Absolute stereochemistry of TT-1 (rasfonin), an α-pyrone-containing natural product from a fungus, Trichurus terrophilus

Article abstract of DOI:10.1016/j.tetlet.2003.09.136

The absolute stereochemistry of TT-1 (1=rasfonin), an α -pyrone-containing natural product from a Fungi Imperfecti, Trichurus terrophilus, was determined as 5R,6R,7S,9R, and 6′S on the basis of synthesis of diastereomers of two fragments of 1 in optically

Full text of DOI:10.1016/j.tetlet.2003.09.136

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 8427–8431
Absolute stereochemistry of TT-1 (rasfonin),
an a-pyrone-containing natural product from a fungus,
Trichurus terrophilus
Kohki Akiyama, Souichi Kawamoto, Haruhiro Fujimoto and Masami Ishibashi*
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Received 4 August 2003; revised 10 September 2003; accepted 12 September 2003
Abstract—The absolute stereochemistry of TT-1 (1=rasfonin), an a-pyrone-containing natural product from a Fungi Imperfecti,
Trichurus terrophilus, was determined as 5R,6R,7S,9R, and 6%S on the basis of synthesis of diastereomers of two fragments of 1
in optically active forms, and comparison of their spectral and optical data with those of natural specimens.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
In 2000, an a-pyrone-containing natural product, TT-1,
was isolated from the ethyl acetate extract of a Fungi
Imperfecti Trichurus terrophilus culture by Fujimoto
and co-workers in our laboratory.1 TT-1 significantly
suppressed proliferation (blastogenesis) of mouse
splenic lymphocytes stimulated with mitogens, con-
canavalin A (Con A) and lipopolysaccharide (LPS),
with IC50 values of 0.7 and 0.5 g/mL, respectively.1
TT-1 was optically active and had a molecular formula
of C25H38O6, and its planar structure was elucidated as
1 consisting of two principal carbon-chains with an
a-pyrone ring on the basis of the spectral data. Almost
at the same time, Hayakawa and co-workers reported
isolation of rasfonin, a new apoptosis inducer in ras-
dependent cells from the fermented mycelia of an
Ascomycete Talaromyces sp. 3656-A1.2 The planar
structure of rasfonin was identical with that of TT-1.
However, the absolute stereochemistry of five chiral
centers of 1 remained undetermined. Here we describe
the determination of the absolute stereochemistry of
five chiral centers of 1 as 5R,6R,7S,9R, and 6%S on the
2. Results and discussion
basis of synthesis of partial structural units (segments A
and B) of 1 in optically active forms and comparison of
their spectral and optical data with those of natural
2.1. Segment B
specimens.
We first studied the synthesis of segment B with 6%S-
configuration as shown in Scheme 1. Triol (2), prepared
from (S)-malic acid,3 was partially protected as di-tert-
butyldimethylsilyl (TBS) ether, followed by tosylation
of the remaining secondary hydroxyl group to afford a
tosylate (3), which was treated with sodium cyanide in
* Corresponding author: Tel./fax: +81-43-290-2913; e-mail:
mish@p.chiba-u.ac.jp
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.09.136

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K. Akiyama et al. / Tetrahedron Letters 44 (2003) 8427–8431
Scheme 1. Reagents and conditions: (a) (i) TBSCl, imidazole, DMF, 0°C, 2 h (73%); (ii) TsCl, pyridine, rt, 72 h (82%); (b) NaCN,
DMSO, 90°C, 3 h (54%); (c) (i) DIBAL, CH2Cl2, −78°C, 2 h; (ii) Ph3PꢀC(CH3)CO2Et, CH2Cl2, rt, 73 h (47% for 2 steps); (d) (i)
DIBAL, CH2Cl2, −78°C, 1 h; (ii) MnO2, CH2Cl2, rt, 14 h; (iii) (EtO)2P(O)CH2CO2Et, NaH, DME, 0°C, 2 h (81% for 2 steps);
(e) (i) TBAF, THF, rt, 2 h (99%); (ii) (S)- or (R)-MTPACl, pyridine, rt, 14 h (71–72%).
