Access to Wieland-Miescher ketone in an enantiomerically pure form by a kinetic resolution with yeast-mediated reduction

Article abstract of DOI:10.1021/jo991192n

Both enantiomers of Wieland-Miescher ketone [3,4,8,8a-tetrahydro-8a- methyl-1,6(2H, 7H)-naphthalenedione], in a highly enantiomerically enriched form, became readily available by a newly developed kinetic resolution with yeast-mediated reduction. From a screening of yeast strains, Torulaspora delbrueckii IFO 10921 was selected. The collected cells of this strain, obtained by an incubation in a glucose medium, smoothly reduced only the isolated carbonyl group of the (S)enantiomer, while the (R)-enantiomer remained intact. Starting from both enantiomers (~70% ee) prepared by an established proline-mediated asymmetric Robinson annulation, the reduction with T. delbrueckii gave the (R)-enantiomer (98% ee) and the corresponding alcohol (4aS,5S)-4,4a,5,6,7,8-hexahydro-5-hydroxy-4a-methyl-2(3H)- naphthalenone (94% ee, 94% de) in preparative scale in nearly quantitative yields. An approach for the asymmetric synthesis of the Wieland-Miescher ketone was also successful. 2-Methyl-2-(3-oxobutyl)-1,3-cyclohexanedione, the prochiral precursor, was reduced with this strain to give a cyclic acetal form of (2S,3S)-3-hydroxy-2-methyl-2-(3-oxobutyl)cyclohexanone, in a stereomerically pure form.

Full text of DOI:10.1021/jo991192n

J . Org. Chem. 2000, 65, 129-135
129
Access to Wiela n d -Miesch er Keton e in a n En a n tiom er ica lly P u r e
F or m by a Kin etic Resolu tion w ith Yea st-Med ia ted Red u ction †
Ken-ichi Fuhshuku, Nobutaka Funa, Tomohiro Akeboshi, Hiromichi Ohta, Hiroyuki Hosomi,
Shigeru Ohba, and Takeshi Sugai*
Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Yokohama 223-8522, J apan
Received J uly 27, 1999
Both enantiomers of Wieland-Miescher ketone [3,4,8,8a-tetrahydro-8a-methyl-1,6(2H,7H)-naph-
thalenedione], in a highly enantiomerically enriched form, became readily available by a newly
developed kinetic resolution with yeast-mediated reduction. From a screening of yeast strains,
Torulaspora delbrueckii IFO 10921 was selected. The collected cells of this strain, obtained by an
incubation in a glucose medium, smoothly reduced only the isolated carbonyl group of the (S)-
enantiomer, while the (R)-enantiomer remained intact. Starting from both enantiomers (∼70% ee)
prepared by an established proline-mediated asymmetric Robinson annulation, the reduction with
T. delbrueckii gave the (R)-enantiomer (98% ee) and the corresponding alcohol (4aS,5S)-4,4a,5,6,7,8-
hexahydro-5-hydroxy-4a-methyl-2(3H)-naphthalenone (94% ee, 94% de) in preparative scale in
nearly quantitative yields. An approach for the asymmetric synthesis of the Wieland-Miescher
ketone was also successful. 2-Methyl-2-(3-oxobutyl)-1,3-cyclohexanedione, the prochiral precursor,
was reduced with this strain to give a cyclic acetal form of (2S,3S)-3-hydroxy-2-methyl-2-(3-oxobutyl)-
cyclohexanone, in a stereomerically pure form.
In tr od u ction
the development of this procedure was, indeed, the reason
this starting material has been so widely applied to
natural product syntheses. While this proline-enamine
cyclization is a very efficient way, there still remains a
drawback to the method; that is, the enantiomeric excess
(ee) of the product is only ∼70%. To obtain an enantio-
merically pure sample from this material, an elegant
procedure by a fractional crystallization has been estab-
lished.4,6 In this procedure, the contaminating undesired
enantiomer can be removed through the preferential
crystallization of its racemic form, which is, however,
accompanied by the loss of an equal amount of the
desired enantiomer (path A, Scheme 1). Moreover, the
success of the fractional crystallization has been found
to depend strongly upon the ee and the chemical purity
of the partially enantiomerically enriched form of 1. Here
we propose an alternative procedure for the kinetic
resolution of the partially enantiomerically enriched form
of 1 using yeast-mediated reduction, which can convert
the undesired enantiomer to the corresponding alcohol
(path B, Scheme 1). This would provide an easier and
more efficient procedure to approach a highly enantio-
merically enriched form of 1, as the newly formed product
resulting from the yeast-mediated transformation can be
separated from the unreacted starting material by a
simple chromatographical analysis.
Enantiomerically enriched forms of Wieland-Miescher
ketone 11 [3,4,8,8a-tetrahydro-8a-methyl-1,6(2H,7H)-
naphthalenedione] have so far been utilized as the
starting materials for the syntheses of naturally occur-
ring products, including terpenoids and steroids.2 For the
efficient preparation of the enantiomerically enriched
form of 1, a method of separation of one enantiomer by
utilizing the formation of an inclusion complex in
trans-4,5-bis(hydroxydiphenylmethyl)-2,2-dimethyl-1,3-dioxa-
cyclopentane, a derivative of tartaric acid, has been
developed.3 On the other hand, much effort has been
devoted to the asymmetric cyclization of its prochiral
precursor, 2-methyl-2-(3-oxobutyl)-1,3-cyclohexanedione
2, and excellent chemical (the use of proline-enamine)4
and biochemical (the use of catalytic antibody)5 proce-
dures have been established. The former method could
be performed in a >100 g scale in the laboratory, and
* Address correspondence to this author. Fax: 81-45-563-5967.
E-mail: sugai@chem.keio.ac.jp.
† The experimental part of this work was taken from the forthcoming
M.S. thesis of K.F. (2001) and the B.S. thesis of N.F. (1998).
(1) Wieland, P.; Miescher, K. Helv. Chim. Acta 1950, 33, 2215-2228.
(2) For recent examples from (R)-1, see the folllowing. (+)-Halena-
quinol: Harada, N.; Sugioka, T.; Ando, Y.; Uda, H.; Kuriki, T. J . Am.
