Jolkinolide D pharmacophore: Synthesis and reaction with biomolecules

Article abstract of DOI:10.1016/j.tet.2003.08.080

To obtain information on the chemical modification of biomolecules with jolkinolide D, a bioactive diterpenoid of plant origin, jolkinolide D pharmacophore was synthesized, and its reactivity toward amino acids, nucleosides, and DNA was investigated.

Full text of DOI:10.1016/j.tet.2003.08.080

Tetrahedron 60 (2004) 7067–7075
Jolkinolide D pharmacophore: synthesis and reaction
with biomolecules
*
Akira Sakakura, Yui Takayanagi, Hiroki Shimogawa and Hideo Kigoshi
Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan
Received 22 May 2003; revised 11 August 2003; accepted 11 August 2003
Available online 25 June 2004
Abstract—To obtain information on the chemical modification of biomolecules with jolkinolide D, a bioactive diterpenoid of plant origin,
jolkinolide D pharmacophore was synthesized, and its reactivity toward amino acids, nucleosides, and DNA was investigated.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
nucleosides, and DNA as nucleophiles and studied the
reactivities of 1.4 We describe herein the synthesis of
jolkinolide D pharmacophore (2) of racemic and optically
active forms and their updated reactivities toward biological
nucleophiles including histidine as an imidazole-containing
amino acid and glutathione as a peptide.
Jolkinolide D (1) is a diterpenoid isolated from Euphorbia
jolkini Boiss in 1974,1 and its stereostructure was recently
determined by X-ray crystallographic analysis.2 It exhibited
cytotoxicity, inhibited tumor invasion into the basement
membrane, and induced apoptosis in tumor cells.2
Jolkinolide D (1) has a g,d-unsaturated-b-hydroxy-a-
methylene lactone unit as the pharmacophore structure,
which suggests that jolkinolide D (1) might irreversibly
alkylate biomolecules such as proteins and DNA in contrast
to popular a-methylene lactones such as in germacrano-
lides, eudesmanolides, guaianolides, and pseudoguaiano-
lides3 that would reversibly alkylate biomolecules (Scheme
1). Because there are no other compounds that have the
pharmacophore, reactivities of the unit toward nucleophiles
have not hitherto been investigated. Generally, cytotoxic
compounds interact with biomolecules, such as enzymes/
proteins and DNA, so we chose amino acids, a peptide,
2. Results and discussion
2.1. Synthesis of jolkinolide D pharmacophore (2)
Racemic jolkinolide D pharmacophore (2) was synthesized
from (^)-6-(tert-butyldimethylsilyloxy)-3-methyl-2-cyclo-
hexenone (3)5 and 2-iodoallylalcohol (4)6 (Scheme 2).
Addition of the vinyllithium reagent, generated from iodide
4 and t-BuLi, to (^)-ketone 3 afforded diastereomeric diols
(^)-5 (56%) and (^)-6 (33%). (^)-Diol 5 could be
transformed into (^)-jolkinolide D pharmacophore (2).
The TBS group of (^)-5 was removed with Bu4NF to give
Scheme 1. Jolkinolide D (1) and its pharmacophore (2).
Keywords: Jolkinolide D; Pharmacophore; Diterpenoid; Synthesis; Reactivity; Alkylation.
*
Corresponding author. Fax: þ81-298-53-4313; e-mail address: kigoshi@chem.tsukuba.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.08.080

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A. Sakakura et al. / Tetrahedron 60 (2004) 7067–7075
Scheme 2. Synthesis of (^)-jolkinolide D pharmacophore (2). Reagents and conditions: (a) t-BuLi, Et2O, 0 8C; (b) Bu4NF, THF, 0 8C; (c) MnO2, CHCl3, rt.
(^)-triol 7 (79%), whose oxidation with MnO2 gave (^)-2
(43%). On the other hand, (^)-diol 6 was converted into
aldehyde 9 by the same procedure (24%). The stereo-
chemistry of the ring juncture in (^)-2 was established by
the small coupling constants of H-12 (ddd, J¼1.3, 3.0,
4.0 Hz).7
2.2. Reactions of jolkinolide D pharmacophore (2) with
amino acids and glutathione
The reactivities of jolkinolide D pharmacophore (2) toward
biomolecules have been investigated to obtain the basic
information on the chemical modification of proteins and
DNA with jolkinolide D (1).
To investigate the difference in reactivities between racemic
and optically active jolkinolide D pharmacophore (2), we
then planned to synthesize (2)-2, which has the same
absolute configuration as natural jolkinolide D (1).
(2)-Jolkinolide D pharmacophore (2) was synthesized
from (2)-acetoxyketone 10, which was prepared by a
reported procedure8 with optimization to obtain the
optically pure sample (34%, .99% ee)9 (Scheme 3).
Hydrolysis of the acetoxy group in (2)-10 with
Initially, the reactions of (^)-2 with three amino acids and a
peptide were examined. A typical amino acid L-alanine,
sulfur-containing L-cysteine and glutathione, and an imida-
zole-containing L-histidine were reacted with an equal
amount of (^)-2 in aqueous THF (pH 7.5). The products
were separated and purified by chromatography (ODS), and
their structures were established by the spectroscopic
method (UV, IR, NMR, 2D NMR, and MS spectra). The
results are summarized in Table 1. (^)-2 reacted with all
amino acids employed here and glutathione to give
alkylated products. The thiol groups in L-cysteine and
glutathione were rapidly alkylated with (^)-2 in high yield
(64% in L-cysteine and 81% in glutathione). In the
alkylation of glutathione, adduct 22, an intermediate to
21, was obtained as a diastereomeric mixture at the 15
position, which supported the alkylation mechanism shown
in Scheme 1. In the case of L-alanine, which has no
nucleophilic functional group in the amino acid residue,
alkylation occurred only at the amino group to afford the
monoalkylated product 14 (53%) and the dialkylated
product 15 (4.3%). L-Histidine afforded three monoalkyl-
ated products, 16 (19%), 17 (26%), and 18 (17%), along
with two dialkylated products 19 (3.8%) and 20 (8.5%). The
product 16 was proved to be alkylated at the amino group,
while the products 17 and 18 were alkylated at the
imidazolyl group. These results indicate that the thiol
group is the most reactive toward (^)-2 among the
nucleophilic groups in L-cysteine and glutathione, and that
the amino group and the imidazolyl group in L-histidine
have similar reactivity toward (^)-2 at pH 7.5. The
alkylated products were obtained as a diastereomeric
mixture because racemic jolkinolide D pharmacophore
10
Sc(OTf)3 followed by protection of the hydroxyl group
with tert-butyldimethylsilyl chloride (TBSCl) and Et3N
afforded (2)-ketone 3 (55%). (2)-Ketone 3 was trans-
formed into (2)-jolkinolide D pharmacophore (2) by the
same procedure as used for the synthesis of (^)-2.
Scheme 3. Synthesis of (2)-jolkinolide D pharmacophore (2). Reagents
and conditions: (a) pig liver esterase, 0.1 M phosphate buffer (pH 7.0),
15 8C; (b) Sc(OTf)3, MeOH–H2O (4:1), rt; (c) TBSCl, Et3N, DMF, rt.

