Alkylbenzoquinone involved in development of cellular slime molds

Article abstract of DOI:10.1021/ol5014119

The structure of the prespore-cell-promoting factor from Dictyostelium discoideum was determined to be 2-hydroxy-5-methyl-6-pentylbenzoquinone. The synthetic compound has prespore-cell-promoting activity similar to the natural one, with half-maximal induction at a concentration as low as 40 pM. It was also found that the factor induces aggregation in an aggregation-deficient mutant of a related species, Polysphodilium violaceum. Both these activities are sensitive to positional isomerism with the 6-methyl-5-pentyl isomer showing no detectable activity.

Full text of DOI:10.1021/ol5014119

Letter
pubs.acs.org/OrgLett
Alkylbenzoquinone Involved in Development of Cellular Slime Molds
Yoshiaki Takaya,*,† Rie Hotta,† Kenshu Fujiwara,‡ Risa Otani,† Yurika Uchiyama,† Mizuki Sakakibara,†
Eri Fukuda,† Masatake Niwa,† Kei Inouye,∥ and Akiko A. Oohata∥,§
†Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan
‡Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
§Biological Laboratory, Kansai Medical University, Hirakata, Osaka 573-1136, Japan
∥Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
S
* Supporting Information
ABSTRACT: The structure of the prespore-cell-promoting factor from
Dictyostelium discoideum was determined to be 2-hydroxy-5-methyl-6-
pentylbenzoquinone. The synthetic compound has prespore-cell-promoting
activity similar to the natural one, with half-maximal induction at a
concentration as low as 40 pM. It was also found that the factor induces
aggregation in an aggregation-deficient mutant of a related species,
Polysphodilium violaceum. Both these activities are sensitive to positional
isomerism with the 6-methyl-5-pentyl isomer showing no detectable activity.
he life cycle of Dictyostelium discoideum consists of
Tgrowth and developmental phases. In the growth phase,
amoebae of this organism multiply by binary fission, and they
initiate development when the food source is exhausted. In
the developmental stage, they aggregate and form multicellular
structures called slugs composed of prespore and prestalk
cells. At fruiting body construction, these cells differentiate
into spores and stalk cells, respectively.
The 34 Mb-genome of D. discoideum has more than 40
genes of putative polyketide synthase.1−3 They are expressed
in a variety of patterns, suggesting their diverse roles in the
life cycle of the organism,4,5 but only several polyketides have
Figure 1. Structures of dictyoquinone (1), DIF-1 (2), and MPDB
(3).
so far been identified.6−9 Of them, (1-(3,5-dichloro-2,6-
dihydroxy-4-methoxyphenyl)-1-hexanone (differentiation-in-
ducing factor-1, DIF-1) (2) and 4-methyl-5-pentylbenzene-
1,3-diol (MPBD) (3) have been isolated from conditioned
medium (CM) and shown to be involved in cell differ-
entiation (Figure 1).4,8,10−13 Here we report on the
identification of a unique alkylbenzoquinone that plays
significant roles in the multicellular development of D.
discoideum and its related species.
In our previous paper, we described a polyketide-like factor
(PLF) with a molecular weight of 208 obtained from CM of
D. discoideum, which plays a key role in the acquisition of
differentiation commitment in early stages of development.14
In this paper, we describe the structure of the factor (2-
hydroxy-5-methyl-6-pentylbenzoquinone), as dictyoquinone
(1), which was determined by spectral analysis and chemical
synthesis, and by their prespore-cell inducing activity.
The molecular formula of 1 was established as C12H16O3 by
HR-ESI-MS (m/z 208.1084 [M]−, calcd m/z 208.1099). The
1H NMR spectrum of 1 showed only aliphatic signals, i.e., two
methyl signals at δ 0.899 (3H, t) and 1.972 (3H, s), a
methylene signal at δ 2.413 (2H, t), and a signal
corresponding to three methylenes at δ 1.27−1.41 (6H, m).
From these signals, only an n-pentyl and a methyl groups are
suggested on the basis of the DQF-COSY spectrum, and
those groups should be attached to sp2 carbons according to
their chemical shifts. On the other hand, six sp3 carbon signals
(δ 32.9, 29.3, 26.7, 23.3, 14.1, and 12.7) were estimated from
cross peaks on a HSQC spectrum. In the heteronuclear
Received: May 17, 2014
Published: June 30, 2014
© 2014 American Chemical Society
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multiplebond correlation (HMBC) spectrum, the methyl
group at δ 1.972 (5-CH3) and the methylene signal at δ
2.413 (H-1′) showed cross peaks at δC 189.2, and they also
correlated with signals around δC 140.6 and δC 143.9 (C-5
and -6). As we reported previously,14 dictyoquinone (1)
showed the absorption at λmax 491 nm, suggesting the
presence of a quinone moiety in this molecule. These pieces
of evidence demonstrated two possible structures (A and B)
for 1, with a pentyl and a methyl groups on one side of the
quinone ring, and a hydroxyl group on the other side (Figure
2). If the correct structure of 1 is one of these suggested,
To determine which of the two structures A and B is
correct, we synthesized both compounds for evaluation of
their biological activities. As shown in Scheme 2, 2,4,5-
trimethoxybenzoic acid (4) was used for starting material for
both compounds. The carboxyl group in 4 was transformed to
a methyl group, followed by introduction of C5 unit to C−6
position to afford compound 7. Boron tribromide treatment
of compound 7 transformed the methoxyl groups to hydroxyl
groups,15 and the consequent phenol 8 was oxidated by
Ag2CO3 to give compound A.16 On the other hand, the