Chem. Soc. 1988, 110, 8483-8487. (-)-Chaparrinone: Grieco, P. A.;
Collins, J . L.; Moher, E. D.; Fleck, T. J .; Gross, R. S. J . Am. Chem.
Soc. 1993, 115, 6078-6093. (+)-Grinderic acid: Paquette, L. A.; Wang,
H.-L. J . Org. Chem. 1996, 61, 5352-5357. (-)-O-Methylshikoccin:
Paquette, L. A.; Backhaus, D.; Braun, R.; Underiner, T. L.; Fuchs, K.
J . Am. Chem. Soc. 1997, 119, 9662-9671. For recent examples from
(S)-1, see the following. (+)-Paspalicine: Smith, A. B., III; Kingery-
Wood, J .; Leenay, T. L.; Nolen, E. G.; Sunazuka, T. J . Am. Chem. Soc.
1992, 114, 1438-1449. (+)-Pyripyropene: A, Nagamitsu, T.; Sunazuka,
T.; Obata, R.; Tomoda, H.; Tanaka, H.; Harigaya, Y.; Omura, S. J . Org.
Chem. 1995, 60, 8126-8127. Paclitaxel: Danishefsky, S. J .; Masters,
J . J .; Young, W. B.; Link, J . T.; Snyder, L. B.; Magee, T. V.; J ung, D.
K.; Isaacs, R. C. A.; Bornmann, W. G.; Alaimo, C. A.; Coburn, C. A.;
Di Grandi, M. J . J . Am. Chem. Soc. 1996, 118, 2843-2859. (+)-
Avarol: An, J .; Wiemer, D. F. J . Org. Chem. 1996, 61, 8775-8779.
(+)-Labd-8(17)-ene-3â,15-diol: Pemp, A.; Seifert, K. Tetrahedron Lett.
1997, 38, 2081-2084.
(4) Eder, U.; Sauer, G.; Wiechert, R. Angew. Chem., Int. Ed. Engl.
1971, 10, 496-497. Hajos, Z. G.; Parrish, D. R. J . Org. Chem. 1974,
39, 1615-1621. Gutzwiller, J .; Buchschacher, P.; Fu¨rst, A. Synthesis
1977, 167-168. Buchschacher, P.; Fu¨rst, A. Org. Synth. 1985, 63, 37-
43. Hajos, Z. G.; Parrish, D. R. Organic. Syntheses; Wiley & Sons:
NewYork, 1990, VII, 368-373.
(5) Zhong, G.; Hoffmann, T.; Lerner, R. A.; Danishefsky, S.; Barbas,
C. F., III. J . Am. Chem. Soc. 1997, 119, 8131-8132. Hoffmann, T.;
Zhong, G.; List, B.; Shabat, D.; Anderson, J .; Gramatikova, S.; Lerner,
R. A.; Barbas, C. F., III. J . Am. Chem. Soc. 1998, 120, 2768-2779.
(6) Harada, N.; Sugioka, T.; Uda, H.; Kuriki, T. Synthesis 1990, 53-
56.
(3) Toda, F.; Tanaka, K. Tetrahedron Lett. 1988, 29, 551-554.
10.1021/jo991192n CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/16/1999

130 J . Org. Chem., Vol. 65, No. 1, 2000
Fuhshuku et al.
Sch em e 1. Wiela n d -Miesch er Keton e 1 a n d th e
P r ep a r a tion of Its En a n tiom er ica lly P u r e F or m s
Sch em e 3. Yea st-m ed ia ted Red u ction of (()-1
w ith T. d elbr u eckii IF O 10921 a n d C. m elibiosica
IAM 4488
Sch em e 2. Red u ction of (()-1 w ith C. fa lca ta
Ta ble 1. In cu ba tion Con d ition s for th e Red u ction of
Ra cem ic Wiela n d -Miesch er Keton e (1)
substrate
concn
(%)
Resu lts a n d Discu ssion
wet cells glucose time convna
entry
(mg/mL)
(%)
(h)
(%)
Eb
Among the various microorganisms which have been
sucessfully applied to the related octalones,7 Prelog and
Acklin reported a microbial approach to obtain an enan-
tiomerically enriched form of 1 from (()-1. They dis-
closed the use of Curvularia falcata, a fungus strain that
reduced only the isolated carbonyl group of 1.8 In their
report, the reduction proceeded in a highly enantiofacially
selective manner, according to the so-called “Prelog rule”.
However, no difference between the rates of the two
enantiomers of 1 was observed, which resulted in the
formation of a mixture of an equal amount of the two
diasteromeric alcohols (4aS,5S)-3a and (4aR,5S)-3a
(Scheme 2).
1
2
3
4
5
0.2
1.0
1.0
1.0
1.0
160
160
80
20
60
0.4
0.4
0.4
0.4
2.0
0.5
1.5
2.5
6.0
2.0
38.3
34.9
26.7
23.5
26.3
87
20
18
16
126
Determined by 1H NMR analysis (270 MHz) of the crude
extract. E was calculated on the basis of the ee of recovered 1
a
b
and conversion.12
two different types of microorganisms, Torulaspora del-
brueckii IFO 10921 and Candida melibiosica IAM 4488
were found to be potent candidates. In the following
section, we describe the stereochemical course of the
reduction mediated by these microorganisms and the
incubation conditions in detail.
T. d elbr u eckii IF O 10921-Med ia ted P r efer en tia l
Red u ction of th e (S)-En a n tiom er . T. delbrueckii IFO
1092111 preferentially reduced the isolated carbonyl
group of (S)-1 to give (4aS,5S)-3a , along with the unre-
acted (R)-1 as shown in Scheme 3. The incubation
conditions were examined as shown in Table 1. The
collected cells of this strain, obtained by incubation in a
conventional glucose medium (5% glucose), were resus-
pended (6% w/v). The reduction proceeded smoothly at
In this context, our first attempt was a screening of
microoganisms from stock cultures and soil samples,
which can preferentially reduce one enantiomer to an-
other, by applying (()-110 as the substrate. Fortunately,
(7) Inayama, S.; Shimizu, N.; Ohkura, T.; Akita, H.; Oishi, T.; Iitaka,
Y. Chem. Pharm. Bull. 1986, 33, 2660-2663. Shimizu, N.; Ohkura,
T.; Akita, H.; Oishi, T.; Iitaka, Y.; Inayama, S. Chem. Pharm. Bull.