A. Sakakura et al. / Tetrahedron 60 (2004) 7067–7075
Table 1. Alkylation of amino acids and glutathione with jolkinolide D pharmacophore (2)a
7069
Products (yields)
L-Cysteine
12 (64%)b
13 (0%)b
12a (38%)c
13a (23%)c
L-Alanine
14 (53%)b
15 (4.3%)b
15a (0%)c
14a (20%)c
L-Histidined
16 (19%)b
17 (26%)b
18 (17%)b
18a (15%)c
19 (3.8%)b
20 (8.5%)b
16a (34%)c
17a (29%)c
19a (5.4%)c
20a (5.0%)c
Glutathione
21 (52%)b
22 (19%)b
23 (10%)b
23a (0%)c
21a (37%)c
22a (60%)c
a
Reaction conditions: 0.2 M jolkinolide D pharmacophore (2) and amino acid (peptide) in aqueous THF, pH 7.5, ambient temperature, and 16 h.
Yields of the products of the reaction with (^)-2.
Yields of the products of the reaction with (2)-2.
Owing to the low solubility of L-histidine to the solvent system, the alkylation experiment was executed in an aqueous THF solution of 0.1 M each of 2 and L-
histidine.
b
c
d
(^)-2 was employed. The diastereomeric ratios of 12, 14,
16, 17, and 21 were ca. 1:1, which were determined by the
1H NMR analysis using 12a, 14a, 16a, 17a, and 21a,
obtained below, as the authentic samples. The yields of
the alkylated products show some difference between the
racemic and optically active 2, however, the 1:1 ratio of
products indicated that the chirality of 2 had essentially no
effect on the alkylation. The differences in yields may be
due to the experimental error in adjusting pH of the reaction
media.
2.3. Reactions of jolkinolide D pharmacophore (2) with
nucleosides and DNA
Four nucleosides, 20-deoxyadenosine, 20-deoxyguanosine,
20-deoxycytidine, and thymidine were reacted with an equal
amount of (^)-jolkinolide D pharmacophore (2) under the
same conditions as those employed for amino acids. The
results are listed in Table 2. The sites of alkylation in the
purine and pyrimidine bases of the nucleosides were deter-
mined by comparison of the difference UV spectra between
alkylated nucleosides and the reference compound 14 with
the UV spectra of the known ethylated nucleosides in acidic,
neutral, and basic solutions (Table 3).11,12 Only in the case
of 20-deoxyguanosine, the alkylation reaction occurred to
afford the product alkylated at the N21 position of guanine,
24. When alkylation of thymidine was executed under basic
conditions (pH 8.7), the product 25, formed by alkylation at
the N23 position of thymine, was obtained in 22% yield.
The alkylation of L-cysteine, L-alanine, L-histidine, and
glutathione by (2)-2 was next investigated. The alkylation
conditions were the same as those employed for (^)-2. The
results are shown in Table 1. (2)-2 showed the same
reactivity toward amino acids and glutathione as (^)-2. The
alkylated products 12a, 14a, 16a, 17a, and 21a could be
obtained as a single isomer.

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A. Sakakura et al. / Tetrahedron 60 (2004) 7067–7075
Table 2. Alkylation of nucleosides with jolkinolide D pharmacophore (2)a
agents. The well-known alkylating agent, dimethyl sulfate,
alkylates guanosine at the N27 position14 and thymidine at
the N23 position15 und0er neutral and aqueous conditions.
N21 Alkylation of 2 -deoxyguanosine was found on
treatment with methyl iodide in dimethyl sulfoxide under
basic conditions.16 In the present study, only the stable
product might be isolated, because the alkylated deoxy-
guanosines at the N27 position are unstable. To the best of
our knowledge, Michael acceptors are well known to react
with amino acid nucleophiles, however, there is little
information so far about the reactions between Michael
acceptors and nucleoside nucleophiles. To clarify the details
of the alkylating profile of 2, the further careful studies are
needed.
Product (yield)
20-Deoxyguanosine
DNA from salmon spermary was reacted with (^)-jolkino-
lide D pharmacophore (2) in aqueous methanol (pH 7.5) at
37 8C for a week. After the enzymatic hydrolysis,13 the
alkylated nucleosides were isolated and characterized as
previously described. Two products 24 (0.48%) and 25
(0.26%) were obtained, which were the same products as in
the reaction of nucleosides with (^)-2, but the yields were
very low.
24 (21%)b
24a (5%, pH 7.5; 52%, pH 8.9)c
Thymidine
2.4. Biological activities of jolkinolide D pharmacophore
25 (22%, pH 8.7)b
25a (18%, pH8.7)c
The preliminary biological tests of (^)-jolkinolide D
pharmacophore (2) indicated that the artificial analog 2
exhibited weaker activities than jolkinolide D (1), concern-
ing to the inhibitory activity against tumor invasion into the
basement membrane and the induction of apoptosis in tumor
cells. These results suggested that the A and B rings in 1 are
important to the biological activities in cells somehow,
maybe because of membrane permeability.
20-Deoxyadenosine
No reaction
No reaction
20-Deoxycytidine
a
Reaction conditions: 0.1 M for 20-deoxyadenosine and 20-deoxycytidine,
0.02 M for 20-deoxyguanosine, 0.2 M for thymidine in aqueous THF, pH
7.5, ambient temperature, and 16 h.
Yields of the products of the reaction with (^)-2.
Yields of the products of the reaction with (2)-2.
b
c
Next, the alkylation of 20-deoxyadenosine, 20-deoxyguano-
sine, 20-deoxycytidine, and thymidine by (2)-2 was
investigated. The results are shown in Table 2, which are
essentially the same as those of the reactions of (^)-2.
Under neutral conditions (pH 7.5), only 20-deoxyguanosine
was alkylated to give 24a (5%). Under basic conditions, 24a
was obtained in a higher yield (pH 8.9, 52%),0and thymidine
was alkyla0ted to give 25a (pH 8.7, 18%). 2 -Deoxyadeno-
sine and 2 -deoxycytidine were not alkylated under either
neutral or basic conditions. The N21 of deoxyguanosine as
well as the N27 is one of the active sites toward alkylating
3. Conclusions
We have synthesized jolkinolide D pharmacophore (2) in
the racemic form (19%, three steps) and the optically pure
form (3.6%, six steps) and have investigated the reactivities
of 2 toward amino acids, nucleosides, and DNA. The order
of reactivity of 2 toward nucleophiles in amino acids at pH
7.5 is the thiol group in L-cysteine and glutathione.the
imidazolyl group in L-histidine, the a-amino groups in
amino acids.the carboxyl groups in amino acids. We also
Table 3. Difference UV spectra of jolkinolide D pharmacophore-nucleoside adducts and UV spectra of ethylated nucleosides
pH 1a pH 7b
pH 13c
Alkylated nucleoside
lmax (nm)
l
min (nm)
lmax (nm)
l
min (nm)
l
max (nm)
l
min (nm)
24
Guanosined
N-1-Ethyl-20-deoxyguanosined
N-7-Ethyl-20-deoxyguanosined
262, 277
256, 277
261, 272 (sh)
257, 275 (sh)
229
228
240
229
257, 272
227
226
237
234
258, 272 (sh)
258–266
258, 270 (sh)
267e
226
232
239
249e
253, 273 (sh)
257, 270 (sh)
257, 278
25
Thymidined
N-3-Ethylthymidined
271
267
269
239
234
240
272
267
269
237
234
240
272
267
270
238
245
240
a
0.1 M HCl/H2O–MeOH (9:1).
H2O–MeOH (9:1).
0.1 M KOH/H2O–MeOH (9:1).
See Ref. 12.
