Hymenoic acid, a novel specific inhibitor of human DNA polymerase λ from a fungus of Hymenochaetaceae sp.

Article abstract of DOI:10.1016/j.bmc.2008.03.021

Hymenoic acid (1) is a natural compound isolated from cultures of a fungus, Hymenochaetaceae sp., and this structure was determined by spectroscopic analyses. Compound 1 is a novel sesquiterpene, trans-4-[(1′E,5′S)-5′-carboxy-1′-methyl-1′-hexenyl]cyclohexanecarboxylic acid. This compound selectively inhibited the activity of human DNA polymerase λ (pol λ) in vitro, and 50% inhibition was observed at a concentration of 91.7 μM. Compound 1 did not influence the activities of the other seven mammalian pols (i.e., pols α, γ, δ, ε, η, ι, and κ), but also showed no effect even on the activity of pol β, which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. This compound also did not inhibit the activities of prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 1 could be a selective inhibitor of eukaryotic pol λ. This compound had no inhibitory activities against two N-terminal truncated pol λ, del-1 pol λ (lacking nuclear localization signal (NLS), BRCA1 C-terminus (BRCT) domain [residues 133-575]), and del-2 pol λ (lacking NLS, BRCT, domain and proline-rich region [residues 245-575]). The compound 1-induced inhibition of intact pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol λ inhibitory mechanism of compound 1 is discussed.

Full text of DOI:10.1016/j.bmc.2008.03.021

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5115–5122
Hymenoic acid, a novel specific inhibitor of human
DNA polymerase k from a fungus of Hymenochaetaceae sp.
Masayuki Nishida,a, Noriko Ida,b, Mao Horio,b Toshifumi Takeuchi,b Shinji Kamisuki,b
Hiroshi Murata,b Kouji Kuramochi,b Fumio Sugawara,b
Hiromi Yoshidaa,c and Yoshiyuki Mizushinaa,c,
*
aLaboratory of Food & Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe,
Hyogo 651-2180, Japan
bDepartment of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan
cCooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
Received 19 February 2008; revised 5 March 2008; accepted 6 March 2008
Available online 10 March 2008
Abstract—Hymenoic acid (1) is a natural compound isolated from cultures of a fungus, Hymenochaetaceae sp., and this structure
was determined by spectroscopic analyses. Compound 1 is a novel sesquiterpene, trans-4-[(10E,50S)-50-carboxy-10-methyl-10-hexe-
nyl]cyclohexanecarboxylic acid. This compound selectively inhibited the activity of human DNA polymerase k (pol k) in vitro,
and 50% inhibition was observed at a concentration of 91.7 lM. Compound 1 did not influence the activities of the other seven
mammalian pols (i.e., pols a, c, d, e, g, i and j), but also showed no effect even on the activity of pol b, which is thought to have
a very similar three-dimensional structure to the pol b-like region of pol k. This compound also did not inhibit the activities of pro-
karyotic pols and other DNA metabolic enzymes tested. These results suggested that compound 1 could be a selective inhibitor of
eukaryotic pol k. This compound had no inhibitory activities against two N-terminal truncated pol k, del-1 pol k (lacking nuclear
localization signal (NLS), BRCA1 C-terminus (BRCT) domain [residues 133–575]), and del-2 pol k (lacking NLS, BRCT domain
and proline-rich region [residues 245–575]). The compound 1-induced inhibition of intact pol k activity was non-competitive with
respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol k inhibitory mechanism of
compound 1 is discussed.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ural compound is a novel sesquiterpene, hymenoic
acid (1), produced by a fungus (Hymenochaetaceae
sp.) (Fig. 1). To our knowledge, there have been no
reports about such natural inhibitors specific to
X-family pols such as b, k and l, except for solanapy-
rone A as a pol b- and k-inhibitor,7 and prunasin as a
pol b-inhibitor,5 which we reported previously. The
compound differed from solanapyrone A in that it
inhibited only pol k among the pols examined to
date.7 No such pol k-specific inhibitors have been
reported.