1987, 35, 429-432. Inayama, S.; Shimizu, N.; Ohkura, T.; Akita, H.;
Oishi, T.; Iitaka, Y. Chem. Pharm. Bull. 1989, 37, 712-717. Shimizu,
N.; Ohkura, T.; Akita, H.; Oishi, T.; Iitaka, Y.; Inayama, S. Chem.
Pharm. Bull. 1989, 37, 1023-1027. Shimizu, N.; Ohkura, T.; Akita,
H.; Oishi, T.; Iitaka, Y.; Inayama, S. Chem. Pharm. Bull. 1989, 37,
2561-2563. Shimizu, N.; Ohkura, T.; Akita, H.; Oishi, T.; Iitaka, Y.;
Inayama, S. Chem. Pharm. Bull. 1991, 39, 2973-2979.
(8) Prelog, V.; Acklin, W. Helv. Chim. Acta 1956, 39, 748-757.
(9) Prelog, V. Pure Appl. Chem. 1964, 9, 119-130. MacLeod, R.;
Prosser, H.; Fikentscher, L.; Lanyi, J .; Mosher, H. S. Biochemistry
1964, 3, 838-846.
(10) Ramachandran, S.; Newman, M. S. Organic Syntheses; Wiley
& Sons: New York, 1973, V, 486-489.
(11) This yeast strain was isolated from soil and identified by
Deutsche Sammlung von Mikroorganismen und Zellikulturen GbmH
(DSMZ). It was deposited at the Institute for Fermentation, Osaka
(IFO), and now available from this Institute, 2-17-85 J uso-honmachi,
Yodogawa-ku, Osaka 532-8686, J apan. For details, see the Experi-
mental Section.

Access to Wieland-Miescher Ketone
J . Org. Chem., Vol. 65, No. 1, 2000 131
Sch em e 4. P r ep a r a tive-Sca le Red u ction of
En a n tiom er ica lly P r e-en h a n ced F or m s of 1 w ith T.
d elbr u eckii IF O 10921
1% of the substrate concentration (Table 1, entries 2-5).
In the best case (Table 1, entry 5), the enantiomeric ratio
(E ) [Vmax(fast)/Km(fast)]/[Vmax(slow)/Km(slow)]), devel-
oped by Sih and co-workers12 as the index for the
efficiency of a kinetic resolution, was 126. Under the
reaction conditions, with an insufficient amount of the
cells (Table 1, entry 4) and/or a low glucose concentration
(Table 1, entry 3), the selectivity became considerably low
(16-20).
C. m elibiosica IAM 4488-Med ia ted P r efer en tia l
Red u ction of th e (R)-En a n tiom er . In contrast to T.
delbrueckii, a yeast strain, C. melibiosica IAM 448813
preferentially reduced the (R)-1 with an E of 15 (Scheme
3). It was noteworthy that the activity and selectivity
were only expressed during the logarithmic growing
phase of the yeast. For this reason, the incubation
conditions were elaborated, and the reduction was initi-
ated by the addition of the substrate several hours later
than the inoculation of the strain to the incubation
medium. The other way was for the incubation to be
carried out for a prolonged period. After the substrate
was mixed with the collected cells in a glucose medium,
the mixture was shaken for 1 week, with the periodic
addition of glucose every day. In this case, the reduction
started suddenly, 4-5 days after the initiation of the
attempted reaction.
Neither procedure described above, however, could be
performed in a highly reproducible manner. In the former
case, the smooth initiation of the reduction seemed to
strongly depend on the cell concentration. If the addition
of substrate was too early, no further growth of the yeast
was observed, probably due to the toxicity of the sub-
strate. On the other hand, the reduction was very slow
when the addition of the substrate was delayed. This
situation compelled us to give up any further attempts
to carry out the reaction on a large scale.
T. delbrueckii, effectively reduced a somewhat sterically
hindered carbonyl group that is located adjacent to a
quaternary carbon atom. This result prompted us to
extend this reduction to other related substrates. The
importance of asymmetrization of prochiral diketones,
which would produce a new quaternary chiral center, has
been pointed out by a number of organic chemists, and
pioneering studies have been reported on the subject of
yeast-mediated asymmetric reductions of cyclic sub-
strates.14,15 Along these lines, the substrate candidates
were selected, as shown in Figure 1. In contrast to the
result on the Wieland-Miescher ketone (1) as described
above, the action of T. delbrueckii showed discrepancy
on other substrates shown in Table 2. As judged from
entries 1-6 (Table 2), it seems that this yeast strain
shows a higher affinity to the substrate bearing 1,3-
dicarbonyl functionalities and its vinylogous structure.
P r ep a r a tive-Sca le Syn th esis of En a n tiom er ica lly
P u r e Wielan d-Miesch er Keton e with T. delbr u eckii.
As mentioned earlier, the reproducibility of the rate of
the reduction and the growth of T. delbrueckii in a large-
scale incubation were very high. This yeast-mediated
reduction was then applied to enantiomerically pre-
enhanced forms of 1, which were prepared by an estab-
lished proline-mediated asymmetric Robinson annula-
tion. The reduction of (R)-1 (70% ee) with T. delbrueckii
gave (R)-1 (97.6% ee) in 82% yield, along with (4aS,5S)-
3a (72.0% de) in 18% yield, as shown in Scheme 4.
Enantiomerically pure (R)-1 was obtained by the single
recrystallization of recovered 1. On the other hand, the
reduction of (S)-1 (70% ee) gave (4aS,5S)-3a (94.4% de)
in 78% yield, along with (R)-1 (18.4% ee) in 22% yield as
shown in Scheme 4. Pure (4aS,5S)-3a was obtained by
the single recrystallization of this material. Both proce-
dures worked very preparatively in several 10 g scales
of the substrate, and the products were obtained in nearly
quantitative yields. Enantiomerically pure (S)-1 was
obtained in a high yield by J ones’ oxidation of (4aS,5S)-
3a followed by single recrystallization of the product.
