Design and synthesis of ladder-shaped tetracyclic, heptacyclic, and decacyclic ethers and evaluation of the interaction with transmembrane proteins

Article abstract of DOI:10.1021/ja801576v

Ladder-shaped polyether (LSP) toxins represented by brevetoxins and Ciguatoxins are thought to bind to transmembrane (TM) proteins. To elucidate the interactions of LSPs with TM proteins, we have synthesized artificial ladder-shaped polyethers (ALPs) containing 6/7/6/6 tetracyclic, 6/7/6/6/7/6/6 heptacyclic, and 6/7/6/6/7/6/6/7/6/6 decacyclic systems, based on the convergent method via α-cyano ethers. The ALPs possessing the simple iterative structure with different numbers of rings would be useful for structure-activity relationship studies on the molecular length, which is supposed to be important when naturally occurring LSPs elicit their toxicity. Two series of ALPs were prepared to evaluate the hydrophilic or hydrophobic effects of the side chains: (i) both sides were functionalized as diols (A series), and (ii) one side remained as diol and the other side was protected as benzyl ethers (B series). To examine the interaction of these ALPs with TM proteins, dissociation of glycophorin A (GpA) dimers into monomers was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The heptacyclic ether (ALP7B) elicited the most potent activity in the presence of 2% SDS buffer, whereas the decacyclic ether (ALP10A) exhibited an intriguing phenomenon to induce precipitation of GpA in a dose-dependent manner, under the low concentration of SDS (0.03%). ALP10A also induced precipitation of integrin α ₁β₁, a TM protein known to form heterodimers in the lipid bilayer membranes. The different activities among the ALPs can be accounted for by the concept of hydrophobic matching that is, lengths of the hydrophobic region including the side chains of ALP7B and ALP10A are ca. 25 A, which match the lengths of the hydrophobic region of α-helical TM proteins, as well as the hydrophobic thickness of lipid bilayer membranes. The concept of the hydrophobic matching would be a clue to understanding the interaction between LSPs and TM proteins, and also a guiding principle to design ALPs possessing potent affinities with TM proteins.

Full text of DOI:10.1021/ja801576v

Published on Web 07/16/2008
Design and Synthesis of Ladder-Shaped Tetracyclic,
Heptacyclic, and Decacyclic Ethers and Evaluation of the
Interaction with Transmembrane Proteins
Kohei Torikai,† Tohru Oishi,*,† Satoru Ujihara,† Nobuaki Matsumori,†
Keiichi Konoki,† Michio Murata,† and Saburo Aimoto‡
Department of Chemistry, Graduate School of Science, Osaka UniVersity, 1-1 Machikaneyama,
Toyonaka, Osaka 560-0043, Japan, and Institute for Protein Research, Osaka UniVersity,
3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received March 3, 2008; E-mail: oishi@chem.sci.osaka-u.ac.jp
Abstract: Ladder-shaped polyether (LSP) toxins represented by brevetoxins and ciguatoxins are thought
to bind to transmembrane (TM) proteins. To elucidate the interactions of LSPs with TM proteins, we have
synthesized artificial ladder-shaped polyethers (ALPs) containing 6/7/6/6 tetracyclic, 6/7/6/6/7/6/6 heptacyclic,
and 6/7/6/6/7/6/6/7/6/6 decacyclic systems, based on the convergent method via R-cyano ethers. The ALPs
possessing the simple iterative structure with different numbers of rings would be useful for structure-activity
relationship studies on the molecular length, which is supposed to be important when naturally occurring
LSPs elicit their toxicity. Two series of ALPs were prepared to evaluate the hydrophilic or hydrophobic
effects of the side chains: (i) both sides were functionalized as diols (A series), and (ii) one side remained
as diol and the other side was protected as benzyl ethers (B series). To examine the interaction of these
ALPs with TM proteins, dissociation of glycophorin A (GpA) dimers into monomers was evaluated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. The heptacyclic ether (ALP7B) elicited the most potent
activity in the presence of 2% SDS buffer, whereas the decacyclic ether (ALP10A) exhibited an intriguing
phenomenon to induce precipitation of GpA in a dose-dependent manner, under the low concentration of
SDS (0.03%). ALP10A also induced precipitation of integrin R1ꢀ1, a TM protein known to form heterodimers
in the lipid bilayer membranes. The different activities among the ALPs can be accounted for by the concept
of “hydrophobic matching” that is, lengths of the hydrophobic region including the side chains of ALP7B
and ALP10A are ca. 25 Å, which match the lengths of the hydrophobic region of R-helical TM proteins, as
well as the hydrophobic thickness of lipid bilayer membranes. The concept of the hydrophobic matching
would be a clue to understanding the interaction between LSPs and TM proteins, and also a guiding principle
to design ALPs possessing potent affinities with TM proteins.
Introduction
to various combinations of the number and sizes of cyclic ethers
ranging from five- to nine-membered rings, and the order of
Ladder-shaped polyether (LSP) compounds are unique natural
products of marine unicellular algae called dinoflagellates
(Figure 1).1 Brevetoxin-B is the first example of LSP, which
was isolated from Karenia breVis (formerly Gymnodinium
breVe) in association with “red tide”, and whose structure was
elucidated in 1981 to be an unprecedented undecacyclic ether.2
More than 50 LSPs have been identified to date including their
congeners, which possess the general structural motif of
continuous trans/syn-fused ether rings as shown in Figure 1;
brevetoxin-A,3 hemibrevetoxin-B,4 brevenal,5 ciguatoxin,6
CTX3C,7 gambieric acid-A,8 gambierol,9 gymnocin-A,10 yes-
sotoxin,11 etc.12 The structural diversity of LSPs is attributed
the ring connection results in considerable skeletal diversity.
These LSPs elicit a broad spectrum of biological activities; for
example, brevetoxins (LD50 > 200 µg/kg, mice, i.p.)13 and
ciguatoxins (LD50 ) 0.25∼3.6 µg/kg, mice, i.p.)6b,7,14 are potent
neurotoxins, while gymnocin-A exhibits cytotoxicity against
P388 mouse leukemia cells (ED50 ) 1.3 µg/mL).10 Gambieric
acid-A is not a mammalian toxin but an antifungal, which is
2000 times more potent than amphotericin B.15 Yessotoxin
induces apoptosis via a mitochondrial signal transduction
pathway.16 Despite the intriguing biological activities, there have
been few mode-of-action studies at the molecular level, chiefly
because of the short supply of materials. Brevetoxins and
ciguatoxins are unusual in that their molecular target has been
† Graduate School of Science.
‡ Institute for Protein Research.
(1) (a) Shimizu, Y. Chem. ReV. 1993, 93, 1685–1698. (b) Yasumoto, T.;
Murata, M. Chem. ReV. 1993, 93, 1897–1909. (c) Scheuer, P. J.