The human genome encodes 16 DNA polymerases
(pols) to conduct cellular DNA synthesis.1 Eukaryotic
cells reportedly contain three replicative types: pols a,
d, and e, mitochondrial pol c, and at least twelve re-
pair types: pols b, d, e, f, g, h, i, j, k, l, r and
REV1.2 We have searched for natural compounds that
selectively inhibit each of these eukaryotic pols, to use
as tools and molecular probes to distinguish pols and
to clarify their biological and in vivo functions.3–10 In
this study, we report a newly found compound that
selectively inhibits only the activity of pol k. The nat-
6'
5'
3
2
OH
4
1'
3'
7'
4'
2'
Keywords: Hymenoic acid; Sesquiterpene; DNA polymerase k; Family
X of DNA polymerases; Enzyme inhibitor.
5
HO
O
3
1
2
*
Corresponding author. Tel.: +81 78 974 1551x3232; fax: +81 78 974
5689; e-mail: mizushin@nutr.kobegakuin.ac.jp
These authors contributed equally to this work.
O
Figure 1. Structure of compound 1 (hymenoic acid).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.021

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M. Nishida et al. / Bioorg. Med. Chem. 16 (2008) 5115–5122
Table 1. 1H and 13C NMR spectral data of compound 1 (hymenoic
acid)
Pol k is a recently described eukaryotic pol belonging to
the pol X family comprising enzymes involved in DNA
repair processes, whose main member is pol b.11 Human
pol k (63.4-kDa) consists of a nuclear localization signal
(NLS) (residues 1–35), a BRCA1 C-terminus (BRCT)
domain (residues 36–132), a proline-rich region (residues
133–244) and a pol b-like region containing a helix–hair-
pin–helix (HhH) and pol X motif (residues 245–575).
The C-terminal part of pol k (residues 244–575) is com-
Position
1
dH (J Hz)
dC
44.4
1
2
2.20 (tt, 12.1, 3.8)
2.05–1.99 (m)
1.48–1.37 (m)
30.5
3
1.78–1.70 (m)
1.34–1.21 (m)
1.83 (tt, 11.8, 3.1)
32.1
posed of a catalytic core which is similar to pol b.12
A
4
47.9
180.1
141.4
14.4
truncated pol k, in which the BRCT motif was deleted
from the N-terminal region (i.e., the C-terminal region
including the pol b-like region), has pol activity.
5
10
10-Me
1.58 (s)
20
30
40
5.16 (t, 7.0)
2.06–1.99 (m)
1.72–1.62 (m)
1.48–1.37 (m)
2.44–2.33 (m)
1.13 (d, 7.0)
123.3
26.4
35.0
In this paper, we report the isolation and structural
determination of a new sesquiterpene, hymenoic acid
(1). We discuss the inhibitory action of compound 1
on various eukaryotic pols and DNA metabolic enzymes
and its binding relation to the enzyme structure of pol k.
50
60
70
40.2
17.7
180.8
Recorded in CD3OD for the residual solvent peak as an internal
standard, and chemical shifts are expressed as d ppm. s, singlet; d,
doublet; t, triplet; tt, triplet of triplets; m, multiplet.
2. Results
2.1. Isolation and cultivation of fungus
The fungal strain, HJ31A, was isolated from a coral col-
lected from Hachijo Island, Japan. The fungus was se-
lected by culturing coral fragments on potato dextrose
agar plates (Difco) and was transferred several times.
The culture was incubated at 25 °C. This fungus was
identified as Hymenochaetaceae sp. by TechnoSuruga
Laboratory Co. Ltd (Shizuoka, Japan). A small agar
plug was then transferred into a 2 L Erlenmeyer flask
containing 1 L of a culture 24 g potato dextrose broth
(Difco). Cultures of HJ31A strain (5 L) were grown
for 49 days without shaking in the dark.
A
OH
HO
O
O
B
H
O
H
H
H
H
H
H
O
H
OH
H
2.2. Extraction and purification of pol k inhibitor from
fungi
H
H
Me
Me
H
HO
H
H
H
The fungal mycelia of HJ31A strain (Hymenochaetaceae
sp.) were removed from the culture broth by filtering
through cheesecloth. The filtrate was extracted with
CH2Cl2. The organic layer was evaporated in vacuo to
obtain 189 mg crude residue. This crude extract was sep-
arated by silica gel column chromatography with tolu-
ene/EtOAc (1:1–1:4, with 2% acetic acid) to give an
active fraction, which inhibited pol activity (i.e., mini-
mum inhibitory concentration was less than 1 mg/ml).
