Synthesis of plakevulin A and structure-activity relationships of its related compounds against DNA polymerases

Article abstract of DOI:10.1016/j.tet.2006.06.023

Synthesis of plakevulin A and structure-activity relationships of its related compounds against DNA polymerases is described. We have achieved a total synthesis and revised the structure of plakevulin A. Several analogues including untenone A, manzamenone

Full text of DOI:10.1016/j.tet.2006.06.023

Tetrahedron 62 (2006) 8006–8015
Synthesis of plakevulin A and structure–activity relationships
of its related compounds against DNA polymerases
Kouji Kuramochi,a Fumiyo Saito,b Ryo Takeuchi,a Tomohiro Era,b Masaharu Takemura,c
Jun’ichi Kobayashi,d Kengo Sakaguchi,a,e Susumu Kobayashib,e and Fumio Sugawaraa,e,
*
aDepartment of Applied Biological Science, Tokyo University of Science (RIKADAI), 2641 Yamazaki, Noda, Chiba 278-8510, Japan
bFaculty of Pharmaceutical Sciences, Tokyo University of Science (RIKADAI), 2641 Yamazaki, Noda, Chiba 278-8510, Japan
cDepartmentofBiology, Faculty ofScience, TokyoUniversityofScience(RIKADAI),1-3 Kagurazaka,Shinjuku-ku, Tokyo162-8601, Japan
dGraduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan
eFrontier Research Center for Genome and Drug Discovery, Tokyo University of Science (RIKADAI), 2641 Yamazaki,
Noda, Chiba 278-8510, Japan
Received 11 May 2006; revised 7 June 2006; accepted 8 June 2006
Available online 27 June 2006
Abstract—Synthesis of plakevulin A and structure–activity relationships of its related compounds against DNA polymerases is described.
We have achieved a total synthesis and revised the structure of plakevulin A. Several analogues including untenone A, manzamenone A,
and optically active plakevulin A, were prepared and tested with an enzyme inhibition assay for mammalian DNA polymerases. The effect
of the methyl ester moiety, and the substituents at the 1- and 4-positions of plakevulin A on DNA polymerase activities are discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
O
OCH3
OH
O
OCH3
OH
1. Introduction
O
5
4
HO
O
1
The cytotoxic oxylipin, plakevulin A (1), was isolated from
an Okinawan sponge Plakortis sp by Kobayashi et al.1 Com-
pound 1 exhibited cytotoxicity against murine leukemia
L1210 and epidermoid carcinoma KB cells (Fig. 1). It was
also reported that 1 inhibited the activities of DNA polymer-
ases (pol) a and g. Although the proposed structure was the
levulinyl ester as depicted for 1a, our synthetic studies and
enzyme-inhibitory assays revealed that the structure of
plakevulin A is actually as shown for 1.2 Recently a synthesis
of optically active (+)-1 has been reported by Honda et al.3
(CH2)15CH3
(CH2)15CH3
O
2
3
Plakevulin A
(1, the revised structure)
Plakevulin A
(1a, the proposed structure)
O
OCH3
(CH2)15CH3
H
O
OCH3
OH
CH3(CH2)15
O
O
OCH3
(CH2)15CH3
H
O
O
OH
Untenone A (2)
Manzamenone A (3)
Untenone A (2), which inhibited the cell proliferation of
L1210 cells, was isolated from the genus Plakortis.4,5 The
structurally related manzamenone A (3), a unique dimeric
fatty acid derivative, was also isolated from the Plakortis.6,7
Untenone A has been considered to be a plausible inter-
mediate in the biosynthetic pathways of manzamenone A
and plakevulin A.1,7 Both 2 and 3 were found to inhibit
mammalian pol a,b, and human terminal deoxynucleotidyl
transferase (TdT).8,9
Figure 1. The revised structure of plakevulin A (1), the proposed structure
of plakevulin A (1a), untenone A (2), and manzamenone (3).
2. Results and discussions
2.1. Synthesis and structural revision of plakevulin A
Our synthetic approach towards the proposed structure of
plakevulin A (1a) was based on the assumed biosynthetic
pathway.1,2,7 The reduction of untenone A (2), followed by
esterification of the resulting alcohol would provide 1a.
Our route to synthesize untenone A was based on the modi-
fied protocol reported by Yamada et al.5b
In this article, we fully describe our synthetic studies and
biological evaluation of a series of plakevulin A analogues.
Keywords: Plakevulin A; Untenone A; Manzamenone A; DNA polymerase.
* Corresponding author. Tel.: +81 4 7124 1501x3400; fax: +81 4 7123
9767; e-mail: sugawara@rs.noda.tus.ac.jp
Protection of the alcohol (ꢀ)-4 with TMSOTf and i-Pr2NEt
gave a TMS ether (ꢀ)-5 (Scheme 1). Methoxycarboxylation
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.06.023

K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
8007
O
OCH3
OTMS
(CH2)15CH3
OH
OTMS
O
a
b
O
O
(CH2)15CH3
(CH2)15CH3
( )-4
( )-6
( )-5
O
O
OCH3
OCH3
OTMS
(CH2)15CH3
c
OTMS
(CH2)15CH3
HO
HO
+
( )-7
( )-8
Scheme 1. Synthesis of the key intermediate (ꢀ)-7. (a) TMSOTf, i-Pr2NEt, CH2Cl2, 0 ꢁC, 90%; (b) LDA, THF/HMPA, then NCCO2CH3, ꢂ42 ꢁC, 63%
(dr¼4.2:1) and (c) DIBAL, CH2Cl2, ꢂ78 ꢁC, 41% for (ꢀ)-7 and 6% for (ꢀ)-8.
of (ꢀ)-5 with LDA and NCCO2CH3 afforded (ꢀ)-6 as a
4.2:1 mixture of inseparable diastereomers in 63% yield.5c
Reduction of (ꢀ)-6 with DIBAL (2 equiv) in CH2Cl2 gave
(ꢀ)-7 and (ꢀ)-8 in 41% and 6% yields, respectively, as the
identified products. In this reaction, b-hydroxyaldehyde was
obtained as the major byproduct, as a mixture that proved
difficult to separate. When 1 equiv of DIBAL was used, no
reaction occurred. Although the use of other reducing agents
such as NaBH4–CeCl3, LiBH4, ZnBH4, and (i-PrO)3Al/
i-PrOH was examined, none or only a trace amount of (ꢀ)-7
was obtained. The stereochemistry of the major isomer (ꢀ)-7
was determined by its NOESY spectrum (Fig. 2a). The
NOESY correlation between H-1 and H-5 in (ꢀ)-7 indicated
the syn relation for H-1/H-5. The anti relation for 4-OTMS
and H-5 in (ꢀ)-7 was determined by the NOESY correla-
tions between H-5 and H-6, and between 4-OTMS and H-23.
hand, the esterification of (ꢀ)-7 with levulinic acid by
EDCI, followed by deprotection of the TMS ether afforded
(ꢀ)-11, the 1-epi-isomer of 1a.
Selected 1H and 13C NMR spectral data of (ꢀ)-1a and
(ꢀ)-11 are summarized in Table 1. As shown in Table 1,
1
the H and 13C NMR spectral data of (ꢀ)-1a and (ꢀ)-11
were different from those of the natural plakevulin A. In par-
ticular, both the proton and carbon signals at C-1 in (ꢀ)-1a
appeared further downfield from those in the natural plake-
vulin A. These observations suggested that the natural plake-
vulin A is not the levulinyl ester, but the delevulinyl form.
Based on these considerations, the removal of the levulinyl
moiety of (ꢀ)-1a was attempted (Scheme 3). Treatment of
(ꢀ)-1a with hydrazine in pyridine and acetic acid gave the
1
alcohol (ꢀ)-1 in 92% yield.11 The H NMR and 13C NMR
The esterification of (ꢀ)-7 was attempted with both inver-
sion and retention of the stereochemistry at C-1 (Scheme 2).
The Mitsunobu esterification of (ꢀ)-7 with levulinic acid
afforded (ꢀ)-9 in 52% yield.10 The observed NOEs between
H-1 and 4-OTMS and between H-1 and H-23 of (ꢀ)-9 indi-
cated that the configuration of 9 was 1S*,4S*,5R* (Fig. 2b).
Deprotection of TMS ether (ꢀ)-9 with TBAF provided the
proposed structure of plakevulin A (1a). On the other
spectral data of synthetic (ꢀ)-1 were in good agreement
with those of the natural plakevulin A except for the peaks
derived from levulinic acid. Therefore the sample of the
natural plakevulin A could be estimated to be an 1:1 mixture
of (+)-1 and levulinic acid. On the other hand, deprotection
of TMS ether (ꢀ)-7 with TBAF afforded (ꢀ)-12 in 64%
1
yield. The H NMR and 13C NMR spectral data of (ꢀ)-12
were actually different from those of 1.
(a)
(b)
2.2. Synthesis of (D)- and (L)-plakevulin A,
and (D)- and (L)-untenone A
23
O
OCH3
23
CO2CH3
5
O
H
OTMS
O
OTMS
Since natural plakevulin A is optically active ([a]2D5 +19 (c
2.0, CHCl3),1 (+)-plakevulin A was synthesized from 13.5b
Compound 13 was prepared from (S)-(tert-butyldimethyl-
silyloxy)-2-cyclopentenone (99% ee by a chiral HPLC),
which was derived from cis-3,5-diacetoxycyclopent-1-ene
HO
H
4
1
4
1
H
R
O
CH2(CH2)14CH3
6
( )-7
( ) (R = C16H33
-9
)
Figure 2. The NOESY correlations for (ꢀ)-7 (left) and (ꢀ)-9 (right).