Imodazole ring-opened 7-ethyl-20-deoxyguanosine.
b
c
d
e

A. Sakakura et al. / Tetrahedron 60 (2004) 7067–7075
7071
demonstrated that 2 alkylated 20-deoxyguanosine at the
N21 position and thymidine at the N23 position and
modified DNA at the same sites in lower yields. The further
studies of biological activities of jolkinolide D pharmaco-
phore are in progress.
aqueous NH4Cl (10 mL), and extracted with EtOAc
(5£25 mL). The combined extracts were washed with
brine (25 mL), dried (Na2SO4), and concentrated. The
residue was purified by column chromatography on silica
gel (20 g, hexane–acetone 3:1!2:1!1:1!1:2) to give
(^)-7 (158 mg, 79%): colorless solid; IR (neat) 3350, 1640,
1
1440, 1170, 1070, 1010, 920 cm21; H NMR (270 MHz,
CDCl3) d 5.36 (d, J¼1.3 Hz, 1H), 5.30 (q, J¼1.3 Hz, 1H),
5.10 (d, J¼1.3 Hz, 1H), 4.32 (s, 2H), 3.85 (dd, J¼3.3,
7.9 Hz, 1H), 2.77 (br s, 2H), 2.17–1.79 (m, 4H), 1.75 (s,
3H). A signal of a hydroxyl proton was not detected; 13C
NMR (CDCl3) d 151.8, 137.4, 126.8, 115.3, 77.6, 74.4, 63.7,
28.9, 27.5, 23.4; MS (FAB) m/z 207 (MþNa)þ; HRMS
(FAB) calcd for C10H16NaO3 [(MþNa)þ] 207.0997, found
207.0996.
4. Experimental
4.1. General
Unless otherwise noted, materials were obtained from
commercial suppliers and used without further purification.
Pig liver esterase (PLE) was purchased from SIGMA as a
lyophilized powder. All reactions involving organometallic
reagents were conducted under a nitrogen atmosphere.
Fuji silysia silica gel BW-820 MH and Nacalai tesque
Cosmosil 75C18-OPN were used for column chroma-
tography. NMR spectra were measured at 270 or
4.1.3. (6)-Triol 8. (^)-Diol 6 (189 mg, 0.630 mmol) was
transformed into (^)-triol 8 (91.4 mg, 80%) by the same
procedure as described for (^)-7. (^)-8: colorless solid; IR
1
(neat) 3370, 1670, 1440, 1380, 1050, 1020, 960 cm21; H
1
500 MHz for H and 67.8 MHz for 13C. Chemical shifts
NMR (270 MHz, CDCl3) d 5.30 (d, J¼1.0 Hz, 1H), 5.26 (s,
1H), 5.20 (d, J¼1.0 Hz, 1H), 4.27 (d, J¼12.5 Hz, 1H), 4.19
(d, J¼12.5 Hz, 1H), 3.79 (t, J¼7.3 Hz, 1H), 3.04 (br s, 2H),
2.29–1.79 (m, 4H), 1.74 (s, 3H). A signal of a hydroxyl
proton was not detected; 13C NMR (CDCl3) d 151.7, 138.6,
124.8, 114.9, 74.7, 72.0, 63.5, 28.6, 25.8, 23.3; MS (FAB)
m/z 207 (MþNa)þ; HRMS (FAB) calcd for C10H16NaO3
[(MþNa)þ] 207.0997, found 207.1013.
are described as d values in ppm relative to TMS. J values
are given in Hz.
4.1.1. (6)-Diols 5 and 6. To a stirred solution of 2-iodo-
allylalcohol (250 mg, 1.4 mmol) in Et2O was added a 1.5 M
solution of t-BuLi in pentane (2.3 mL, 3.4 mmol) at
278 8C. The mixture was stirred at 0 8C for 2.5 h, and
then a solution of (^)-ketone 3 (100 mg, 0.45 mmol) in
Et2O (1 mL) was added dropwise. The resulting mixture
was stirred at 0 8C for 1 h. After the addition of methanol
(0.8 mL) and H2O (5 mL), the reaction mixture was
extracted with Et2O (3£5 mL). The combined extracts
were washed with brine (5 mL), dried (Na2SO4), and
concentrated. The residue was purified by column chroma-
tography on silica gel (4 g, hexane–Et2O 2:1!1:1!1:2) to
give (^)-5 (76 mg, 56%) and (^)-6 (44 mg, 33%). (^)-5:
colorless oil; IR (neat) 3410, 1470, 1090, 1060, 890,
840 cm21; 1H NMR (270 MHz, CDCl3) d 5.33 (s, 1H), 5.30
(d, J¼1.7 Hz, 1H), 5.08 (d, J¼1.7 Hz, 1H), 4.38 (d, J¼
12.2 Hz, 1H), 4.27 (d, J¼12.2 Hz, 1H), 3.95 (t, J¼6.9 Hz,
1H), 3.25 (br s, 1H), 3.04 (br s, 1H), 2.14–1.82 (m, 4H),
1.78 (s, 3H), 0.96 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H); 13C
NMR (CDCl3) d 149.0, 136.5, 125.9, 117.7, 78.0, 65.1, 28.9,
27.5, 25.9, 23.0, 18.2, 24.5, 24.5. A signal of one of
quaternary carbons was not detected; MS (FAB) m/z 321
(MþNa)þ; HRMS (FAB) calcd for C16H30NaO3Si
[(MþNa)þ] 321.1862, found 321.1878. (^)-6: colorless
4.1.4. (6)-Jolkinolide D pharmacophore (2). To a stirred
solution of (^)-7 (50 mg, 0.27 mmol) in CHCl3 (5 mL) was
added MnO2 (0.12 g, 1.37 mmol) at ambient temperature.
The mixture was stirred at ambient temperature for 1 h. To
the mixture was added MnO2 (0.12 g, 1.37 mmol), and the
mixture was stirred at ambient temperature for 1 h. Further,
MnO2 (0.12 g, 1.37 mmol) was added, and the mixture was
stirred at ambient temperature for 1 h. The reaction mixture
was filtered through a pad of Celite, and the residue was
washed with CHCl3. The filtrate and washings were
combined and concentrated. The residue was purified by
column chromatography on silica gel (9 g, hexane–EtOAc
2:1!1:1) to give (^)-2 (4.4 mg) and a diastereomeric
mixture of hemiacetals (27.8 mg). To a stirred solution of the
diastereomeric mixture of hemiacetals (27.8 mg) in CHCl3
(5 mL) was added MnO2 (0.13 g, 1.53 mmol) at ambient
temperature. The mixture was stirred at ambient tempera-
ture for 3 h. The reaction mixture was filtered through a pad
of Celite, and the residue was washed with CHCl3. The
filtrate and washings were combined and concentrated. The
residue was purified by column chromatography on silica
gel (0.5 g, hexane–EtOAc 2:1!1:1) to give (^)-2
(22.9 mg). Total yield: 27.3 mg (56%). (^)-2: colorless
solid; UV (CH3CN) lmax 223 nm (1 5500); IR (neat) 3400,
1
oil; IR (neat) 3390, 1470, 1100, 1010, 900, 840 cm21; H
NMR (270 MHz, CDCl3) d 5.25 (d, J¼1.3 Hz, 1H), 5.21 (s,
1H), 5.10 (d, J¼1.3 Hz, 1H), 4.29 (dd, J¼4.3, 13.2 Hz, 1H),
4.09 (dd, J¼7.9, 13.2 Hz, 1H), 3.86 (dd, J¼4.0, 6.9 Hz, 1H),
2.62 (dd, J¼4.3, 7.9 Hz, 1H), 2.14–1.75 (m, 4H), 1.73 (s,
3H), 0.90 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H). A signal of a
hydroxyl proton was not detected; 13C NMR (CDCl3) d
151.2, 137.1, 124.6, 115.0, 75.5, 71.8, 64.4, 27.7, 26.8, 25.8,
23.3, 18.1, 24.1, 24.5; MS (FAB) m/z 321 (MþNa)þ;
HRMS (FAB) calcd for C16H30NaO3Si [(MþNa)þ]
321.1862, found 321.1886.