The active fraction was concentrated in vacuo to yield
compound 1 (11.5 mg).
Figure 2. (A) Selected COSY (bold lines) and HMBC (1H ! 13C)
(arrows) correlations and (B) key NOESY (dashed lines) correlations
in compound 1 (hymenoic acid).
groups. Taken together, the structure of 1 was deter-
mined to be 4-(-50-carboxy-10-methyl-10-hexenyl)cyclo-
hexanecarboxylic acid.
The relative stereochemistry of 1 was determined by
NOESY analysis (Fig. 2B). NOESY correlations
between axial H-1 and axial H-3 protons, and those
between axial H-4 and axial H-2 protons indicated a
1,4-trans relationship between the substituents bonded
to the cyclohexane ring. The geometry of the double
bond at C-10 was determined to be in an E-orientation
by NOESY correlations between H-4 and H-20, and
between 10-Me and H-30 protons.
2.3. Structure determination of pol k inhibitor
The molecular formula of 1 was determined by high res-
olution electrospray ionization mass spectrometry to be
C15H24O4. 1H and 13C NMR data for 1 are summarized
in Table 1. COSY and HMBC spectra suggested that 1
has 1,4-disubstituted cyclohexane and 6-substituted-2-
methyl-5-heptanonyl moiety as partial structures
(Fig. 2A). The IR absorption at 1705 cmꢀ1 and two car-
bon resonances at d 180.8 and 180.1 (C-70 and C-5,
respectively) showed the presence of two carboxy
Compound 1 was an optically active compound, since
22
D
CH3OH). To elucidate the absolute configuration at
the optical rotation value of 1 was ½aꢁ +16.3 (c 1.0,

M. Nishida et al. / Bioorg. Med. Chem. 16 (2008) 5115–5122
5117
C-50, compound 1 was reduced to the corresponding
diol 2, which was converted to its Mosher esters 3
(Scheme 1). The absolute configuration at C-50, where
a methyl group was located, was elucidated on the basis
of chemical shift differences and signal patterns of the
two geminal protons at C-50.13,14 Reduction of 1 with
LiAlH4 in THF gave 2. Esterification of 2 with
(S)- and (R)-MTPACl gave bis-(R)- and bis-(S)-MTPA
esters (3a and 3b, respectively). Absolute configuration
at C-50 was deduced by differences in the chemical shift
and the signal pattern of the two geminal protons at
C-70 in 3a and 3b.13,14 CH2 protons at C-70 in 3a
appeared as adjacent signals at d 4.16 (Fig. 3A). On
the other hand, CH2 protons at C-70 in 3b appeared as
two separate signals at d 4.24 and d 4.08 (Fig. 3B). These
results indicates that the absolute configuration at C-50
was S. Thus, compound 1 was determined to be trans-
4-[(10E,50S)-50-carboxy-10-methyl-10-hexenyl]cyclohex-
anecarboxylic acid, and named hymenoic acid.
2.4. Effects of compound 1 on the activities of mammalian
DNA polymerases and other DNA metabolic enzymes
First, the isolated compound 1 was investigated as to
whether it inhibits the activities of nine mammalian pols,
pols a to j. As shown in Figure 4A, compound 1 at
200 lM inhibited the activity of human pol k, but had
no influence at all on the activities of nuclear replicative
pols such as calf pol a, human pol d and human pol e,
human mitochondrial replicative pol c, or repair-related
pols such as rat pol b, human pols g, i and j. This com-
pound also inhibited the activity of plant (i.e., rice) pol k
to the same extent as it inhibited human pol k. Com-
pound 1 had no inhibitory effect on fish (i.e., cherry sal-
mon) pol d, insect (i.e., fruit fly) pols a, d and e, plant
(i.e., cauliflower) pol a, or prokaryotic pols such as the
Klenow fragment of Escherichia coli pol I, T4 pol and
Taq pol. The compound did not inhibit the activities
of other DNA metabolic enzymes, such as calf primase
of pol a, human immunodeficiency virus type-1 (HIV-
1) reverse transcriptase, human telomerase, T7 RNA
polymerase, mouse inosine 50-monophosphate (IMP)
dehydrogenase (type II), human topoisomerases I and
II, T4 polynucleotide kinase and bovine deoxyribonucle-
ase I either (Fig. 4B). These results suggested that com-
pound 1 could selectively inhibit the activity of
eukaryotic pol k.