Tetrahedron 1994, 50, 3–18. (d) Murata, M.; Yasumoto, T. Nat. Prod.
Rep. 2000, 17, 293–314. (e) Yasumoto, T. Chem. Rec. 2001, 1, 228–
242. (f) Daranas, A. H.; Norte, M.; Ferna´ndez, J. J. Toxicon 2001,
39, 1101–1132.
(3) (a) Shimizu, Y.; Chou, H.-N.; Bando, H.; Van Duyne, G.; Clardy,
J. C. J. Am. Chem. Soc. 1986, 108, 514–515. (b) Pawlak, J.; Tempesta,
M. S.; Golik, J.; Zagorski, M. G.; Lee, M. S.; Nakanishi, K.; Iwashita,
T.; Gross, M. L.; Tomer, K. B. J. Am. Chem. Soc. 1987, 109, 1144–
1150.
(2) Lin, Y.-Y.; Risk, M.; Ray, S. M.; Engen, D. V.; Clardy, J.; Golik, J.;
James, J. C.; Nakanishi, K. J. Am. Chem. Soc. 1981, 103, 6773–6775.
9
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2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 10217–10226 10217

A R T I C L E S
Torikai et al.
identified;17 the toxins share a common binding site on an R
subunit of voltage-sensitive sodium channels (VSSCs), with very
high affinity as shown by their dissociation constants in the
nanomolar-subnanomolar range. Because of the structural
similarity, molecular targets of LSPs are considered to be
transmembrane (TM) proteins including ion channels or recep-
tors. For mode-of-action studies of LSPs, most of which are
hardly obtainable from natural sources, total synthesis of the
LSPs, in which remarkable progress has been made in the recent
decade,18 should be crucial. As a matter of fact, by using
synthetic specimens,19,20 it has been revealed that gambierol
selectively inhibits the voltage-gated K+ channels in mouse taste
cells, while CTX3C is ineffective against the same channels,
although markedly affecting the ion currents through voltage-
gated Na+ channels.21 Furthermore, gambierol has recently been
shown to be a TRPV1 (transient receptor potential vanilloid 1)
antagonist.22
It is reported that the binding of PbTx-3 (a brevetoxin-B
derivative) to VSSCs was competitively inhibited by the addition
of not only ciguatoxin (Ki ) 0.15 nM) but also gambieric acid-A
(Ki ) 0.11 µM) and gambierol (Ki ) 1.4 µM) while their
affinities are 104∼106 times lower than that of ciguatoxin.23
Although it is suggested that there is a significant relationship
between the size of the polycyclic region and the inhibitory
activity,23,24 yessotoxin did not inhibit the binding despite the
same number of rings as brevetoxin-B (undecacyclic).23 In
addition, it is reported that the Ki value of a small pentacyclic
LSP, brevenal,5 is 1.85 µM, which is comparable to that of
gambierol (heptacyclic). Therefore, not only the size of the
polycyclic region (number of rings), but also molecular shape
and functional groups of the LSP should be taken into
consideration upon estimating the affinity of LSPs to VSSCs.
The structural diversity of natural LSPs, differing in length
(including the number of rings and side chains), shape (the order
of connected ring sizes), and functional groups, has been an
obstacle for quantitative structure-activity relationship (SAR)
studies. To sort out the size effects of the polycyclic region
from other factors on the biological activities, it is necessary to
prepare model compounds as molecular probes that are com-
posed of a consistent ring sequence with different length, which
are unavailable from natural sources. Recently, syntheses of
model compounds mimicking LSPs have been reported by
Martin,25 Oguri,26 Tachibana,27 and our groups,28,29 for the
purpose of elucidating the molecular basis for biological
(4) Prasad, A. V. K.; Shimizu, Y. J. Am. Chem. Soc. 1989, 111, 6476–
6477.
(5) Bourdelais, A. J.; Jacocks, H. M.; Wright, J. L. C.; Bigwarfe, P. M.,
Jr.; Baden, D. G. J. Nat. Prod. 2005, 68, 2–6.
(6) (a) Murata, M.; Legrand, A.-M.; Ishibashi, Y.; Yasumoto, T. J. Am.
Chem. Soc. 1989, 111, 8929–8931. (b) Murata, M.; Legrand, A.-M.;
Ishibashi, Y.; Fukui, M.; Yasumoto, T. J. Am. Chem. Soc. 1990, 112,
4380–4386. (c) Satake, M.; Morohashi, A.; Oguri, H.; Oishi, T.;
Hirama, M.; Harada, N.; Yamamoto, T. J. Am. Chem. Soc. 1997, 119,
11325–11326. (d) Yasumoto, T.; Igarashi, T.; Legrand, A.-M.; Cruchet,
P.; Chinain, M.; Fujita, T.; Naoki, H. J. Am. Chem. Soc. 2000, 122,
4988–4989. (e) Lewis, R. J.; Sellin, M.; Poli, M. A.; Norton, R. S.;
MacLeod, J. K.; Sheil, M. M. Toxicon 1991, 29, 1115–1127.
(7) (a) Satake, M.; Murata, M.; Yasumoto, T. Tetrahedron Lett. 1993,
34, 1975–1978. (b) Satake, M.; Fukui, M.; Legrand, A.-M.; Cruchet,
P.; Yasumoto, T. Tetrahedron Lett. 1998, 39, 1197–1198.
(8) (a) Nagai, H.; Torigoe, K.; Satake, M.; Murata, M.; Yasumoto, T.;
Hirota, H. J. Am. Chem. Soc. 1992, 114, 1102–1103. (b) Nagai, H.;
Murata, M.; Torigoe, K.; Satake, M.; Yasumoto, T. J. Org. Chem.
1992, 57, 5448–5453. (c) Morohashi, A.; Satake, M.; Nagai, H.;
Oshima, Y.; Yasumoto, T. Tetrahedron 2000, 56, 8995–9001.
(9) (a) Satake, M.; Murata, M.; Yasumoto, T. J. Am. Chem. Soc. 1993,
115, 361–362. (b) Morohashi, A.; Satake, M.; Yasumoto, T. Tetra-
hedron Lett. 1999, 40, 97–100.
(19) (a) Total syntheses of gambierol, see: Fuwa, H.; Sasaki, M.; Satake,
M.; Tachibana, K. Org. Lett. 2002, 4, 2981–2984. (b) Fuwa, H.;
Kainuma, N.; Tachibana, K.; Sasaki, M. J. Am. Chem. Soc. 2002, 124,
14983–14992. (c) Kadota, I.; Takamura, H.; Sato, K.; Ohno, A.;
Matsuda, K.; Yamamoto, Y. J. Am. Chem. Soc. 2003, 125, 46–47. (d)
Kadota, I.; Takamura, H.; Sato, K.; Ohno, A.; Matsuda, K.; Satake,
M.; Yamamoto, Y. J. Am. Chem. Soc. 2003, 125, 11893–11899. (e)
Johnson, H. W. B.; Majumder, U.; Rainier, J. D. J. Am. Chem. Soc.