1760, 1670, 1270, 1150, 1010, 950 cm21
;
1H NMR
(270 MHz, CDCl3) d 6.27 (s, 1H), 5.85 (s, 1H), 5.24 (m,
1H), 4.49 (ddd, J¼1.3, 3.0, 4.0 Hz, 1H), 2.22–2.07 (m, 2H),
1.98–1.84 (m, 2H), 1.74 (s, 3H), a signal of a hydroxyl
proton was not detected; 13C NMR (CDCl3) d 168.8, 142.1,
140.0, 121.9, 121.1, 81.1, 72.8, 23.8, 23.7, 22.4; MS (FAB)
m/z 203 (MþNa)þ, 181 (MþH)þ; HRMS (FAB) calcd for
C10H12NaO3 [(MþNa)þ] 203.0684, found 203.0685.
4.1.2. (6)-Triol 7. To a stirred solution of (^)-5 (323 mg,
1.09 mmol) in THF (12 mL) was added a 1.0 M solution of
Bu4NF in THF (2.18 mL, 2.18 mmol) at 0 8C. The mixture
was stirred at 0 8C for 30 min, diluted with saturated
4.1.5. (6)-Aldehyde 9. To a stirred solution of (^)-8

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A. Sakakura et al. / Tetrahedron 60 (2004) 7067–7075
(6.2 mg, 0.030 mmol) in CHCl3 (5 mL) was added MnO2
(61.2 mg, 0.70 mmol) at ambient temperature. The mixture
was stirred at ambient temperature for 16 h. The reaction
mixture was filtered through a pad of Celite, and the residue
was washed with CHCl3. The filtrate and washings were
combined and concentrated. The residue was purified by
column chromatography on silica gel (2 g, hexane–EtOAc
2:1!1:1) to give (^)-9 (1.5 mg, 24%): colorless oil; UV
(CH3CN) lmax 209 nm (sh, 1 4800); IR (neat) 3410, 2840,
1690, 1620, 1390, 1060 cm21; 1H NMR (270 MHz, CDCl3)
d 9.60 (s, 1H), 6.59 (s, 1H), 6.22 (s, 1H), 5.24 (s, 1H), 3.90
(m, 1H), 2.52 (d, J¼5.9 Hz, 1H), 2.18–1.81 (m, 4H), 1.76
(s, 3H); 13C NMR (CDCl3) d 197.5, 140.5, 124.4, 72.3, 29.9,
28.0, 25.5, three signals of quaternary carbons were not
detected; MS (FAB) m/z 205 (MþNa)þ; HRMS (FAB)
calcd for C10H14NaO3 [(MþNa)þ] 205.0841, found
205.0820.
4.1.10. (2)-Jolkinolide D pharmacophore (2). The
experimental procedure followed was as described for
compound (^)-2. (2)-2: colorless solid; [a]2D2¼2181 (c
0.10, CHCl3).
Reaction of (^)-2 with L-cysteine. L-Cysteine (3.8 mg,
31 mmol) was dissolved in H2O (0.10 mL), and the pH was
adjusted to 7.5 by adding a trace amount of 1 M NaOH. To
the aqueous solution was added a solution of (^)-2 (5.6 mg,
31 mmol) in THF (0.03 mL). The mixture was stirred at
ambient temperature for 16 h and then concentrated. The
residue was purified by column chromatography on ODS
silica gel (0.5 g, MeOH–H2O 1:10!1:4) to give 12
(5.6 mg, 64%): colorless solid; UV (H2O–MeOH 9:1)
l
max 283 nm (1 11,000); IR (neat) 3450, 1740, 1650, 1495,
1390, 1340, 1040 cm21; H NMR (500 MHz, CD3OD) d
6.56 (s, 0.5H), 6.54 (s, 0.5H), 4.94 (dd, J¼5.1, 13.2 Hz, 1H),
3.74 (m, 1H), 3.54 (d, J¼13.5 Hz, 0.5H), 3.53 (d, J¼
13.5 Hz, 0.5H), 3.46 (d, J¼13.5 Hz, 1H), 3.18 (dd, J¼4.0,
14.7 Hz, 0.5H), 3.16 (dd, J¼3.8, 14.4 Hz, 0.5H), 2.90 (dd,
J¼8.2, 14.7 Hz, 0.5H), 2.89 (dd, J¼9.4, 14.4 Hz, 0.5H),
2.55–2.36 (m, 3H), 1.96 (s, 3H), 1.67–1.53 (m, 1H);
MS (FAB) m/z 284 (MþH)þ; HRMS (FAB) calcd for
C13H17NNaO4S [(MþNa)þ] 306.0776, found 306.0784.
1
4.1.6. (2)-6-Acetoxy-3-methyl-2-cyclohexenone (10). To
a stirred solution of (^)-6-acetoxy-3-methyl-2-cyclohexe-
none (10) (2.4 g, 14.3 mmol) in DMSO (10 mL) was added
0.1 M phosphate buffer (pH 7.0, 250 mL) at 15 8C, and the
mixture was stirred for 30 min. After PLE (50 mg) was
added, the resulting mixture was stirred at 15 8C for 5 h. The
reaction mixture was extracted with Et2O (3£200 mL). The
combined extracts were washed with brine, dried (Na2SO4),
and concentrated. The residual oil was purified by column
chromatography on silica gel (50 g, hexane–Et2O 1:2) to
give (2)-10 (620 mg, 34%) along with (þ)-6-hydroxy-3-
methyl-2-cyclohexenone (11) (372 mg, 16%). (2)-10:
colorless oil; [a]2D0¼2163 (c 1.0, CHCl3).
4.1.11. Reaction of (2)-2 with L-cysteine. (2)-2 (5.1 mg,
28 mmol) and L-cysteine (3.4 mg, 28 mmol) were reacted at
pH 7.5 for 16 h, to give 12a (3.0 mg, 34%) and 13a (1.9 mg,
1
22%). 12a: H NMR (270 MHz, CD3OD) d 6.55 (s, 1H),
4.94 (dd, J¼5.1, 14.9 Hz, 1H), 3.77 (dd, J¼3.8, 8.6 Hz, 1H),
3.55 (d, J¼13.5 Hz, 1H), 3.46 (d, J¼13.5 Hz, 1H), 3.18 (dd,
J¼4.3, 14.9 Hz, 1H), 2.91 (dd, J¼8.4, 14.9 Hz, 1H), 2.52–
2.36 (m, 3H), 1.96 (s, 3H), 1.63 (m, 1H). 13a: UV (H2O–
MeOH 9:1) lmax 282 nm (1 7400); IR (neat) 3440, 1760,
4.1.7. (2)-6-(tert-Butyldimethylsilyloxy)-3-methyl-2-
cyclohexenone (3). To a stirred solution of (2)-10
(1.58 g, 9.40 mmol) in a 1:4 mixture of MeOH and H2O
(10 mL) was added scandium triflate (920 mg, 1.86 mmol)
at ambient temperature. The mixture was stirred at ambient
temperature for 66 h, diluted with H2O (20 mL), and
extracted with EtOAc (3£25 mL). The combined extracts
were washed with brine (10 mL), dried (Na2SO4), and
concentrated to give the crude hydroxyketone (2)-11
(1.58 g). The product was used for the next reaction without
purification. The analytical sample of (2)-11 was prepared
by column chromatography on silica gel (hexane–Et2O
1:1): colorless oil; [a]2D5¼2164 (c 1.08, CHCl3). To a
solution of the crude (2)-11 (1.58 g) in DMF (11 mL) was
added Et3N (2.2 mL, 16 mmol) and TBSCl (2.1 g, 14 mmol)
at ambient temperature. The mixture was stirred at ambient
temperature for 1 h, diluted with H2O (14 mL), and
extracted with Et2O (3£14 mL). The combined extracts
were washed with brine (14 mL), dried (Na2SO4), and
concentrated. The residual oil was purified by column
chromatography on silica gel (60 g, hexane–Et2O
15:1!10:1) to give (2)-3 (1.22 g, 55% from (2)-10):
colorless oil; [a]2D1¼289.3 (c 1.0, CHCl3).