Figure 3. (A) Proton signals of H-5 and H-70 in bis-(R)-MTPA ester
(3a) and (B) proton signals of H-5 and H-70 in bis-(S)-MTPA ester
(3b).
2.5. Inhibitory effect of compound 1 on full-length or
fragments of human pol k
Figure 5 shows the inhibition dose–response curves of
compound 1 against intact or truncated human pol k.
Full-length pol k (residues 1–575) and the N-terminal
deleted versions, del-1 pol k (133–575) and del-2 pol k
(245–575), were prepared. Since a C-terminal pol b-like
region containing pol X motif in pol k is essential for
DNA polymerization activity,12 these truncated pol k
have pol activity. The inhibition of intact pol k was
dose-dependent, with 50% inhibition observed at doses
of 91.7 lM. On the other hand, compound 1 did not
influence the activity of two truncated pol k, del-1 (lack-
ing NLS and BRCT domain) and del-2 (lacking NLS,
BRCT domain and proline-rich region). These results
suggested that the N-terminal region of pol k might be
important for inhibition of the activity of DNA poly-
merization by compound 1.
OH
LiAlH4
THF
1
HO
2.6. Mode of inhibition of pol k by compound 1
2
Next, to elucidate the mechanism by which compound 1
inhibited full-length pol k (residues 1–575), the extent of
inhibition as a function of the DNA template-primer or
dNTP substrate concentration was studied (Table 2). In
kinetic analysis, poly(dA)/oligo(dT)12–18 and deoxythy-
midine 50-triphosphate (dTTP) were used as the synthe-
sized DNA template-primer and deoxynucleoside
50-triphosphate (dNTP) substrate, respectively. Double
reciprocal plots of the results showed that the com-
(
S
)-(–)- or (
R
)-(+)-MTPACl
OR
DMAP
7'
RO
Pyridine
5
3a: R = (
R
)-MTPA
3b: R = (
S)-MTPA
Scheme 1. Synthesis of MTPA esters (3a and 3b).

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M. Nishida et al. / Bioorg. Med. Chem. 16 (2008) 5115–5122
A
Calf DNA polymerase α
Rat DNA polymerase β
Human DNA polymerase γ
Human DNA polymerase δ
Human DNA polymerase ε
Human DNA polymerase η
Human DNA polymerase ι
Human DNA polymerase κ
Human DNA polymerase λ (Full-length: 1-575)
Human DNA polymerase λ (Del-1: 133-575)
Human DNA polymerase λ (Del-2: 245-575)
Cherry salmon DNA polymerase δ
Fruit fly DNA polymerase α
Fruit fly DNA polymerase δ
Fruit fly DNA polymerase ε
Cauliflower DNA polymerase α
Rice DNA polymerase λ
E. coli DNA polymerase I
T4 DNA polymerase
Taq DNA polymerase
DNA polymerase activity (%)
Calf primase of DNA polymerase α
B
HIV-1 reverse transcriptase
Human telomerase
T7 RNA polymerase
Mouse IMP dehydrogenase (type II)
Human DNA topoisomerase I
Human DNA topoisomerase II
T4 Polynucleotide kinase
Bovine deoxyribonuclease I
Relative activity (%)
Figure 4. Effect of compound 1 (hymenoic acid) on the activities of various DNA polymerases and other DNA metabolic enzymes. (A) Pols from
mammals and various species, (B) other DNA metabolic enzymes. Compound 1 (200 lM each) was incubated with each enzyme (0.05 U). % of
relative activity. Enzymatic activity was measured as described previously.3,4,6,22 Enzyme activity in the absence of the compound was taken as 100%.