2005, 127, 848–849. (f) Majumder, U.; Cox, J. M.; Johnson, H. W. B.;
Rainier, J. D. Chem.sEur. J. 2006, 12, 1736–1746. (g) Johnson,
H. W. B.; Majumder, U.; Rainier, J. D. Chem.sEur. J. 2006, 12, 1747–
1753.
(10) Satake, M.; Shoji, M.; Oshima, Y.; Naoki, H.; Fujita, T.; Yasumoto,
T. Tetrahedron Lett. 2002, 43, 5829–5832.
(11) Murata, M.; Kumagai, M.; Lee, J. S.; Yasumoto, T. Tetrahedron Lett.
1987, 28, 5869–5872.
(12) (a) C-CTX: Lewis, R. J.; Vernoux, J.-P.; Brereton, I. M. J. Am. Chem.
Soc. 1998, 120, 5914–5920. (b) gymnocin-B: Satake, M.; Tanaka, Y.;
Ishikura, Y.; Oshima, Y.; Naoki, H.; Yasumoto, T. Tetrahedron Lett.
2005, 46, 3537–3540. (c) prymnecin : Igarashi, T.; Satake, M.;
Yasumoto, T. J. Am. Chem. Soc. 1999, 121, 8499–8511. (d) maito-
toxin: Murata, M.; Naoki, H.; Iwashita, T.; Matsunaga, S.; Sasaki,
M.; Yokoyama, A.; Yasumoto, T. J. Am. Chem. Soc. 1993, 115, 2060–
2062.
(20) Total synthesis of CTX3C, see: (a) Hirama, M.; Oishi, T.; Uehara,
H.; Inoue, M.; Maruyama, M.; Oguri, H.; Satake, M. Science 2001,
294, 1904–1907. (b) Inoue, M.; Uehara, H.; Maruyama, M.; Hirama,
M. Org. Lett. 2002, 4, 4551–4554. (c) Inoue, M.; Miyazaki, K.; Uehara,
H.; Maruyama, M.; Hirama, M. Proc. Natl. Acad. Sci. U.S.A. 2004,
101, 12013–12018. (d) Inoue, M.; Hirama, M. Acc. Chem. Res. 2004,
37, 961–968. (e) Hirama, M. Chem. Rec. 2005, 5, 240–250.
(21) (a) Yamaoka, K.; Inoue, M.; Miyahara, H.; Miyazaki, K.; Hirama,
M. Br. J. Pharmacol. 2004, 142, 879–889. (b) Ghiaroni, V.; Sasaki,
M.; Fuwa, H.; Rossini, G. P.; Scalera, G.; Yasumoto, T.; Pietra, P.;
Bigiani, A. Toxicol. Sci. 2005, 85, 657–665. (c) Ghiaroni, V.; Fuwa,
H.; Inoue, M.; Sasaki, M.; Miyazaki, K.; Hirama, M.; Yasumoto, T.;
Rossini, G. P.; Scalera, G.; Bigiani, A. Chem. Senses 2006, 31, 673–
680. (d) Cuypers, E.; Abdel-Mottaleb, Y.; Kopljar, I.; Rainier, J. D.;
Raes, A. L.; Snyders, D. J.; Tytgat, J. Toxicon 2008, 51, 974–983.
(22) Cuypers, E.; Yanagihara, A.; Rainier, J. D.; Tytgat, J. Biochem.
Biophys. Res. Commun. 2007, 361, 214–217.
(13) (a) Baden, D. G. FASEB J. 1989, 3, 1807–1817. (b) Dechraoui, M.-
Y.; Naar, J.; Pauillac, S.; Legrand, A.-M. Toxicon 1999, 37, 125–
143.
(14) (a) Lewis, R. J.; Sellin, M.; Poli, M. A.; Norton, R. S.; MacLeod,
J. K.; Sheil, M. M. Toxicon 1991, 29, 1115–1127. (b) Vernoux, J.-P.;
Lewis, R. J. Toxicon 1997, 35, 889–900.
(15) Nagai, H.; Mikami, Y.; Yazawa, K.; Gonoi, T.; Yasumoto, T. J.
Antibiot. 1993, 46, 520–522.
(16) Korsnes, M. S.; Hetland, D. L.; Espenes, A.; Tranulis, M. A.; Aune,
T. Toxicol. in Vitro 2006, 20, 1077–1087.
(17) (a) Catterall, W. A.; Risk, M. Mol. Pharmacol. 1981, 19, 345–348.
(b) Trainer, V. L.; Baden, D. G.; Catterall, W. A. J. Biol. Chem. 1994,
269, 19904–19909. (c) Dechraoui, M.-Y.; Naar, J.; Pauillac, S.;
Legrand, A.-M. Toxicon 1999, 37, 125–143.
(23) Inoue, M.; Hirama, M.; Satake, M.; Sugiyama, K.; Yasumoto, T.
Toxicon 2003, 41, 469–474.
(24) Gawley, R. E.; Rein, K. S.; Jeglitsch, G.; Adams, D. J.; Theodorakis,
E. A.; Tiebes, J.; Nicolaou, K. C.; Baden, D. G. Chem. Biol. 1995, 2,
533–541.
(18) Reviews on the total synthesis of ladder-shaped polyethers, see: (a)
Fuwa, H.; Sasaki, M. Curr. Opin. Drug DiscoVery DeV. 2007, 10,
784–806. (b) Nakata, T. Chem. ReV. 2005, 105, 4314–4347. (c) Inoue,
M. Chem. ReV. 2005, 105, 4379–4405. (d) Kadota, I.; Yamamoto, Y.
Acc. Chem. Res. 2005, 38, 423–432. (e) Sasaki, M.; Fuwa, H. Synlett
2004, 1851–1874. (f) Inoue, M. Org. Biomol. Chem. 2004, 2, 1811–
1817. (g) Marmsa¨ter, F. P.; West, F. G. Chem.sEur. J. 2002, 8, 4346–
4353. (h) Evans, P. A.; Delouvrie, B. Curr. Opin. Drug DiscoVery
DeV. 2002, 5, 986–999. (i) Mori, Y. Chem.sEur. J. 1997, 3, 849–
852. (j) Alvarez, E.; Candenas, M.-L.; Pe´rez, R.; Ravelo, J. L.; Mart´ın,
J. D. Chem. ReV. 1995, 95, 1953–1980.
(25) Candenas, M. L.; Pinto, F. M.; Cintado, C. G.; Morales, E. Q.; Brouard,
I.; D´ıaz, M. T.; Rico, M.; Rodr´ıguez, E.; Rodr´ıguez, R. M.; Pe´rez, R.;
Pe´rez, R. L.; Mart´ın, J. D. Tetrahedron 2002, 58, 1921–1942.