DMSO to give a nitrile (4) with inversion of configura-
tion.4,5 Reduction of the nitrile (4) with DIBAL fol-
lowed by the Wittig reaction with (1-carbethoxy-
ethylidene)triphenylphosphorane afforded the trisubsti-
tuted unsaturated ester (5). The ester (5) was reduced
with DIBAL, followed by oxidation with manganese
dioxide to give an aldehyde, which was immediately
subjected to Horner–Emmons reaction with triethyl
phosphonoacetate in the presence of sodium hydride to
afford the di-TBS ether of segment B (6). The geometry
of two double bonds contained in 6 was shown to be
both E from the observation of NOE (H-2% and H3-9%;
H3-9% and H2-10%; H-3% and H-5%), the coupling constant
(J2%,3%=15.6 Hz), and high-field resonance of the C-9%
methyl carbon (lC 12.7). Deprotection of the TBS
groups of 6 followed by treatment with (S)- and (R)-
MTPA chloride in pyridine gave di-(R)- and di-(S)-
MTPA esters of segment B (7 and 8), respectively. On
the other hand, di-(R)- and di-(S)-MTPA esters (9 and
10, respectively) of natural product TT-1 (1) were also
prepared by treatment with (R)- and (S)-MTPA acid
with DCC and pyridine in CH2Cl2, respectively. Com-
parison of the 1H NMR chemical shift data of the
MTPA esters (7/8 and 9/10) is summarized in Table 1.
Even though the alcohol moieties of the C-1% ester
group are different between 7/8 (=ethyl group) and
ene protons on the C-10% position of di-(R)-MTPA
esters (7 and 9) resonated equivalently at lH 4.20 (2H),
while the C-10% methylene protons of di-(S)-MTPA
esters (8 and 10) were observed unequivalently [8: 4.12
(1H, dd, J=10.6 and 5.6 Hz) and 4.26 (1H, dd, J=10.6
and 6.8 Hz); 10: 4.11 (1H, dd, J=10.9 and 5.6 Hz) and
4.25 (1H, dd, J=10.9 and 6.5 Hz)].6 These results
implied that the synthetic segment B derivative (6) had
the same absolute configuration at the C-6% position as
that of natural product (1). If the synthetic compound
had the opposite absolute configuration, 1H NMR
chemical shift data of synthetic di-(R)- and (S)-MTPA
esters had to be identical with those of natural di-(S)-
and (R)-MTPA esters, respectively. Since the synthetic
compound (6) had the 6%S-configuration, the absolute
configuration of C-6% position of 1 was concluded as S.
2.2. Segment A
Compound 1 contained four chiral centers in segment
A, and two of them (C-5 and C-6) are located vicinally
on the a-pyrone ring. The coupling constant (J5,6=2.5
Hz) and observation of NOE between H-5 and H-6
suggested that these two hydrogens (H-5 and H-6) were
cis.1
1
9/10 (=segment A), it was quite apparent that the H
The remaining two other chiral centers (C-7 and C-9)
consisted in a 1,3-dimethyl system in an acyclic side
chain. As a result of literature search7,8 and analysis of
synthetic compounds prepared by us9 (Fig. 1), we pro-
