Determination of absolute stereochemistry, total synthesis, and evaluation of peptides from the myxomycete Physarum melleum

Article abstract of DOI:10.1016/j.bmcl.2007.11.005

The absolute stereochemistry of melleumin A (1) and B (2), novel peptide compounds isolated from the myxomycete Physarum melleum, was determined by synthesis of their segments and by a modified Mosher's method. Total synthesis of melleumin B (2) was achie

Full text of DOI:10.1016/j.bmcl.2007.11.005

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 95–98
Determination of absolute stereochemistry, total synthesis,
and evaluation of peptides from the myxomycete Physarum melleum
Shuwa Hanazawa, Midori A. Arai, Xiaofan Li and Masami Ishibashi*
Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan
Received 13 September 2007; revised 31 October 2007; accepted 1 November 2007
Available online 5 November 2007
Abstract—The absolute stereochemistry of melleumin A (1) and B (2), novel peptide compounds isolated from the myxomycete
Physarum melleum, was determined by synthesis of their segments and by a modified Mosher’s method. Total synthesis of melleumin
B (2) was achieved by a stereoselective method, which provided further evidence for all the absolute stereochemistries of melleumin
B (2). The Wnt signal inhibitory activities of 2 and its 10R-epimer 19 were evaluated. Compound 19 showed moderate inhibition of
Wnt signal transcription, which suggests that melleumin analogues might be useful as Wnt signal inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Myxomycetes (true slime molds) are an unusual group
of primitive organisms that may be assigned to one of
the lowest classes of eukaryotes. Our analysis of myxo-
mycetes has led us to isolate new compounds.1–3 A novel
peptide lactone, melleumin A (1), and its seco acid
methyl ester, melleumin B (2), were isolated from cul-
tured plasmodium of the myxomycete Physarum
melleum (Fig. 1).1 The stereochemistry of 2 at the chiral
centers was established as 3S, 10S, and 11R configura-
tions. However, the absolute stereochemistry of the
C-4 position of 2 remained undetermined. Here, we de-
scribe the determination of the absolute stereochemistry
at the C-4 position of 2 on the basis of the synthesis of
partial structures of 2 in optically active forms. The total
synthesis of melleumin B (2) provided unequivocal evi-
dence for all the stereochemistry of melleumin B (2).
The 10R-epimer of melleumin B (19) exhibited Wnt sig-
nal inhibition.
Figure 1. Structure of melleumin A (1) and B (2). Stereochemistry at
the C-4 position was determined in this study.
Melleumin A (1) and B (2) consist of four residues,
p-methoxybenzoic acid (pMBz), L-threonine (L-Thr),
glycine (Gly), and an unusual amino acid, a tyrosine-
attached acetic acid (TyrA).
compared them by HPLC analysis with the unit ob-
tained from hydrolysis of melleumin B (2).
Synthesis of the two diastereomers of the TyrA unit is
shown in Scheme 1. L-Tyrosine methyl ester hydrochlo-
ride was protected with t-butoxycarbonyl (Boc) ester to
give 3.4 Reduction of the protected tyrosine 3 with DI-
BAL gave aldehyde 4, which was treated with a lithium
enolate of ethyl acetate to give two diastereomers 5a and
5b.5–7 Their stereochemistries were determined by the
modified Mosher’s method (see Supplementary data,
Fig. 1).8 Hydrolysis and deprotection of the Boc group
of 5a and 5b gave 7a and 7b, respectively.
The undefined chiral center at the C-4 position was in-
cluded in TyrA unit. To determine the absolute configu-
ration, we synthesized two diastereomers of TyrA and
Keywords: Myxomycete; Physarum melleum; Melleumin; Peptide; Wnt
signal inhibitor.
*
Corresponding author. Tel./fax: +81 43 290 2913; e-mail: mish@
p.chiba-u.ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.005