(26) (a) Oguri, H.; Oomura, A.; Tanabe, S.; Hirama, M. Tetrahedron Lett.
2005, 46, 2179–2183. (b) Oguri, H.; Tanabe, S.; Oomura, A.; Umetsu,
M.; Hirama, M. Tetrahedron Lett. 2006, 47, 5801–5805.
(27) Sasaki, M.; Tachibana, K. Tetrahedron Lett. 2007, 48, 3181–3186.
(28) Torikai, K.; Yari, H.; Mori, M.; Ujihara, S.; Matsumori, N.; Murata,
M.; Oishi, T. Bioorg. Med. Chem. Lett. 2006, 16, 6355–6359.
9
10218 J. AM. CHEM. SOC. VOL. 130, NO. 31, 2008

Synthesis of Artificial Ladder-Shaped Polyethers
A R T I C L E S
Figure 1. Structures of representative ladder-shaped polyethers from marine origin.
activities. However, the number of rings is limited to the
pentacyclic or the heptacyclic system. Herein, we report design
and systematic synthesis of artificial ladder-shaped polyethers
(ALPs) with tetracyclic, heptacyclic, and decacyclic systems
encompassing iterative 6/7/6 tricycles. Studies on the evaluation
of the interaction with TM proteins are also reported.
the other side was equipped with hydroxy groups (termed as
ALPA series) or benzyloxy groups (ALPB series) to assess the
hydrophilic or hydrophobic effects of the side chains; (iii) these
ALPs can be efficiently synthesized in a convergent manner.
Significant advancements in the construction of polycyclic
ether systems based on the one-pot strategies have been made
by epoxide-opening cascades30 and two-directional elongation.31
Recently, we have reported the synthesis of a tetracyclic model
compound (Figure 3A) by using Takeda cyclization,28 and the
Results
Design and Synthesis of Artificial Ladder-Shaped Polyethers.
We designed 6/7/6/6 tetracyclic, 6/7/6/6/7/6/6 heptacyclic, and
6/7/6/6/7/6/6/7/6/6 decacyclic ALPs (1∼3, Figure 2), which
possess a common iterative 6/7/6 ring system with different
molecular length, by the following reasons: (i) a trans-fused
6/7/6 ring system mimics the partial framework frequently
occurring in natural LSPs such as yessotoxin, gambierol, and
gymnocin-A, with placing angular methyl(s) to improve solubil-
ity of the ALPs in the lipid membranes; (ii) one of the terminal
sides is functionalized as diol to increase water solubility and
(30) (a) Tokiwano, T.; Fujiwara, K.; Murai, A. Synlett 2000, 335–338. (b)
Valentine, J. C.; McDonald, F. E.; Neiwert, W. A.; Hardcastle, K. I.
J. Am. Chem. Soc. 2005, 127, 4586–4587. (c) Simpson, G. L.; Heffron,
T. P.; Merino, E.; Jamison, T. F. J. Am. Chem. Soc. 2006, 128, 1056–
1057. (d) Vilotijevic, I.; Jamison, T. F. Science 2007, 317, 1189–
1192.
(31) (a) Nicolaou, K. C.; McGarry, D. G.; Sommers, P. K. J. Am. Chem.
Soc. 1990, 112, 3696–3697. (b) Ravelo, J. L.; Regueiro, A.; Martin,
J. D. Tetrahedron Lett. 1992, 33, 3389–3392. (c) Clark, J. S.; Hamelin,
O. Angew. Chem., Int. Ed. 2000, 39, 372–374. (d) Matsuo, G.;
Kawamura, K.; Hori, N.; Matsukura, H.; Nakata, T. J. Am. Chem.
Soc. 2004, 126, 14374–14736. (e) Clark, J. S.; Kimber, M. C.;
Robertson, J.; McErlean, C. S. P.; Wilson, C. Angew. Chem., Int. Ed.
2005, 44, 6157–6162.
(29) Torikai, K.; Yari, H.; Murata, M.; Oishi, T. Heterocycles 2006, 70,
161–167.
9
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A R T I C L E S
Torikai et al.
Scheme 1. Synthetic Strategy Based on the Convergent Method
via R-Cyano Ethers
Figure 2. Structures of artificial ladder-shaped polyethers (ALPs).
system being obtained from [m] and [n] ring fragments (m, n:
number of rings of the fragments). However, it was uncertain
whether the R-cyano ether method was applicable for synthesiz-
ing a large molecule such as a decacyclic system.
Synthesis of the 6/7/6/6 tetracyclic ALP (1) based on the
R-cyano ether method32 is depicted in Scheme 2. Tetrahydro-
pyran derivative 4b34,37 was chosen as a pivotal intermediate
for synthesizing the ALPs. The dibenzylether 4b and tetraol 4a
derived from 4b by means of hydrogenolysis were used as
monocyclic model compounds termed as ALP1B (4b) and
ALP1A (4a), respectively, to compare with the ALPs (1-3).
Condensation of diol 532 and aldehyde 634 derived from 4b
according to the reported procedure, respectively, proceeded
smoothly by the action of Sc(OTf)3 to give seven-membered
cyclic acetal in 95% yield. Regioselective opening of the acetal
was achieved by treatment with TMSCN in the presence of
Sc(OTf)3 at room temperature for one hour to form R-cyano
ether 7 (53%) with the recovery of the starting material (44%),
which was recycled three times to provide 7 in 82% total yield.
Although prolonged reaction time of the acetal cleavage resulted
in removal of the PMB group, it would be overcome by using
2-naphthylmethyl (NAP) group instead of PMB.34 Sequential
conversion of primary alcohol 7 into terminal olefin 8 via
2-nitrophenyl selenide was unsuccessful under the standard
conditions.38 After considerable experimentation, we found that
the addition of MS4A is requisite for conversion of 7 into the
Figure 3. Structures of model compounds.
methodology was applied to a heptacyclic ether on the basis of
the double reaction strategy.29 However, construction of the
seven-membered ring and alkylation to introduce angular methyl
groups were problematic in terms of yield and stereoselectivity
to give 6/7-cis-fused product as a major component (Figure 3B).
In this study, we envisaged a convergent strategy18 via R-cyano
ethers (R-cyano ether method) developed in our laboratory32
for the synthesis of the ALPs (1-3), which was successfully
applied to the convergent synthesis of the CDEF-33 and FGHI-
ring34 fragments of yessotoxin. As illustrated in Scheme 1, a
6/7/6/6-fused system possessing an angular methyl group (A),
would be derived from R-cyano ether (D) via ring closing
metathesis (RCM)35,36 for the construction of the seven-
membered ring, followed by mixed thioacetal (C) formation
and alkylation of the corresponding sulfone (B). The key
intermediate (D) could be retrosynthetically dissected into diol
(E) and aldehyde (F). In this strategy, two rings are constructed
through the coupling of fragments, therefore, [m + n + 2] ring
(36) (a) For reviews of RCM, see: Grubbs, R. H.; Miller, S. J.; Fu, G. C.