O
OCH3
O
OCH3
OTMS
(CH2)15CH3
O
O
O
OH
O
O
O
b
d
a
c
(CH2)15CH3
O
O
( )-1a
( )-9
( )-7
O
OCH3
O
OCH3
OH
O
OTMS
O
(CH2)15CH3
(CH2)15CH3
O
O
( )-10
( )-11
Scheme 2. Synthesis of the proposed structure of (ꢀ)-plakevulin A (1a) and (ꢀ)-11. (a) levulinic acid, DIAD, PPh3, toluene, 52%; (b) TBAF, THF, 0 ꢁC, 94%;
(c) levulinic acid, EDCI, DMAP, 1,4-dioxane, 83% and (d) TBAF, THF, 0 ꢁC, 49%.

8008
K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
Table 1. The selected 1H and 13C NMR spectral data of the natural plake-
vulin A, (ꢀ)-1a, and (ꢀ)-11
(ꢂ)-5 in 90% yield (99% ee by a chiral HPLC). Treatment of
(ꢂ)-5 with LDA followed by NCCO2CH3 in THF/HMPA
gave 6 in a 4.2:1 diastereomeric mixture.
23
23
22
22
O
O
OCH3
OCH3
O
4'
O
4'
5
4
5
4
5'
3'
1'
5'
3'
1'
OH
OH
O
O
Reduction of 6 with DIBAL afforded (ꢂ)-7 and (+)-8 in 36%
and 5%, respectively (Scheme 5). Esterification of (ꢂ)-7
with p-nitrobenzoic acid under Mitsunobu conditions gave
(+)-15 in 61% yield. Methanolysis of the p-nitrobenzoate
15 and spontaneous migration of TMS group provided
1-O-TMS ether 16. Finally desilylation of (+)-16 with
TBAF afforded (+)-plakevulin A (1) in 73% yield. The opti-
cal rotation of our synthetic (+)-1 ([a]2D1 +27.1 (c 0.55,
CHCl3)) was slightly higher than that of natural 1 ([a]D25
+19 (c 2.0, CHCl3)),1 and almost the same value as that
of the synthetic (+)-1 reported by Honda et al. ([a]2D2 +24.1
(c 0.6, CHCl3)).3 Compound (+)-1 was also obtained by
desilylation of (+)-8 with TBAF.
2'
1
2'
1
(CH2)15CH3
(CH2)15CH3
O
O
2
3
6
23
2
3
6
23
( )-1a (the proposed structure)
1H NMR Natural plakevulin A 1a
( )-11
11
d (m, Hz)
d (m, Hz)
d (m, Hz)
1
5.34 ddd, 5.2,
1.7, 1.5
6.04 ddd, 4.4,
1.5, 0.8
5.82 br d, 6.9
2
3
5
6
23
20
30
50
5.92 dd, 5.6, 1.8
5.85 dd, 5.6, 1.5
2.82 d, 5.2
1.81 m
5.91 dd, 5.4, 1.5 5.90 dd, 5.6, 2.1
5.94 dd, 5.4, 0.8 6.08 d, 5.6
2.96 d, 4.4
1.80 m
3.76 s
3.22 dd, 6.9, 1.0
1.65 m
3.75 s
3.78 s
2.63 t, 6.4
2.75 t, 6.4
2.20 s
2.56 m
2.75 m
2.18 s
2.55 t, 6.5
2.72 m
2.19 m
Since deprotection of TMS ether 6 could give optically
active (ꢂ)-untenone A (2), deprotection of 6 was attempted
(Scheme 6). First, treatment of 6 with TBAF in THF gave 2
as a racemic form ([a]2D3w0 (c 0.25, CHCl3)). Although the
formation of the acylic b-ketoester could not be observed
in this reaction, the basic conditions would induce the retro-
aldol reaction of the b0-hydroxy-b-ketoester and promote the
racemization of 2. Thus deprotection of 6 was performed
under acidic conditions. Treatment of 6 with a catalytic
amount of concd HCl in methanol gave optically active
(ꢂ)-untenone A (2) in 94% yield. The optical rotation of
our synthetic (ꢂ)-2 ([a]D23 ꢂ71.3 (c 0.94, CHCl3)) was iden-
tical with those reported in the literature ([a]2D7 ꢂ73.3 (c 1.2,
CHCl3))5c by Asami et al., and [a]D26 ꢂ79.7 (c 1.0, CHCl3) by
Honda et al.3
13C NMR
1
2
3
4
78.2
135.7
136.9
84.9
80.9
131.5
139.8
85.3
76.8
129.4
142.2
83.4
5
6
60.6
40.6
57.7
40.8
53.9
40.4
22
23
10
20
30
40
50
172.7
52.1
177.6
27.6
37.7
171.8
52.2
172.4
27.9
37.8
171.5
52.1
172.0
28.0
37.7
206.5
29.7
206.3
29.8
206.3
29.8
O
OCH3
OH
O
OCH3
OH
O
(ꢂ)-Plakevulin A (1) ([a]2D1 ꢂ25.7 (c 0.10, CHCl3)) and
(+)-untenone A (2) ([a]D23 +72.2 (c 0.50, CHCl3)) were pre-
pared starting from 175b according to the same procedures
(Scheme 7).
a
b
O
HO
HO
(CH2)15CH3
(CH2)15CH3
O
( )-1a
( )-1
O
OCH3
OTMS
(CH2)15CH3
O
OCH3
OH
2.3. Preparation of ( )-untenone A, ( )-manzamenone
A, and untenone A derivatives
HO
(CH2)15CH3
( )-12
( )-7
In order to examine the structure–activity relationships, we
prepared a number of untenone A derivatives.9
Scheme 3. Synthesis of the alcohol (ꢀ)-1 and (ꢀ)-12. (a) NH2NH2$H2O,
pyridine/AcOH, 92% and (b) TBAF, THF, 64%.
(ꢀ)-Untenone A (2) was prepared from (ꢀ)-6, from which in
turn (ꢀ)-manzamenone A (3) was prepared by heating via
a unique biogenetic pathway reported by Whitehead et al.
(Scheme 8).7
(Scheme 4).12 Desilylation of 13 with TBAF afforded (ꢂ)-
14 in 82% yield. Oxidation of (ꢂ)-14 with Jones reagent, fol-
lowed by protection of the tert-alcohol as a TMS ether gave
OH
OH
(CH2)15CH3
OH
(CH2)15CH3
TBSO
a
HO
O
b
(CH2)15CH3
13
(–)-14
(–)-4
O
OCH3
OTMS
(CH2)15CH3
OTMS
(CH2)15CH3
O
O
c
d
(–)-5
6
(dr = 4.2:1)
Scheme 4. Synthesis of the key intermediate 6. (a) TBAF, THF, 84%; (b) Jones reagent, acetone, 77%; (c) TMSOTf, i-Pr2NEt, CH2Cl2, 0 ꢁC, 92% and (d) LDA,
THF/HMPA, then NCCO2CH3, ꢂ42 ꢁC, 69% (dr¼4.2:1).

K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
8009
O
OCH3
OTMS
(CH2)15CH3
O
OCH3
OTMS
O
OCH3
OTMS
(CH2)15CH3
(+)-8
a
O
HO
HO
+
(CH2)15CH3
6
(–)-7
(dr = 4.2 : 1)
O
OCH3
OTMS
O
OCH3
OH
O2N
c
b
(–)-7
O
TMSO
(CH2)15CH3
(CH2)15CH3
O
(+)-15
(+)-16
O
OCH3
OH
d
HO
e
(+)-8
(+)-1
(CH2)15CH3
(+)-1
Scheme 5. Synthesis of (+)-plakevulin A (1). (a) DIBAL, CH2Cl2, ꢂ78 ꢁC, 36% for (ꢂ)-7 and 5% for (+)-8; (b) DIAD, PPh3, p-nitrobenzoic acid, THF, 61%;
(c) NaOCH3, CH3OH, 81%; (d) TBAF, THF, 73% and (e) TBAF, THF, 52%.
O
OCH3
H
O
OCH3
OTMS
(CH2)15CH3
O
OCH3
OH
(CH2)15CH3
O
OCH3
OH
TBAF
THF
CH3(CH2)15
O
O
O
a
b
(CH2)15CH3
O
( )-6
OCH3
(CH2)15CH3
6
H
OH
O
( )-2
[α]D23 = 0 (c 0.25, CHCl3)
(dr = 4.2:1)
O
( )-2
Scheme 8. Synthesis of (ꢀ)-2 and (ꢀ)-3. (a) concd HCl, CH3OH and (b) 6.