An Attem p t for th e Asym m etr ic Syn th esis of
En a n tiom er ica lly P u r e Wiela n d -Miesch er Keton e
w ith T. d elbr u eckii. The above-mentioned yeast strain,
(14) Zhou, W.-S.; Huang, D.-Z.; Wang, Z.-Q. Kexue Tongbao 1980,
25, 741-742. Zhou, W.-S.; Huang, D.-Z.; Dong, Q.-C.; Zhuang, Z.-P.;
Wang, Z.-Q. Chem. Nat. Prod. Proc. Sino-Am. Symp. 1980, 299-301.
Zhou, W.-S.; Huang, D.-Z.; Dong, Q.-C.; Wang, Z.-Q. Huaxue Xuebao
1982, 40, 648-656. Zhou, W.-S.; Zhuang, Z.-P.; Wang, Z.-Q.Huaxue
Xuebao 1982, 40, 666-669. Brooks, D. W.; Grothaus, P. G.; Irwin, W.
L. J . Org. Chem. 1982, 47, 2820-2821. Lu, Y.; Barth, G.; Kieslich, K.;
Strong, P. D.; Duax, W. L.; Djerassi, C. J . Org. Chem. 1983, 48, 4549-
4554. Brooks, D. W.; Mazdiyasni, H.; Chakrabarti, S. Tetrahedron Lett.
1984, 25, 1241-1244. Yanai, M.; Sugai, T.; Mori, K. Agric. Biol. Chem.
1985, 49, 2373-2377. Dai, W.-M.; Zhou, W.-S. Tetrahedron 1985, 41,
4475-4482. Mori, K.; Mori, H. Tetrahedron 1985, 41, 5487-5493.
Brooks, D. W.; Mazdiyasni, H.; Sallay, P. J . Org. Chem. 1985, 50,
3411-3414. Brooks, D. W.; Woods, K. W. J . Org. Chem. 1987, 52,
2036-2039. Brooks, D. W.; Madiyasni, H.; Grothaus, P. G. J . Org.
Chem. 1987, 52, 3223-3232. Mori, K.; Fujiwhara, M. Tetrahedron
1988, 44, 343-354. Mori, K.; Mori, H. Organic Syntheses; Wiley &
Sons: New York, 1993, VIII, 312-315. Sakakibara, M.; Ogawa-Uchida,
A. Biosci. Biotechnol. Biochem. 1995, 59, 1300-1303.
(12) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J . J . Am. Chem.
Soc. 1982, 104, 7294-7299.
(13) This yeast strain is available from the IAM culture collection,
Institute of Molecular Cell Biosciences at the University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-0032, J apan.
(15) Zhou, W.-S.; Wang, Z.-Q.; Zhang, H.; Fei, R.-Y.; Zhuang, Z.-P.
Huaxue Xuebao 1985, 43, 168-173.

132 J . Org. Chem., Vol. 65, No. 1, 2000
Fuhshuku et al.
Sch em e 5. Red u ction of 2 w ith T. d elbr u eckii
IF O 10921
F igu r e 1. Candidate of substituted cyclohexenones for asym-
metric reduction with T. delbrueckii IFO 10921.
Ta ble 2. Red u ction of Ca r bon yl Com p ou n d s w ith T.
d elbr u eckii
wet cells time convn yield
syn/anti
entry substratea (mg/mL) (h)
(%)
(%) of the productb
1
2
3
4
5
6
(()-1
60
300
60
60
60
2
20
24
24
72
24
26
44
0
23
37
100
26
24
1.4:98.6
1:1.9
(()-4
tion of the major product from the incubation mixture,
at the appropriate stage, was examined. To our surprise,
the major product was not identical to that which was
expected, the (2R,3S) form of 3-hydroxy-2-methyl-2-(3-
oxobutyl)cyclohexanone (8a ) that had been reported as
the product by the reduction of 2 with brewers’ yeast.15
5
6
7
2
20
21
60
1:2.5
1:5.9
>99.9:0.1
60
a
b
Substrate concentration ) 1.0%. Syn means that the sub-
stituent R and the substituent hydroxyl group are located in a
syn relationship.
1
By the elucidation of its H NMR spectrum, the structure
of the product was suggested to be 9 (Scheme 5).
Ta ble 3. Red u ction of P r och ir a l Dik eton e 2 w ith
T. d elbr u eckii
Of the three carbonyl groups of 2, one on the carbocyclic
skeleton was preferentially reduced, and the initially
formed hydroxy diketone spontaneously cyclized via
intramolecular hemiacetal formation to give 9. Due to
the acid-labile nature of 9, the recovery of the product
from the incubation broth was best (60%) in the following
workup procedure: the crude product was roughly puri-
fied by chromatography through a short column of silica
gel and subsequently recrystallized as shown in entry 5
(Table 3). The crystalline nature made the isolation of 9
possible in a highly pure state, and the relative config-
uration of 9 was unambiguously determined, by X-ray
crystallographic analysis, to be (1R*,6R*,7R*), as shown
in Figure 2.
time
(h)
yield
(%)
method of
entry
purificationa
1
2
3
4
5
6
16
16
12
20
24
36
29
52
46
57
60
51
A
B
B
B
B
B
a
A: Crude product was extensively chromatographed over silica
gel for purification; B. Crude product was roughly chromato-
graphed and then recrystallized for purification.
The reduction of monoketone (Table 2, entry 2) was very
slow, and the product was obtained only in very low yield
as a diastereomeric mixture. The introduction of a
carboxymethyl group also had a deleterious effect on the
reducing enzyme system of this yeast (Table 2, entry 3).
The reduction worked by the protection of the carboxyl
group as its esters in entries 4 and 5 (Table 2), while no
high diastereomeric selectivity was observed in either
case.