posed a hypothesis on the relative stereochemistry of
NMR chemical shifts of synthetic and natural di-(R)-
MTPA esters (7 and 9, respectively) coincided well with
1
each other, and the H NMR chemical shifts of syn-
thetic and natural di-(S)-MTPA esters (8 and 10,
respectively) were also parallel. Particularly, the methyl-
1
acyclic 1,3-dimethyl systems as follows. The H NMR
1
Table 1. Comparison of H NMR data of segment B parts of synthetic and natural MTPA esters (7–10) in CDCl3
Position
Synthetic di-(R)-MTPA ester Natural di-(R)-MTPA ester
Synthetic di-(S)-MTPA ester
(8)
Natural di-(S)-MTPA ester
(10)
(7)
(9)
2%
3%
5%
6%
7%
8%
5.80 (d, J=15.6)
7.20 (d, J=15.6)
5.48 (d, J=10.1)
2.95 (m)
1.62 (m) and 1.93 (m)
4.11 (m)
5.78 (d, J=15.7)
7.23 (d, J=15.7)
5.53 (d, J=10.2)
2.95 (m)
1.62 (m) and 1.93 (m)
4.09 (m)
5.80 (d, J=15.9)
7.20 (d, J=15.9)
5.50 (d, J=10.1)
2.91 (m)
1.61 (m) and 1.91 (m)
4.06 (m)
5.78 (d, J=15.7)
7.23 (d, J=15.7)
5.55 (d, J=9.0)
2.90 (m)
1.60 (m) and 1.91 (m)
4.04 (m)
4.33 (quint, J=5.4)
1.58 (3H, s)
4.33 (quint, J=5.4)
1.57 (3H, s)
4.41 (quint, J=5.4)
1.58 (3H, s)
4.40 (quint, J=5.4)
1.57 (3H, s)
9%
10%
4.20 (2H, d, J=6.1)
4.20 (2H, m)
4.12 (dd, J=10.6, 5.6)
4.26 (dd, J=10.6, 6.8)
4.11 (dd, J=10.9, 5.6)
4.25 (dd, J=10.9, 6.5)

K. Akiyama et al. / Tetrahedron Letters 44 (2003) 8427–8431
8429
Figure 1. 1H NMR chemical shifts of methylene protons located between two methyl-bearing methine carbons in acyclic
1,3-dimethyl systems (in CDCl3).7–9
Scheme 2. Reagents and conditions: (a) (i) benzyl 2,2,2-trichloroacetimidate, TfOH, cyclohexane/CH2Cl2 (1:1), rt, 4 h; (ii)
NaOHaq, MeOH, rt, 1 h (68% for 2 steps); (iii) (COCl)2, DMSO, Et3N, CH2Cl2, −78°C, 2 h; (iv) (EtO)2P(O)CH2CO2Et, NaH,
DME, 0°, 2h (91% for 2 steps); (b) AD-mix-b, CH3SO2NH2, t-BuOH/H2O (1:1), rt, 40 h (78%); (c) (CH3)2C(OCH3)2, TsOH,
acetone, rt, 1 h (96%); (d) (i) LiAlH4, THF, rt, 0.5 h; (ii) TBSCl, imidaole, DMF, rt, 2 h (94% for 2 steps); (iii) H2, Pd/C, EtOH,
rt, 18 h (91%); (iv) I2, Ph3P, imidazole, benzene, rt, 2 h (97%); (e) 2-bromo-cis-2-butene, Li, THF, 0°C to rt, 18 h (58%); (f) (i)
TBAF, THF, rt, 1 h; (ii) (COCl)2, DMSO, Et3N, CH2Cl2, −78°C, 2 h; (iii) (CF3CH2O)2P(O)CH2CO2Me, KHMDS, 18-crown-6,
THF, −78°C, 2 h (75% for 3 steps); (g) TsOH, MeOH (87%); (h) AD-mix-a, CH3SO2NH2, t-BuOH/H2O (1:1), rt, 43 h (82%).
ously described for 1,3-diol or 1,3-dimethoxy sys-
tems.10,11 In the case of natural product (1), the
corresponding two methylene protons on C-8 were
observed non-equivalently (lH 1.04 and 1.20; Dl=
0.16), suggesting that the relative stereochemistry of the
1,3-dimethyl groups on C-7 and C-9 was syn. From the
discussions described above, 5,6-cis and 7,9-syn were
proposed for the relative stereochemistry of segment A.