( )-3
O
OCH3
OH
O
OCH3
(CH2)15CH3
O
OCH3
OMOM
(CH2)15CH3
conc. HCl
6
O
CH3OH
94%
a
(dr = 4.2:1)
(CH2)15CH3
O
O
(–)-2
[α]D23 = -71.3 (c 0.94, CHCl3)
lit. [α]D26 = -79.7 (c 0.94, CHCl3)
20
( )-19
O
OCH3
(CH2)15CH3
Scheme 6. Deprotection of TMS ether of 6. (a) TBAF, THF and (b) concd
HCl (cat), CH3OH, 94%.
b
O
( )-19
Hydrogenation of the double bond and elimination of the
methoxymethyloxy group occurred by treatment of (ꢀ)-
195b with 10% Pd on carbon under an H2 atmosphere to
yield 20 (Scheme 9). On the other hand, treatment of
(ꢀ)-19 with Pd(OH)2 under an H2 atmosphere afforded
(ꢀ)-21. Compound (ꢀ)-21 was produced by further hydro-
genation and the isomerization of the b-ketoester. The stereo-
chemistry of (ꢀ)-21 was established by the NOESY
( )-21
Scheme 9. Synthesis of (ꢀ)-20 and (ꢀ)-21. (a) H2, Pd/C, EtOAc, quant. and
(b) H2, Pd(OH)2/C, EtOAc, quant.
experiment (Fig. 3). The NOESY correlations between
H-3a and H-5, H-3a and H-6, and H-5 and H-6, and the
NOESY correlation between H-3b and H-4 indicated the
(CH2)15CH3
(CH2)15CH3
a
OH
(CH2)15CH3
OH
b
O
TBSO
HO
OH
(+)-4
17
(–)-18
O
OCH3
O
OCH3
(CH2)15CH3
OH
(CH2)15CH3
OH
O
HO
(+)-2
(–)-1
Scheme 7. Synthesis of (ꢂ)-1 and (+)-2. (a) TBAF, THF, 77% and (b) Jones reagent, acetone, 0 ꢁC, 66%.

8010
K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
Table 3. The IC50 values for enzymatic inhibition of DNA polymerase
a (pol a) and b (pol b), and human terminal deoxynucleotidyl transferase
(TdT) by (ꢀ)-2, (ꢀ)-3, (ꢀ)-4, (ꢀ)-19, 20, and (ꢀ)-21
CO2CH3
CO2CH3
5
H
5
H
O
O
Compounds
IC50 (mM)
4
1
1
3
3
R
CH2(CH2)14CH3
Pol a
4.3
1.9
>200
5.9
17
20
Pol b
TdT
16
2.5
>200
18
6
H
H
(ꢀ)-2
(ꢀ)-3
(ꢀ)-4
(ꢀ)-19
20
57
3.2
>200
9.3
107
90
( )-21 (R=C16H33
)
( )-21
Figure 3. The NOESY correlations for (ꢀ)-21.
129
84
(ꢀ)-21
anti-relation for H-4/H-5. Reduction of the double bond
of (ꢀ)-19 was unsuccessful by hydrogenation or 1,4-reduc-
tion. Hydrogenation of (ꢀ)-19 with Rh–Al2O3 and PtO2
gave (ꢀ)-21. The use of other conditions (NaBH4/MeOH,
Mg/MeOH, and CuCl, PhMe2SiH/DMF, etc.) gave complex
mixture.
We newly carried out DNA polymerase assays using pol
a and pol b in order to test the influence of the chirality of
plakevulin A (1) and untenone A (2) on the inhibitory activi-
ties. Table 4 shows the inhibitory effects of (+)-1, (ꢂ)-1,
(+)-2, and (ꢂ)-2 against pol a and pol b. The inhibitory
activities of (ꢂ)-1 against these enzymes (IC50¼49 mM for
pol a, IC50¼72 mM for pol b) were slightly stronger than
those of (+)-1 (IC50¼137 mM for pol a, IC50¼189 mM for
pol b). In contrast, there are no significant differences in
the inhibitory activities between (+)-2 and (ꢂ)-2.
2.4. Structure–activity relationships of synthetic
derivatives for inhibition of DNA polymerases
Synthetic (ꢀ)-1, (ꢀ)-1a, (ꢀ)-11, and (ꢀ)-12 were tested
with an enzyme inhibition assay for mammalian DNA poly-
merases a (pol a) and b (pol b). Table 2 shows the value of
50% inhibitory concentrations of these compounds. Com-
pound (ꢀ)-1 inhibited pol a (IC50¼61 mM) and weakly
inhibited pol b (IC50¼179 mM), whereas the levulinyl ester
(ꢀ)-1a did not influence pol a and pol b at concentrations
lower than 200 mM. Interestingly, although (ꢀ)-12 had no
influence on pol a and pol b at concentrations lower than
200 mM, the levulinyl ester (ꢀ)-11 inhibited the activity
of pol a (IC50¼66 mM) and pol b (IC50¼132 mM). These
results indicate that the stereochemistry and its functionality
at C-1 greatly influenced the inhibitory activities against pol
a and pol b.
Table 4. The IC50 values for enzymatic inhibition of DNA polymerase
a (pol a) and b (pol b) by (+)-1, (ꢂ)-1, (+)-2, and (ꢂ)-2
Compounds
IC50(mM)
Pol a
Pol b
(+)-1
(ꢂ)-1
(+)-2
(ꢂ)-2
137
49
13
19
189
72
91
54
Table 2. The IC50 values for enzymatic inhibition of DNA polymerase
a (pol a) and b (pol b) by (ꢀ)-1, (ꢀ)-1a, (ꢀ)-11, and (ꢀ)-12
3. Conclusion
Compounds
IC50(mM)
We have achieved a total synthesis of the proposed structure
of plakevulin A (1a). However, the 1H and 13C NMR spectral
data of 1a were different from those of natural plakevulin A.
The chemical shifts of the proton and the carbon at C-1 of 1a
especially deviated downfield from those of natural plake-
vulin A. Thus 1a was converted into the corresponding alco-
hol (1) by removal of the levulinyl ester. The NMR data of
1 was identical with that of the natural product.
Pol a
Pol b
(ꢀ)-1
66
>200
61
179
>200
132
(ꢀ)-1a
(ꢀ)-11
(ꢀ)-12
>200
>200
The inhibitory activities of (ꢀ)-2, (ꢀ)-3, (ꢀ)-4, (ꢀ)-19, 20,
and (ꢀ)-21 against pol a, pol b, and TdT are summarized in
Table 3. We found that synthetic (ꢀ)-untenone A (2) pos-
sessed selective inhibitory activity against the enzymes
(IC50¼4.3 mM for pol a, IC50¼57 mM for pol b, and
IC50¼16 mM for TdT). (ꢀ)-Manzamenone A (3) was found
to have strong inhibitory activity against all of these enzymes
in the micromolar range. The b-hydroxyketone (4) showed
no inhibitory activity against pol a, pol b, and TdT. Meth-
oxymethyl-protected untenone A (19) showed inhibitory
activity against the enzymes in the submicromolar and
micromolar range, but exhibited nonselective inhibitory
activity against the enzymes when compared to (ꢀ)-2. Both
the a,b-unsaturated b-ketoester (20) and the saturated deoxy-
genated derivative (21) showed weaker inhibitory activities
against polymerases than untenone A (2). These results indi-
cate that the methyl ester moiety and the substituents at C-4
affected the inhibitory activities against pol a, pol b, and TdT.
We have prepared optically active plakevulin A (1) and
untenone A (2) according to the modified protocol reported
by Yamada et al. Compounds (+)-1, (ꢂ)-1, (+)-2, and (ꢂ)-2
were prepared and tested with an enzyme inhibition assay
for mammalian DNA polymerases a (pol a) and b (pol b).
Several analogues were also prepared in order to examine
the structure–activity relationships of 1 in the inhibition of
DNA polymerases. We found that the methyl ester of 1
was important for the inhibitory activity and that the sub-
stituents at C-1 and C-4 greatly influenced the activity.
Although the inhibitory activity of (ꢂ)-1 against pol a and
pol b was slightly more potent than that of (+)-1, there
were no significant differences in the inhibitory activity
between (+)-2 and (ꢂ)-2. Among the synthetic analogues,
manzamenone (3) showed the most potent activity against
pol a, pol b, and TdT.

K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
8011
4. Experimental
oil. 1H NMR (600 MHz, CDCl3) major isomer: d 7.46 (1H,
d, J¼5.8 Hz), 6.27 (1H, d, J¼5.8 Hz), 3.69 (3H, s), 3.38 (1H,
s), 1.87 (1H, m), 1.69 (1H, m), 1.35–1.25 (28H, br m), 0.88
(3H, t, J¼6.8 Hz), 0.11 (9H, s), minor isomer: d 7.55 (1H, d,
J¼5.8 Hz), 6.19 (1H, d, J¼5.8 Hz), 3.76 (3H, s), 3.51 (1H,
s), 1.78 (1H, m), 1.66 (1H, m), 1.35–1.25 (28H, br m),
0.88 (3H, t, J¼6.8 Hz), 0.14 (9H, s); 13C NMR (100 MHz,
CDCl3) major isomer: d 201.4, 167.6, 164.6, 133.5, 81.3,
62.0, 51.9, 41.6, 31.9, 29.7, 29.63 (ꢃ4), 29.60, 29.58,
29.54, 29.48, 29.4, 29.3, 24.1, 22.6, 14.1, 2.2 (ꢃ3), minor
isomer: d 200.6, 168.6, 165.9, 131.7, 83.6, 64.8, 52.1,
38.3, 31.9, 29.9, 29.7, 29.6 (ꢃ6), 29.5 (ꢃ3), 23.8, 22.6,
14.1, 1.9 (ꢃ3); IR (neat) 2925, 2853, 1750, 1718, 1464,
1436, 1340, 1315, 1252, 1151, 1103, 1051, 1009, 941,
757 cmꢂ1; HRMS calcd for C26H48O2NaSi ([M+Na]+)
475.3214, found 475.3189.
4.1. General
1H and 13C NMR were recorded on a JEOL JNM-LD400, or
on a BRUKER DXR400 or DRX600. Chemical shifts were
reported in d, parts per million (ppm), relative to TMS as an
internal standard or calibrated using residual undeuterated
solvent as an internal reference. IR spectra were recorded
on a JASCO FT/IR-410 spectrometer. Mass spectra were
obtained on API QSTAR Pulsar i spectrometer. Optical rota-
tions were measured on a JASCO P-1030 digital polarimeter.
