trisporoids were
obtained in a single operation and high yields (ca. 95%, GC-
MS).4,37 The required organozincates were obtained from the
corresponding iodides without the need for protecting groups.38
This minimized the number of transformations on the generally
acid- and base-sensitive trisporoid backbone. For example, the
trisporoids 2 and 4 were available from 11 or 12 by alkylation
with an organozincate derived from 4-iodo-butan-2-one.39 The
same strategy was followed to generate the secondary alcohols
(27) Naora, H.; Ohnuki, T.; Nakamura, A. Bull. Chem. Soc. Jpn. 1988,
61, 2859-2863.
(28) McMurry, J. E.; Kocovsky, P. Tetrahedron Lett. 1984, 25, 4187-
4190.
Under the same conditions, growing cultures of the (+)-
mating type produced the corresponding trisporic acid type C
in acceptable yield (ca. 10%). With respect to the CD spectra
of 6 and methyltrisporate C, the pro-S methyl group at C1 of
the cyclohexene was converted into the -COOH group,
consistent with the established absolute stereochemistry of the
(29) Kim, T. H.; Asaka, Y. Chem. Express 1991, 6, 125-128.
(30) Guirado, A.; Moss, G. P.; Utley, J. H. P. J. Chem. Soc., Chem.
Commun. 1987, 41-42.
(31) Oritani, T.; Yamashita, K. Phytochemistry 1983, 22, 1909-1912.
(32) Haag, A.; Eschenmoser, W.; Eugster, C. H. HelV. Chim. Acta 1980,
63, 10-15.
(33) Jurado-Gonzalez, M.; Sullivan, A. C.; Wilson, J. R. H. Tetrahedron
Lett. 2003, 44, 4283-4286.
(39) Irifune, S.; Kibayashi, T.; Ishii, Y.; Ogawa, M. Synthesis-Stuttgart
1988, 366-369.
(34) Barrero, A. F.; Alvarez-Manzaneda, E. J.; Alvarez-Manzaneda, R.;
Chahboun, R.; Meneses, R.; Aparicio, M. Synlett 1999, 713-716.
(35) Takahashi, S.; Oritani, T.; Yamashita, K. Tetrahedron 1988, 44,
7081-7088.
(40) Kobayashi, Y.; Kumar, G. B.; Kurachi, T.; Acharya, H. P.;
Yamazaki, T.; Kitazume, T. J. Org. Chem. 2001, 66, 2011-2018.
(41) Ernst, B.; Wagner, B. HelV. Chim. Acta 1989, 72, 165-171.
(42) Mori, K.; Maemoto, S. Liebigs Ann. Chem. 1987, 863-869.
(43) Akita, H. Chem. Pharm. Bull. 1983, 31, 1796-1799.
(44) Wo¨stemeyer, J. Eur. J. Biochem. 1985, 146, 443-448.
(45) Sutter, R. P.; Whitaker, J. P. Naturwissenschaften 1981, 68, 147-
148.
(36) Sarett, L. H.; Arth, G. E.; Lukes, R. M.; Beyler, R. E.; Poos, G. I.;
Johns, W. F.; Constantin, J. M. J. Am. Chem. Soc. 1952, 74, 4974-4976.
(37) Knochel, P.; Perea, J. J. A.; Jones, P. Tetrahedron 1998, 54, 8275-
8319.
(38) Huo, S. Q. Org. Lett. 2003, 5, 423-425.
1368 J. Org. Chem., Vol. 72, No. 4, 2007

Efficient Generation of a Trisporoid Library
SCHEME 4. Building Block Strategy for the Synthesis of the Early Trisporoids of the Series A, B, and Ca
a Reagents and conditions: (a) (i) (BrCH2PPh3)+Br-, LiHMDS, THF, -78 °C for 1 h, room temperature for 12 h, (ii) KOH, MeOH, room temperature,
6 h; (b) MnO2, CH2Cl2, room temperature, 12 h; (c) and (f) Bu2Zn, Pd[PPh3]4, THF, -78 °C to room temperature, 3-12 h; (d) and (g) 4-iodobutan-2-one-
zincate, Pd[PPh3]4, THF, -78 °C to room temperature, 3-12 h; (e) and (h) (i) ((4-iodobutan-2-yloxy)(tert-butyl)dimethylsilane)-zincate, Pd[PPh3]4, THF,
-78 °C to room temperature, 3-12 h, (ii) aq HF in MeCN (0.1%), room temperature, 12 h.
SCHEME 5. Synthesis of [2H3]-â-C18-Ketone (19)a
trisporoids into trisporic acids. By using cultures of either the
(+)- or (-)-mating types, the transformation can be terminated
at the level of the trisporols or trisporic acids, respectively.
Moreover, the conversion of the â-C18-ketone (1) or downstream
intermediates such as 4, 5, and 6 establishes these compounds
as intermediates en route to the terminal trisporic acids.