Acc. Chem. Res. 1995, 28, 446–452. (b) Blechert, S. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 2036–2056. (c) Grubbs, R. H.; Chang, S.
Tetrahedron 1998, 54, 4413–4450. (d) Armstrong, S. K. J. Chem. Soc.,
Perkin Trans. 1998, 1, 371–388. (e) Fu¨rstner, A. Angew. Chem., Int.
Ed. 2000, 39, 3012–3043. (f) Trnka, T. M.; Grubbs, R. H. Acc. Chem.
Res. 2001, 34, 18–29. (g) Grubbs, R. H. Tetrahedron 2004, 60, 7117–
7140. (h) Deiters, A.; Martin, S. F. Chem. ReV. 2004, 104, 2199–
2238. (i) Nicolaou, K. C.; Bulger, P. G.; Sarlah, D. Angew. Chem.,
Int. Ed. 2005, 44, 4490–4527.
(32) Oishi, T.; Watanabe, K.; Murata, M. Tetrahedron Lett. 2003, 44, 7315–
7319.
(33) Oishi, T.; Suzuki, M.; Watanabe, K.; Murata, M. Heterocycles 2006,
69, 91–98.
(37) Nicolaou, K. C.; Hwang, C.-K.; Duggan, M. E.; Nugiel, D. A.; Abe,
Y.; Reddy, K. B.; DeFrees, S. A.; Reddy, D. R.; Awartani, R. A.;
Conely, S. R.; Rutjes, F. P. J. T.; Theodorakis, E. A. J. Am. Chem.
Soc. 1995, 117, 10227–10238.
(34) Watanabe, K.; Suzuki, M.; Murata, M.; Oishi, T. Tetrahedron Lett.
2005, 46, 3991–3995.
(35) (a) Fu, G. C.; Nguyen, S. T.; Grubbs, R. H. J. Am. Chem. Soc. 1993,
115, 9856–9857. (b) Schwab, P.; Grubbs, R. H.; Ziller, J. W. J. Am.
Chem. Soc. 1996, 118, 100–110.
(38) Grieco, P. A.; Gilman, S.; Nishizawa, M. J. Org. Chem. 1976, 41,
1485–1486.
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Synthesis of Artificial Ladder-Shaped Polyethers
A R T I C L E S
Scheme 2. Synthesis of the Tetracyclic ALPs (1)a
a Reagents and conditions: (a) H2, Pd(OH)2/C, THF, room temperature (rt), 3.5 d, 92%; (b) (t-Bu)2Si(OTf)2, 2,6-lutidine, DMF, 0 °C, 20 min; (c) H2,
Pd(OH)2/C, THF, rt, 18 h, 97% (two steps); (d) p-MeOC6H4CH(OMe)2, CSA, CH2Cl2, rt, 4.5 h, 91%; (e) DIBALH, CH2Cl2, -78 to -11 °C, 2 h; (f) MsCl,
Et3N, CH2Cl2, 0 °C, 20 min; (g) NaCN, 18-crown-6, DMF, 80 °C, 4 h, 81% (three steps); (h) DIBALH, CH2Cl2, -67 to -25 °C, 1.5 h, 86%; (i) Sc(OTf)3,
benzene, rt, 40 min, 95%; (j) TMSCN, Sc(OTf)3, CH2Cl2, rt, 1 h, 82% (after three cycles); (k) o-NO2-C6H4SeCN, n-Bu3P, MS4A, THF, rt, 1 h; (l) mCPBA,
1,2-dichloroethane, rt, 15 min, then NaHCO3, 60 °C, 25 min, 74% (two steps); (m) DIBALH, toluene, -78 °C, 1 h, 71%; (n) tetravinyltin, MeLi, THF, -78
to -62 °C, 12 min, 90%; (o) Dess-Martin periodinane, CH2Cl2, rt, 1 h, 97%; (p) Grubbs cat. (2nd generation) 11, toluene, reflux, 15 min, 95%; (q) H2,
PtO2, AcOEt, rt, 3.5 h; (r) Dess-Martin periodinane, CH2Cl2, rt, 2 h, 96% (two steps); (s) DBU, toluene, reflux, 39 h, 93%; (t) DDQ, H2O, CH2Cl2, rt, 3.5 h,
68%, (epimer 11%); (u) EtSH, Zn(OTf)2, CH2Cl2, 0 to 14 °C, 2.5 h, 86%; (v) mCPBA, CH2Cl2, rt, 30 min, then Me3Al, -40 to -20 °C, 1 h, 91%; (w)
TBAF, THF, rt, 24 h, 85%; (x) H2, Pd(OH)2/C, THF, rt, 10 h, quant.
corresponding selenide, and mCPBA was superior to hydrogen
peroxide in the oxidation of the selenide giving selenoxide. Thus,
olefin 8 was successfully obtained in 74% yield for two steps.
Reduction of the nitrile 8 with DIBALH gave aldehyde 9, which
was converted into enone 10 by treatment with vinyllithium
followed by Dess-Martin oxidation39 of the resulting allylic
alcohol (62%, three steps). RCM of the diene 10 by the ac-
tion of second generation Grubbs catalyst 11 (3.5 mol %)40
proceeded smoothly in toluene at reflux to afford the seven-
membered cyclic enone in 95% yield. Hydrogenation of the
enone using PtO2 gave a mixture of saturated ketones with
concomitant formation of alcohols by overreaction, which was
readily oxidized to give ketone 12 and 13 as a mixture of
diasteomers in a 1:1 ratio (96%, two steps). Conversion of the
undesired epimer 13 into 12 was achieved by treatment with
DBU in toluene at 110 °C (12:13 ) 5:1), and the structure of
12 was confirmed by NOE experiments. Removal of the PMB
group using DDQ (at this stage, the minor epimer was removed
by silica gel column chromatography), followed by treatment
with EtSH in the presence of Zn(OTf)241 yielded cyclic mixed
thioacetal 14 (58%, two steps). Stereoselective introduction of
an angular methyl group42 was successfully achieved through
oxidation of mixed thioacetal 14 with mCPBA toward sulfone,
and one-pot treatment of the reaction mixture with Me3Al to
afford 15 (91%) as a single isomer. The configuration of the
angular methyl group was unambiguously determined by NOE
experiments. Removal of the silyl group of 15 with TBAF
yielded 1b (ALP4B) in 85% yield. Thus, 6/7/6/6 tetracyclic
ALP4B was synthesized from monocyclic building blocks (5
and 6) in 14% overall yield for 14 steps (87% average yield).
Hydrogenolysis of the benzyl ether 1b afforded tetraol 1a
(ALP4A) quantitatively.