Melting points were determined with Yanaco MP-3S melt-
ing point apparatus. Column chromatography was carried
out on Fuji Silisia PSQ100B. Analytical thin-layer chroma-
tography (TLC) was performed on precoated Merck silica
gel 60 F254 plates, and compounds were visualized by UV
illumination (254 nm) or heating at 150 ꢁC after spraying
phosphomolybdic acid in ethanol. THF was distilled from
sodium/benzophenone. CH2Cl2 was distilled from P2O5.
HMPA and diisopropylamine were distilled from CaH2.
All other solvent and reagents were obtained from commer-
cial sources and used without further purification. Organic
extracts were dried over Na2SO4, filtered, and concentrated
using a rotary evaporator. Involatile oils and solids were
vacuum dried.
4.1.3. (1RS,4SR,5RS)-4-Hexadecyl-1-hydroxy-5-methoxy-
carbonyl-4-trimethylsiloxy-2-cyclopentene, ( )-7. To a
solution of (ꢀ)-6 (200 mg, 0.44 mmol) in CH2Cl2 (5 mL) was
added DIBAL (940 mL of a 0.95 M solution in CH2Cl2,
0.89 mmol) at ꢂ78 ꢁC. After the mixture was stirred at
ꢂ78 ꢁC for 2.5 h, the mixture was diluted with Et2O. Then
1.0 mL of MeOH, followed by Celite was added, and the
mixture was stirred at rt for 1 h. The mixture was filtrated
through Celite and the filtrate was concentrated. The residue
was purified by silica gel column chromatography (9:1/4:1
hexane/EtOAc) to give both (ꢀ)-7 (82 mg, 41%) and (ꢀ)-8
4.1.1. 4-Hexadecyl-4-trimethylsiloxy-2-cyclopenten-1-
one, ( )-5. To a solution of (ꢀ)-5 (637 mg, 1.98 mmol)
and i-Pr2NEt (690 mL, 3.96 mmol) in CH2Cl2 (20 mL) was
added TMSOTf (390 mL, 2.18 mmol) at 0 ꢁC and the mix-
ture was stirred at 0 ꢁC for 15 min. Then the mixture was
quenched by the addition of satd aq NaHCO3 and extracted
with ether. The organic layer was washed with H2O and
brine, dried (Na2SO4), and concentrated. The residue was
purified by silica gel column chromatography (10:1
hexane/EtOAc) to give TMS ether (703 mg, 90%) as a white
solid. 1H NMR (600 MHz, CDCl3) d 7.44 (1H, d, J¼5.7 Hz),
6.11 (1H, d, J¼5.7 Hz), 2.50 (2H, m), 1.68 (2H, m), 1.31–
1.25 (28H, br m), 0.88 (3H, t, J¼6.9 Hz), 0.11 (9H, s); 13C
NMR (100 MHz, CDCl3) d 206.9, 166.9, 132.8, 81.3, 49.6,
41.9, 31.9, 29.9, 29.68 (ꢃ4), 29.65 (ꢃ2), 29.61, 29.55,
29.5, 29.4, 24.3, 22.7, 14.1, 2.1 (ꢃ3); IR (KBr) 2925,
2853, 1726, 1591, 1465, 1407, 1341, 1253, 1200, 1077,
938, 841, 756 cmꢂ1; HRMS calcd for C24H46O2NaSi
([M+Na]+) 417.3159, found 417.3171.
1
(12 mg, 6%) as colorless oil. H NMR (400 MHz, CDCl3)
d 5.94 (1H, dd, J¼5.6 Hz, 2.0 Hz), 5.84 (1H, d, J¼5.6 Hz),
4.72 (1H, m), 4.08 (1H, d, J¼9.5 Hz), 3.70 (3H, s), 3.30
(1H, d, J¼7.1 Hz), 1.72 (1H, m), 1.58 (1H, m), 1.32–1.22
(28H, br m), 0.88 (3H, t, J¼7.2 Hz), 0.09 (9H, s); 13C
NMR (100 MHz, CDCl3) 172.3, 136.9, 135.2, 87.9, 75.0,
56.7, 51.5, 42.5, 31.9, 29.8, 29.68 (ꢃ4), 29.65 (ꢃ2), 29.61,
29.57, 29.5, 29.3, 24.3, 22.7, 14.1, 2.2 (ꢃ3); IR (neat)
3511, 3018, 2926, 2854, 1717, 1466, 1438, 1415, 1360,
1253, 1176, 1099, 1067, 991, 843, 668 cmꢂ1; HRMS calcd
for C26H50O4NaSi ([M+Na]+) 477.3370, found 447.3380.
4.1.4. (1SR,4SR,5RS)-4-Hexadecyl-1-hydroxy-5-methoxy-
carbonyl-4-trimethylsiloxy-2-cyclopentene, ( )-8. 1H NMR
(400 MHz, CDCl3) d 6.09 (1H, d, J¼6.0 Hz), 5.97 (1H, dd,
J¼6.0 Hz, 2.4 Hz), 4.96 (1H, m), 4.03 (1H, d, J¼4.0 Hz),
3.77 (3H, s), 3.07 (1H, d, J¼5.6 Hz), 1.64 (2H, m), 1.32–
1.25 (28H, br m), 0.88 (3H, t, J¼7.2 Hz), 0.11 (9H, s); 13C
NMR (100 MHz, CDCl3) 173.5, 140.7, 132.7, 88.2, 74.4,
58.8, 51.7, 40.7, 31.9, 30.0, 29.7 (ꢃ6), 29.65, 29.6 (ꢃ2),
29.4, 23.9, 22.7, 14.1, 2.1 (ꢃ3); IR (neat) 3471, 3018, 2926,
4.1.2. 4-Hexadecyl-5-methoxycarbonyl-4-trimethyl-
siloxy-2-cyclopenten-1-one, ( )-6. To a solution of diiso-
propylamine (560 mL, 3.92 mmol) in THF (13 mL) was
added n-BuLi (2.5 mL of a 1.58 M solution in hexane,
3.92 mmol) at 0 ꢁC and the mixture was stirred at 0 ꢁC for
10 min. The mixture was cooled to ꢂ78 ꢁC. A solution of
(ꢀ)-5 (703 mg, 1.78 mmol) in THF/HMPA (10:1, 5 mL)
was added to the mixture at ꢂ78 ꢁC and the mixture was
stirred at ꢂ78 ꢁC for 80 min. Then methyl cyanoformate
(340 mL, 4.27 mmol) was added and the mixture was stirred
at ꢂ45 ꢁC for 55 min. The mixture was quenched by the
addition of satd NH4Cl and extracted with ether. The organic
layer was washed with brine, dried (Na2SO4), and concen-
trated. The residue was purified by silica gel column chro-
matography (4:1 hexane/EtOAc) to give methyl ester
(505 mg, 63%) as a 4.2:1 diasteromeric mixture as colorless
2854, 1712, 1464, 1439, 1355, 1253, 1215, 1095, 843 cmꢂ1
;
HRMS calcd for C26H50O4NaSi ([M+Na]+) 477.3370, found
447.3392.
4.1.5. (1SR,4SR,5RS)-4-Hexadecyl-1-levuloyloxy-5-
methoxycarbonyl-4-trimethylsiloxy-2-cyclopentene, ( )-9.
To a solution of (ꢀ)-7 (3.3 mg, 7.3 mmol), levulinic acid
(7.0 mL, 68.4 mmol) and PPh3 (19.2 mg, 73.2 mmol) was
added DIAD (40% in toluene, 3.7 mL, 7.32 mmol). The reac-
tion mixture was stirred for 2 h. The resulting mixture was
concentrated in vacuo. The residue was purified by silica
gel chromatography (hexane/AcOEt 4:1) to afford (ꢀ)-9
1
(2.0 mg, 52%) as a white solid. Mp¼32–33 ꢁC. H NMR
(600 MHz, CDCl3) d 6.12 (1H, d, J¼4.6 Hz), 5.93 (1H, d,

8012
K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
J¼5.7 Hz), 5.91 (1H, d, J¼5.7 Hz), 3.70 (3H, s), 2.95 (1H,
d, J¼4.6 Hz), 2.74 (2H, m), 2.55 (2H, m), 2.18 (3H, s), 1.79
(2H, br m), 1.34–1.26 (28H, br m), 0.88 (3H, t, J¼6.0 Hz),
0.04 (9H, s); 13C NMR (100 MHz, CDCl3) d 206.4,
172.3, 170.8, 138.8, 132.1, 87.9, 80.8, 59.0, 51.7, 42.1,
37.9, 31.9, 29.9, 29.9, 29.7 (ꢃ5), 29.7 (ꢃ2), 29.6, 29.6,
29.4, 27.9, 24.6, 22.7, 14.1, 2.0 (ꢃ3); IR (film) 3020, 2927,
added TBAF (40 mL of 1.0 M solution in THF, 40 mmol) at
0 ꢁC. The reaction mixture was stirred at 0 ꢁC for 10 min.