Experimental Section
Formation of Zincates, Representative Procedure.38 (4-Iodo-
butan-2-one)-zincate. In a dry 25 mL Schlenk tube were placed
under argon dry DMAP (4 mL), I2 (63.5 mg, 0.25 mmol), and Zn
dust (477.5 mg, 7.58 mmol). The mixture was stirred at room
temperature until the red color of the I2 disappeared. 4-Iodo-butan-
2-one (1 g, 5.05 mmol) was added to the 0 °C cold solution, and
stirring was continued for 15 min. For the (4-iodobutan-2-yloxy)-
(tert-butyl)dimethylsilane, the zincate formation was performed at
40 °C for 3 h. Completion of the metal insertion was indicated by
GC-MS analysis of the hydrolyzed and iodinated mixture.
Wittig Olefination, General Procedure. 2-(4-Brom-3-methyl-
buta-1,3-dienyl)-1,3,3-trimethylcyclohex-2-ene (9). A suspension
of bromomethyltriphenylphosphonium bromide (13.2 g, 30.3 mmol)
in dry THF (60 mL) was stirred at -78 °C, and a freshly prepared
solution of LiN(SiMe3)2 (28.5 mL, 1.03 M in THF) was slowly
added. To achieve complete deprotonation, the suspension was
stirred for 30 min at -78 °C and allowed to warm to 0 °C for
30 min. After recooling to -78 °C, â-ionone (8) (3.89 g, 18.2
mmol) in dry THF (5 mL) was slowly added via syringe. After
1 h, the mixture was allowed to reach room temperature and stirring
was continued overnight. The solution was hydrolyzed with aq NH4-
Cl, extracted with 3 × 50 mL of Et2O, and dried with Na2SO4, and
the solvent was removed under reduced pressure. The triphen-
ylphosphanoxide was precipitated with pentane and removed by
filtration over Alox N using petroleum ether for elution. Because
the product readily decomposed upon contact with chromatographic
materials, the crude bromide was used for further transformations.
Yield: ∼4.47 g (16.6 mmol; 82%).
a Reagents and conditions: (a) CH3OD, N-ethyl-diisopropylamine, 3 days;
(b) 18, Pd(Ac)2, but-3-en-2-one, PPh3, Et3N, THF, 60 °C, 120 h.
SCHEME 6. Feeding Experiments with Growing Cultures
of the (-)-Mating Type of B. trispora
family of trisporoids. Because the trisporic acids represent the
last products of the biosynthetic sequence, the earlier trisporols
21 and 22 have the same absolute configuration.
In summary, we have shown that growing cultures of
zygomycete fungi can be successfully used to transform early
3-((1E,3E/Z)-4-Bromo-3-methylbuta-1,3-dienyl)-2,4,4-trimeth-
ylcyclohex-2-one (12). The same procedure as described for 9 was
followed with bromomethyltriphenylphosphonium bromide (13.2
g, 30.4 mmol), dry THF (60 mL), LiN(SiMe3)2 (28.5 mL, 1.03 M
J. Org. Chem, Vol. 72, No. 4, 2007 1369

Schachtschabel and Boland
in THF), and 1-acetoxy-â-ionone 10 (5.1 g, 18.2 mmol in 5 mL of
dry THF). The triphenylphosphanoxide was precipitated with
pentane and removed by filtration on Alox N using petroleum ether/
ether (6:1, v/v) for elution. A solution of 3-((1E,3E/Z)-4-bromo-
3-methylbuta-1,3-dienyl)-2,4,4-trimethylcyclohex-2-enylacetate (5.0
g, 15.3 mmol) in MeOH (20 mL) was alkalinized with methanolic
KOH (10 M). After stirring for 6 h, aq NH4Cl was added and the
water phase was extracted with Et2O. The extract was dried (Na2-
SO4), and the solvent was evaporated under reduced pressure. Flash
filtration on Alox N with petroleum ether/ether (1:1, v:v) afforded
11 as a yellow oil. 11 (4.0 g, 14 mmol) was dissolved in CH2Cl2
(100 mL), and MnO2 (28 g, 0.45 mol) was added. The suspension
was stirred for 12 h. The MnO2 was filtered over celite, and after
removal of solvent, the ketone 12 was used without further
purification for alkylation. Yield from 10: ∼70%.
(m, 1H), 1.60-1.67 (m, 1H), 1.69 (s, 3H), 1.70-1.77 (m, 1H),
1.87-1.96 (m, 1H), 2.08 (s, 3H), 2.30 (s, 3H), 5.21-5.24 (t, 3J )
4.77 Hz, 1H), 6.10-6.14 (d, 3J ) 16.44 Hz, 1H), 7.15-7.19 (d, 3J
) 16.44 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 18.2, 21.2, 25.1,
27.3, 27.4, 28.7, 34.5, 34.7, 71.9, 129.9, 133.3, 141.7, 142.0, 170.8,
198.0. EI-MS [M]+: 235 (29), 208 (19), 190 (100), 175 (80), 161
(12), 151 (16), 147 (46), 133 (42), 123 (15), 119 (36), 109 (42),
105 (24), 91 (25), 77 (12), 65 (7), 55 (10). HRMS (EI) m/z calcd
for C15H22O3 235.133420, found 235.133965.