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S. Hanazawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 95–98
Scheme 1. Reagents and conditions: (a) DIBAL, toluene, À78 ꢁC, 6 min; (b) EtOAc, LDA, THF, À78 ꢁC, 2 h, 11% (two steps), 5a/5b = 54/46; (c)
LiOH, THF/MeOH/H2O = 3/1/1, rt, 1 h; (d) TFA, CH2Cl2, rt, 1 h.
Comparison of the reverse-phase HPLC retention time
between melleumin B (2) hydrolysate and the synthetic
TyrA unit (7a and 7b) was performed. The retention
time of 7a matched that of the melleumin B (2) hydroly-
sate (see Supplementary data, Fig. 2).9 Melleumin A (1)
and B (2) have the same stereochemistry because treat-
ment of 1 with 28% MeONa afforded 2.1 The result sug-
gested that the stereochemistry of melleumin A (1) and
B (2) has the same structure as 7a (3S,4S) and all the
absolute stereochemistries of melleumin A (1) and B
(2) were determined as 3S, 4S, 10S, and 11R.
TBSCl,10 followed by deprotection of the Boc group
with TBSOTf11 to give a free amine 11. This method
generally requires two steps and purification after each
step. This step was improved as a one-pot reaction
according to procedure reported by Corey et al. First,
9a was treated with TBSOTf for cleavage of the Boc
group, and then 2,6-lutidine was added to give the
TBS ether.12 The reaction mixture, containing free
amine 11, was evaporated and the residue was sub-
jected to coupling with Fmoc-Gly to give compound
12 with a good yield (78%, two steps from 9a). Subse-
quently, deprotection of the Fmoc group gave the de-
sired TyrA-Gly unit 13.
Next, we studied the total synthesis of melleumin B (2).
As shown in Scheme 2, the protected TyrA units (9a and
9b) were prepared from 3 in the same manner as 3 to 5a
and 5b, and the phenolic hydroxyl group was protected
with benzyl (Bn) ether.4 The absolute stereochemistries
of 9a and 9b were confirmed by comparison of 1H
NMR spectral data with 5a and 5b after cleavage of
the Bn group.
The L-Thr-pMBz unit 16 was prepared from the known
compound 14.13 MOM protection and hydrolysis of the
methyl ester gave the desired L-Thr-pMBz unit 16
(Scheme 3).
Coupling of the TyrA-Gly unit 13 with the L-Thr-
pMBz unit 16 gave tripeptide 17 with a good
yield (67%, two steps from 12) including the 10R-
Compound 9a, having the desired stereochemistry of
the TyrA unit, was protected with TBS ether using
epimer 18 (17/18 = 92/8, determined by 1H NMR),
Scheme 2. Reagents and conditions: (a) BnBr, K2CO3, acetone, reflux, 24 h, 99%; (b) DIBAL, toluene, À78 ꢁC, 0.5 h, 86%; (c) EtOAc, LDA, THF,
À78 ꢁC, 2 h, 80%, 9a/9b = 1/1; (d) TBSCl, imidazole, DMF, rt, 54 h, 81%; (e) TBSOTf, CH2Cl2, 2 h; (f) TBSOTf, CH2Cl2, 0 ꢁC, 2 h, then 2,6-lutidine,
2 h; (g) Fmoc-Gly, EDC, HOBt, 4-DMAP, CH2Cl2, rt, overnight, 78% (two steps from 9a); (h) piperidine, CH2Cl2, 0 ꢁC, 1 h.

S. Hanazawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 95–98
97
(2/19 = 91/9, determined by reversed-phase HPLC).14
Melleumin B (2) and 19 were separated as a pure form
1
by reversed-phase HPLC.15 The H and 13C NMR and
FABMS spectra of synthetic 2 were compared with
those of a natural product specimen, showing that they
were completely identical (see Supplementary data).
This result provided unequivocal evidence of the whole
structure of melleumin B (2) as well as melleumin A
(1), including absolute stereochemistry.1
Scheme 3. Reagents and conditions: (a) MOMCl, iPr2NEt, CH2Cl2, rt,
48 h, 97%; (b) LiOH, THF/H2O = 1/1, 0 ꢁC, 45 min, 95%.
We examined the Wnt signal inhibitory activity of the
two synthesized compounds (2 and 19) using a lucifer-
ase reporter gene assay.16 Wnt signaling activates gene
transcription by forming a complex between DNA-
binding proteins of the Tcf/LEF family and b-catenin.
SuperTOP-Flash, a b-catenin-responsive reporter plas-
mid with multiple TCF-binding sites (CCTTTGATC),
was activated in cells.17,18 SuperFOP-Flash has eight
mutated TCF-binding sites (CCTTTGGCC), therefore
and we used a mixture of 17 and 18 for next steps
(Scheme 4).
Deprotection of the TBS and MOM groups on the tri-
peptide, followed by hydrolysis of the ethyl ester with
LiOH, afforded the seco acid, which was converted to
a methyl ester with TMS-diazomethane. The benzyl
group was finally removed by hydrogenolysis to give
melleumin B (2) as a mixture with its 10R-epimer 19
a
selective inhibitor would not show enhanced
Scheme 4. Reagent and conditions: (a) EDC, HOOBt, 4-DMAP, CH2Cl2, 0 ꢁC, overnight, 67% (two steps from 12), 17/18 = 92/8; (b) TBAF, THF,
rt, 3 h; (c) HCl, EtOH, 60 ꢁC, 45 min; (d) LiOH, THF/H2O = 1/1, 0 ꢁC, 45 min; (e) TMS-diazomethane, MeOH, rt, overnight; (f) H2, 10% Pd/C,
MeOH, rt, overnight, 56% (five steps), 2/19 = 91/9.
Figure 2. Wnt signal inhibitory activity: (A) SuperTOP-Flash transfected cells inhibition by 2; (B) SuperTOP-Flash transfected cells inhibition by 19;
(C) TOP/FOP-Flash reporter activation ratio of 19.

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S. Hanazawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 95–98
transcription in SuperFOP-Flash transfected cells;
thus, these cells provide a negative control for the as-
say and the TOP/FOP-Flash reporter activation ratio
provides a measure of the selectivity of Wnt signal
inhibition.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.11.005.
The results are shown in Figure 2, along with the cell
viability in order that a decrease in cell number should
References and notes
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We thank Professor Jeremy Nathans (John Hopkins
Medical School) for kindly providing STF cells (293 cell
line stably transfected with SuperTOP-Flash by Profes-
sor R.T. Moon) and Professor Randall T. Moon (Uni-
versity of Washington) for the SuperTOP-Flash
reporter plasmid. This work was supported by Grants-
in-Aid from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan (Nos. 18032020 and
19310136 to M.I. and No. 18790012 to M.A.A.).
M.A.A. also gratefully acknowledges financial support
from the Uehara Memorial Foundation, and S.H. also
gratefully acknowledges financial support from the
Nanohana Competition 2007 for students (Chiba Uni-
versity Venture Business Laboratory).
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