The resulting mixture was quenched with water, and
extracted with Et2O. The combined extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel chromatography
(5:1/2:1 hexane/EtOAc) to afford (ꢀ)-11 (3.5 mg, 49%)
as a white solid. Mp¼79–81 ꢁC. 1H NMR (600 MHz,
CDCl3) d 6.08 (1H, d, J¼5.6 Hz), 5.90 (1H, dd, J¼5.6 Hz,
2.1 Hz), 5.82 (1H, dd, J¼6.9 Hz, 1.5 Hz), 4.06 (1H, s),
3.75 (3H, s), 3.22 (1H, dd, J¼6.9 Hz, 1.0 Hz), 2.72 (2H,
m), 2.55 (2H, t, J¼6.5 Hz), 2.19 (3H, s), 1.65 (2H, m),
1.33–1.24 (28H, br m), 0.88 (3H, t, J¼6.7 Hz); 13C NMR
(100 MHz, CDCl3) d 206.3, 172.0, 171.5, 142.2, 129.4,
83.4, 76.8, 53.9, 52.1, 40.4, 37.7, 31.9, 29.9, 29.8, 29.7
(ꢃ4), 29.6 (ꢃ2), 29.6, 29.6, 29.5, 29.4, 28.0, 24.1, 22.7,
14.1; IR (film) 3505, 3020, 2926, 2854, 1722, 1519, 1465,
1439, 1408, 1359, 1216, 1158, 1076, 1030, 929, 757,
669 cmꢂ1; HRMS calcd for C28H48O6Na ([M+Na]+)
503.3343, found 503.3311.
2854, 1737, 1437, 1361, 1160, 1084, 846, 669 cmꢂ1
;
HRMS calcd for C31H56O6Na [(M+Na)+] 575.3738, found
575.3766.
4.1.6. (1SR,4SR,5RS)-4-Hexadecyl-4-hydroxy-1-levuloyl-
oxy-5-methoxycarbonyl-2-cyclopentene (the proposed
structure of plakevulin A), ( )-1a. To a solution of (ꢀ)-9
(5.5 mg, 9.9 mmol) in dry THF (0.5 mL) was added dropwise
a solution of TBAF in THF (20 mL of 1.0 M solution in THF,
20 mmol) at 0 ꢁC. The reaction mixture was stirred at 0 ꢁC
for 30 min. The resulting mixture was quenched with water,
and extracted with Et2O. The combined extracts were
washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel chro-
matography (hexane/AcOEt¼5:1 to 2:1) to afford (ꢀ)-1a
4.1.9. ( )-Plakevulin A ( )-1. To a solution of (ꢀ)-1a
(1.1 mg, 2.6 mmol) in dry pyridine (1.0 mL) was added a
solution hydrazine monohydrate (2.0 mL, 41.2 mmol) in
pyridine/AcOH (3:2, 1.0 mL). The reaction mixture was
stirred for 20 min. The resulting mixture was quenched with
water, and extracted with EtOAc. The combined extracts
were washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by silica gel chro-
matography (1:1 hexane/EtOAc) to afford (ꢀ)-1 (0.9 mg,
1
(4.5 mg, 94%) as a white solid. Mp¼45–47 ꢁC. H NMR
(600 MHz, CDCl3) d 6.04 (1H, ddd, J¼4.4 Hz, 1.5 Hz,
0.8 Hz), 5.94 (1H, dd, J¼5.4 Hz, 0.8 Hz), 5.91 (1H, dd,
J¼5.4 Hz, 1.5 Hz), 3.76 (3H, s), 2.96 (1H, d, J¼4.4 Hz),
2.75 (2H, m), 2.56 (2H, m), 2.30 (1H, s), 2.18 (3H, s),
1.80 (2H, m), 1.34–1.26 (28H, br m), 0.88 (3H, t,
J¼6.8 Hz); 13C NMR (100 MHz, CDCl3) d 206.3, 172.4,
171.8, 139.8, 131.5, 85.3, 80.9, 57.7, 52.2, 40.8, 37.8,
31.9, 29.9, 29.8, 29.7 (ꢃ5), 29.7 (ꢃ2), 29.6, 29.6, 29.4,
27.9, 24.2, 22.7, 14.1; IR (film) 3482, 3018, 2925, 2854,
1739, 1462, 1438, 1363, 1265, 1201, 1160, 1020, 759,
667 cmꢂ1; HRMS calcd for C28H48O6Na ([M+Na]+)
503.3343, found 503.3307.
92%) as
a
white solid. Mp¼80–82 ꢁC. 1H NMR
(600 MHz, CDCl3) d 5.94 (1H, dd, J¼5.7 Hz, 1.8 Hz),
5.84 (1H, dd, J¼5.7 Hz, 1.5 Hz), 5.34 (1H, m), 3.79 (3H,
s), 2.83 (1H, d, J¼5.3 Hz), 2.45 (1H, s), 2.00 (1H, br s),
1.81 (2H, m), 1.37–1.22 (28H, br m), 0.88 (3H, t,
J¼6.9 Hz); 13C NMR (100 MHz, CDCl3) d 172.6, 137.0,
135.7, 84.8, 78.2, 60.5, 52.1, 40.6, 31.9, 29.9, 29.7 (ꢃ5),
29.7 (ꢃ2), 29.6, 29.5, 29.4, 24.5, 22.7, 14.1; IR (film)
3445, 3019, 2927, 2855, 1724, 1520, 1465, 1439, 1374,
1041, 928, 759, 669 cmꢂ1; HRMS calcd for C23H42O4Na
([M+Na]+) 405.2975, found 405.2972.
4.1.7. (1RS,4SR,5RS)-4-Hexadecyl-1-levuloyloxy-5-
methoxycarbonyl-4-trimethylsiloxy-2-cyclopentene, ( )-
10. To a solution of (ꢀ)-7 (30 mg, 66 mmol) and levulinic
acid (13.5 mL, 132 mmol) in 1,4-dioxane (0.7 mL) were
added EDCI (25.3 mg, 132 mmol) and DMAP (0.8 mg,
6.6 mmol) at rt. The mixture was stirred at rt for 2 h. The
resulting mixture was quenched with water, and extracted
with Et2O. The combined extracts were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel chromatography (hexane/
AcOEt¼20:1 to 2:1) to afford (ꢀ)-10 (30.3 mg, 83%) as
white wax and the recovered (ꢀ)-7 (3.8 mg, 13%). 1H
NMR (600 MHz, CDCl3) d 6.01 (1H, d, J¼5.8 Hz), 5.87
(1H, dd, J¼5.8 Hz, 2.0 Hz), 5.61 (1H, br d, J¼7.2 Hz), 3.64
(3H, s), 3.39 (1H, d, J¼7.2 Hz), 2.73 (2H, m), 2.58 (2H,
m), 2.18 (3H, s), 1.69 (1H, m), 1.58 (1H, m), 1.34–1.26
(28H, br m), 0.88 (3H, t, J¼6.7 Hz), 0.10 (9H, s); 13C NMR
(100 MHz, CDCl3) d 206.5, 172.5, 168.7, 139.8, 129.9,
87.2, 75.9, 56.7, 51.2, 42.3, 37.8, 31.9, 29.8, 29.7 (ꢃ4),
29.6 (ꢃ3), 29.6, 29.6, 29.5, 29.3, 28.1, 24.3, 22.7, 14.1, 2.0
(ꢃ3); IR (film) 3022, 2926, 2854, 1744, 1464, 1436, 1359,
1252, 1158, 1098, 919, 843, 667 cmꢂ1; HRMS calcd for
C31H56O6SiNa ([M+Na]+) 575.3738, found 575.3777.
4.1.10. (1RS,4SR,5RS)-4-Hexadecyl-1,4-dihydroxy-5-
methoxycarbonyl-2-cyclopentene, ( )-12. To a solution
of (ꢀ)-7 (8.9 mg, 19.5 mmol) in THF (2 mL) was added
TBAF (30 mL of 1.0 M solution in THF, 30 mmol) at 0 ꢁC.
The reaction mixture was stirred at 0 ꢁC for 10 min. The
resulting mixture was quenched with water, and extracted
with Et2O. The combined extracts were washed with
brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography (hexane/
AcOEt 5:1 to 2:1) to afford (ꢀ)-12 (4.8 mg, 64%) as white
wax. 1H NMR (400 MHz, CDCl3) d 6.09 (1H, dd,
J¼5.6 Hz, 2.4 Hz), 6.03 (1H, d, J¼5.6 Hz), 4.82 (1H, m),
3.80 (3H, s), 3.50 (1H, s), 3.04 (1H, br d, J¼8.0 Hz), 2.99
(1H, d, J¼6.1 Hz), 1.73 (2H, m), 1.30–1.25 (28H, br m),
0.88 (3H, t, J¼6.8 Hz); 13C NMR (100 MHz, CDCl3)
d 173.0, 140.0, 134.7, 83.9, 75.8, 55.0, 52.0, 39.3, 31.9,
29.9, 29.7 (ꢃ4), 29.6 (ꢃ2), 29.6, 29.6, 29.5, 29.3, 24.4,
22.7, 14.1; IR (film) 3440, 3014, 2925, 2854, 1718, 1464,
4.1.8. (1RS,4SR,5RS)-4-Hexadecyl-4-hydroxy-1-levuloyl-
oxy-5-methoxycarbonyl-2-cyclopentene, ( )-11. Toa solu-
tion of (ꢀ)-10 (8.2 mg, 15 mmol) in dry THF (0.5 mL) was
1440, 1357, 1214, 1176, 1064, 931, 759, 667 cmꢂ1
;
HRMS calcd for C23H42O4Na ([M+Na]+) 405.2975, found
405.2989.

K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
8013
4.1.11. (1S,4R)-(L)-4-Hexadecyl-1,4-dihydroxy-2-cyclo-
pentene, (L)-14. To a solution of 13 (2.6 g, 4.7 mmol) in
THF (25 mL) was added TBAF (4.7 mL of a 1.0 M solution
in THF, 4.7 mmol) at 0 ꢁC and the mixture was stirred at rt
for 30 min. Then the mixture was quenched by the addition
of H2O and extracted with EtOAc. The organic layer was
washed with brine, dried (Na2SO4), and concentrated. The
residue was purified by silica gel column chromatography
(4:1 hexane/EtOAc) to give diol (1.3 g, 84%) as a white
solid. Mp¼79–80 ꢁC; [a]D23 ꢂ45.9 (c 0.80, CHCl3).