chemical shifts of the methylene protons located
between the two methyl-bearing methine carbons in
acyclic 1,3-dimethyl systems may be diagnostic for the
relative stereochemistry (syn or anti ) of 1,3-dimethyl
systems: ‘the two methylene protons of syn
diastereomers were magnetically non-equivalent (Dl=
0.1–0.6), while those of anti diastereomers resonated
equivalently (Dl=0).’ Similar arguments were previ-

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K. Akiyama et al. / Tetrahedron Letters 44 (2003) 8427–8431
1
Table 2. Comparison of H and 13C NMR data of natural and synthetic 5-membered lactones (13 and 14) in CDCl3
Position
Synthetic 13 lH
Natural 13 lH
Synthetic 14 lH
Synthetic 13 lC
Natural 13 lC
Synthetic 14 lC
2
3
4
5
6
7
8
–
–
–
172.7
172.7
122.9
153.4
85.6
75.1
33.5
172.9
122.5
154.2
84.1
76.2
34.7
6.19 (dd, J=5.8, 2.1) 6.19 (dd, J=5.8, 2.1) 6.18 (dd, J=5.8, 2.1) 122.9
7.43 (dd, J=5.8, 1.5) 7.43 (dd, J=5.8, 1.4) 7.46 (dd, J=5.8, 2.1) 153.3
5.11 (dt, J=5.8, 1.8) 5.11 (dt, J=5.8, 1.6) 5.16 (quint, J=2.1)
3.50 (brs)
1.82 (m)
1.06 (m)
85.5
75.1
33.4
41.1
3.50 (brs)
1.82 (m)
1.06 (m)
3.47 (brs)
1.86 (m)
1.14 (m)
41.2
40.1
1.47 (m)
1.47 (m)
1.50 (m)
9
10
1.65 (m)
1.69 (m)
1.66 (m)
1.70 (m)
1.66 (m)
1.69 (m)
27.8
47.5
27.9
47.5
28.5
47.0
2.00 (dd, J=12.8,
4.1)
2.00 (dd, J=12.9,
4.1)
2.06 (dd, J=12.7,
4.2)
11
12
13
14
15
16
–
–
–
134.2
120.2
13.3
14.7
20.0
15.5
134.3
120.2
13.4
14.8
20.1
15.6
134.4
120.2
13.3
16.6
20.9
15.6
5.18 (q, J=6.7)
1.57 (d, J=6.7)
1.03 (d, J=6.7)
0.79 (d, J=6.3)
1.55 (s)
5.18 (q, J=6.7)
1.57 (d, J=6.7)
1.03 (d, J=6.6)
0.79 (d, J=6.3)
1.55 (s)
5.18 (d, J=6.7)
1.58 (d, J=6.7)
1.05 (d, J=6.6)
0.85 (d, J=6.3)
1.56 (s)
Thus, four diastereomers (11 and 12, and their enan-
tiomers) remain to be likely out of sixteen possibilities
for the absolute configuration of segment A. We then
studied the synthesis of two possible diastereomers (11
and 12) as optically active forms to determine the
absolute configurations by comparison of their spectral
and optical data with those of the natural specimen. In
our synthetic studies as shown in Scheme 2, the
diastereomers (13 and 14) with a 5-membered lactone
were eventually obtained in place of 11 and 12 with a
6-membered lactone. On the other hand, the 5-mem-
bered lactone (13) was also obtained by hydrolysis of
natural product (1). We therefore were able to complete
our purpose of determination of the stereochemistry of
segment A by using the data of these 5-membered
lactones.
oxidation and Still’s variant of the Horner–Emmons
reaction19 afforded the Z-unsaturated ester (21, J3,4
11.7 Hz). Treatment of 21 with TsOH in methanol
afforded the 5-membered lactone (13) and no 6-mem-
bered lactone (11) was obtained.20,21
=
The other diastereomer 14 with 5S,6S,7S,9R-configura-
tion was also prepared by similar procedures from the
diastereomeric diol (22) which was obtained through
asymmetric dihydroxylation of the unsaturated ester
(16) using AD-mix-a.16
On the other hand, alkaline hydrolysis of natural
product (1) was carried out [(i) 0.1 N KOH–Et2O (1:4),
rt, 28 h; (ii) HCl/MeOH (pH 3.5), rt, 18 h], and it was
found that a 5-membered lactone was obtained in 44%
1
yield for the segment A part.22 The H NMR chemical
shift data of the 5-membered lactone obtained from the
natural specimen were compared with those of the two
diastereomers (13 and 14) to reveal that the relative
stereochemistry of 5-membered lactone obtained from
the natural specimen was identical with the
diastereomer (13) (Table 2). The optical rotations of
natural-13 and synthetic-13 were found to be both
dextrorotatory, and both of them showed positive Cot-
ton effects in their CD spectra,23 indicating that natu-
ral-13 and synthetic-13 were optically identical (not
enantiomers). Thus, the absolute configurations of TT-
1 (1=rasfonin) were established as 5R,6R,7S,9R, and
6%S (Fig. 2).
The diastereomer 13 with 5R,6R,7S,9R-configurations
was prepared as shown in Scheme 2. The known
monoacetate (15), which was prepared from methyl
dimethylmalonate12,13 through enantioselective acetyla-
tion with lipase AK,7,14 was converted into an E-unsat-
urated ester (16, J4,5=15.6 Hz)15 through four-step
reactions [(1) benzylation, (2) hydrolysis, (3) Swern
oxidation, and (4) Horner–Emmons reaction]. The
asymmetric dihydroxylation of ester (16) with AD-mix-
b16 led to the a,b-dihydroxy ester (17), which was
protected with an acetonide to give 18. The LiAlH4
reduction of 18, followed by protection with TBS ether,
hydrogenolysis of benzyl ether, and treatment with
iodine and triphenylphosphine afforded the iodide (19).