Synthesis of Trisporins, General Procedure. 3-((1E,3E/Z)-
(R,S)-7-(tert-Butyldimethyl-silanoyl)-3-methylocta-1,3-dienyl)-
2,4,4-trimethylcyclohex-2-enone. To a -78 °C cold solution of
the vinyl halide 12 (200 mg, 0.71 mmol) and Pd[PPh3]4 (46.2 mg,
40 µmol) in absolute THF (20 mL) was added the zincate solution
(2.2 mL, 2.2 mmol, 1 M). The mixture was allowed to come to
room temperature within 3 h, and stirring was continued for 12 h.
The reaction mixture was quenched with saturated NH4Cl solution
(25 mL) and extracted with Et2O (3 × 20 mL). After removal of
the solvent, the oil was purified on silica with petroleum ether/
ether (6:1,v:v). Yield: 227 mg (0.58 mmol, 82%). IR [cm-1]: 2958,
(3E,5E,7E/Z)-6-Methyl-8-(2,6,6-trimethylcyclohex-1-enyl)-
octa-3,5,7-trien-2-one (â-C18-Ketone) (1). In absolute THF (25
mL) were placed palladium(II) acetate (12 mg, 56 µmol), PPh3 (60
mg, 0.23 mmol), the vinyl halide (9) (300 mg), triethylamine (180
µL, 1.34 mmol), and but-3-en-2-one (237 mg, 1.35 mmol). The
mixture was heated for 120 h at 60 °C. The reaction was quenched
with saturated NH4Cl solution (30 mL) and extracted with Et2O (3
× 30 mL) and dried (Na2SO4), and the solvent was removed under
reduced pressure. Purification was achieved on silica using
petroleum ether/ether (9:1,v:v) for elution. Yield: 241 mg (84%)
as a mixture of E/Z-isomers (7:3). IR [cm-1]: 2927, 2863, 1659,
1588, 1449, 1360, 1253, 975. 1H NMR (400 MHz, CDCl3) δ 1.03
(s, 6H), 1.45-1.49 (m, 2H), 1.58-1.67 (m, 2H), 1.71 and 1.73 (s,
1
2929, 2857, 1663, 1465, 1254, 1135, 1091, 1032, 835, 775. H
NMR (400 MHz, CDCl3) δ 0.01 and 0.02 (2s, 6H), 0.85-0.87 (m,
9H), 1.10-1.11 (m, 2H), 1.16-1.17 (m, 6H), 1.43-1.49 (m, 2H),
1.80-1.87 (m, 9H), 2.10-2.13 (m, 1H), 2.25-2.27 (m, 1H), 2.47-
3
2.51 (m, 2H), 3.75-3.79 (m, 1H), 5.49-5.52 (t, J ) 7.44 Hz,
0.8H), 5.55-5.59 (t, 3J ) 7.20 Hz, 0.2H), 6.03-6.06 (d, 3J ) 16.10
3
3
Hz, 0.2H), 6.13-6.16 (d, J ) 16.22 Hz, 0.8H), 6.18-6.21 (d, J
) 16.10 Hz, 0.2H), 6.60-6.63 (d, 3J ) 16.22 Hz, 0.8H). 13C NMR
(100 MHz, CDCl3) δ -4.8, -4.4, 13.8, 18.1, 20.1, 23.9, 25.81,
25.84, 27.50, 27.53, 34.2, 35.6, 37.2, 39.9, 68.1, 121.9, 124.7, 129.6,
131.5, 133.3, 135.5, 141.3, 161.9, 199.5. EI-MS [M]+: 390 (48),
375 (7), 333 (22), 258 (30), 243 (100), 231 (19), 215 (14), 201
(15), 187 (26), 185 (21), 173 (21), 159 (29), 145 (15), 133 (9), 119
(14), 103 (12), 91 (7), 75 (47). HRMS (EI) m/z calcd for C24H42O2-
Si 390.295409, found 390.295766.
3
3H), 2.01-2.05 (t, J ) 6.33 Hz, 2H), 2.06 (s, 3H), 2.29 (s, 3H),
3
6.05-6.18 (m, 2.8H), 6.37-6.43 (2d, J ) 15.8 Hz, 1H), 6.6-
3
3
7.72 (d, J ) 15.8 Hz, 0.2H), 7.53 and 7.60 (dd, J ) 11.92 Hz
and J ) 12.28 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 13.1,
3
19.1, 21.7, 27.6, 28.9, 33.1, 34.3, 39.6, 126.2, 127.7, 128.6, 129.3,
130.9, 131.2, 132.4, 136.7, 137.5, 138.0, 139.2, 144.5, 145.5, 198.4.
EI-MS [M]+ 258 (100), 243 (61), 215 (34), 199 (14), 189 (26),
187 (24), 185 (24), 175 (28), 173 (39), 159 (54), 157 (26), 145
(47), 143 (26), 133 (24), 131 (26), 123 (22), 119 (28), 115 (18),
109 (28), 107 (18), 105 (27), 93 (12), 91 (26), 81 (10), 77 (11).
HRMS (EI) m/z calcd for C18H26O 258.198366, found
258.197555.