1H NMR (400 MHz, CDCl3) d 5.91 (1H, dd, J¼5.6 Hz,
2.0 Hz), 5.88 (1H, d, J¼5.6 Hz), 4.67 (1H, m), 2.55 (2H,
br s), 2.41 (1H, dd, J¼14.3 Hz, 7.0 Hz), 1.73 (1H, d,
J¼14.3 Hz, 3.1 Hz), 1.58 (2H, m), 1.30–1.25 (28H, br m),
0.88 (3H, t, J¼6.7 Hz); 13C NMR (100 MHz, CDCl3)
d 140.0, 134.9, 84.1, 75.3, 48.0, 40.5, 31.9, 30.0, 29.68
(ꢃ4), 29.65 (ꢃ3), 25.59, 29.57, 29.4, 24.3, 22.7, 14.1; IR
(KBr) 3317, 2918, 2849, 1469, 1360, 1302, 1213, 1159,
was added to the mixture at ꢂ78 ꢁC and the mixture was
stirred at ꢂ78 ꢁC for 15 min. Then methyl cyanoformate
(170 mL, 2.14 mmol) was added and the mixture was stirred
at ꢂ45 ꢁC for 2 h. The mixture was quenched by the addition
of H2O and extracted with EtOAc. The organic layer was
washed with brine, dried (Na2SO4), and concentrated. The
residue was purified by silica gel column chromatography
(4:1 hexane/EtOAc) to give 6 (245 mg, 69%) as a 4.2:1 dia-
steromeric mixture as colorless oil. The 1H NMR, 13C NMR,
and IR data of 6 were identical with those of (ꢀ)-6.
4.1.15. (1R,4S,5R)-(L)-4-Hexadecyl-1-hydroxy-5-meth-
oxycarbonyl-4-trimethylsiloxy-2-cyclopentene, (L)-7.
To a solution of 6 (125 mg, 0.28 mmol) in CH2Cl2 (5 mL)
was added DIBAL (620 mL of a 0.94 M solution in
CH2Cl2, 0.58 mmol) at ꢂ78 ꢁC. After the mixture was
stirred at ꢂ78 ꢁC for 1 h, the mixture was diluted with
Et2O. Then 1.0 mL of MeOH, followed by Celite was added,
and the mixture was stirred at rt for 1 h. The mixture was fil-
trated through Celite and the filtrate was concentrated. The
residue was purified by silica gel column chromatography
(9:1/4:1 hexane/EtOAc) to give both (ꢂ)-7 (45.5 mg,
36%) and (+)-8 (6.8 mg, 5%) as colorless oil. [a]2D3 ꢂ12.7
(c 0.45, CHCl3).
1086, 1054, 995, 971, 935, 873, 825, 783, 722, 603 cmꢂ1
;
HRMS calcd for C21H40O2Na ([M+Na]+) 347.2920, found
347.2916.
4.1.12. (4R)-(L)-4-Hexadecyl-4-hydroxy-2-cyclopenten-
1-one, (L)-4. To
a
solution of (ꢂ)-14 (503 mg,
1.55 mmol) in acetone (15 mL) was added the Jones reagent
(0.5 mL) in portions. Then the mixture was quenched by the
addition of 2-propanol (0.5 mL) and diluted with EtOAc and
H2O. The organic layer was separated and washed with H2O,
brine, dried (Na2SO4), and concentrated. The residue was
purified by silica gel column chromatography (3:1 hexane/
EtOAc) to give (ꢂ)-4 (383 mg, 77%) as a white solid. The
product was recrystallized from Et2O to obtain colorless
crystals. White solid, mp¼52–53 ꢁC; colorless crystal,
mp¼45–46 ꢁC; [a]D23 ꢂ54.5 (c 1.10, CHCl3). 1H NMR
(400 MHz, CDCl3) d 7.39 (1H, d, J¼5.7 Hz), 6.09 (1H, d,
J¼5.7 Hz), 2.53 (1H, d, J¼18.6 Hz), 2.41 (1H, d,
J¼18.6 Hz), 2.07 (1H, br s), 1.71 (2H, m), 1.35–1.23
(28H, br m), 0.85 (3H, t, J¼6.6 Hz); 13C NMR (100 MHz,
CDCl3) d 206.9, 165.8, 133.3, 79.2, 48.8, 40.3, 31.9, 29.8,
29.7 (ꢃ3), 29.63 (ꢃ2), 29.61, 29.59, 29.5, 29.4, 29.3, 24.2,
22.7, 14.1; IR (KBr) 3418, 3017, 2925, 2854, 1715, 1466,
4.1.16. (1S,4S,5R)-4-Hexadecyl-1-hydroxy-5-methoxy-
carbonyl-4-trimethylsiloxy-2-cyclopentene, (D)-8. Col-
orless oil. [a]2D1 +54.0 (c 0.33, CHCl3).
4.1.17. (1S,4S,5R)-(D)-4-Hexadecyl-5-methoxycarbonyl-
1-(4-nitrobenzoyl)-4-trimethylsiloxy-2-cyclopentene,
(D)-15. To a solution of (ꢂ)-7 (30.1 mg, 66.2 mmol), p-
nitrobenzoic acid (58.0 mg, 347 mmol) and PPh3 (93.2 mg,
355 mmol) in THF (1.5 mL) was added DIAD (180 mL of
a 40% solution in toluene, 356 mmol) at rt and the mixture
was stirred at rt for 10 min. The mixture was quenched by
the addition of H2O and extracted with EtOAc. The organic
layer was washed with brine, dried (Na2SO4), and concen-
trated. The residue was purified by silica gel column chro-
matography (30:1 hexane/EtOAc) to give (+)-15 (24.2 mg,
61%) as a white solid. Mp¼85–86 ꢁC; [a]D21 +117.3 (c
1
1404, 1339, 1215, 1067 cmꢂ1
;
HRMS calcd for
1.47, CHCl3). H NMR (400 MHz, CDCl3) d 8.27 (2H, d,
C21H38O2Na ([M+Na]+) 345.2764, found 345.2765.
J¼6.8 Hz), 8.17 (2H, d, J¼6.8 Hz), 6.42 (1H, m), 6.07
(1H, dd, J¼5.8 Hz, 1.9 Hz), 6.03 (1H, dd, J¼5.8 Hz,
1.4 Hz), 3.75 (3H, s), 3.15 (1H, d, J¼4.8 Hz), 1.84 (2H,
m), 1.33–1.24 (28H, br m), 0.88 (3H, t, J¼6.8 Hz), 0.09
(9H, s); 13C NMR (100 MHz, CDCl3) d 170.2, 164.3,
150.6, 139.7, 135.4, 131.5, 130.8 (ꢃ2), 123.5 (ꢃ2), 87.9,
82.1, 59.1, 51.8, 42.1, 31.9, 29.9, 29.7 (ꢃ6), 29.64 (ꢃ2),
29.61, 29.3, 24.7, 22.7, 14.1, 2.0 (ꢃ3); IR (KBr) 2919,
2850, 1744, 1720, 1606, 1525, 1333, 1274, 1116, 968,
4.1.13. (4R)-(L)-4-Hexadecyl-4-trimethylsiloxy-2-cyclo-
penten-1-one, (L)-5. To a solution of (ꢂ)-4 (311 mg,
0.96 mmol) and i-Pr2NEt (480 mL, 2.75 mmol) in CH2Cl2
(5 mL) was added TMSOTf (250 mL, 1.38 mmol) at 0 ꢁC
and the mixture was stirred at 0 ꢁC for 15 min. Then the mix-
ture was quenched by the addition of H2O and extracted with
CH2Cl2. The organic layer was washed with brine, dried
(Na2SO4), and concentrated. The residue was purified by
silica gel column chromatography (10:1 hexane/EtOAc) to
give (ꢂ)-5 (351 mg, 92%) as a white solid. Mp¼31–32 ꢁC;
[a]2D3 ꢂ16.0 (c 1.17, CHCl3).
844, 721 cmꢂ1
;
HRMS calcd for C33H53NO7NaSi
([M+Na]+) 626.3483, found 626.3510.
4.1.18. (1S,4S,5R)-( )-4-Hexadecyl-4-hydroxy-5-meth-
oxycalbonyl-1-trimethylsiloxy-2-cyclopentene, (D)-16.
To a solution of (+)-15 (26.5 mg, 43.9 mmol) in THF–
MeOH (1/1, 3 mL) was added NaOMe (52.0 mL of a 1 M so-
lution in MeOH, 52.0 mmol) at rt and the mixture was stirred
at rt for 20 min. The mixture was quenched by the addition
of H2O and extracted with EtOAc. The organic layer was
washed with brine, dried (Na2SO4), and concentrated. The
residue was purified by silica gel column chromatography
4.1.14. 4-Hexadecyl-5-methoxycarbonyl-4-trimethyl-
siloxy-2-cyclopenten-1-one (6). To a solution of diisopro-
pylamine (250 mL, 1.76 mmol) in THF (5 mL) was added
n-BuLi (1.1 mL of a 1.57 M solution in hexane, 1.73
mmol) at 0 ꢁC and the mixture was stirred at 0 ꢁC for
10 min. The mixture was cooled to ꢂ78 ꢁC. A solution of
(ꢂ)-5 (309 mg, 0.78 mmol) in THF/HMPA (10:1, 4.4 mL)

8014
K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
(5:1 hexane/EtOAc) to TMS ether (16.2 mg, 81%) as color-
J¼6.8 Hz); 13C NMR (100 MHz, CDCl3) d 139.6, 135.9,
85.1, 76.1, 48.1, 41.6, 31.9, 30.0, 29.7 (ꢃ6), 29.64 (ꢃ2),
29.58, 29.3, 24.4, 22.7, 14.1; IR (KBr) 3330, 2919, 2850,
1465, 1406, 1346, 1268, 1190, 1160, 1126, 1103, 1045,
915, 867, 783, 722 cmꢂ1; HRMS calcd for C21H40O2Na
([M+Na]+) 347.2920, found 347.2913.
