Design and synthesis of an artificial ladder-shaped polyether that interacts with glycophorin A

Article abstract of DOI:10.1016/j.bmcl.2006.09.004

Ladder-shaped polyether (LSP) compounds, such as brevetoxins and ciguatoxins, are thought to interact with transmembrane (TM) proteins. As a model LSP compound, we designed and synthesized an artificial tetracyclic ether (1) and evaluated its interaction

Full text of DOI:10.1016/j.bmcl.2006.09.004

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6355–6359
Design and synthesis of an artificial ladder-shaped polyether
that interacts with glycophorin A
Kohei Torikai, Hiroshi Yari, Megumi Mori, Satoru Ujihara, Nobuaki Matsumori,
Michio Murata and Tohru Oishi*
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
Received 5 August 2006; revised 31 August 2006; accepted 2 September 2006
Available online 20 September 2006
Abstract—Ladder-shaped polyether (LSP) compounds, such as brevetoxins and ciguatoxins, are thought to interact with transmem-
brane (TM) proteins. As a model LSP compound, we designed and synthesized an artificial tetracyclic ether (1) and evaluated its
interaction with glycophorin A (GpA), a membrane protein known to dimerize or oligomerize between membrane-integral a-helical
domains. Model compound 1 was found to induce the dissociation of oligomeric GpA in a similar manner to natural LSPs when
examined by SDS–PAGE. The results suggest that even an artificial tetracyclic ether possesses the ability to interact with TM pro-
teins, presumably through the intermolecular hydrogen bonds (Ca–HÁ Á ÁO) with the GXXXG motif.
Ó 2006 Elsevier Ltd. All rights reserved.
Ladder-shaped polyether (LSP) compounds,1 including
brevetoxin B,2 ciguatoxin,3 yessotoxin,4 and gymnocin-
A5 (Fig. 1), which are unique products of Dinophyceae
and Haptophyceae, are composed of continuous trans-
fused cyclic ethers and are known to possess potent tox-
icity. Although more than 50 naturally occurring LSPs
have been identified, there have been few molecular
mode-of-action studies, chiefly due to the short supply
of materials. Brevetoxins and ciguatoxins are unusual
in that their molecular target has been identified;6 the
toxins share a common binding site on an a subunit of
voltage-sensitive sodium channels (VSSCs), with very
high affinity (their dissociation constants are in the
nanomolar–subnanomolar range). Because VSSCs are
comprised of 24 transmembrane (TM) helices, mem-
brane-integral a-helices are considered to be the inter-
acting motif of LSPs. Although other LSPs (e.g.,
gambierol7 and gambieric acid A8) show weak interac-
tion with VSSCs,9 they are thought to bind to different
molecular targets upon exerting their toxicity. When a
polyether matches the binding part of a membrane pro-
tein, its binding affinity is greatly enhanced, probably
due to an arrangement of polyether oxygen atoms as
hydrogen bond acceptors,9 because the distance between
the neighboring skeletal oxygen atoms on the same side
10
˚
of the LSPs is consistent with the helix pitch (ca. 5 A).
Recently, we developed an assay system for evaluating
the interaction between LSPs and membrane-integral
a-helices using glycophorin A (GpA), a heavily glycosyl-
ated TM protein occurring in erythrocyte membrane
which is known to form dimers and oligomers.11 The
activities of LSPs were evaluated on the basis of dissoci-
ation of the GpA oligomers and dimers (Fig. 2A), which
was detected by SDS–PAGE analysis. Brevetoxin B and
yessotoxin were found to induce dissociation of oligo-
meric GpA into the corresponding dimers or monomers.
The structure of the TM region of dimeric GpA in both
aqueous detergent micelles and lipid bilayers has been
determined by NMR, as illustrated in Figure 2B.12
Weak hydrogen bonds between the Ca–H of glycine
and a carbonyl oxygen (Ca–HÁ Á ÁO)13 were suggested
to play an important role in stabilizing the dimeric struc-
ture (G79-I76 and G83-V80), while the residual side
chains synergistically contribute to stabilization of the
complex by van der Waals contact. Recently, the
GXXXG sequence motif (the so-called glycine zipper)14
has commonly been found to mediate TM helix oligo-
merization. We postulate that the GXXXG sequence
may be a candidate for an interacting motif with LSPs,
whose oxygen array possibly acts as a series of HB
acceptors. According to this hypothesis, a tetracyclic
LSP should possess enough length to interact with the
GXXXG motif to induce dissociation of the oligomeric
Keywords: Ladder-shaped polyether; Artificial ladder-shaped polyether;
Transmembrane protein; Glycophorin A; GXXXG motif.
*
Corresponding author. Tel.: +06 6850 5775; fax: +06 6850 5785;
e-mail: oishi@ch.wani.osaka-u.ac.jp
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.004