Data are shown as means SEM of four-independent experiments.
pound 1-induced inhibition of intact pol k activity was
non-competitive with respect to both the DNA tem-
plate-primer and the dNTP substrate. For the DNA
template-primer, the apparent Michaelis constant (Km)
was unchanged at 2.38 lM, whereas 75.0, 58.6 and
48.9% decreases in maximum velocity (Vmax) were ob-
served in the presence of 20, 40 and 60 lM of compound
1, respectively. The Km for the dNTP substrate was un-
changed at 1.18 lM, and the Vmax for the dNTP sub-
strate decreased from 52.6 to 27.0 pmol/h in the
presence of 60 lM of compound 1. Inhibition constant
(Ki) values, obtained from Dixon plots, were found to

M. Nishida et al. / Bioorg. Med. Chem. 16 (2008) 5115–5122
5119
3. Discussion
100
80
60
40
20
0
As described in this report, we found a novel potent
inhibitor specific to human pol k from a fungus,
Hymenochaetaceae sp. This natural compound was
found to be a sesquiterpene, hymenoic acid (1).
Pol k belongs to the pol X family,11,15,16 which includes
pols b, k, l, terminal deoxynucleotidyltransferase, yeast
pol IV, mitochondrial pol b, nuclear pol b from protozo-
ans and 20-kDa aflican swine fever virus pol X.17–23
Family X of pols is composed of a NLS, a BRCT do-
main, a proline-rich region, and a pol b-like region con-
taining two HhHs and a pol X motif (Fig. 6). However,
pol b (residues 1–335) lacks an NLS, a BRCT domain
and a proline-rich region. Human pol k shares 54%,
47% and 30% homology to human pol b, l and yeast
pol IV, respectively. Interestingly, compound 1 inhibited
only the activities of full-length pol k (residues 1–575)
among the eukaryotic pols and other DNA metabolic
enzymes tested, although it did not inhibit the activities
of the N-terminal deleted pol k, such as del-1 (residues
133–575) and del-2 of pol k (residues 245–575), or pol
b (Fig. 5). The compound 1-induced inhibition of intact
pol k was non-competitive with respect to both the
DNA template-primer and the dNTP substrate (Table
2), indicating that compound 1 did not directly bind to
either the DNA template-binding site or the dNTP sub-
strate-binding site of pol k. Since both the DNA tem-
plate-primer and dNTP substrate-binding sites are
present at pol X motif in the pol b-like region of the
C-terminal pol k,11 compound 1 must not directly bind
to the pol b-like core. These results suggested that com-
pound 1 might bind to the N-terminal region of pol k
(residues 1–132) including the NLS and the BRCT do-
main directly, and subsequently inhibited enzymes hav-
ing the pol b-like region of pol k (Fig. 6).
0
50
100
150
200
Compound 1 (μM)
Figure 5. Dose–response curves of compound 1 (hymenoic acid) for
human pol k inhibition. The enzymes used (0.05 U each) were full-
length (residues 1–575) human pol k (closed circle), del-1 (residues
133–575) of human pol k (open triangle), and del-2 (residues 245–575)
of human pol k (open square). Pol activities were measured as
described in Section 4. Pol activity in the absence of the compound was
taken as 100%. Data are shown as means SEM of three-independent
experiments.
Table 2. Kinetic analysis of the inhibitory effects of Compound 1
(hymenoic acid) on the activity of human DNA polymerases k (full-
length, residues 1–575) as a function of the DNA template-primer dose
and the nucleotide substrate concentration
a
a
DNA
substrate
Compound Km Vmax Kib
Inhibitory
(lM) (pmol/h) modea (lM)
1 (lM)
Template-
primerc
0
2.38 83.3
62.5
48.8
52.8
Non-
competitive
20
40
60
Although the biochemical function of pol k is unclear as
yet, pol k appears to work in a similar manner to pol b.22
Pol b, which is widely known to have roles in the short-
patch base excision repair (BER) pathway,24–29 plays an
essential role in neural development.30 Recently, pol k
was found to contain 50-deoxyribose-5-phosphate
(dRP) lyase activity, but no apurinic/apyrimidinic (AP)
lyase activity,28 and to be able to substitute pol b in
in vitro base excision repair (BER), suggesting that pol
k also participates in BER. Northern blot analysis indi-
cated that transcripts of pol b were abundantly expressed
in the testis, thymus and brain in rats,31 but pol k was
efficiently transcribed mostly in the testis.11 The reason
why the testis and thymus require pol b activity has been
suggested; both organs have DNA repair and recombi-
nation systems for meiotic crossing over and immuno-
globulin production,32,33 and the systems require the
polymerase. The roles of pol b in the brain are unknown
as yet; therefore, pol k as well as pol b may also have a
role in the testis. Since the DNA repair system at meiotic
prophase requires pol b activity, the system must contain
a process similar to BER. The system may also require
pol k activity, and pol k may be an essential enzyme
for nucleotide excision repair (NER). In this connection,
the fact that the molecular target of the bio-antimutagen
40.8
Nucleotided
substrate
0
1.18 52.6
40.0
32.3
44.7
Non-
competitive
20
40
60
27.0
a These data were obtained from Lineweaver–Burk plot.