2,4,4-Trimethyl-3-((1E/Z)-3-methylocta-1,3-dienyl)cyclohex-
2-enone (Trisporin A) (13). Following the general procedure for
trisporins, 12 (200 mg, 0.71 mmol) and Pd[PPh3]4 (46.2 mg, 40
µmol) were dissolved in absolute THF (20 mL) and the Bu2Zn
solution (2.2 mL, 2.2 mmol, 1 M) was added. After removal of the
solvent, the oil was purified on silica with petroleum ether/ether
(9:1, v/v). Yield: 159 mg (0.61 mmol, 86%) as a mixture of E/Z
isomers (1:1). IR [cm-1]: 2958, 2860, 1666, 1586, 1461, 1353,
[1,1-2H3]-(3E)-4-(2,6,6-Trimethylcyclohex-1-enyl)but-3-en-2-
one (18). â-Ionone (8) (500 mg, 2.6 mmol) and ethyl-diisopropyl-
amine (100 µL) were dissolved in deuterated methanol (5 mL) and
stirred for 3 days. D2O (10 mL) was added, and the aqueous phase
was extracted with Et2O (5 × 10 mL). The unified organic extracts
were dried (Na2SO4), and the solvent was removed under reduced
1
1333, 1308, 1198, 1093, 1031, 968. H NMR (400 MHz, CDCl3)
δ 0.89-0.93 (m, 3H), 1.17 and 1.18 (2s, 6H), 1.29-1.43 (m, 4H),
1.81-1.86 (m, 8H), 2.13-2.21 (m, 2H), 2.47-2.52 (m, 2H), 5.50
(t, 3J ) 7.19 Hz, 0.5H), 5.56 (t, 3J ) 7.37 Hz, 0.5H), 6.04 (d, 3J )
16.19 Hz, 0.5H), 6.13-6.22 (d, 3J ) 16.07 Hz, 1H), 6.62 (d, 3J )
16.19 Hz, 0.5H). 13C NMR (100 MHz, CDCl3) δ 12.0, 13.7, 13.9,
20.1, 22.2, 22.4, 27.2, 27.5, 28.2, 31.6, 32.0, 34.2, 35.6, 37.3, 121.9,
124.5, 129.6, 131.5, 133.5, 133.7, 135.9, 141.4, 161.7, 199.4. EI-
MS [M]+: 260 (70), 245 (38), 227 (13), 217 (23), 204 (36), 203
(73), 190 (18), 189 (100), 175 (27), 171 (12), 163 (24), 161 (45),
148 (15), 147 (45), 145 (13), 134 (14), 133 (88), 131 (12), 119
(68), 117 (11), 107 (13), 105 (27), 93 (11), 91 (29), 81 (13), 79
(11), 77 (13), 69 (14), 55 (13). HRMS (EI) m/z calcd for C18H28O
260.214016, found 260.213158.
2
pressure. Yield: 496 mg (2.5 mmol; 98%, >97% H). IR [cm-1]:
2931, 2866, 2252, 1687, 1662, 1607, 1457, 1297, 1262, 1176, 1027,
1
980. H NMR (400 MHz, CDCl3) δ 1.06 (s, 6H), 1.48 (m, 2H),
3
3
1.62 (m, 2H), 1.76 (s, 3H), 2.06 (t, J ) 6.3 Hz, 2H), 6.11 (d, J
) 16.4 Hz, 1H), 7.26 (d, 3J ) 16.4 Hz, 1H). 13C NMR (100 MHz,
CDCl3) δ 18.9, 21.7, 26.2-26.8, 28.8, 33.5, 34.0, 39.7, 131.6, 135.9,
136.0, 143.2, 198.8. EI-MS [M]+: 195 (8), 181 (16), 180 (100),
179 (16), 162 (5), 149 (6), 138 (10), 133 (9), 125 (7), 121 (5), 119
(6), 107 (6), 105 (6), 95 (6), 93 (11), 91 (10), 79 (6), 77 (7), 69
(4), 65 (4), 55 (5). HRMS (EI) m/z calcd for C13H17D3O 195.170246,
found 195.170017.
2,4,4-Trimethyl-3-(oxobut-1-enyl)cyclohex-2-enylacetate (10).