1630, 1390, 1100, 1010 cm21
;
1H NMR (500 MHz,
CD3OD) d 5.59 (s, 0.4H), 5.43 (s, 0.6H), 4.45 (s, 1H),
3.83–3.60 (m, 1H), 3.17 (m, 1H), 2.97 (m, 2H), 2.88–2.73
(m, 1H), 2.58–2.35 (m, 1H), 2.20–1.78 (m, 4H), 1.75 (s,
1.8H), 1.74 (s, 1.2H); MS (FAB) m/z 302 (MþH)þ; HRMS
(FAB) calcd for C13H20NO5S [(MþH)þ] 302.1062, found
302.1056.
4.1.12. Reaction of (6)-2 with L-alanine. L-Alanine
(2.5 mg, 28 mmol) was dissolved in H2O (0.10 mL), and
the pH was adjusted to 7.5 by adding a trace amount of 1 M
NaOH. To the aqueous solution was added a solution of
(^)-2 (5.0 mg, 28 mmol) in THF (0.03 mL) at ambient
temperature. After stirring for 16 h, the reaction mixture
was diluted with H2O (3 mL) and extracted with CHCl3
(3£3 mL). The aqueous layer was concentrated to give a
solid, which was purified by column chromatography on
ODS silica gel (0.5 g, MeOH–H2O 1:20!1:10!1:4!1:1)
and preparative HPLC (Develosil ODS-HG-5, 20£250 mm,
CH3CN–0.02 M aqueous NH4OAc, 8.0 mL/min) to give 14
(3.7 mg, 53%). The combined CHCl3 extracts were dried
(Na2SO4) and concentrated to give a solid, which was
purified by column chromatography on silica gel (0.5 g,
hexane–acetone 3:1!2:1!1:1) to give 15 (0.5 mg, 4%)
along with recovered (^)-2 (0.1 mg, 2%). 14: colorless
solid; UV (H2O–MeOH 9:1) lmax 279 nm (1 16,000); IR
(KBr) 3510, 3360, 1760, 1665, 1590, 1400, 1365,
4.1.8. (2)-Diol 5. The experimental procedure followed was
as described for compound (^)-5. (2)-5: colorless oil;
[a]2D2¼22.5 (c 0.10, CHCl3).
4.1.9. (2)-Triol 7. The experimental procedure followed
was as described for compound (^)-7. (2)-7: colorless
solid; [a]2D2¼14 (c 0.10, CHCl3).
1335 cm21
;
1H NMR (270 MHz, CD3OD) d 6.61 (s,
0.5H), 6.57 (s, 0.5H), 5.05 (dd, J¼5.3, 13.9 Hz, 1H),

A. Sakakura et al. / Tetrahedron 60 (2004) 7067–7075
7073
4.03–3.73 (m, 2H), 3.54 (m, 1H), 2.55–2.43 (m, 3H), 2.00
(s, 3H), 1.95–1.65 (m, 1H), 1.48 (d, J¼6.6 Hz, 3H); MS
(FAB) m/z 252 (MþH)þ; HRMS (FAB) calcd for
C13H18NO4 [(MþH)þ] 252.1236, found 252.1240. 15:
colorless solid; UV (H2O–MeOH 9:1) lmax 281 nm (1
12,000); IR (neat) 1745, 1650, 1460, 1395, 1340,
(t, J¼7.0 Hz, 0.5H), 4.45 (t, J¼7.0 Hz, 0.5H), 3.60–3.38
(m, 2H), 2.63–2.41 (m, 6H), 2.02 (s, 3H), 2.01 (s, 3H),
1.75–1.60 (m, 2H); MS (FAB) m/z 480 (Mþ); HRMS
(FAB) calcd for C26H30N3O6 [(MþH)þ] 480.2135, found
480.2138. 20: colorless solid; UV (H2O–MeOH 9:1) lmax
280 (1 17,000); IR (neat) 3480, 1740, 1660, 1430, 1340,
1
1
1035 cm21; H NMR (500 MHz, CDCl3) d 6.38 (s, 1H),
1200, 1140, 1040 cm21; H NMR (500 MHz, CD3OD) d
6.37 (s, 1H), 4.80–4.76 (m, 2H), 3.59–3.51 (m, 1H), 3.44–
3.26 (m, 4H), 2.50–2.38 (m, 6H), 1.96 (s, 6H), 1.75–1.53
(m, 2H), 1.38 (d, J¼7.0 Hz, 1.5H), 1.36 (d, J¼7.0 Hz,
1.5H); MS (FAB) m/z 414 (MþH)þ; HRMS (FAB) calcd for
C23H28NO6 [(MþH)þ] 414.1917, found 414.1889.
9.02 (s, 1H), 7.57 (s, 1H), 6.61 (s, 1H), 6.53 (s, 1H), 5.13 (s,
2H), 5.06 (dd, J¼5.2, 13.0 Hz, 1H), 5.05 (dd, J¼5.2,
13.0 Hz, 1H), 4.42–4.34 (m, 1H), 4.08 (d, J¼12.7 Hz, 1H),
4.04 (d, J¼12.7 Hz, 1H), 3.56–3.38 (m, 2H), 2.61–2.43 (m,
6H), 2.01 (s, 3H), 2.00 (s, 3H), 1.74–1.63 (m, 2H); MS
(FAB) m/z 480 (MþH)þ; HRMS (FAB) calcd for
C26H30N3O6 [(MþH)þ] 480.2135, found 480.2138.
4.1.13. Reaction of (2)-2 with L-alanine. (2)-2 (5.1 mg,
28 mmol) and L-alanine (2.5 mg, 28 mmol) were reacted at
pH 7.5 for 16 h, to give 14a (1.4 mg, 20%) along with
4.1.15. Reaction of (2)-2 with L-histidine. (2)-2
(10.5 mg, 58 mmol) and L-histidine (9.0 mg, 58 mmol)
were reacted at pH 7.5 for 16 h to give 16a (6.3 mg,
34%), 17a (5.3 mg, 29%), 18a (2.7 mg, 15%), 19a (1.5 mg,
1
recovered (2)-2 (3.5 mg, 69%): 14a: H NMR (270 MHz,
CD3OD) d 6.60 (s, 1H), 5.04 (dd, J¼4.9, 13.0 Hz, 1H),
4.00–3.78 (m, 2H), 3.55 (m, 1H), 2.55–2.43 (m, 3H), 2.00
(s, 3H), 1.70 (m, 1H), 1.49 (d, J¼6.5 Hz, 3H).