Finally, we attempted the T. delbrueckii-catalyzed
reduction of 2-methyl-2-(3-oxobutyl)-1,3-cyclohexanedione
(2, entry 6). The reaction was carefully monitored by thin-
layer chromatographic analysis, because all of the three
carbonyl groups were susceptible to reduction and the
product would be a complex mixture. The exploration of
both the incubation conditions and the workup procedure
is summarized in Table 3. One major product was
observed along with the progress of incubation (Table 3,
entries 1-5), which, after prolonged incubation, began
to decrease (Table 3, entry 6), allowing other more polar
materials to become dominant. The workup and purifica-
The next task was to determine the absolute configu-
ration of 9. This was accomplished by a chemical cor-
relation of 9 to (1S,8aR)-3b as shown in Scheme 6. First,
hemiacetal 9 was treated with acetic anhydride and
pyridine to give acetate 8b. In this case, the acetylation
occurred almost exclusively on the secondary hydroxy
group with a concomitant hemiacetal ring-opening reac-
tion. The acid-catalyzed Robinson annulation reaction of
8b was performed according to the reported procedure,15
which resulted in bicyclic acetate 3b. At this stage, the
sign of rotation of 3b ([R]20 -93.0 (chloroform)) was
D
compared to that of an authentic sample ([R]20 -92.0
D
(chloroform)) derived from (4aR,5S)-3b, which was ob-
tained by a C. melibiosica-catalyzed reduction of 1. As
the ee of (1S,8aR)-3b was confirmed to be >99%, by a
chiral stationary phase HPLC analysis, it was revealed
that the stereoselectivity of the reduction of prochiral
triketone 2 was high.

Access to Wieland-Miescher Ketone
J . Org. Chem., Vol. 65, No. 1, 2000 133
glucose medium (containing glucose (50 mg), malt extract (50
mg), peptone (70 mg), yeast extract (50 mg), KH2PO4 (30 mg),
and K2HPO4 (20 mg), at pH 6.5, total volume of 10 mL) for 2
days at 30 °C. Then to the mixture were added (()-1 (20 mg)
and glucose (100 mg), and the mixture was shaken on a
reciprocal shaker (250 cpm) for several days at 30 °C. The
progress of the reduction was confirmed by TLC analysis (silica
gel, developed with hexanes/ethyl acetate 1:2). Each reaction
mixture was worked up separately, in the same way as
described below. The reaction mixture was filtered through a
Celite pad and extracted with ethyl acetate. The combined
organic layer was washed with brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo. The recovered
substrate was purified by preparative TLC (hexanes/ethyl
acetate 1:2, Rf ) 0.3-0.4). The ee of 1 was estimated from
HPLC analysis. HPLC (column, Chiralcel OJ , 0.46 cm × 25
cm, hexane/2-propanol 9:1; flow rate of 0.5 mL/min): tR ) 49.6
min for (R)-1, 54.7 min for (S)-1.16
Iden tification of Micr oor gan ism . T. delbrueckii IFO10921
was identified11 to the species level as T. delbrueckii Lindner
based on morphological and physiological characteristics.
Morphology: colony on potato dextrose agar butyrous, smooth,
creme colored. Cells: globose, mostly 5 µm φ; budding multi-
polar. Pseudomycelium and true mycelium absent; no sexual
reproduction detected. Good growth at 37 °C. Utilization of
C- and N-sources: anaerobic, glucose +; aerobic, glucose +;
R-methyl glycoside -; galactose -; salicin -; sorbose +;
cellobiose -; rhamnose -; maltose -; dulcit -; lactose -;
inositol -; melibiose -; mannitol +; sucrose +; sorbitol +;
trehalose +; glycerol -; inulin +; erythritol -; melezitose -;
D-arabinose -; raffinose +; L-arabinose -; starch -; ribose -;
xylitol +; D-xylose -; gluconate -; L-xylose -; 2-keto-gluconate
+; adonitol -; 5-keto-gluconate -; nitrate -. From these
results, the strain was identified as T. delbrueckii Lindner.
F igu r e 2. Structure of 9 determined by X-ray analysis.
Sch em e 6. Deter m in a tion of th e Absolu te
Con figu r a tion of 9
Red u ction of (()-1 w ith T. d elbr u eckii IF O10921. A
small portion of the yeast cells of T. delbrueckii IFO10921
grown on the agar-plate culture was aseptically inoculated into
a glucose medium (containing glucose (5.0 g), peptone (2.0 g),
yeast extract (0.5 g), KH2PO4 (0.3 g), and K2HPO4 (0.2 g), at
pH 6.5, total volume of 100 mL) and then incubated for 2 days
at 30 °C. The wet cells were harvested by centrifugation (3 000
rpm) and washed with phosphate buffer (0.1 M, pH 6.5). The
combined wet cells (6.0 g) were resuspended in a reaction
medium (containing glucose (2.0 g), phosphate buffer (0.1 M,
pH 6.5), total volume of 100 mL) in a 500 mL shaking culture
(Sakaguchi) flask, together with (()-1 (1.02 g, 5.72 mmol),
and shaken on a reciprocal shaker (186 cpm) for 2 h at 30 °C.
The reaction mixture was filtered through a Celite pad and
extracted with ethyl acetate. The combined organic layer was
washed with brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo. The residue was charged on a silica
gel column (50 g). Elution with hexanes/ethyl acetate (3:1 to
2:1) afforded (R)-1 (759 mg, 74%): IR ν˜max 1710, 1665, 1615
cm-1 1H NMR (270 MHz, CDCl3) δ 1.44 (3H, s), 1.61-1.79
;
(1H, m), 2.06-2.20 (3H, m), 2.35-2.54 (4H, m), 2.65-2.78 (2H,
m), 5.85 (1H, d, J ) 1.8 Hz). Its IR and NMR spectra were
identical to those reported previously.4 On the basis of HPLC
analysis, the ee of (R)-1 was estimated to be 34.8%.
Further elution of the column (hexanes/ethyl acetate 1:1)
afforded (4aS,5S)-3a (275 mg, 26%): IR ν˜max 3420, 1655, 1615,
1
1055 cm-1; H NMR (270 MHz, CDCl3) δ 1.19 (3H, s), 1.31-
Con clu sion
1.49 (1H, m), 1.62-1.93 (5H, m), 2.13-2.50 (5H, m), 3.41 (1H,
The two T. delbrueckii-catalyzed approaches, kinetic
resolution and asymmetrization, worked very well in a
preparative scale. These two methodologies would be new
entries to the preparation of useful starting materials in
an enantiomerically pure form toward natural product
synthesis.
(16) The authors observed an inversion of the retention time of (R)-1
and (S)-1 on Chiralcel OJ . First, a commercial new column shows good
separation by eluting with hexane/2-propanol (9:1). The repeatedly
used column tended to showed low separation and sometimes, surpris-
ingly, an inverted tR (min) of 40.8 for (S)-1 and 43.5 for (R)-1 by eluting
with hexane/2-propanol (15:1). However, washing the exhausted
column with ethanol again showed a sharp separation of two enanti-
omers, tR (min) ) 49.6 for (S)-1 and 54.7 for (R)-1. From this example,
we concluded that the condition of the chiral stationary phase column
should always be maintained and those who use the elution pattern
on chiral stationary phase for the determination of the absolute
configuration should be very careful. We strongly recommend the
combination of the measurement of the sign of the optical rotation.