b These data were obtained from Dixon plot.
c That is, poly(dA)/oligo(dT)12–18.
d That is, dTTP.
be 52.8 and 44.7 lM for the DNA template-primer and
dNTP substrate, respectively. Because the Ki value was
smaller for the dNTP substrate than for the DNA tem-
plate-primer, the affinity of compound 1 is greater for
the enzyme–nucleotide substrate binary complex than
for the enzyme–DNA template-primer complex. When
activated DNA (i.e., DNA with gaps digested by bovine
dexoyribonuclease I) and four dNTPs were used as the
DNA template-primer and dNTP substrates, respec-
tively, the mode of inhibition of pol k by compound 1
was the same as that with the synthesized DNA tem-
plate-primer (data not shown).

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M. Nishida et al. / Bioorg. Med. Chem. 16 (2008) 5115–5122
Compound 1 binding
Compound 1
inhibitory
activity
Pol β-like region
NLS
15
HhH HhH
Pol X motif
1
36
133
245
pol λ
Full-length:
BRCT
Proline-rich
++
132
244
575
575
133
pol λ
Del-1:
-
245
pol λ
Del-2:
-
-
575
1
Pol β:
335
Figure 6. Schematic representation of pol k and b of the pol X family. The NLS, BRCT domain, proline-rich region, HhH and pol X motif are
indicated. The pol b-like region includes two HhHs and a pol X motif. The inhibitory activity of compound 1 against these pols is indicated below,
‘++’ is IC50 value = <100 lM, ‘+’ is IC50 value = 100 to 200 lM, and ‘ꢀ’ is IC50 value = >200 lM.
was a pol k inhibitor is of great interest. Bio-antimutagen
may lead to blockage of the mis-match error in BER,
NER and translesion synthesis of DNA-damaged cells.
To determine why a bio-antimutagen is a pol k-specific
inhibitor, we are at present analyzing the structure and
function of pol k using an inhibitor.
purified, and prokaryotic pols and other DNA meta-
bolic enzymes were purchased as described in our previ-
ous report.3,4,6 The intact and truncated human pol k
(full-length, del-1 and del-2 of pol k) was constructed
as described by Shimazaki et al.34 The activities of all
pols and other DNA metabolic enzymes were measured
as described in previous reports.3,4,6,35 The substrates of
the pols were poly(dA)/oligo(dT)12–18 and dTTP as the
DNA template-primer and dNTP substrate, respec-
tively. Compound 1 was dissolved in dimethylsulfoxide
(DMSO) at various concentrations and sonicated for
30 s. The sonicated samples (4 ll) were mixed with
16 ll of each pol enzyme (final amount, 0.05 U) in
50 mM Tris–HCl (pH 7.5) containing 1 mM dithiothre-
itol, 50% glycerol and 0.1 mM EDTA, and kept at 0 °C
for 10 min. These inhibitor–enzyme mixtures (8 ll) were
added to 16 ll of each standard enzyme reaction mix-
ture, and incubation was carried out at 37 °C for
60 min, except for Taq pol, which was incubated at
74 °C for 60 min. Activity without the inhibitor was con-
sidered to be 100%, and the remaining activity at each
concentration of the inhibitor was determined relative
to this value. One unit of pol activity was defined as
the amount of enzyme that catalyzed the incorporation
of 1 nmol of dNTP (i.e., dTTP) into the synthetic
DNA template-primer (i.e., poly(dA)/oligo(dT)12–18, A/
T = 2/1) in 60 min at 37 °C under normal reaction con-
ditions for each enzyme.3,4
Compound 1 could potently inhibit only the activity of
human and plant pol k, and the inhibitory effect of com-
pound 1 on pol k from other species was not tested;
therefore, we will try to investigate it in further study.