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K. Torikai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6355–6359
Figure 1. Structures of brevetoxin B, ciguatoxin, yessotoxin, and gymnocin-A.
GpA (Fig. 2C). Therefore, we designed an artificial lad-
der-shaped polyether (ALP) 1,10,15,16 taking account of
the following points: (i) a trans-fused 6/6/7/6 tetracyclic
ether system mimics the partial framework of yessotox-
in, gambierol, and gymnocin-A; (ii) the number of ether
rings is consistent with that of hemibrevetoxin B, which
is the shortest natural LSP; (iii) one of the terminal sides
is functionalized with hydroxy groups to increase water
solubility, and the other side with benzyl ethers to retain
sufficient lipophilicity to access the hydrophobic TM re-
gion; (iv) compound 1 could be synthesized expeditious-
ly from fragments 2 and 3 in a convergent manner
(Scheme 1). Herein we report the synthesis of ALP 1,
and evaluation by SDS–PAGE of its interaction with
GpA based on the dissociation of GpA dimers and
oligomers.
tively to give primary alcohol 8, which was converted to
corresponding iodide 9. Side-chain elongation of 9 was
achieved by means of alkylation with lithium enolate
prepared from t-butyl acetate, followed by saponifica-
tion of the resulting ester to afford 3.
We then moved on to coupling of the fragments and
synthesis of the 6/6/7/6 tetracyclic ether 1 as depicted
in Scheme 3. Condensation of alcohol 2 with carboxylic
acid 3 by treating with EDCÆHCl in the presence of
DMAP and CSA18 afforded the ester 10. The p-me-
thoxybenzylidene acetal was converted to di(t-butyl)sil-
ylene to yield 11 at 86% for three steps. The next task
was intramolecular carbonyl olefination mediated by a
low-valent titanium complex (Takeda reaction),19,20
one of the critical points in the present synthesis. Dith-
ioacetal 11 was treated with Cp2Ti[P(OEt)3]2 to yield
dihydropyran 12, which was immediately subjected to
a hydroboration-oxidation sequence to afford a separa-
ble mixture of the desired alcohol 13a (28%) and diaste-
reomer 13b (16%). Oxidation of the alcohols 13a and
13b furnished ketone 14a (93%) and 14b (65%), respec-
tively, and the undesired 14b was converted to 14a by
treating with DBU at 50 °C. Removal of the PMB group
of 14a using DDQ gave a hydroxy-ketone, which was
Synthesis of 1 commenced with the preparation of frag-
ments 2 and 3 from known tetrahydropyran derivatives
5 and 7 (Scheme 2).16,17 Oxidation of the primary alco-
hol 5 followed by treatment of the resulting aldehyde
with PhSSPh and n-Bu3P gave dithioacetal 6, and
removal of the TBS group of 6 afforded the secondary
alcohol 2. For carboxylic acid 3, reductive cleavage of
the p-methoxybenzylidene acetal 7 proceeded regioselec-

K. Torikai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6355–6359
6357
Figure 2. Hypothetical model of LSP-GpA interaction. (A) Illustrative
pathway of dissociation of GpA oligomers to dimers or monomers. (B)
Schematic drawing of the partial structure of GpA dimer reported by
Smith et al. (C) Hypothetical model of complexation of a GpA
monomer with a LSP.
Scheme 2. Reagents and conditions: (a) Dess-Martin periodinane,
CH2Cl2, rt, quant.; (b) PhSSPh, n-Bu3P, neat, rt; (c) TBAF, THF, rt,
74% (two steps); (d) DIBAL, CH2Cl2, À78 to À10 °C, 80%; (e) I2,
PPh3, imidazole, THF, rt, 90%; (f) t-BuOAc, LDA, THF, HMPA, À73
to À15 °C, 78%; (g) KOH, MeOH, THF, H2O, reflux, 97%.
removal of the silyl group of 17 with TBAF furnished
the tetracyclic ether 123 (69%), whose stereochemistry
was unambiguously determined by NOE experiments.
We then turned our attention to evaluation of the inter-
action of 1 with GpA based on dissociating activity by
SDS–PAGE (Fig. 3).11 As shown in lane 1, intact
GpA exists as oligomers in various aggregated states,
with a significant amount of dimers. When GpA was
treated with desulfated yessotoxin (dsYTX, lanes 2–4)
the oligomers dissociated dose-dependently to form di-
mers, and a considerable amount of monomers were
also formed (lanes 3 and 4). Meanwhile, treatment of
GpA with 1 induced the dissociation of oligomers, as
expected (lanes 6 and 7), and the dissociation activity
of 1 was comparable to that of dsYTX. It is noteworthy
that no significant interaction was observed for monocy-
clic ether 1824 (Fig. 4) possessing the same functional
groups as 1 (lanes 8 and 9). As already reported, other
artificial polyether compounds, such as polyethylene
glycol (lanes 10 and 11) and 18-crown-6, did not show
the dissociation activity.11 These results strongly suggest
that the ladder-shaped skeleton, in which the inter-
atomic distances of the neighboring two oxygen atoms
on the same side are fixed at a similar distance to the he-
lix pitch, plays an important role in the interaction with
TM proteins. These results may be a clue to understand-
ing the mode-of-action of natural LSP toxins.
Scheme 1. Synthesis plan for the artificial ladder-shaped polyether 1.
treated with TMSSEt in the presence of TMSOTf to af-
ford hydroxy dithioacetal 15 (87%). In contrast to the
six-membered ring system,21 a mixed thioacetal was
not obtained under these reaction conditions. The for-
mation of the seven-membered ring was then examined
by means of Nicolaou’s method.22 Dithioacetal 15 was
treated with AgClO4 in the presence of silica gel in nitro-
methane at room temperature to afford mixed thioacetal
16a and epimer16b in a 1:1.5 ratio (42%); these were sep-
arable by silica gel chromatography. The final methyla-
tion leading to tetracyclic ether 1 was performed using
16a. Oxidation of mixed thioacetal 16a with mCPBA to-
ward sulfone, followed by treatment with Me3Al, affor-
ded trans/syn-fused 17 as a single isomer (52%). Finally,
In conclusion, an artificial ladder-shaped polyether (1)
was designed with the aim of evaluating its interaction
with transmembrane protein glycophorin A, and