The heptacyclic ALP 2 was synthesized in an analogous
sequence as ALP 1 based on the R-cyano ether method, starting
from diol 16 prepared from tetracyclic ether 15 (Scheme 3).
Coupling of the diol 16 with aldehyde 6 by means of acetal
formation and regioselective cleavage to provide R-cyano ether
17, followed by construction of seven-membered ring through
RCM and epimerization afforded ketone 18. Subsequent six-
membered ring formation was successfully achieved in a
stereoselective manner (confirmed by NOE experiments of 19)
to afford 6/7/6/6/7/6/6 heptacyclic 2b (ALP7B). The overall
yield from 16 and 6 was 6.6%, and the average yield was
calculated to be 82% for 14 steps. Removal of the benzyl groups
of 2b gave tetraol 2a (ALP7A).
(39) (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155–4156. (b)
Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899.
(40) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1,
953–956.
(41) (a) Fuwa, H.; Sasaki, M.; Tachibana, K. Tetrahedron 2001, 57, 3019–
3033. (b) Nicolaou, K. C.; Veale, C. A.; Hwang, C.-K.; Hutchinson,
J.; Prasad, C. V. C.; Ogilvie, W. W. Angew. Chem., Int. Ed. Engl.
1991, 30, 299–303.
(42) (a) Nicolaou, K. C.; Duggan, M. E.; Hwang, C.-K. J. Am. Chem. Soc.
1986, 108, 2468–2469. (b) Nicolaou, K. C.; Prasad, C. V. C.; Hwang,
C.-K.; Duggan, M. E.; Veale, C. A. J. Am. Chem. Soc. 1989, 111,
5321–5330.
9
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A R T I C L E S
Torikai et al.
Scheme 3. Synthesis of the Heptacyclic ALPs (2)a
system which is the largest ALP to date possessing iterative
structure. These ALPs with different number of rings comprising
a 6/7/6 iterative sequence are useful molecular probes for SAR
studies on the interaction with TM proteins and biological
activities.
Evaluation of the Interaction between ALPs and TM
Proteins. To evaluate the interaction of the ALPs with
TM proteins, glycophorin A (GpA), a heavily glycosylated TM
protein occurring in erythrocyte membrane was selected. GpA
is known to form a dimer43 in membrane environments by
interaction mainly between R-helical TM domain (73 ITLIIF-
GVMAGVIGTILLISYGI 95) in which the GXXXG motif44
(glycine zipper)45 is considered to be important for dimerization.
GpA is water soluble but tends to aggregate in water even in
the presence of detergents. Bormann et al.46 have reported that
GpA migrated on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) as oligomers or dimers, which can
be dissociated into monomers by peptides corresponding to the
TM domain (Figure 4). The dissociation can thus be accounted
for by direct binding of the peptides to the TM part of GpA.
We utilized this method to evaluate the interaction with LSPs
in place of the R-helical peptides on the basis of the SDS-PAGE
analysis47 and found that LSPs such as brevetoxin-B and
yessotoxin as well as the tetracyclic ALP possessing benzyl
ethers (Figure 3A)28 induced dissociation of oligomeric GpA
into its dimers and monomers. Under the same conditions, the
ALPB series possessing benzyl ether moieties (1b, 2b, and 3b)
were incubated (24 nmol, 1.5 mM) with GpA (2.6 pmol, 0.16
µM) in buffer containing 0.03% SDS (Figure 5A). As shown
in lane 1 of Figure 5A, intact GpA exists as a mixture of
oligomers and dimers. When GpA was treated with ALP1B
(lane 2), oligomer bands decreased and dimer bands appeared.
When GpA was treated with ALP4B (lane 3), the oligomer
bands disappeared completely, and a considerable amount of
monomers (38 kDa) as well as dimers emerged. Contrary to
our expectation, ALP7B did not induce dissociation of GpA
dimers on the SDS-PAGE (lane 4). For the ALP10B, even the
dissociation of GpA oligomers did not occur, while a smaller
amount of ALP10B (12 nmol, 0.75 mM) than ALP1B-7B (24
nmol, 1.5 mM) was incubated because of its low solubility in
0.03% SDS buffer (critical micelle concentration of SDS:
∼0.25%). Therefore, GpA was incubated with ALPB series in
2% SDS buffer (Figure 5B). As shown in lane 1 of Figure 5B,
intact GpA exists predominantly as dimers with small amount
of monomers. When GpA was treated with ALP1B (lane 2),
ALP4B (lane 3), and ALP7B (lane 4), the monomer band
increased with increasing the number of rings, whereas ALP10B
(lane 5) exhibited no significant effect.
a Reagents and conditions: (a) H2, Pd(OH)2/C, THF, rt, 6 h, quant.; (b)
Sc(OTf)3, benzene, rt, 1 h, 79%; (c) TMSCN, Sc(OTf)3, CH2Cl2, rt, 80
min, 67%; (d) o-NO2-C6H4SeCN, n-Bu3P, MS4A, THF, rt, 40 min; (e)
mCPBA, 1,2-dichloroethane, rt, 30 min, then NaHCO3, 60 °C, 30 min, 71%
(two steps); (f) DIBALH, CH2Cl2, -78 °C, 50 min, 63%; (g) tetravinyltin,
MeLi, THF, -78 to -67 °C, 20 min, 91%; (h) Dess-Martin periodinane,
CH2Cl2, rt, 50 min, 92%; (i) Grubbs cat. (2nd generation) 11, toluene, reflux,
20 min, 95%; (j) H2, PtO2, AcOEt, rt, 2.5 h, then Dess-Martin periodinane,
CH2Cl2, rt, 75 min, 94%; (k) DBU, toluene, reflux, 64 h, 82%; (l) DDQ,
H2O, CH2Cl2, rt, 50 min, 59%; (m) EtSH, Zn(OTf)2, CH2Cl2, 0 °C to rt,
3 h, 79%; (n) mCPBA, CH2Cl2, 0 °C, 30 min, then Me3Al, -55 to -32
°C, 40 min, quant.; (o) HF·pyridine, THF, 0 to 10 °C, 3 h, quant.; (p) H2,
Pd(OH)2/C, THF, rt, 10 h, quant.
The remaining task is synthesis of the decacyclic ALP (3),
the most challenging molecule in the present study (Scheme
4). In an analogous sequence as the synthesis of ALPs 1 and 2,
coupling of diol 16 and aldehyde 20 (prepared from 1a by the
identical procedure from 4b to 6, Scheme 2) provided R-cyano
ether 21, and conversion to decacyclic ether 23 via ketone 22
proceeded smoothly irrespective of the size of the synthetic
intermediates. Although it was not an easy task to determine
the configuration of the newly formed angular methyl group of
ALP 3 because of the overlapped NMR signals due to its
iterative structure, the 920 MHz 1H NMR was of great use for
structure determination, which enabled the NOE experiments
by selective irradiation of the individual angular methyl groups
of 23 (Supporting Information). Removal of the silyl group of
23 afforded 6/7/6/6/7/6/6/7/6/6 decacyclic 3b (ALP10B). The
average yield from 16 and 3b for 14 steps was 83% (6.8% total
yield), which is comparable to that of the heptacyclic ALP.