Exp er im en ta l Section
Scr een in g of Micr oor ga n ism s. The screening of micro-
organisms was performed as follows: The microorganisms
from stock cultures and soil samples were incubated in a

134 J . Org. Chem., Vol. 65, No. 1, 2000
Fuhshuku et al.
dd, J ) 4.3, 11.4 Hz (4aS,5S)), 3.65 (1H, dd, J ) 2.5, 2.6 Hz
(4aR,5S)), 5.78 (1H, d, J ) 1.2 Hz). Its IR and NMR spectra
were identical with those reported previously.17 J udging from
the area of signals at δ 3.43 for (4aS,5S)-3a and δ 3.65 for
(4aR,5S)-3a , the de of (4aS,5S)-3a was estimated to be 96.2%.
A small portion of (4aS,5S)-3a was oxidized with J ones’
reagent and was converted to (S)-1. On the basis of HPLC
analysis, the ee of (S)-1 was estimated to be 97.8%.
basis of the NMR spectrum, the de of (4aS,5S)-3a was
estimated to be >99.9%.
A small portion of (4aS,5S)-3a was oxidized with J ones’
reagent and was converted to (S)-1 as a solid. On the basis of
HPLC analysis, the ee of (S)-1 was estimated to be 99.0%.
Further purification by recrystallization from hexanes/ethyl
acetate afforded (S)-1 as needles: mp 45.0-47.0 °C; [R]25
)
D
+95.5 (c 0.98, toluene). Its IR and NMR spectra were idential
with those of (R)-1 as described above. On the basis of HPLC
analysis, the ee of (S)-1 was estimated to be >99.9%.
(4a R,5S)-Hexa h yd r o-5-h yd r oxy-4a -m eth yl-4,4a ,5,6,7,8-
h exa h yd r o-2(3H)-n a p h th a len on e (4a R,5S)-3a . A small
portion of the yeast cells of C. melibiosica IAM4488 grown on
the agar-plate culture was aseptically inoculated into a glucose
medium (containing glucose (1.0 g), peptone (2.0 g), yeast
extract (0.5 g), KH2PO4 (0.3 g), K2HPO4 (0.2 g), at pH 6.5, total
volume of 100 mL), and the culture was shaken on a reciprocal
shaker (186 cpm) at 30 °C. After 12 h, (()-1 (198 mg, 1.11
mmol) was added to the mixture, and it was shaken for 68 h
at 30 °C. The extraction and workup were performed as
described above. The residue was charged on a silica gel
column (20 g). Elution with hexane/ethyl acetate (3:1 to 1:1)
afforded (S)-1 (129 mg, 65%) as a solid. On the basis of HPLC
analysis, the ee of (S)-1 was estimated to be 39.2%. Its IR and
NMR spectra were identical with those of (R)-1, as described
above.
(R)-3,4,8,8a -Tet r a h yd r o-8a -m et h yl-1,6(2H ,7H )-n a p h -
th a len ed ion e (R)-1. The large-scale incubation of T. del-
brueckii IFO10921 was carried out as follows: A seed culture
(100 mL) was prepared in the same glucose medium as
described above for 30 h at 30 °C. The resulting mixture was
aseptically poured into another glucose medium (containing
glucose (100 g), peptone (40 g), yeast extract (10 g), KH2PO4
(6.0 g), K2HPO4 (4.0 g), at pH 6.5, total volume of 2000 mL) in
a 5000 mL Erlenmeyer cultivating flask with two internal
projections and further incubated for 40 h at 30 °C. At this
stage, its OD (660 nm) reached 61-67. The wet cells were
harvested by centrifugation (5 000 rpm) and washed with
phosphate buffer (0.1 M, pH 6.5). The combined wet cells (60
g) were resuspended in a reaction medium (containing glucose
(20 g), phosphate buffer (0.1 M, pH 6.5), total volume of 1000
mL) in a 5000 mL Erlenmeyer cultivating flask with two
internal projections, together with (R)-1 (70% ee, 10.0 g, 56.1
mmol), and shaken on a gyrorotary shaker (186 rpm) for 6 h
at 30 °C. The reaction mixture was filtered through a Celite
pad and extracted with ethyl acetate. The combined organic
layer was washed with brine, dried over anhydrous sodium
sulfate, and concentrated in vacuo. The residue was charged
on a silica gel column (450 g). Elution with hexanes/ethyl
acetate (3:1 to 1:1) afforded (R)-1 (8.22 g, 82%) as a solid. On
the basis of HPLC analysis, the ee of (R)-1 was estimated
97.6%.
Further elution of the column (hexanes/ethyl acetate 1:1)
afforded (4aR,5S)-3a (62 mg 31%) as a solid. J udging from the
NMR spectrum, as described above, the de of (4aR,5S)-3a was
estimated to be 87.4%. Further purification by two recrystal-
lizations from hexane/ethyl acetate afforded (4aR,5S)-3a as
needles: mp 92.4-92.8 °C (lit.8 mp 94-95 °C); [R]20D ) -108.5
(c 1.04, benzene) (lit.8 [R]25 ) -111 (c 1.3, benzene)); IR ν˜max
D
3435, 1645, 1605, 1065 cm-1
;
1H NMR (270 MHz, CDCl3) δ
1.23 (3H, s), 1.46-1.53 (1H, m), 1.66-2.11 (5H, m), 2.23-2.65
(5H, m), 3.41 (1H, dd, J ) 4.3, 11.4 Hz (4aS,5S)), 3.65 (1H,
dd, J ) 2.5, 2.6 Hz (4aR,5S)), 5.85 (1H, d, J ) 1.7 Hz). Its IR
and NMR spectra were identical with those reported previ-
ously.18 On the basis of the NMR spectrum, the de of (4aR,5S)-
3a was estimated to be >99.9%. Anal. Calcd for C11H16O2: C,
73.30; H, 8.95. Found: C, 73.29; H, 8.91.