To a solution of 1,4-benzoquinone (2 g, 18.5 mmol) and â-ionone
(8) (3.55 g, 18.5 mmol) in acetic acid (60 mL) were added
palladiumbis(trifluoroacetate) (300 mg, 0.93 mmol) and o-meth-
oxyacetophenone (560 mg, 3.7 mmol). The mixture was heated to
70 °C for 12 h. NaOH solution (200 mL, 6 N) was added, and the
water phase was extracted with 5 × 50 mL of Et2O. The unified
organic extracts were washed with 30 mL of saturated NaHCO3
solution. The crude product was purified on silica (petroleum ether/
ether (2:1; v:v)). Yield: 3.93 g (15.7 mmol; 85%). IR [cm-1]: 2966,
2,4,4-Trimethyl-3-((1E/Z)-3-methyl-7-oxo-octa-1,3-dienyl)cy-
clohex-2-enone (Trisporin B) (4). The reaction was performed
according to the general procedure for trisporins with 12 (100 mg,
0.35 mmol) and Pd[PPh3]4 (23.1 mg, 20 µmol) in absolute THF
(20 mL), and 4-iodobutan-2-onezincate (2.2 mL, 2.2 mmol, 1 M)
was added. The crude product was purified over silica using
petroleum ether/ether (2:1,v/v) for elution. Yield: 78 mg (0.28
mmol, 81%) as a mixture of E/Z isomers (2:1). IR [cm-1]: 2925,
1
2867, 1716, 1662, 1353, 1334, 1199, 1160, 1095, 1032, 969. H
NMR (400 MHz, CDCl3) δ 1.15 and 1.16 (s, 6H), 1.80-1.86 (m,
1
2934, 2862, 1722, 1677, 1451, 1366, 1245, 1019, 962, 860. H
NMR (400 MHz, CDCl3) δ 1.08 (s, 3H), 1.04 (s, 3H), 1.42-1.48
8H), 2.12 and 2.14 (2s, 3H), 2.39-2.56 (m, 6H), 5.47-5.50 (t, 3J
3
) 6.52 Hz, 0.65H), 5.42-5.45 (t, J ) 7.56 Hz, 0.35H), 6.06-
1370 J. Org. Chem., Vol. 72, No. 4, 2007

Efficient Generation of a Trisporoid Library
6.09 (d, 3J ) 16.03 Hz, 0.35H), 6.15-6.20 (2d, 3J ) 16.25 Hz, 3J
) 16.03 Hz, 1H), 6.58-6.62 (d, 3J ) 16.25 Hz, 0.65H). 13C NMR
(100 MHz, CDCl3) δ 12.1, 13.7, 20.1, 22.7, 27.5, 29.9, 34.3, 35.7,
37.3, 43.0, 43.6, 122.8, 125.5, 129.6, 130.9, 132.7, 133.1, 134.4,
140.7, 161.5, 199.4, 207.9. EI-MS [M]+: 274 (100), 259 (27), 241
(16), 231 (62), 217 (21), 203 (51), 201 (55), 200 (29), 199 (44),
189 (29), 185 (23), 175 (31), 173 (32), 161 (33), 157 (30), 147
(40), 145 (29), 133 (53), 119 (52), 105 (37), 91 (48), 79 (25), 77
(29), 69 (26), 55 (36). HRMS (EI) m/z calcd for C18H26O2
274.193280, found 274.193399.
(400 MHz, CDCl3) δ 0.91 and 1.01 (2s, 6H), 1.38-1.44 (m, 1H),
1.59-1.73 (m, 3H), 1.73-1.94 (m, 7H), 2.13 and 2.14 (2s, 3H),
2.38-2.43 (m, 2H), 2.47-2.54 (m, 2H), 3.96-4.00 (m, 1H), 5.28
3
3
3
and 5.37 (2t, J ) 7.03 Hz, J ) 7.09 Hz, 1H), 5.92-6.07 (3d, J
) 16.01 Hz, 3J ) 16.01 Hz, 3J ) 16.26 Hz, 1.5H), 6.38-6.42 (d,
3J ) 16.26 Hz, 0.5H). 13C NMR (100 MHz, CDCl3) δ 12.2, 18.6,
20.3, 21.7, 22.5, 27.3, 28.4, 28.9, 29.7, 29.9, 34.4, 43.3, 43.8, 70.1,
123.9, 126.8, 127.9, 129.3, 129.9, 130.7, 133.1, 138.4, 142.2, 208.5.
EI-MS [M]+: 276 (15), 258 (64), 243 (7), 225 (6), 218 (10), 215
(15), 200 (16), 187 (21), 185 (100), 173 (26), 171 (13), 170 (14),
163 (11), 159 (53), 157 (57), 147 (23), 145 (43), 144 (24), 143
(32), 142 (14), 141 (12), 135 (17), 133 (24), 131 (26), 129 (23),
128 (20), 122 (15), 121 (31), 119 (50), 117 (16), 115 (17), 109
(16), 107 (28), 105 (40), 95 (26), 93 (22), 91 (36), 81 (17), 79
(21), 77 (20), 55 (13). HRMS (EI) m/z calcd for C18H28O2
276.208930, found 276.207939.
3-((1E/Z)-7-Hydroxy-3-methylocta-1,3-dienyl)-2,4,4-trimeth-
ylcyclohex-2-enone (Trisporin C) (14). To a solution of 3-((1E,3E/
Z)-(R,S)-7-(tert-butyldimethyl-silanoyl)-3-methylocta-1,3-dienyl)-
2,4,4-triethylcyclohex-2-enone (20 mg, 0.051 mmol) in MeCN (10
mL) was added HF (100 µL, aq 40%). The solution was stirred for
6 h and quenched with water (20 mL). The aqueous phase was
extracted with Et2O (3 × 20 mL), dried (Na2SO4), and purified on
Florisil using petroleum ether/ether (1:1, v/v) for elution. Yield:
13.3 mg (0.047 mmol, 95%) as a mixture of E/Z isomers (3:2). IR
[cm-1]: 3428, 2963, 2925, 2865, 1657, 1452, 1355, 1334, 1310,
1093, 969. 1H NMR (400 MHz, CDCl3) δ 1.15 and 1.16 (2s, 6H),
1.17 and 1.21 (2d, 3H), 1.45-1.60 (m, 2H), 1.81-1.88 (m, 8H),
2.16 (s, 1H), 2.21-2.34 (m, 2H), 2.47-2.51 (m, 2H), 2.47-2.51
3-((1E,3E)-7-Hydroxy-3-methylocta-1,3-dienyl)-2,4,4-trimeth-
ylcyclohex-2-enol (4-Dihydrotrisporin C) (16). Prepared according
to the synthetic procedure for 15, from 11 (200 mg, 0.7 mmol)
with Pd[PPh3]4 (46.2 mg, 40 µmol) in absolute THF (20 mL) and
(4-iodobutan-2-yloxy)(tert-butyl)dimethylsilane-zincate (2.2 mL, 2.2
mmol, 1 M). After the workup procedure, the crude product was
diluted with MeCN (10 mL), containing HF (100 µL, aq 40%).