Finally, hydrogenolysis of benzyl ether 3b gave tetraol 3a
(ALP10A).
To investigate whether the ALPs interact with the R-helical
TM domain of GpA, a peptide composed of 29 residues (GpA-
TM: 70 EPEITLIIFGVMAGVIGTILLISYGIRRL 98) was
(43) (a) Lemmon, M. A.; Flanagan, J. M.; Hunt, J. F.; Adair, B. D.;
Bormann, B.-J.; Dempsey, C. E.; Engelman, D. M. J. Biol. Chem.
1992, 267, 7683–7689. (b) Lemmon, M. A.; Flanagan, J. M.; Treutlein,
H. R.; Zhang, J.; Engelman, D. M. Biochemistry 1992, 31, 12719–
12725. (c) Langosch, D.; Brosing, B.; Kolmar, H.; Fritz, H.-J. J. Mol.
Biol. 1996, 263, 525–530.
(44) Liu, Y.; Engelman, D. M.; Gerstein, M. Genome Biol. 2002, 3, 1–12.
(45) Kim, S.; Jeon, T.-J.; Oberai, A.; Yang, D.; Schmidt, J. J.; Bowie, J. U.
Proc. Natl. Acad. Sci. USA 2005, 102, 14278–14283.
Thus, systematic synthesis of ALPs composed of the tetra-
cyclic, heptacyclic, and decacyclic systems has been successfully
achieved in a convergent manner. The R-cyano ether method
proved to be a powerful strategy, not only for coupling of small
fragments, but also for large segments giving decacyclic ether
(46) Bormann, B.-J.; Knowles, W. J.; Marchesi, V. T. J. Biol. Chem. 1989,
264, 4033–4037.
(47) Mori, M.; Oishi, T.; Matsuoka, S.; Ujihara, S.; Matsumori, N.; Murata,
M.; Satake, M.; Oshima, Y.; Matsushita, N.; Aimoto, S. Bioorg. Med.
Chem. 2005, 13, 5099–5103.
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Synthesis of Artificial Ladder-Shaped Polyethers
A R T I C L E S
Scheme 4. Synthesis of the Decacyclic ALPs (3)a
a Reagents and conditions: (a) p-MeO-C6H4CH(OMe)2, CSA, CH2Cl2, rt, 5 h, 92%; (b) DIBALH, CH2Cl2, -62 to -13 °C, 2 h; (c) MsCl, Et3N, CH2Cl2,
0 °C, 10 min; (d) NaCN, 18-crown-6, DMF, 80 °C, 5 h, 94% (three steps); (e) DIBALH, CH2Cl2, -76 to -45 °C, 50 min, 93%; (f) Sc(OTf)3, benzene, rt,
6.5 h, 85%; (g) TMSCN, Sc(OTf)3, CH2Cl2, rt, 80 min, 61%; (h) o-NO2-C6H4SeCN, n-Bu3P, MS4A, THF, rt, 30 min; (i) mCPBA, 1,2-dichloroethane, rt,
15 min, then NaHCO3, 60 °C, 24 min, 85% (two steps); (j) DIBALH, CH2Cl2, -83 to -50 °C, 35 min, 70%; (k) tetravinyltin, MeLi, THF, -78 to -63 °C,
30 min, 94%; (l) Dess-Martin periodinane, CH2Cl2, rt, 1.5 h, 99%; (m) Grubbs cat. (2nd generation) 11, toluene, reflux, 15 min, 86%; (n) H2, PtO2, AcOEt,
rt, 3.5 h, then Dess-Martin periodinane, CH2Cl2, rt, 30 min, 90%; (o) DBU, toluene, reflux, 21 h, 60%; (p) DDQ, H2O, CH2Cl2, rt, 2 h, 83%; (q) EtSH,
Zn(OTf)2, CH2Cl2, 0 °C to rt, 1.5 h, 65%; (r) mCPBA, CH2Cl2, 0 °C, 20 min, then Me3Al, -52 to -20 °C, 1 h, 96%; (s) HF ·pyridine, THF, 0 °C to rt, 3.5 h,
98%; (t) H2, Pd(OH)2/C, THF, MeOH, rt, 4 days, 62%.
Figure 4. Illustrative pathways of dissociation of GpA dimer into monomer
and GpA/GpA-TM complex.
Figure 6. SPR sensorgram for the binding of different concentrations of
ALP7B to GpA-TM.
Table 1. Dissociation Constants of ALPB Series against GpA-TM
Determined by SPR
entry
ALP
KD (µM)
1
2
3
4
ALP1B
ALP4B
ALP7B
ALP10B
2.4 × 103
2.4 × 102
48
NDa
a Not determined.
solubility in the running buffer containing DMSO (1%) and
Tween20 (400 µM). Dose dependent changes in sensorgrams
were observed with ALP7B (Figure 6) as well as ALP1B and
ALP4B (Figures S1 and S2, Supporting Information). By
comparing KD values, affinities of ALPs to GpA-TM increased
with increasing the number of rings (Table 1), which cor-
responded to the results of SDS-PAGE in Figure 5B.
Figure 5. SDS-PAGE of GpA in the presence of ALPB series in SDS
buffer. (A) GpA (2.6 pmol, 0.16 µM) alone (lane 1), in the presence of 24
nmol (1.5 mM) of ALP1B (lane 2), ALP4B (lane 3), ALP7B (lane 4), and
12 nmol of ALP10B (lane 5) in 0.03% SDS buffer. (B) GpA (2.6 pmol,
0.16 µM) alone (lane 1), in the presence of 24 nmol (1.5 mM) of ALP1B
(lane 2), ALP4B (lane 3), ALP7B (lane 4), and ALP10B (lane 5) in 2%
SDS buffer. GpA was visualized by silver staining.
Then GpA was treated with ALPA series with tetraols in 2%
SDS buffer, however, no dissociation of the dimers was
synthesized using solid-phase methods,48 and the interaction
between ALP and GpA-TM was analyzed by using surface
plasmon resonance (SPR) techniques.49 The GpA-TM was
coupled to the biosensor surface, and sensorgrams of ALPB
series were recorded except for ALP10B because of the low
(48) Smith, S. O.; Song, D.; Shekar, S.; Groesbeek, M.; Ziliox, M.; Aimoto,
S. Biochemistry 2001, 40, 6553–6558.
(49) (a) Fa¨gerstam, L. G.; Frostell-Karlsson, Å.; Karlsson, R.; Persson, B.;
Ro¨nnberg, I. J. Chromatogr. 1992, 597, 397–410. (b) Johnsson, B.;
Loefaas, S.; Lindquist, G. Anal. Biochem. 1991, 198, 268–277.