The iodide (19) was treated with the alkenyllithium
reagent17 derived from 2-bromo-cis-2-butene to give the
E-alkene [20, lC 13.4 (C-13) and 15.5 (C-16)18]. Depro-
tection of the TBS group of 20 followed by Swern
Figure 2. Structure of 1 including absolute stereochemistry.

K. Akiyama et al. / Tetrahedron Letters 44 (2003) 8427–8431
8431
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Acknowledgements
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We are grateful to Amano Pharmaceutical Co. for a
generous gift of lipase AK and to Dr. Toshiaki Nagata
(Takeda Chemical Industries, Ltd.) and Professor
Katsuo Ohkata (Hiroshima University) for valuable
discussions. This work was partly supported by a
Grant-in-Aid from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
15. The numberings of the synthetic intermediates also refer
to those of the natural product (1).
16. Sharpless, K. B.; Amberg, W.; Beller, M.; Chen, H.;
Hartung, J.; Kawanami, Y.; Lubben, D.; Manoury, E.;
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18. The 13C NMR chemical shifts for the corresponding
methyl groups of 3-methyl-2Z-hexene [lC 12.0 (C-1) and
22.1 (3-Me)] and 3-methyl-2E-hexene [lC 12.0 (C-1) and
14.2 (3-Me)]: Dorman, D. E.; Jautelat, M.; Roberts, J. D.
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4. The nitrile (4) was transformed into (2S)-1-benzyloxy-2-
methylbutane (23) by 6 steps [(1) DIBAL, CH2Cl2,
−78°C, 2 h, (2) NaBH4, EtOH, rt, 1 h (67% in 2 steps),
(3) NaH, BnBr, DMF, 0°C, 3 h (66%), (4) TBAF, THF,
rt, 1 h (98%), (5) MsCl, pyridine, rt, 4 h (72%), (6)
19. Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24,
4405–4408.
20. The 13C NMR of the 5-membered lactone (13) showed
signals due to the 5-membered lactone moiety at lC 172.7
(C-2), 122.9 (C-3), 153.4 (C-4), 85.5 (C-5), and 75.0 (C-6).
The 13C NMR data in the literature21 for the correspond-
ing positions of the 5-membered and 6-membered lac-
tones (24 and 25) are as follows: 24 (5-membered): lC
172.8 (C-2), 122.3 (C-3), 153.8 (C-4), 85.7 (C-5), and 74.7
(C-6); 25 (6-membered): lC 162.9 (C-2), 123.8 (C-3),
142.6 (C-4), 66.2 (C-5), and 80.6 (C-6).
LiAlH4, THF, rt,
1 h (87%)]. (2S)-1-benzyloxy-2-
methylbutane (23) was also prepared from commercially
available (2S)-2-methyl-1-butanol by 1 step [NaH, BnBr,
DMF, 0°C, 3 h (72%)]. The optical rotation data were as
follows: 23 from nitrile (4): [h]2D7 +4.2 (c 0.21, CHCl3); 23
from commercially available (2S)-2-methyl-1-butanol:
[h]2D7 +4.3 (c 2.1, CHCl3); literature value of the enan-
tiomer, (2R)-1-benzyloxy-2-methylbutane (ent-23): [h]D27
−4.5 (c 3.23, CHCl3).5 Thus, the absolute configuration of
the nitrile (4) generated through inversion of configura-
tion was established.
21. Sa´nchez-Sancho, F.; Valverde, S.; Herrado´n, B. Tetra-
hedron: Asymmetry 1996, 7, 3209–3246.
22. The product corresponding to segment B was not isolated
at this time from the hydrolysis products of 1.
23. Synthetic 13: [h]D24 +31 (c 0.30, CHCl3); CD (MeOH) Do
+5.2 (214 nm); Natural 13: [h]2D4.5 +87 (c 0.76, CHCl3);
CD (MeOH) Do +16.2 (215 nm).
5. White, J. D.; Johnson, A. T. J. Org. Chem. 1994, 59,
3347–3358.