compound B was prepared as follows. The carboxyl group in
4 was reduced to an aldehyde 9, and then it was transformed
to a pentyl group to give compound 10. A methyl group was
incorporated into 10 to give compound 12. Subsequently,
compound B was obtained in a manner similar to that for
1
compound A (Scheme 3). However, as the H NMR spectra
of the synthetic A and B are almost identical, comparison of
the NMR data alone was not sufficient for identification of the
natural compound (1).
Dictyoquinone (1) promotes prespore cell differentiation in
submerged culture conditions at low cell densities in the
presence of the prespore-inducing PsiA glycoprotein17,18 and
also promotes DIF-dependent stalk cell differentiation in
vitro.14 The synthesized compounds A and B were tested for
their in vitro prespore- and stalk cell-promoting activities
under respective assay conditions. As shown in Figure 3,
compound A showed prespore-promoting activity, with half-
maximal induction at a concentration as low as 40 pM,
whereas compound B had no detectable activity even at the
highest concentration tested. In addition, compounds A but
not B promoted stalk cell differentiation in vitro (Figure SI-1
in SI).
We also examined whether compounds A and B have the
D-factor activity. D-factor is a small molecule required for the
initiation of aggregation in a related species Polysphodilium
violaceum; starved cells of the D-factor nonproducing aggA
mutants of P. violaceum aggregate and form fruiting bodies
normally if supplied with D-factor.19 Its structure is
unresolved but reported to have structural features very
similar to those of dictyoquinone (1).20 We used the aggA
mutant strain A586 of P. violaceum to test compounds A and
B, as well as crude dictyoquinone preparations from D.
discoideum, for their D-factor activity. As shown in Figure 4,
only compound A rescued the mutant phenotype. Crude
Figure 2. HMBC correlations and possible structures (A and B) for
dictyoquinone (1).
another proton signal corresponding to a proton on
benzoquinone ring (H-3) should be observed on the 1H
NMR spectrum, though no other signal was detected. This is
accounted for by H−D exchange on C-3 position of the
compound in methanol-d4, which was used as a solvent to
measure NMR spectra since we used aqueous methanol to
purify this compound (1) without any information on the
structure (Scheme 1).
Scheme 1. H−D Exchange on C-3
Scheme 2. Synthesis of Compound A
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Scheme 3. Synthesis of Compound B
none (1) for its activities suggested that its action is mediated
by binding to a specific receptor.
Structural comparison of dictyoquinone (1) with MPBD
(3) suggests that they are metabolites of the same
biosynthetic pathway, possibly the former being an oxidation
product of the latter. In support of this possibility, synthetic
MPBD (3) treated with Frem
́
y’s salt21 gave dictyoquinone (1)
in good yield (Scheme 4). Further studies are required to
determine the biosynthetic pathways of these compounds and
their relationship.
Scheme 4. Transformation of MPBD (3) into
Dictyoquinone (1) by Fremy’s Salt
́
Figure 3. Dose−response curves for the effects of compounds A and
B on prespore cell differentiation. The results shown are the means
and SDs of three independent experiments.
In conclusion, we have determined the structure of an
alkylbenzoquinone, dictyoquinone (1), which plays significant
roles in the multicellular development of D. discoideum and
possibly in other species of the Dictyostelid slime mold. In
multicellular organisms, besides ubiquinone and plastoqui-
none, naturally occurring benzoquinone compounds have
been shown to have various biological functions: e.g.,
antioxidation,22 antimicrobial defense,23 signal transmis-
sion,24,25 and allelopathic interaction.26 Our results point to
the possibility of benzoquinone compounds playing a role as
signaling molecules in the control of development.
Figure 4. Effects of compounds A and B on the development of the
aggregation-defective mutant A586 of P. violaceum. Small pieces of
filter paper containing 4.8 pmol of compound A, compound B, or
DMSO as a solvent control were placed around the starved A586
cells spotted on agar plate. Cells responded to compound A alone to
form fruiting bodies (arrow). Higher magnification image of the
fruiting bodies is shown to the right. Aggregation was never observed
around the filter paper of compound B or DMSO.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details of the syntheses of compounds A and B
and the bioassays. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
dictyoquinone preparations also had the D-factor activity
(data not shown).
From these results, the molecular structure of dictyoqui-
none (1) was determined to be 2-hydroxy-5-methyl-6-
pentylbenzoquinone (structure A). The isomeric specificity
and the extremely low concentrations required of dictyoqui-
■
*E-mail: ytakaya@meijo-u.ac.jp.
Notes
The authors declare no competing financial interest.
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(21) Carlson, B. W.; Miller, L. L. J. Am. Chem. Soc. 1985, 107,
479−485.
ACKNOWLEDGMENTS
■
K.I. is grateful to Dr. Mike Hanna of Rensselaer Polytechnic
Institute for providing the mutant strain of P. violaceum and
for sharing unpublished results. We thank Dr. Ted Cox of
Princeton University for his useful advice. Thanks are due to
Drs. Katsuo Asakura and Kazuko Tanaka of JEOL Co., Ltd.,
for measuring NMR and MS, respectively.
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