1
less oil. [a]2D3 +73.2 (c 1.0, CHCl3). H NMR (600 MHz,
CDCl3) d 5.92 (1H, dd, J¼5.8 Hz, 1.7 Hz), 5.80 (1H, dd,
J¼5.8 Hz, 1.7 Hz), 5.45 (1H, m), 3.71 (3H, s), 2.81 (1H, d,
J¼5.5 Hz), 2.21 (1H, br s), 1.81 (2H, m), 1.30–1.25 (28H,
br m), 0.88 (3H, t, J¼6.8 Hz), 0.02 (9H, s); 13C NMR
(100 MHz, CDCl3) d 171.4, 136.3, 136.2, 87.8, 77.7, 62.5,
51.6, 41.6, 31.9, 29.9, 29.69 (ꢃ5), 29.65 (ꢃ2), 29.6 (ꢃ2),
29.4, 24.9, 22.7, 14.1, 2.0 (ꢃ3); IR (film) 3444, 2925,
2854, 1734, 1463, 1438, 1359, 1250, 1216, 1082, 937,
844 cmꢂ1; HRMS calcd for C26H50O4NaSi ([M+Na]+)
477.3370, found 477.3356.
4.1.22. (4S)-(D)-4-Hexadecyl-4-hydroxy-2-cyclopenten-1-
one, (D)-4. To a solution of (ꢂ)-18 (505 mg, 1.55 mmol) in
acetone (30 mL) was added the Jones reagent (0.6 mL) in
portions. The mixture was stirred at rt for 30 min. Then the
mixture was quenched by the addition of 2-propanol
(1.0 mL) and diluted with EtOAc and H2O. The organic layer
was separated and washed with H2O, brine, dried (Na2SO4),
and concentrated. The residue was purified by silica gel
column chromatography (3:1 hexane/EtOAc) to give (+)-4
(332 mg, 66%) as a white solid. The product was recrystal-
lized from Et2O to obtain colorless crystals. Mp¼45–46 ꢁC;
[a]2D0 +51.9 (c 1.10, CHCl3).
4.1.19. (D)-Plakevulin A (1). To a solution of (+)-18
(14.4 mg, 31.7 mmol) in THF (1.0 mL) was added TBAF
(40.0 mL of 1.0 M solution in THF, 40.0 mmol) and the mix-
ture was stirred at rt for 1 h. The mixture was quenched by
the addition of H2O and extracted with EtOAc. The organic
layer was washed with brine, dried (Na2SO4), and concen-
trated. The residue was purified by silica gel column chro-
matography (2:1 hexane/EtOAc) to give (+)-plakevulin A
(8.9 mg, 73%) as colorless wax. The product was recrystal-
lized from hexane/EtOAc to obtain white solid. Colorless
wax, mp¼61–62 ꢁC; White solid, mp¼73–74 ꢁC; [a]2D1
4.1.23. (4S)-(D)-4-Hexadecyl-4-trimethylsiloxy-2-cyclo-
penten-1-one, (D)-5. White solid, mp¼30–31 ꢁC; [a]D21
+16.1 (c 1.27, CHCl3).
1
+27.1 (c 0.55, CHCl3). The H NMR, 13C NMR, and IR
4.1.24. (1S,4R,5S)-(D)-4-Hexadecyl-1-hydroxy-5-
methoxycarbonyl-4-trimethylsiloxy-2-cyclopentene,
(D)-7. Colorless oil; [a]2D2 +12.7 (c 0.67, CHCl3).
data of (+)-1 were identical with those of (ꢀ)-1.
4.1.20. (L)-Untenone A (2). To a solution of 6 (18.7 mg,
0.041 mmol) in MeOH (1 mL) was added one drop of concd
HCl (ca. 10 mL) at rt and the mixture was stirred at rt for
15 min. The mixture was quenched by the addition of H2O
and extracted with EtOAc. The organic layer was washed
with brine, dried (Na2SO4), and concentrated. The residue
was purified by silica gel column chromatography (9:1
hexane/EtOAc with 1% AcOH) to give (ꢂ)-untenone A (2)
(14.7 mg, 94%) as colorless wax. The product was recrystal-
lized from hexane to obtain white solid. Colorless wax,
mp¼w30 ꢁC; White solid, mp¼60–62 ꢁC; [a]2D3 ꢂ71.3
4.1.25. (1R,4R,5S)-4-Hexadecyl-1-hydroxy-5-methoxy-
carbonyl-4-trimethylsiloxy-2-cyclopentene, (L)-8. Color-
less oil; [a]2D1 ꢂ52.4 (c 0.10, CHCl3).
4.1.26. (1R,4R,5S)-(L)-4-Hexadecyl-5-methoxycar-
bonyl-1-(4-nitrobenzoyl)-4-trimethylsiloxy-2-cyclo-
pentene, (L)-15. White solid, mp¼82–83 ꢁC; [a]D19 ꢂ111.7
(c 0.55, CHCl3).
4.1.27. (1R,4R,5S)-(L)-4-Hexadecyl-4-hydroxy-5-
methoxycalbonyl-1-trimethylsiloxy-2-cyclopentene,
(L)-16. Colorless oil; [a]2D1 ꢂ73.3 (c 0.25, CHCl3).
1
(c 0.94, CHCl3). H NMR (400 MHz, CDCl3) d 7.53 (1H,
d, J¼5.7 Hz), 6.19 (1H, d, J¼5.7 Hz), 3.80 (3H, s), 3.65
(1H, br s), 3.47 (1H, s), 1.81 (1H, m), 1.70 (1H, m), 1.32–
1.25 (28H, br m), 0.88 (3H, t, J¼6.9 Hz); 13C NMR
(100 MHz, CDCl3) d 200.0, 169.1, 167.1, 132.3, 79.9, 60.8,
52.9, 40.4, 31.9, 29.74, 29.67 (ꢃ4), 29.64, 29.62, 29.58,
29.5, 29.4, 29.3, 23.8, 22.7, 14.1; IR (neat) 3451, 2919,
2850, 1740, 1712, 1467, 1437, 1321, 1256, 1154, 1035,
817, 763, 721 cmꢂ1; HRMS calcd for C23H41O4Na
([M+Na]+) 381.3004, found 381.3012.
4.1.28. (L)-Plakevulin A (1). Colorless wax, mp¼60–
62 ꢁC; white soild, mp¼70–71 ꢁC; [a]2D1 ꢂ25.7 (c 0.10,
CHCl3).
4.1.29. (D)-Untenone A (2). White solid, mp¼60–62 ꢁC;
[a]2D3 +72.2 (c 0.50, CHCl3).
4.1.30. ( )-Manzamenone A (3). Compound (ꢀ)-2 (20 mg,
47 mmol) was heated at w70 ꢁC for 24 h. The residue was
purified by PTLC (hexane/EtOAc/AcOH 150:60:1) to give
4.1.21. (1S,4S)-(L)-4-Hexadecyl-1,4-dihydroxy-2-cyclo-
pentene, (L)-18. To a solution of 17 (326 mg, 0.74 mmol)
in THF (6 mL) was added TBAF (0.92 mL of a 1.0 M solu-
tion in THF, 0.92 mmol) at 0 ꢁC and the mixture was stirred
at rt for 7 h. Then the mixture was quenched by the addition
of H2O and extracted with EtOAc. The organic layer was
washed with brine, dried (Na2SO4), and concentrated. The
residue was purified by silica gel column chromatography
(4:1 hexane/EtOAc) to give (ꢂ)-18 (185 mg, 77%) as a white
1
(ꢀ)-3 (3.7 mg, 20%). H NMR (600 MHz, CDCl3) d 6.18
(1H, br s), 3.88 (3H, s), 3.62 (1H, m), 3.55 (3H, s), 3.51
(1H, d, J¼5.9 Hz), 3.20 (1H, dd, J¼7.7 Hz, J¼6.2 Hz),
3.13 (1H, m), 2.93 (1H, t, J¼8.3 Hz), 2.45 (1H, m), 2.19
(2H, m), 1.60–1.26 (56H, br m), 0.88 (6H, t, J¼6.6 Hz).
4.1.31. 2-Methoxycarbonyl-3-hexadecyl-2-cyclopenten-
1-one (20). A solution of (ꢀ)-19 (2.5 mg, 5.9 mmol) and
10% Pd on carbon (0.7 mg) in EtOAc (1 mL) was stirred
at rt under an H2 atmosphere for 14.5 h. The mixture was fil-
trated through Celite and washed with EtOAc. The solvent
was removed under a reduced pressure. The residue was
1
solid. Mp¼83 ꢁC; [a]D23 ꢂ28.7 (c 0.68, CHCl3). H NMR
(600 MHz, CDCl3) d 5.91 (1H, dd, J¼5.6 Hz, 2.0 Hz),
5.87 (1H, dd, J¼5.6 Hz, 1.2 Hz), 5.04 (1H, m), 2.30 (1H,
dd, J¼14.0 Hz, 6.8 Hz), 1.81 (1H, dd, J¼14.0 Hz, 4.0 Hz),
1.73–1.63 (4H, m), 1.30–1.26 (28H, br m), 0.88 (3H, t,

K. Kuramochi et al. / Tetrahedron 62 (2006) 8006–8015
8015
2. Saito, F.; Takeuchi, R.; Kamino, T.; Kuramochi, K.; Sugawara,
F.; Sakaguchi, K.; Kobayashi, S.; Tsuda, M.; Kobayashi, J.