Further purification by recrystallization from hexanes/ethyl
acetate afforded (R)-1 as needles: mp 48.6-49.4 °C (lit.4 mp
49-50 °C); [R]27D ) -95.2 (c 1.03, toluene) (lit.4 [R]25D ) +97.3
(c 1.0 toluene) for (S)-1). Its IR and NMR spectra were identical
with those of (R)-1 as descrived above. On the basis of HPLC
analysis, the ee of (R)-1 was estimated to be 99.5%. Anal. Calcd
for C11H14O2: C, 74.13; H, 7.92. Found: C, 73.80; H, 8.06.
Further elution of the column (hexanes/ethyl acetate 1:1)
afforded (4aS,5S)-3a (1.84 g, 18%). Its IR and NMR spectra
were identical with those of (4aS,5S)-3a as descrived above.
On the basis of the NMR spectrum, the de of (4aS,5S)-3a was
estimated to be 72.0%.
(4a S,5S)-4,4a ,5,6,7,8-Hexa h yd r o-5-h yd r oxy-4a -m eth yl-
2(3H)-n a p h th a len on e (4a S,5S)-3a . The large-scale incuba-
tion of T. delbrueckii IFO10921 and the reaction were carried
out in a manner similar to the procedure as described above.
The combined wet cells (60 g) were resuspended in a reaction
medium (containing glucose (20 g), phosphate buffer (0.1 M,
pH 6.5), total volume of 1000 mL) in a 5000 mL Erlenmeyer
cultivating flask with two internal projections, together with
(S)-1 (70% ee, 10.0 g, 56.1 mmol), and shaken on a gyrorotary
shaker (186 rpm) for 14 h at 30 °C. The extraction and workup
were performed as described above. The residue was charged
on a silica gel column (450 g). Elution with hexanes/ethyl
acetate (3:1 to 1:1) afforded (R)-1 (2.21 g, 22%) as a solid. Its
IR and NMR spectra were identical with those of (R)-1 as
described above.On the basis of HPLC analysis, the ee of (R)-1
was estimated to be 18.4%.
(1S,3S,6S)-3-Hydr oxy-2-oxa-3,6-dim eth yl-bicyclo[4.4.0]-
7-d eca n on e (1S,3S,6S)-9. The incubation of T. delbrueckii
IFO10921 was carried out in a manner similar to the procedure
as described above. The combined wet cells (6.0 g) were
resuspended in a reaction medium (containing glucose (2.0 g),
phosphate buffer (0.1 M, pH 6.5), total volume of 100 mL) in
a 500 mL shaking culture (Sakaguchi) flask, together with 2
(1.01 g, 5.14 mmol), and shaken on a reciprocal shaker (186
cpm) for 24 h at 30 °C. The reaction mixture was extracted
with ethyl acetate. The combined organic layer was washed
with brine, dried over anhydrous sodium sulfate, and concen-
trated in vacuo. The residue was charged on a silica gel column
(60 g). Elution with hexanes/ethyl acetate (4:1 to 1:1) afforded
crude (1S,3S,6S)-9 as a solid. Further purification by recrys-
tallization from hexanes/ethyl acetate afforded (1S,3S,6S)-9
(544 mg) as needles. The remaining mixture was charged on
a silica gel column (20 g). Elution with hexanes/ethyl acetate
(4:1 to 1:1) and subsequent recrystallization afforded further
(1S,3S,6S)-9 (68.9 mg). The combined yield of (1S,3S,6S)-9
(613 mg) was 60%. This was employed for the next step
without further purification.
Recrystallization from hexanes/ethyl acetate gave an ana-
lytical sample of (1S,3S,6S)-9 as needles: mp 75.4-76.2 °C;
Further elution of the column (hexanes/ethyl acetate 1:1)
afforded (4aS,5S)-3a (7.91 g, 78%) as a solid. On the basis of
the NMR spectrum, the de of (4aS,5S)-3a was estimated to
be 94.4%.
[R]24 ) -117.9 (c 0.96, chloroform); IR ν˜max 3460, 1690, 1080
D
1
cm-1; H NMR (400 MHz, CDCl3) δ 1.13 (3H, s), 1.35 (3H, s),
1.41-1.48 (1H, m), 1.57-1.63 (2H, m), 1.71-1.74 (1H, m),
1.80-1.86 (1H, m), 1.88 (1H, s), 2.04-2.18 (3H, m), 2.24-2.29
Further purification by recrystallization from hexanes/ethyl
acetate afforded (4aS,5S)-3a as needles: mp 44.0-46.2 °C
(1H, m), 2.49-2.58 (1H, m), 4.21 (1H, dd, J ) 2.2, 2.2 Hz); 13
C
(lit.17 mp 45-48 °C); [R]24 ) +191.6 (c 1.05, benzene) (lit.8
D
NMR (100 MHz, CDCl3) δ 21.2, 24.4, 26.4, 27.5, 30.4, 32.0,
37.9, 47.2, 75.3, 95.8, 214.3. Anal. Calcd for C11H18O3: C, 66.64;
H, 9.15.
[R]25D ) +203 (c 1.55 benzene)). Its IR and NMR spectra were
identical with those of (4aS,5S)-3a as described above. On the
(18) Tables of atomic parameters, bond lengths, and bond angles
for 9 have been deposited with the Cambridge Crystallographic Data
Centre.
(17) Yeo, S.-K.; Hatae, N.; Seki, M.; Kanematsu, K. Tetrahedron
1995, 51, 3499-3506.

Access to Wieland-Miescher Ketone
J . Org. Chem., Vol. 65, No. 1, 2000 135
Found: C, 66.89; H, 9.09. Single-crystal X-ray diffraction
measurement was performed with a Rigaku AFC-7R four-circle
diffractometer with Mo KR radiation (λ ) 0.710 73 Å). The
crystal data are as follows: C11H18O3, Mr ) 198.26, orthor-
hombic, P212121, a ) 11.420(5) Å, b ) 15.451(5) Å, c ) 6.126-
(3) Å, V ) 1080.9(9) Å3, Z ) 4, µ ) 0.087 mm-1, Dx ) 1.218
Mg m-3, R ) 0.054 for 1051 reflections.18 The molecular
structure is shown in Figure 2.