Since pol species specificity was extremely high, this ses-
quiterpene could be a useful molecular tool as a pol k-
specific inhibitor in studies to determine the precise roles
of pol k in vitro, and also might be useful to develop a
drug design strategy for cancer chemotherapy agents
for clinical radiation therapy or cancer chemotherapy.
4. Experimental
4.1. Materials
Nucleotides and chemically synthesized DNA template-
primers such as poly(dA), oligo(dT)12–18, and [3H]dTTP
(43 Ci/mmol) were purchased from GE Healthcare Bio-
Sciences (Little Chalfont, UK). All other reagents were
of analytical grade and were purchased from Wako
Chemical Industries (Osaka, Japan).
4.3. Instrumental analyses
4.2. DNA polymerase and other DNA metabolic enzymes
assays
1H and 13C NMR spectra were recorded on a Bruker
400 MHz spectrometer (Avance DRX-400). Samples
were prepared in CD3OD, and the residual solvent peak
(d 3.30) and d 49.0 (ppm) from CD3OD were used as
Pols from mammals, a fish (i.e., cherry salmon), an in-
sect (i.e., fruit fly) and a plant (i.e., cauliflower) were

M. Nishida et al. / Bioorg. Med. Chem. 16 (2008) 5115–5122
5121
internal references for 1H and13C NMR spectra, respec-
tively. Chemical shifts were expressed in d (ppm) relative
to the reference, and coupling constants (J) were ex-
pressed in Hz.
(1H, m), 1.29–1.13 (2H, m), 1.03 (2H, m), 0.92 (3H, d,
J = 6.8 Hz).
4.4.4. Bis-(S)-MTPA ester (3b). To an solution of 2
(3 mg, 12.5 lmol) and DMAP (0.2 mg, 1.64 lmol) in
pyridine (1 ml) was added (R)-(ꢀ)-MTPACl (47 lL,
252 lmol) at rt, and the mixture was stirred at rt for
3 h. The mixture was concentrated and the residue
was purified by silica gel chromatography (CHCl3/
MeOH = 99:1) to yield 3b (9.5 mg, quant.) as a color-
less oil. LR-ESIMS m/z 695.0 [M+Na]+, 1H NMR
(400 MHz, CDCl3); d 7.55–7.49 (4H, m), 7.44–7.38
(6H, m), 4.24 (1H, dd, J = 10.7, 5.6 Hz), 4.18 (1H,
dd, J = 10.7, 6.1 Hz), 4.09 (1H, dd, J = 10.7, 6.6 Hz),
4.08 (1H, dd, J = 10.7, 6.4 Hz), 3.57–3.54 (6H, m),
2.06–1.92 (2H, m), 1.89–1.80 (1H, m), 1.81–1.74 (2H,
m), 1.78–1.68 (1H, m), 1.72–1.65 (2H, m), 1.70–1.60
(1H, m), 1.67–1.60 (1H, m), 1.54 (3H, s), 1.43–1.33
(1H, m), 1.26–1.14 (2H, m), 1.03 (2H, m), 0.92 (3H,
d, J = 6.8 Hz).
Optical rotations were recorded on a JASCO P-1010
digital polarimeter at room temperature.
Infrared spectra (IR) were recorded on a JASCO FT/IR-
410 spectrometer, and were reported as wave numbers
(cmꢀ1).
Electrospray ionization mass spectra (ESIMS) were ob-
tained on an Applied Biosystems mass spectrometer
(APIQSTAR pulsar i) under conditions of high resolu-
tion (HR) or low resolution (LR), using poly (ethylene
glycol) as an internal standard.