Tetrahedron Lett. 2004, 45, 8069–8071.
3. Mizutani, H.; Watanabe, M.; Honda, T. Synlett 2005, 793–796.
4. (a) Ishibashi, M.; Takeuchi, S.; Kobayashi, J. Tetrahedron Lett.
1993, 34, 3749–3750; (b) Ishibashi, M.; Kobayashi, J. Kagaku
To Seibutsu 1993, 31, 659–664.
5. Total synthesis of untenone A: (a) Takeda, K.; Nakayama, I.;
Yoshii, E. Synlett 1994, 178; (b) Miyaoka, H.; Watanuki, T.;
Saka, Y.; Yamada, Y. Tetrahedron 1995, 51, 8749–8756; (c)
Asami, M.; Ishizaki, T.; Inoue, S. Tetrahedron Lett. 1995, 36,
1893–1894; (d) Kuhn, C.; Skaltounis, L.; Monneret, C.;
Florent, J.-C. Eur. J. Org. Chem. 2003, 2585–2595. See also
Refs. 3 and 7.
purified by silica gel column chromatography (4:1 hexane/
EtOAc) to give 20 (1.8 mg, 100%) as colorless oil. 1H
NMR (600 MHz, CDCl3) d 3.84 (3H, s), 2.76 (2H, t,
J¼7.9 Hz), 2.68 (2H, m), 2.49 (2H, m), 1.57 (4H, m), 1.36
(1H, m), 1.26–1.32 (23H, br m), 0.88 (3H, t, J¼7.0 Hz);
13C NMR (100 MHz, CDCl3) d 203.8, 189.0, 163.8, 51.8,
34.9, 32.7, 31.9, 30.4, 29.7 (ꢃ7), 29.6, 29.6, 29.5, 29.4,
29.3, 27.7, 22.7, 14.1; IR (film) 3020, 2926, 2854, 1739,
1714, 1620, 1465, 1437, 1361, 1295, 1259, 1216, 1155,
1026, 758, 667 cmꢂ1; HRMS calcd for C23H40O3Na
([M+Na]+) 387.2869, found 387.2868.
4.1.32. (2RS,3SR)-2-Methoxycarbonyl-3-hexadecyl-
cyclopentanone, ( )-21. A solution of (ꢀ)-19 (8.4 mg,
20 mmol) and 20% Pd(OH)2 on carbon (2.3 mg) in EtOAc
(1 mL) was stirred at rt under an H2 atmosphere for 26 h.
The mixture was filtrated through Celite and washed with
EtOAc. The solvent was removed under a reduced pressure.
The residue was purified by silica gel column chromato-
graphy (4:1 hexane/EtOAc) to give (ꢀ)-21 (7.1 mg, 100%)
as a white solid. Mp¼38–41 ꢁC. 1H NMR (600 MHz,
CDCl3) d 3.76 (3H, s), 2.83 (1H, d, J¼11.2 Hz), 2.57 (1H,
m), 2.42 (1H, dd, J¼8.3 Hz, 18.7 Hz), 2.32 (1H, m), 2.23
(1H, m), 1.54 (1H, m), 1.47 (1H, m), 1.43 (1H, m), 1.36
(1H, m), 1.31–1.27 (27H, br m), 0.88 (3H, t, J¼6.9 Hz);
13C NMR (100 MHz, CDCl3) d 212.1, 170.1, 61.9, 52.4,
41.5, 38.5, 35.0, 31.9, 29.7 (ꢃ7), 29.6, 29.6, 29.5, 29.4,
27.4, 27.2, 22.7, 14.1; IR (film) 3021, 2927, 2855, 1754,
6. Tsukamoto, S.; Takeuchi, S.; Ishibashi, M.; Kobayashi, J.
J. Org. Chem. 1992, 57, 5255–5260.
7. (a) Al-Busafi, S.; Drew, M. G. B.; Sanders, T.; Whitehead, R. C.
Tetrahedron Lett. 1998, 39, 1647–1650; (b) Al-Busafi, S.;
Whitehead, R. C. Tetrahedron Lett. 2000, 41, 3467–3470; (c)
Al-Busafi, S.; Doncaster, J. R.; Drew, M. G. B.; Regan,
A. C.; Whitehead, R. C. J. Chem. Soc., Perkin Trans. 1 2002,
476–484; (d) Doncaster, J. R.; Ryan, H.; Whitehead, R. C.
Synlett 2003, 651–654; (e) Etchells, L. L.; Sardarian, A.;
Whitehead, R. C. Tetrahedron Lett. 2005, 46, 2803–2807; (f)
Doncaster, J. R.; Etchells, L. L.; Kershaw, N. M.; Nakamura,
R.; Ryan, H.; Takeuchi, R.; Sakaguchi, K.; Sardarian, A.;
Whitehead, R.C. Bioorg. Med.Chem. Lett.2006, 16, 2877–2881.
8. Perpelescu, M.; Tsuda, M.; Suzuki; Yoshida, S.; Kobayashi, J.
Nat. Med. 2004, 58, 86.
9. Saito, F.; Takeuchi, R.; Kamino, T.; Kuramochi, K.; Sugawara,
F.; Sakaguchi, K.; Kobayashi, S. Bioorg. Med. Chem. Lett.
2004, 14, 1975–1977.
10. Mitsunobu, O. Synthesis 1981, 1–28.
11. van Boom, J. H.; Burger, P. M. J. Tetrahedron Lett. 1976, 17,
4875–4878.
1726, 1463, 1439, 1216, 1129, 927, 759, 669 cmꢂ1
;
HRMS calcd for C23H42O3Na ([M+Na+]) 389.3026, found
389.3039.
4.2. DNA polymerase assay13
Calf pol a was purified by immuno-affinity column chroma-
tography as described previously.14 Recombinant rat pol
b was purified based on the method described by Date
et al.15 Recombinant human TdT was purified as described
by Ibe et al.16 The synthesized compounds were dissolved
in dimethyl sulfoxide (DMSO) at various concentrations and
sonicated for 1 min. Five micro liters of these compounds in
40% DMSO were mixed with 10 ml of 2ꢃ reaction mixture
(100 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 2 mM dithio-
threitol, 15% glycerol, 250 mg/ml activated DNA, 100 mM
each of dATP, dGTP, and dCTP, and 0.2 mM [3H]dTTP)
and 5 ml of each enzyme (0.01 units). These mixtures were
incubated on ice for 10 min, then at 37 ꢁC for 60 min. The
amount of incorporated [3H]dTMP into activated DNAwith-
out inhibitors was considered 100%. One unit of the activity
was defined as the amount of enzyme that catalyzes the
incorporation of 1 nmol of deoxyribonucleotide triphos-
phates into template DNA in 60 min at 37 ꢁC.
12. (a) Laumen, K.; Schneider, M. Tetrahedron Lett. 1984, 25,
5875–5878; (b) Johnson, C. R.; Bis, S. J. Tetrahedron Lett.
1992, 33, 7287–7290.
13. (a) Mizushina, Y.; Tanaka, N.; Yagi, H.; Kurosawa, T.; Onoue,
M.; Seto, H.; Horie, T.; Aoyagi, N.; Yamaoka, M.; Matsukage,
A.; Yoshida, S.; Sakaguchi, K. Biochim. Biophys. Acta 1996,
1308, 256–262; (b) Mizushina, Y.; Yagi, H.; Tanaka, N.;
Kurosawa, T.; Seto, H.; Katsumi, K.; Onoue, M.; Ishida, H.;
Iseki, A.; Nara, T.; Morohashi, K.; Horie, T.; Onomura, Y.;
Narusawa, M.; Aoyagi, N.; Takami, K.; Yamaoka, M.; Inoue,
Y.; Matsukage, A.; Yoshida, S.; Sakaguchi, K. J. Antibiot.
(Tokyo) 1996, 49, 491–492; (c) Mizushina, Y.; Yoshida, S.;
Matsukage, A.; Sakaguchi, K. Biochim. Biophys. Acta 1997,
1336, 509–521.
14. (a) Tamai, K.; Kojima, K.; Hanaichi, T.; Masaki, S.; Suzuki,
M.; Umekawa, H.; Yoshida, S. Biochim. Biophys. Acta 1988,
950, 263–273; (b) Takemura, M. Biochim. Biophys. Acta.
2002, 1571, 151–156.
15. Date, T.; Yamaguchi, M.; Hirose, F.; Nishimoto, Y.; Tanihara,
K.; Matsukage, A. Biochemistry 1988, 27, 2983–2990.
16. Ibe, S.; Fujita, K.; Toyomoto, T.; Shimazaki, N.; Kaneko, R.;
Tanabe, A.; Takebe, I.; Kuroda, S.; Kobayashi, T.; Toji, S.;
Tamaki, K.; Yamamoto, H.; Koiwai, O. Genes Cells 2001, 6,
815–824.
References and notes
1. Tsuda, M.; Endo, T.; Perpelescu, M.; Yoshida, S.; Watanabe,
K.; Fromont, J.; Mikami, Y.; Kobayashi, J. Tetrahedron 2003,
59, 1137–1141.