(1S,2S)-2-Met h yl-3-oxo-2-(3-oxob u t yl)cycloh exyl Ac-
eta te (1S,2S)-8b. A mixture of (1S,3S,6S)-9 (746 mg, 3.76
mmol), anhydrous pyridine (6 mL), and acetic anhydride (6
mL) was stirred for 6 days at room temperature and further
stirred for 2 days at 35 °C. The reaction mixture was poured
into water and acidified to pH 2 by adding 2 M hydrochloric
acid. The reaction mixture was extracted with ethyl acetate.
The combined organic layer was successively washed with 2
M hydrochloric acid, water, saturated aqueous sodium hydro-
gen carbonate solution, and brine; dried over anhydrous
sodium sulfate; and concentrated in vacuo. The residue was
charged on a silica gel column (20 g). Elution with hexanes/
ethyl acetate (15:1 to 4:1) afforded (1S,2S)-8b (847 mg, 94%)
as a solid. This was employed for the next step without further
purification.
Further purification by a bulb-to-bulb distillation afforded
(1S,8aR)-3b as an oil: bp 160 °C/1.8 mmHg (bath tempera-
ture); [R]20 ) -93.0 (c 1.58, chloroform); IR ν˜max 1730, 1670,
D
1620, 1240 cm-1; H NMR (270 MHz, CDCl3) δ 1.31 (3H, s),
1
1.51 (1H, ddd, J ) 3.3, 5.0, 13.2 Hz), 1.69-2.01 (4H, m), 2.06
(3H, s), 2.19 (1H, ddd, J ) 5.3, 13.5, 13.5 Hz), 2.29-2.56 (4H,
m), 4.85 (1H, dd, J ) 2.6, 2.6 Hz), 5.84 (1H, d, J ) 1.3 Hz); 13
C
NMR (100 MHz, CDCl3) δ 20.4, 21.2, 21.8, 25.9, 30.9, 31.7,
33.9, 39.5, 76.9, 126.3, 166.3, 170.1, 198.6. Anal. Calcd for
C
13H18O3: C, 70.24; H, 8.16. Found: C, 70.17; H, 8.00.
For the determination of the absolute configuration of the
product, the corresponding (1S,8aR)-3b was prepared in the
following manner: A mixture of (4aR,5S)-3a (199 mg, 1.10
mmol), anhydrous pyridine (2 mL), and acetic anhydride (1
mL) was stirred for 2 days at room temperature. The reaction
mixture was subsequently poured into water and acidified to
pH 2 by adding 2 M hydrochloric acid. The reaction mixture
was extracted with ethyl acetate. The combined organic layer
was washed with 2 M hydrochloric acid, water, saturated
aqueous sodium hydrogen carbonate solution, and brine; dried
over anhydrous sodium sulfate; and concentrated in vacuo. The
residue was charged on a silica gel column (15 g). Elution with
hexanes/ethyl acetate (10:1 to 4:1) afforded (1S,8aR)-3b (242
Recrystallization from hexanes/ethyl acetate gave an ana-
lytical sample of (1S,2S)-8b as needles: mp 48.4-48.8 °C;
mg, 99%), [R]20 ) -92.0 (c 0.64, chloroform).
D
For the determination of the ee of the product, the corre-
sponding (()-(1S*,8aS*)-3b was prepared in the reported
procedure from (()-1.20,21
[R]20 ) +42.9 (c 1.03, chloroform); IR ν˜max 1735, 1715, 1235
D
cm-1
;
1H NMR (400 MHz, CDCl3) δ 1.08 (3H, s), 1.64-2.09
(6H, m), 2.05 (3H, s), 2.12 (3H, s), 2.20-2.41 (4H, m), 4.87 (1H,
dd, J ) 3.3, 6.7 Hz). Its IR and NMR spectra were identical
with those of (1R,2R)-8b, reported previously.19 Anal. Calcd
for C13H20O4: C, 64.98; H, 8.39. Found: C, 65.06; H, 8.48.
(1S,8a R)-1,2,3,4,6,7,8,8a -oct a h yd r o-8a -m et h yl-6-oxo-
n a p h th yl Aceta te (1S,8a R)-3b. A mixture of (1S,2S)-8b (60.8
mg, 0.253 mmol), benzene (6 mL), and a catalytic amount of
p-toluenesulfonic acid was stirred under reflux for 2 days with
an azeotropic removal of water. The reaction mixture was
poured into saturated aqueous sodium hydrogen carbonate
solution and extracted with ethyl acetate. The combined
organic layer was successively washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo. The
residue was charged on a silica gel column (3 g). Elution with
hexanes/ethyl acetate (10:1 to 1:1) afforded (1S,8aR)-3b (47.0
mg, 84%). The ee of (1S,8aR)-3b was estimated from HPLC
analysis. HPLC (column, Chiralcel OJ ; hexane/2-propanol 9:1;
flow rate of 0.5 mL/min]: tR ) 41.1 min for (1R,8aS)-3b, 46.7
min for (1S,8aR)-3b. Accordingly, the ee of (1S,8aR)-3b was
estimated to be >99.9%.
Ack n ow led gm en t. This work was supported by a
Grant-in-Aid for Scientific Research (Grant No. 0923-
1244) from the Ministry of Education, Science, Sports
and Culture, J apan. We express our sincere thanks to
Professor Isao Kuwajima of Kitasato Institute for his
encouragement throughout this study. The authors also
thank Professor Susumu Kobayashi, Kenji Mori, Dr.
Hirosato Takikawa, and Mr. Arata Yajima of the
Science University of Tokyo for their discussion on the
application of this microorganism-mediated synthesis.
Mr. Masahiro Yokoyama’s help with the preparation of
the racemic substrate is also acknowledged with thanks.
J O991192N
(20) Ward, D. E.; Rhee, C. K.; Zoghaib, W. M. Tetrahedron Lett.
1988, 29, 517-520. Ward, D. E.; Rhee, C. K. Can. J . Chem. 1989, 67,
1206-1211.
(21) Shimizu, T.; Hiranuma, S.; Nakata, T. Tetrahedron Lett. 1996,
37, 6145-6148.
(19) Renouf, P.; Poirier, J .-M.; Duhamel, P. J . Chem. Soc., Perkin
Trans. 1 1997, 1739-1745.