4.4. Structure determination
4.4.1. Hymenoic acid (trans-4-[(10E,50S)-50-carboxy-10-
methyl-10-hexenyl]cyclohexanecarboxylic acid) (1). Yel-
22
D
2934, 2861, 1705, 1518, 1454, 1419, 1258, 1041, 925,
low oil; ½aꢁ +16.3 (c 1.0, CH3OH); IR (film) mmax
Acknowledgments
759, 670 cmꢀ1
;
HR-ESIMS m/z found 267.1601
We are grateful for the donations of calf pol a by Dr. M.
Takemura of Tokyo University of Science (Tokyo, Ja-
pan), rat pol b by Dr. A. Matsukage of Japan Women’s
University (Tokyo, Japan), human pol c by Dr. M. Suzu-
ki of Nagoya University School of Medicine (Nagoya, Ja-
pan), human pols d and e by Dr. K. Sakaguchi of Tokyo
University of Science (Chiba, Japan), human pols g and i
by Dr. F. Hanaoka and Dr. C. Masutani of Osaka Uni-
versity (Osaka, Japan), human pol j by Dr. H. Ohmori
and Dr. E. Ohashi of Kyoto University (Kyoto, Japan),
and human pol k by Dr. O. Koiwai and Dr. N. Shimazaki
of Tokyo University of Science (Chiba, Japan).
This work was supported in part by a Grant-in-Aid for
Kobe-Gakuin University Joint Research (A), and the
‘Academic Frontier’ Project for Private Universities:
matching fund subsidy from the Ministry of Education,
Science, Sports, and Culture of Japan (MEXT), 2006–
2010, (H.Y. and Y.M.). Y.M. acknowledges a Grant-
in-Aid for Young Scientists (A) (No. 19680031) from
MEXT, Grants-in-Aid from the Nakashima Founda-
tion (Japan), Foundation of Oil & Fat Industry kaikan
(Japan), and The Salt Science Research Foundation,
No. 08S3 (Japan).
[MꢀH]ꢀ, calcd for C15H23O4: 267.1616; 1H and 13C
NMR data, see Table 1.
4.4.2. (5E)-6-[trans-4-(Hydroxymethyl)cyclohexyl]-2-meth-
ylhept-5-en-1-ol (2). To a solution of 1 (18.0 mg,
67.5 lmol) in THF (10 ml) was added LiAlH4
(25.0 mg, 675 lmol) at rt, and the mixture was stirred
at rt for 1 h. The reaction was quenched by the addition
of EtOAc and Na2SO4, filtrated through Celite and
washed with EtOAc. The filtrate was concentrated,
and the residue was purified by silica gel chromatogra-
phy (toluene/EtOAc = 1:1) to yield 2 (10.2 mg, 57%) as
a colorless oil. LR-ESIMS m/z 266.9 [M+Na]+, 1H
NMR (400 MHz, CDCl3); d 6.81 (1H, t, J = 6.8 Hz),
3.51 (1H, dd, J = 10.2, 5.8 Hz), 3.46 (2H, d,
J = 6.0 Hz), 3.43 (1H, dd, J = 10.2, 6.3 Hz), 2.11–1.93
(2H, m), 1.87–1.81 (2H, m), 1.83–1.73 (1H, m), 1.76–
1.70 (2H, m), 1.67–1.60 (1H, m), 1.58 (3H, s), 1.49–
1.39 (2H, m), 1.23 (2H, dq, J = 11.9, 3.1 Hz), 1.19–
1.11 (1H, m), 0.98 (2H, dq, J = 11.9, 3.1 Hz), 0.93
(3H, d, J = 6.6 Hz); 13C NMR (100 MHz, CDCl3); d
140.0, 122.6, 68.7, 68.3, 47.2, 40.3, 35.4, 33.2, 31.1
(2C), 29.6 (2C), 25.1, 16.5, 14.4.
4.4.3. Bis-(R)-MTPA ester (3a). To an solution of 2
(3 mg, 12.5 lmol) and DMAP (0.2 mg, 1.64 lmol) in
pyridine (1 ml) was added (S)-(+)-MTPACl (23 lL,
123.2 lmol) at rt, and the mixture was stirred at rt for
20 h. The mixture was concentrated and the residue
was purified by silica gel chromatography (toluene/
EtOAc = 2:1) to yield 3a (1.1 mg) as a colorless oil.
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