The solution was stirred for 6 h and quenched with water (20 mL).
The aqueous phase was extracted with Et2O (3 × 20 mL), dried
(Na2SO4), and purified on Florisil using petroleum ether/ether (1:
1, v/v) for elution. Yield: 140 mg (0.5 mmol, 72%). The compound
is very sensitive to light and oxygen and should be handled under
argon. Due to the high sensitivity, 16 was derivatized with MSTFA
to the corresponding bistrimethylsilylether. IR [cm-1]: 2957, 2917,
2849, 1266, 838, 742. 1H NMR (400 MHz, CDCl3) δ 0.11 (s, 9H),
3
(m, 2H), 3.77-3.85 (m, 1H), 5.49-5.52 (t, J ) 7.32 Hz, 0.6H),
3
3
5.55-5.58 (t, J ) 7.33 Hz, 0.4H), 6.04-6.07 (d, J ) 16.13 Hz,
0.4H), 6.15 and 6.21 (2d, 3J ) 16.26 Hz, 3J ) 16.13 Hz, 0.4H and
0.6H), 6.61-6.64 (d, 3J ) 16.26 Hz, 0.6H). 13C NMR (100 MHz,
CDCl3) δ 12.1, 13.8, 20.1, 23.5, 23.9, 24.8, 27.5, 34.2, 35.6, 37.1,
38.6, 39.1, 67.5, 122.3, 124.9, 131.9, 132.7, 133.1, 133.7, 134.8,
141.1, 161.9, 199.6. EI-MS [M]+: 276 (100), 243 (37), 232 (32),
220 (39), 205 (33), 203 (29), 201 (26), 187 (29), 173 (32), 163
(39), 162 (25), 161 (32), 159 (30), 147 (38), 145 (31), 133 (50),
121 (25), 119 (48), 107 (21), 105 (35), 93 (28), 91 (45), 81 (25),
79 (27), 77 (25), 69 (31), 55 (38). HRMS (EI) m/z calcd for
C18H28O2 276.208930, found 276.209442.
3
0.15 (s, 9H), 0.97 (s, 3H), 1.02 (s, 3H), 1.15-1.16 (d, J ) 5.95
Hz, 3H), 1.43 (s, 3H), 1.53-1.57 (m, 2H), 1.61-1.69 (m, 3H),
1.71 and 1.74 (2s, 3H), 1.96-2.06 (m, 1H), 2.09-2.23 (m, 2H),
3
3.78-3.81 (m, 1H), 4.01-4.05 (m, 1H), 5.33 (t, J ) 6.52 Hz,
Representative Procedure for 4-Dihydrotrisporins. 2,4,4-
Trimethyl-3-((1E,3E/Z)-3-methylocta-1,3-dienyl)cyclohex-2-
enol (4-Dihydrotrisporin A) (15). To a -78 °C cold solution of
11 (200 mg, 0.7 mmol) and Pd[PPh3]4 (46.2 mg, 40 µmol) in
absolute THF (20 mL) was added zincate solution (2.2 mL, 2.2
mmol, 1 M). The mixture was allowed to come to room temperature
within 60 min, and stirring was continued for 48 h. The reaction
mixture was quenched with saturated NH4Cl solution (25 mL) and
extracted with Et2O. After removal of the solvent, the oil was
purified on Florisil with petroleum ether/ether (2:1, v/v) for elution.
Yield: 144 mg (0.55 mmol, 79%) as mixture of E/Z-isomers (19:
1) after chromatography. The compound is very sensitive to light
and oxygen and should be handled under argon. IR [cm-1]: 3331,
2930, 2860, 1454, 1022, 965, 740. 1H NMR (400 MHz, CDCl3) δ
0.87-0.92 (m, 3H), 1.00 and 1.03 (2s, 6H), 1.30-1.36 (m, 4H),
1.39-1.44 (m, 1H), 1.57 (s, 1H), 1.61-1.94 (m, 9H), 2.12-2.16
0.3H), 5.40 (t, 3J ) 7.56 Hz, 0.7H), 5.96 (d, 3J ) 16.03 Hz, 0.7H),
6.02 and 6.04 (2d, 3J ) 16.03 Hz, 3J ) 16.26 Hz, 1H), 6.43 (d, 3J
) 16.26 Hz, 0.3H). 13C NMR (100 MHz, CDCl3) δ 12.3, 14.1,
23.8, 23.9, 24.8, 29.7, 30.3, 31.9, 35.2, 39.9, 39.9, 68.1, 71.1, 123.7,
125.5, 129.8, 131.0, 131.6, 133.9, 135.0, 138.6, 141.2. EI-MS [M]+:
422 (82), 407 (8), 366 (2), 351 (2), 332 (6), 317 (26), 311 (11),
289 (5), 277 (55), 261 (40), 247 (16), 237 (60), 224 (85), 211 (100),
197 (30), 187 (30), 171 (49), 157 (29), 143 (40), 129 (14), 117
(24), 107 (10), 95 (9), 73 (84), 55 (6). HRMS (EI) m/z calcd for
C24H46O2Si2 422.303638, found 422.303223.