1
5.4%), and 20a (1.4 mg, 5.0%). 16a: H NMR (270 MHz,
CD3OD) d 8.85 (s, 1H), 7.49 (s, 1H), 6.54 (s, 1H), 5.05 (dd,
J¼5.1, 13.2 Hz, 1H), 4.30 (dd, J¼6.2, 6.8 Hz, 1H), 4.04 (s,
2H), 3.50 (dd, J¼6.2, 14.9 Hz, 1H), 3.42 (dd, J¼6.8,
14.9 Hz, 1H), 2.60–2.40 (m, 3H), 1.99 (s, 3H), 1.71 (m,
1H). 17a: 1H NMR (270 MHz, CD3OD) d 8.93 (s, 1H), 7.50
(s, 1H), 6.60 (s, 1H), 5.11 (s, 2H), 5.04 (dd, J¼5.7, 13.8 Hz,
1H), 4.29 (t, J¼6.8 Hz, 1H), 3.39 (dd, J¼6.8, 14.9 Hz, 1H),
2.58–2.41 (m, 3H), 2.01 (s, 3H), 1.68 (m, 1H). A signal of a
proton (b-position of L-histidine) was overlapped with the
4.1.14. Reaction of (6)-2 with L-histidine. L-Histidine
(9.0 mg, 58 mmol) was dissolved in H2O (0.50 mL), and the
pH was adjusted to 7.5 by adding a trace amount of 1 M
NaOH. To the aqueous solution was added a solution of
(^)-2 (11 mg, 58 mmol) in THF (0.08 mL). The mixture
was stirred at ambient temperature for 16 h and concen-
trated. The residue was purified by column chromatography
on silica gel (ODS 0.8 g, MeOH–0.1 M aqueous TFA
1:20!1:5!1:1) and preparative HPLC (Develosil ODS-
HG-5, 20£250 mm, CH3CN–0.2 M aqueous TFA 15:85,
8.0 mL/min) to give 16 (3.6 mg, 19%), 17 (4.9 mg, 26%), 18
(3.2 mg, 17%), 19 (1.1 mg, 3.8%), and 20 (2.5 mg, 8.5%).
16: colorless oil; UV (H2O–MeOH 9:1) lmax 280 (1 7100);
1
solvent signals. 18a: H NMR (270 MHz, CD3OD) d 8.91
(s, 1H), 7.61 (s, 1H), 5.20 (s, 1H), 4.61 (dd, J¼6.5, 14.0 Hz,
1H), 4.52 (dd, J¼7.6, 14.0 Hz, 1H), 4.50 (dd, J¼4.6,
11.3 Hz, 1H), 4.27 (t, J¼7.0 Hz, 1H), 2.29–1.98 (m, 3H),
1.89–1.74 (m, 1H), 1.72 (s, 3H). Signals of three protons
(H-14 of jolkinolide D pharmacophore and b-position of
L-histidine) were overlapped with the solvent signals. 19a:
1H NMR (270 MHz, CD3OD) d 9.16 (s, 1H), 7.58 (s, 1H),
6.59 (s, 1H), 6.56 (s, 1H), 5.15 (s, 2H), 5.12 (s, 2H), 5.10–
4.97 (m, 2H), 4.29 (m, 1H), 2.63–2.39 (m, 6H), 2.02 (s, 3H),
2.01 (s, 3H), 1.83–1.55 (m, 2H). Signals of two protons
(b-position of L-histidine) were overlapped with the solvent
1
IR (neat) 1730, 1720, 1670, 1650, 1200, 1140 cm21; H
NMR (500 MHz, CD3OD) d 8.89 (s, 1H), 7.51 (s, 1H), 6.54
(s, 1H), 5.06 (dd, J¼5.1, 13.7 Hz, 1H), 4.40 (m, 1H), 4.09 (s,
2H), 3.57 (dd, J¼5.1, 15.5 Hz, 0.5H) 3.53 (dd, J¼4.8,
16.9 Hz, 0.5H), 3.45 (m, 1H), 2.61–2.44 (m, 3H), 2.00 (s,
3H), 1.75–1.64 (m, 1H); MS (FAB) m/z 318 (MþH)þ;
HRMS (FAB) calcd for C16H20N3O4 [(MþH)þ] 318.1454,
found 318.1467. 17: colorless oil; UV (H2O–MeOH 9:1)
1
signals. 20a: H NMR (270 MHz, CD3OD) d 8.76 (s, 1H),
l
max 279 (1 5900); IR (neat) 3450, 1730, 1680, 1440, 1200,
7.45 (s, 1H), 6.58 (s, 1H), 6.52 (s, 1H), 5.07 (s, 2H), 5.03
(dd, J¼4.6, 13.0 Hz, 2H), 4.10 (m, 1H), 4.01 (d, J¼13.5 Hz,
1H), 3.93 (d, J¼13.5 Hz, 1H), 2.61–2.43 (m, 6H), 2.01 (s,
3H), 1.99 (s, 3H), 1.74–1.63 (m, 1H). Signals of two
protons (b-position of L-histidine) were overlapped with the
solvent signals.
1140 cm21; H NMR (500 MHz, CD3OD) d 8.98 (s, 1H),
7.52 (s, 1H), 6.60 (s, 1H), 5.04 (dd, J¼5.1, 13.6 Hz, 1H),
5.12 (s, 2H), 4.31 (t, J¼6.7 Hz, 1H), 3.40 (dd, J¼6.7,
15.9 Hz, 1H), 2.61–2.43 (m, 3H), 2.01 (s, 3H), 1.73–1.62
(m, 1H). A signal of a proton (b-position of histidine) was
overlapped with the solvent signals; MS (FAB) m/z 318
(MþH)þ; HRMS (FAB) calcd for C16H20N3O4 [(MþH)þ]
318.1454, found 318.1478. 18: colorless solid; UV (H2O–
MeOH 9:1) lmax 267 (1 2100); IR (neat) 3440, 1770, 1680,
1
4.1.16. Reaction of (6)-2 with glutathione. Glutathione
(17 mg, 55 mmol) was dissolved in H2O (0.20 mL), and the
pH was adjusted to 7.5 by adding a trace amount of 1 M
NaOH. To the aqueous solution was added a solution of
(^)-2 (10 mg, 55 mmol) in THF (0.06 mL). The mixture
was stirred at ambient temperature for 16 h and concen-
trated. The residue was purified by column chromatography
on silica gel (ODS 0.6 g, MeOH–0.1 M aqueous TFA
1:10!1:9!1:8!1:7!1:3!1:1) and preparative HPLC
(Develosil ODS-HG-5, 20£250 mm, CH3CN–0.2 M aqu-
eous TFA 82:18, 8.0 mL/min) to give 21 (17 mg, 52%), 22
(6.3 mg, 19%), 23 (4.2 mg, 10%) as the TFA salt. 21:
colorless oil; UV (H2O–MeOH 9:1) lmax 283 (1 16,000);
IR (neat) 3380, 1730, 1660, 1650, 1540, 1430, 1200,
1640, 1430, 1200, 1140, 1000 cm21; H NMR (500 MHz,
1
CD3OD) d 8.75 (s, 1H), 7.54 (s, 1H), 5.39 (s, 1H), 4.51 (m,
2H), 4.43 (dd, J¼8.2, 14.3 Hz, 1H), 4.16 (m, 1H), 3.41 (dd,