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K. Torikai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6355–6359
Figure 3. SDS–PAGE of GpA in the presence of natural and artificial
polyethers. GpA (2.6 pmol, 98 ng) alone (lane 1); in the presence of
2.4 nmol (lane 2), 24 nmol (lane 3), and 72 nmol (lane 4) of desulfated
yessotoxin (dsYTX); in the presence of 2.4 nmol (lane 5), 24 nmol (lane
6), and 72 nmol (lane 7) of ALP 1; in the presence of 24 nmol (lane 8)
and 72 nmol (lane 9) of ALP 18; in the presence of 24 nmol (lane 10)
and 72 nmol (lane 11) of PEG. GpA was visualized by silver staining.
Figure 4. Structure of monocyclic ether 18.
that even the tetracyclic ALP possesses the ability to in-
duce dissociation of glycophorin A oligomers to form
dimers and monomers. In order to elicit the precise
mechanism of molecular recognition, synthesis of more
ingeniously designed ALPs is in progress in our
laboratory.
Acknowledgments
We are grateful to Dr. Keiichi Konoki in our laboratory
for discussion. This work was supported by Grant-
in-Aid for Exploratory Research (No. 15655033), for
Scientific Research on Priority Areas (No. 16073211)
from MEXT.
References and notes
Scheme 3. Reagents and conditions: (a) EDCÆHCl, DMAP, CSA,
CH2Cl2, reflux; (b) TsOH, H2O, MeOH, rt; (c) t-Bu2Si(OTf)2, 2,6-
lutidine, DMF, 0 °C, 86% (three steps); (d) Cp2Ti[P(OEt)3]2, MS4A,
THF, rt; (e) BH3 ÆTHF, 0 °C to rt, then NaBO3, H2O, THF, reflux (13a,
28%; 13b, 16% for two steps); (f) Dess-Martin periodinane, CH2Cl2, rt,
93% (for 13a–14a); TPAP, NMO, MS4A, CH2Cl2, rt, 65% (for 13b–14b);
(g) DBU, toluene, 50 °C, 47%; (h) DDQ, H2O, CH2Cl2, 0 °C to rt, 63%;
(i) TMSSEt, TMSOTf, CH2Cl2, À78 to À40 °C, 87%; (j) AgClO4,
NaHCO3, SiO2, MS4A, CH3NO2, rt, 90 min, 42% (16a:16b = 1:1.5,
separable); (k) mCPBA, NaHCO3, CH2Cl2, rt; (l) Me3Al, CH2Cl2, À50
to À20 °C, 52% (two steps); (m) TBAF, THF, rt, 69%.
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J = 11.0 Hz, Bn), 4.52 (1H, d, J = 12.5 Hz, Bn), 4.35 (1H,
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3.76 (1H, dd, J = 11.5, 4.0 Hz, H1), 3.71 (1H, dd, J = 12.0,
1.0 Hz, H17), 3.67 (1H, ddd, J = 9.0, 9.0, 4.5 Hz, H3), 3.57
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11.0, 5.0 Hz, H13), 3.43 (1H, ddd, J = 12.0, 11.0, 5.0 Hz,
H15), 3.30 (1H, m, H12), 3.22 (2H, m, H2, H16), 3.17 (1H,
dd, J = 10.5, 4.5 Hz, H9), 3.10 (1H, ddd, J = 12.5, 12.5,
4.0 Hz, H6), 2.96 (1H, ddd, J = 12.5, 12.5, 4.5 Hz, H5),
2.42 (1H, ddd, J = 12.5, 5.0, 5.0 Hz, H14eq), 2.37 (1H, ddd,
J = 12.0, 4.5, 4.5 Hz, H4eq), 2.13 (1H, m, H11), 2.03 (1H,
dd, J = 12.5, 4.0 Hz, H7eq), 1.83 (1H, m, H10), 1.74 (2H,
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