Biotransformation of 20 to Trisporol B (21) and Trisporol
C (22). Cells of the (-)-mating type of Blakeslea trispora were
precultivated on a SUP medium for 3 days. The grown mycelia
were sterile filtered off from the medium and placed on a maltose
medium. [2H3]-Trisporin B (20) (10 mg) was added and incubated
for 48 h. The mycelia were filtered off, and the broth was adjusted
to pH 8 with NaOH and extracted with chloroform/i-propanol (20:
1, v/v). After drying and removal of the solvent, the products were
purified and separated by chromatography on silica with petroleum
ether/ether (1:1, v/v). Yield: 1.0 mg (ca. 10%, 1:1 mixture of [2H3]-
21 and [2H3]-22). The substance is very sensitive to light and oxygen
and should be handled under argon. The spectra were identical with
the literature data18,21 except for a singlet at 1.81 ppm owing to
substitution by deuterium atoms.
3
3
(m, 2H), 3.99-4.01 (t, J ) 4.23 Hz, 1H), 5.35-5.38 (t, J)7.67
3
3
Hz, 1H), 6.01-6.04 (d, J ) 16.02 Hz, 1H), 6.42-6.45 (d, J )
16.02 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ13.9, 18.6, 20.3,
22.3, 27.1, 27.4, 28.5, 29.0, 32.2, 34.5, 34.7, 70.3, 125.8, 129.2,
130.7, 131.4, 131.9, 142.3. EI-MS [M - 18]+: 244 (100), 229
(50), 201 (22), 187 (45), 173 (56), 159 (68), 145 (71), 131 (29),
119 (44), 105 (17), 91 (14). HRMS (EI) m/z calcd for C18H28
244.219101, found 244.218601.
(7E/Z)-8-(3-Hydroxy-2,6,6-trimethylcyclohex-1-enyl)-6-meth-
ylocta-5,7-dien-2-one (4-Dihydrotrisporin B) (2). Following the
procedure for 15, 2 was prepared from 11 (200 mg, 0.7 mmol),
Pd[PPh3]4 (46.2 mg, 40 µmol) in absolute THF (20 mL), and
4-iodobutan-2-one-zincate (2.2 mL, 2.2 mmol, 1 M). The crude
product was purified over silica using petroleum ether/ether (6:1,
v/v) for elution. Yield: 133 mg (0.48 mmol, 69%) as a mixture of
E/Z isomers (1:1). The compound is very sensitive to light and
oxygen and should be handled under argon. IR [cm-1]: 3411, 2958,
Isolation of Trisporic Acids. Cells of the (+)- and (-)-mating
type of Blakeslea trispora were separately precultivated on a SUP
medium for 3 days. The grown mycelium was sterile filtered off
from the media and both were placed together on maltose media
and shaken for the following 6 days. The mycelium was removed,
and the medium was adjusted to pH 8 with NaOH and extracted
with chloroform/i-propanol (20:1, v/v) for removal of the precursors.
Next, the medium was adjusted to pH 2 and extracted with
chloroform/isopropanol (20:1, v/v). After drying and removal of
1
2926, 2853, 1713, 1449, 1360, 1266, 1159, 1023, 964. H NMR
J. Org. Chem, Vol. 72, No. 4, 2007 1371

Schachtschabel and Boland
the solvent, the products were purified and separated on silica with
petroleum ether/ether (1:1, v/v).
3H), 1.40 (s, 3H), 1.46-1.51 (m, 2H), 1.65-1.79 (m, 3H), 1.82
(s, 3H), 1.93 (s, 3H), 1.95-2.03 (m, 1H), 2.13-2.28 (m, 2H), 3.65
(s, 3H), 3.76-3.80 (m, 1H), 4.14-4.16 (t, 3J ) 5.49 Hz, 1H), 5.39-
2-((1E,3E)-7-Hydroxy-3-methylocta-1,3-dienyl)-1,3-dimethyl-
4-oxocyclohex-2-enecarboxylic Acid (Trisporic Acid C, As
Methyl Ester). IR [cm-1]: 3445, 2922, 2852, 1730, 1660, 1454,
3
3
5.42 (t, J ) 7.56 Hz, 1H), 6.17-6.20 (d, J ) 16.26 Hz, 1H),
3
6.45-6.49 (d, J ) 16.26 Hz, 1H). 13C NMR (100 MHz, CDCl3)
1
1351, 1308, 1250, 1188, 1134, 1097, 1024, 969. H NMR (400
δ 17.5, 20.1, 23.6, 23.8, 24.3, 28.3, 32.5, 39.4, 46.1, 52.1, 67.5,
70.2, 125.3, 128.5, 130.6, 132.7, 134.4, 135.1, 177.9. EI-MS [M
- 18]+: 322 (1), 304 (85), 289 (18), 245 (7), 229 (9), 204 (100),
189 (18), 173 (11), 171 (12), 161 (27), 145 (26), 133 (24), 125
(20), 119 (57), 105 (34), 91 (32), 85 (36), 55 (67). HRMS (EI) m/z
calcd for C19H28O3 322.214410, found 322.214348.