J¼5.2, 8.2 Hz, 1H), 3.30–3.24 (m, 2H), 2.17–2.07 (m, 2H),
1.99–1.88 (m, 2H), 1.81 (s, 3H); MS (FAB) m/z 336
(MþH)þ; HRMS (FAB) calcd for C16H22N3O5 [(MþH)þ]
336.1559, found 336.1568. 19: colorless solid; UV (H2O–
MeOH 9:1) lmax 280 (1 18,000); IR (KBr) 3450, 1750,
1680, 1440, 1210, 1040 cm21 1H NMR (500 MHz,
;
CD3OD) d 9.19 (s, 1H), 7.61 (s, 1H), 6.58 (m, 1H), 6.56
(s, 1H), 5.15 (s, 2H), 5.12 (s, 2H), 5.08–4.95 (m, 2H), 4.46

7074
A. Sakakura et al. / Tetrahedron 60 (2004) 7067–7075
1
1140 cm21; H NMR (270 MHz, CD3OD) d 6.49 (s, 1H),
4.65 (dd, J¼5.1, 9.0 Hz, 0.5H), 4.63 (dd, J¼5.1, 9.0 Hz,
0.5H), 4.04 (t, J¼5.1 Hz, 1H), 3.92 (s, 2H), 3.45 (br s, 2H),
3.03 (dd, J¼5.1, 14.0 Hz, 0.5H), 3.02 (dd, J¼5.1, 14.0 Hz,
0.5H), 2.77 (dd, J¼9.0, 14.0 Hz, 0.5H), 2.76 (dd, J¼9.0,
14.0 Hz, 0.5 Hz), 2.57 (t, J¼6.9 Hz, 2H), 2.53–2.36 (m,
3H), 2.36–2.08 (m, 2H), 1.95 (s, 3H), 1.80–1.45 (m, 1H), A
signal of a proton (H-12) was overlapped with the solvent
signals; MS (FAB) m/z 470 (MþH)þ; HRMS (FAB) calcd
for C20H28N3O8S [(MþH)þ] 470.1597, found 470.1598. 22:
colorless solid; IR (neat) 3320, 1740, 1670 1540, 1430,
(^)-2 (1.8 mg, 36%). 24: colorless solid; UV (H2O–MeOH
9:1) lmax 257 (1 10,000), 272 nm (1 11,900); IR (neat) 3330,
1730, 1640, 1580, 1540, 1440, 1100 cm21 1H NMR
;
(270 MHz, CD3OD) d 7.96 (s, 1H), 6.77 (s, 1H), 6.25 (dd,
J¼6.2, 7.3 Hz, 1H), 5.12 (d, J¼15.1 Hz, 1H), 4.99 (dd,
J¼5.1, 14.0 Hz, 1H), 4.69 (d, J¼15.1 Hz, 1H), 4.51 (ddd,
J¼3.1, 3.5, 6.2 Hz, 1H), 3.97 (ddd, J¼3.5, 3.5, 4.1 Hz, 1H),
3.76 (dd, J¼3.5, 12.2 Hz, 1H), 3.72 (dd, J¼4.1, 12.2 Hz,
1H), 2.68 (ddd, J¼6.2, 7.3, 13.5 Hz, 1H), 2.47–2.39 (m,
3H), 2.33 (ddd, J¼3.1, 6.2, 13.5 Hz, 1H), 1.94 (s, 3H),
1.71–1.60 (m, 1H); MS (FAB) m/z 430 (MþH)þ; HRMS
(FAB) calcd for C20H24N5O6 [(MþH)þ] 430.1727, found
430.1708.
1200, 1140, 1050, 1010 cm21
;
1H NMR (270 MHz,
CD3OD) d 5.58 (s, 0.3H), 5.42 (s, 0.7H), 4.73–4.58 (m,
1H), 4.46–4.31 (m, 1H), 4.03–3.85 (m, 3H), 3.20–3.02 (m,
2H), 2.98–2.66 (m, 3H), 2.58 (m, 2H), 2.36–2.02 (m, 5H),
2.02–1.80 (m, 1H), 1.77 (s, 2.1H), 1.74 (s, 0.9H); MS
(FAB) m/z 488 (MþH)þ; HRMS (FAB) calcd for
C20H30N3O9S [(MþH)þ] 488.1703, found 488.1715. 23:
colorless solid; UV (H2O–MeOH 9:1) lmax 281 (1 17,000);
4.1.19. Reaction of (2)-2 with 20-deoxyguanosine (pH
7.5). (2)-2 (5.0 mg, 28 mmol) and 20-deoxyguanosine
(7.9 mg, 28 mmol) were reacted at pH 7.5 for 16 h to give
24a (0.6 mg, 5%) along with recovered (2)-2 (4.1 mg,
1
81%). 24a: H NMR (270 MHz, CD3OD) d 7.95 (s, 1H),
IR (neat) 3340, 1740, 1660, 1200, 1030 cm21; H NMR
6.76 (s, 1H), 6.25 (dd, J¼6.2, 7.6 Hz, 1H), 5.12 (d, J¼
15.9 Hz, 1H), 4.99 (dd, J¼5.1, 14.0 Hz, 1H), 4.69 (d, J¼
15.9 Hz, 1H), 4.51 (ddd, J¼3.0, 3.0, 6.2 Hz, 1H), 3.98 (ddd,
J¼3.0, 3.5, 4.1 Hz, 1H), 3.78 (dd, J¼3.5, 12.2 Hz, 1H), 3.70
(dd, J¼4.1, 12.2 Hz, 1H), 2.69 (ddd, J¼6.2, 7.6, 13.5 Hz,
1H), 2.52–2.40 (m, 3H), 2.34 (ddd, J¼3.0, 6.2, 13.5 Hz,
1H), 1.93 (s, 3H), 1.64 (m, 1H).
1
(270 MHz, CD3OD) d 6.54 (br s, 1H), 6.49 (s, 1H), 5.06 (dd,
J¼5.3, 13.4 Hz, 1H), 4.98–4.91 (m, 1H), 4.63 (dd, J¼5.1,
9.2 Hz, 0.5H), 4.62 (dd, J¼5.1, 9.2 Hz, 0.5H), 4.08 (dd,
J¼5.4, 6.3 Hz, 1H), 4.02 (s, 2H), 3.93 (s, 2H), 3.45 (s, 2H),
3.04 (dd, J¼5.1, 14.0 Hz, 0.5H), 3.03 (dd, J¼5.1, 14.0 Hz,
0.5H), 2.77 (dd, J¼9.2, 14.0 Hz, 0.5H), 2.76 (dd, J¼9.2,
14.0 Hz, 0.5H), 2.66 (t, J¼6.5 Hz, 2H), 2.62–2.37 (m, 6H),
2.32–2.18 (m, 2H), 2.00 (s, 3H), 1.95 (s, 3H), 1.83–1.55
(m, 2H); MS (FAB) m/z 632 (MþH)þ; HRMS (FAB) calcd
for C30H38N3O10S [(MþH)þ] 632.2278, found 632.2274.
4.1.20. Reaction of (2)-2 with 20-deoxyguanosine (pH
8.9). (2)-2 (5.0 mg, 28 mmol) and 20-deoxyguanosine
(7.9 mg, 28 mmol) were reacted at pH 8.9 for 16 h to give
24a (7.1 mg, 52%) along with recovered (2)-2 (2.1 mg,
42%).
4.1.17. Reaction of (2)-2 with glutathione. (2)-2 (10 mg,
55 mmol) and glutathione (17 mg, 55 mmol) were reacted at
pH 7.5 for 16 h to give 21a (20 mg, 60%) and 22a (12 mg,
37%) as the TFA salt, respectively. 21a: 1H NMR
(270 MHz, CD3OD) d 6.47 (s, 1H), 4.64 (dd, J¼5.4,
8.9 Hz, 1H), 4.04 (t, J¼5.9 Hz, 1H), 3.91 (s, 2H), 3.51 (d,
J¼13.5 Hz, 1H), 3.40 (d, J¼13.5 Hz, 1H), 3.02 (dd, J¼5.4,
14.3 Hz, 1H), 2.71 (dd, J¼8.9, 14.3 Hz, 1H), 2.54 (t,
J¼6.8 Hz, 2H), 2.53–2.36 (m, 3H), 2.20 (m, 2H), 1.93 (s,
3H), 1.60 (m, 1H). A signal of a proton (H-12 of jolkinolide
D pharmacophore) was overlapped with the solvent signals.
4.1.21. Reaction of (6)-2 with thymidine (pH 8.7).
Thymidine (5.9 mg, 24 mmol) was dissolved in H2O
(0.1 mL), and the pH was adjusted to 8.7 by adding a
trace amount of 1 M NaOH. To the aqueous solution was
added a solution of (^)-2 (4.4 mg, 24 mmol) in THF
(0.03 mL) at ambient temperature. After stirring for 16 h,
the reaction mixture was diluted with H2O (3 mL) and
extracted with EtOAc (3£3 mL). The aqueous layer was
concentrated to give a solid, which was purified by column
chromatography on silica gel (ODS, 0.5 g, MeOH-H2O
1:10!1:3!1:2!1:1) to give impure 25 (1.9 mg). The
combined organic extracts were dried and concentrated
to give a solid, which was purified by column chroma-
tography on silica gel (ODS, 0.5 g, MeOH–H2O
1:10!1:3!1:2!1:1) to give impure 25 (4.7 mg) along
with recovered (^)-2 (1.7 mg, 39%). Further purification of
the combined impure 25 by preparative HPLC (Develosil
ODS-HG-5, 20£250 mm, MeOH–H2O 40:60!45:55,
linear gradient, 50 min, 5.0 mL/min) gave 25 (2.1 mg,
22%). 25: colorless solid; UV (H2O–MeOH 9:1) lmax
274 nm (1 18,000); IR (neat) 3430, 1740, 1700, 1650, 1340,
1
22a: H NMR (270 MHz, CD3OD) d 5.58 (s, 0.33H), 5.42
(s, 0.67H), 4.71–4.58 (m, 1H), 4.48–4.31 (m, 1H), 4.10–
3.99 (m, 1H), 3.93 (s, 2H), 3.20–3.02 (m, 2H), 2.97–2.68
(m, 3H), 2.68–2.50 (m, 2H), 2.33–2.01 (m, 5H), 2.01–1.80
(m, 1H), 1.77 (s, 2H), 1.73 (s, 1H).
4.1.18. Reaction of (6)-2 with 20-deoxyguanosine.
20-Deoxyguanosine (7.9 mg, 28 mmol) was dissolved in
H2O (1.25 mL), and the pH was adjusted to 7.5 by adding a
trace amount of 1 M NaOH. To the aqueous solution was
added a solution of (^)-2 (5.0 mg, 28 mmol) in THF
(0.03 mL) at ambient temperature. After stirring for 16 h,
the reaction mixture was diluted with H2O (3 mL) and
extracted with a 2:1 mixture of hexane and Et2O (3£3 mL).
The aqueous layer was concentrated to give a solid, which
was purified by column chromatography on ODS silica gel
(0.5 g, MeOH–H2O 1:4!1:3!1:2!1:1!2:1) to give 24
(2.5 mg, 21%). The combined organic extracts were dried
and concentrated to give a solid, which was purified by
column chromatography on silica gel (0.5 g, CHCl3–
acetone–MeOH 10:1:0!5:5:1!3:3:1!2:2:1) to recover
1
1050 cm21; H NMR (500 MHz, CDCl3) d 7.40 (q, J¼
1.2 Hz, 1H), 6.38 (s, 0.5H), 6.37 (s, 0.5H), 6.18 (t, J¼
6.5 Hz, 0.5H), 6.17 (t, J¼6.5 Hz, 0.5H), 4.93 (d, J¼14.7 Hz,
0.5H), 4.91 (d, J¼14.7 Hz, 0.5H), 4.79 (d, J¼14.7 Hz,
0.5H), 4.77 (d, J¼14.7 Hz, 0.5H), 4.74 (dd, J¼4.9, 13.5 Hz,
1H), 4.60 (m, 1H), 4.01 (ddd, J¼3.3, 3.3, 3.3 Hz, 1H), 3.94
(br d, J¼12.4 Hz, 1H), 3.86 (br d, J¼12.4 Hz, 1H), 2.56
(br s, 1H), 2.44–2.33 (m, 5H), 1.93 (s, 3H), 1.91 (s, 3H),
1.63–1.58 (m, 1H), 1.57 (br s, 1H); MS (FAB) m/z 405

A. Sakakura et al. / Tetrahedron 60 (2004) 7067–7075
7075
(MþH)þ; HRMS (FAB) calcd for C20H25N2O7 [(MþH)þ]
for Bioorganic Research, Shorai Foundation for Science and
Technology, Yamada Science Foundation, the Fujisawa
Foundation, University of Tsukuba Research Projects, and
Wako Pure Chemical Industries, Ltd. The IR and NMR
spectra were recorded at the Chemical Analysis Center,
University of Tsukuba.
405.1662, found 405.1667.
4.1.22. Reaction of (2)-2 with thymidine (pH 8.7). (2)-2
(4.1 mg, 23 mmol) and thymidine (5.5 mg, 23 mmol) were
reacted at pH 8.7 for 16 h to give 25a (1.0 mg, 18%) along
with recovered (2)-2 (3.5 mg, 84%). 25a: 1H NMR
(500 MHz, CDCl3) d 7.35 (q, J¼0.9 Hz, 1H), 6.38 (s, 1H),
6.17 (t, J¼6.7 Hz, 1H), 4.92 (d, J¼14.9 Hz, 1H), 4.76 (d,
J¼14.9 Hz, 1H), 4.74 (dd, J¼5.3, 13.7 Hz, 1H), 4.60 (m,
1H), 3.98 (ddd, J¼3.2, 3.5, 3.5 Hz, 1H), 3.92 (br d, J¼
12.2 Hz, 1H), 3.81 (br d, J¼12.2 Hz, 1H), 2.51–2.27 (m,
5H), 1.93 (s, 3H), 1.91 (s, 3H), 1.73–1.44 (m, 1H).
References and notes
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4.1.23. Reaction of (6)-2 with DNA. DNA from salmon
spermary (30 mg, 0.10 mmol in nucleotide) was dissolved
in H2O (1.5 mL), and the pH was adjusted to 7.5 by adding a
trace amount of 1 M NaOH. To the aqueous solution was
added a solution of (^)-2 (17.4 mg, 0.10 mmol) in MeOH
(0.15 mL) at 37 8C. After stirring at 37 8C for a week, the
reaction mixture was diluted with H2O (3 mL) and extracted
with EtOAc (3£3 mL). The aqueous layer was concentrated
to give a solid, which was hydrolyzed with deoxyribo-
nuclease I (Takara, 1830 unit) in an aqueous buffer [3.6 mL,
50 mM Tris–HCl (pH 9.0), 5 mM MgCl2, 10 mM 2-mer-
captoethanol] at 37 8C overnight. To the reaction mixture
were added snake venom phosphodiesterase (Worthington
Biochemical Corporation, 63.5 unit) and bacterial alkaline
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by preparative HPLC (Develosil ODS-HG-5, 20£250 mm,
MeOH–H2O 40:60!45:55, linear gradient, 50 min,
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0.26%) along with 2 -deoxyadenosine, 20-deoxyguanosine,
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extracts were dried and concentrated to recover (^)-2
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This work was supported in part by a Grant-in-Aid for
Priority Area from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, Suntory Institute
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