3
MHz, CDCl3) δ 1.21-1.22 (d, J ) 6.3 Hz, 3H), 1.42-1.55 (m,
2H), 1.56 (s, 3H), 1.70 (s, 1H), 1.86 (s, 3H), 1.92-1.98 (m, 4H),
2.15-2.25 (m, 1H), 2.28-2.35 (m, 1H), 2.37-2.45 (m, 1H), 2.50-
2.56 (m, 2H), 3.68 (s, 3H), 3.75-3.86 (m, 1H), 5.60-5.64 (t, 3J )
3
3
6.3 Hz, 1H), 6.42-6.38 (d, J ) 16.34 Hz, 1H), 6.82-6.86 (d, J
) 16.34 Hz, 1H). 13C NMR (100 MHz, CDCl3) 12.1, 14.1, 19.8,
22.6, 23.7, 33.2, 34.5, 39.4, 46.7, 52.5, 67.5, 124.7, 132.2, 132.5,
132.6, 134.7, 152.4, 177.0, 197.7. EI-MS [M]+: 320 (100), 305
(10), 287 (12), 276 (12), 261 (11), 247 (8), 243 (13), 233 (5), 227
(14), 220 (46), 215 (14), 202 (15), 201 (18), 199 (14), 187 (34),
175 (22), 173 (35), 162 (25), 161 (25), 159 (45), 147 (33), 145
(31), 135 (25), 133 (20), 131 (22), 119 (25), 107 (21), 105 (28), 93
(23), 91 (37), 85 (13), 81 (26), 79 (23), 77 (21), 55 (36). HRMS
(EI) m/z calcd for C19H28O4 320.198760, found 320.199165. CD
spectra were identical with the reported data.14
Acknowledgment. We thank Dr. S. Bartram and S. Lorenz
for EI high-resolution mass spectra, M. Richter for help with
the cultivation of the fungi, Prof. James D. White for providing
the MS spectra of trisporol A and B. Financial support by the
Deutsche Forschungsgemeinschaft Priority Program 1152, “Evo-
lution of Metabolic Diversity” (Schi495/3), and the Max Planck
Society are gratefully acknowledged.
Supporting Information Available: Infrared spectra, mass
spectra, and 1H NMR and 13C NMR spectra of compounds 1, 2, 4,
10, 13-15, 17-19, and 3-((1E,3E/Z)-(R,S)-7-(tert-butyldimethyl-
silanoyl)-3-methylocta-1,3-dienyl)-2,4,4-trimethylcyclohex-2-
enone, 2,4,4-trimethyl-3-((1E,3E)-3-[2H3]-ethylocta-1,3-dienyl)-
cyclohex-2-enone, 2,4,4-trimethyl-3-((1E,3E/Z)-3-[2H3]-methylocta-
1,3-dienyl)cyclohex-2-enol, methyl-2-((1E,3E)-7-hydroxy-3-methyl-
octa-1,3-dienyl)-1,3-dimethyl-4-oxocyclohex-2-enecarboxylate, and
methyl-4-hydroxy-2-((1E,3E)-7-hydroxy-3-methylocta-1,3-dienyl)-
1,3-dimethylcyclohex-2-enecarboxylate. This material is available
free of charge via the Internet at http://pubs.acs.org.
Methyl-4-hydroxy-2-((1E,3E)-7-hydroxy-3-methylocta-1,3-di-
enyl)-1,3-dimethylcyclohex-2-enecarboxylate (4-Dihydrometh-
yltrisporate C). Isolated trisporic acid C (50 mg, 0.16 mmol) was
derivatized with diazomethane and diluted in dry THF (5 mL), and
NaBH4 (1.5 mg, 0.04 mmol) was added and stirred for 30 min.
HCl (0.1 M) was slowly added until the precipitate was dissolved.
The aqueous phase was extracted with ether and dried, and the
solvent was evaporated under reduced pressure. The product was
purified by chromatography on Florisil with petroleum ether/ether
(1:1, v/v) for elution. Yield: 46.8 mg (0.14 mmol, 91%). IR [cm-1]:
3382, 2932, 2869, 1714, 1455, 1377, 1260, 1197, 1122, 1018,
1
3
911. H NMR (400 MHz, CDCl3) δ 1.18-1.19 (d, J ) 5.95 Hz,
JO062236C
1372 J. Org. Chem., Vol. 72, No. 4, 2007