9
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A R T I C L E S
Torikai et al.
Figure 9. 3D structures of SDS, ALP4B, ALP7B, ALP10A, and GpA-
TM (from left).
edly, an intriguing phenomenon was observed that some
precipitates formed when GpA was treated with ALP10A. After
centrifugation, the resulting precipitates were dissolved in 6%
SDS buffer. The supernatant and the solubilized precipitates
were subjected to SDS-PAGE, respectively (Figure 7A, lanes
4 and 8). The same procedure was also performed with ALP1A
(lanes 1 and 5), ALP4A (lanes 2 and 6), and ALP7A (lanes 3
and 7). As shown in Figure 7A, only ALP10A turned out to
induce sedimentation of GpA. The effect is dose dependent,
and more than 150 times of ALP10A is required to elicit the
activity (Figure S5, Supporting Information). It is noteworthy
that the GpA pellet obtained by the treatment with ALP10A in
0.03% SDS buffer, was no longer soluble in a buffered aqueous
solution in the absence of SDS, while intact GpA is highly
soluble in water. Then, we ascertained whether the ALPB series
cause precipitation of GpA under the low concentration of SDS
(0.03%), and the same procedure as for ALPA series was
performed with ALP1B (Figure 7B, lanes 1 and 5), ALP4B
(lanes 2 and 6), ALP7B (lanes 3 and 7), and ALP10B (lanes 4
and 8). Among the ALPB series, ALP7B elicited the most potent
precipitation-inducing activity, and the results are consistent with
the GpA-dissociation activity in 2% SDS buffer (Figure 5B),
while the activity of ALP7B is lower than that of ALP10A.
To examine whether the precipitate-inducing activity of
ALP10A is specific for GpA, integrin R1ꢀ1 was subjected to
the same experiment. Integrin R1ꢀ1 is known to form a
heterodimer in membrane environment by interaction between
R-helical TM domains (R1: 966 LWVILLSAFAGLLLLMLLI-
LALW 988; ꢀ1: 966 IIPIVAGVVAGIVLIGLALLLIW 988)
containing a glycine zipper motif (one of the G’s is substituted
with A or S).50 Analogously, ALP10A induced sedimentation
of integrin R1ꢀ1 (Figure S6, Supporting Information). For bovine
serum albumin, a water soluble protein without a TM domain,
no precipitation formed by ALP10A in 0.03% SDS.
Figure 7. SDS-PAGE of GpA in the presence of ALPs in 0.03% SDS
buffer. Supernatant of the centrifuged sample was loaded in lanes 1 to 4,
and the residue dissolved in 6% SDS buffer was loaded in lanes 5 to 8,
respectively. (A) GpA (2.6 pmol, 0.16 µM) in the presence of 24 nmol
(1.5 mM) of ALP1A (lanes 1 and 5), ALP4A (lanes 2 and 6), ALP7A (lanes
3 and 7), and 12 nmol (0.75 mM) of ALP10A (lanes 4 and 8). (B) GpA
(2.6 pmol, 0.16 µM) in the presence of 24 nmol (1.5 mM) of ALP1B (lanes
1 and 5), ALP4B (lanes 2 and 6), ALP7B (lanes 3 and 7), and 12 nmol
(0.75 mM) of ALP10B (lanes 4 and 8). GpA was visualized by silver
staining.
Figure 8. Hypothetical pathways of (A) dissociation of GpA dimers to
monomers (ALP7B, 2% SDS) and (B) sedimentation of GpA (ALP10A,
0.03% SDS).
Discussion
observed (Figure S3, Supporting Information). We postulated
that ALPA series, both sides of which were functionalized with
polar hydroxy groups, were prevented from permeation into SDS
micelles. Therefore, GpA was incubated with ALPA series under
the low concentration of SDS (0.03%), but no dissociation of
GpA occurred (Figure S4, Supporting Information). Unexpect-
The present SAR studies of the ALPs revealed that ALP7B
elicited notable activity to induce dissociation of GpA dimers
(50) Lin, X.; Tan, S. M.; Law, S. K. A.; Torres, J. Proteins 2006, 63, 16–
23.
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Synthesis of Artificial Ladder-Shaped Polyethers
A R T I C L E S
Figure 10. Comparison of the molecular length of ALPs, GpA-TM, and continuum electrostatic model of a biological membrane environment.42
to monomers in the presence of 2% SDS (in SDS micelles),
while ALP10A exhibited intriguing phenomenon to induce
sedimentation of GpA and integrin R1ꢀ1 under the low
concentration (0.03%) of SDS (Figure 8). ALP7B also induced
precipitation of GpA in 0.03% SDS buffer but the efficacy is
lower than ALP10A. Although the precise mechanism is
unknown on the dissociation of GpA dimers or the formation
of the precipitates containing GpA or integrins, both the
dissociation- and precipitation-inducing activity against the TM
proteins would be related to the interaction between ALPs and
TM region of the proteins as supported by SPR experiments.
We postulated that differences in efficacy among the ALPs can
be correlated with the concept of “hydrophobic matching”,51
since SDS micelles are expected to provide membrane environ-
ments similar to those of lipid bilayers, particularly for
hydrophobic interactions with TM regions. Comparison of the
molecular lengths of the ALPs and TM region of GpA (GpA-
TM) was performed by a MacroModel program (Figure 9) as
schematically illustrated in Figure 10. The molecular length was
also compared with a five-slab of continuum electrostatic model
of a biological membrane environment reported by Sengupta
et al.,52 in which the membrane model is composed of a low
dielectric constant (ꢀ ) 2) region corresponding to the alkyl
chains of lipids, high dielectric constant (ꢀ ) 10) region
corresponding to the polar head groups of lipids, and bulk water
(ꢀ ) 80) phase. As shown in Figure 10, it is obvious that the
length of the hydrophobic region of ALP7B including the benzyl
ethers (ca. 25 Å) matches that of low dielectric constant region
of membrane when the polar hydroxy groups are placed into
the slab of high dielectric constant. The hydrophobic nature of
the benzyl groups is also considered to assist insertion of ALP7B
into lipid membranes. In analogy with other membrane proteins,
the amino acid sequence of GpA-TM matches the continuum
electrostatic model; thus, the hydrophobic region (I73-I95) lies
between the polar amino acid residues (E70, E72 and R96, R97)
residing at both ends of the R-helix. For ALP10A, not only the
hydrophobic (polycyclic ether skeleton) but also hydrophilic
(hydroxy groups) region can be placed in the five slab areas in
a similar manner to that of GpA-TM. On the other hand, the
length of the hydrophobic region of ALP7A is not long enough,
and that of ALP10B is too long in comparison with that of GpA-
TM, which could account for their weak dissociation- or
precipitation-inducing activity. Looking back at the structures
of naturally occurring LSPs (Figure 1), we noticed that
ciguatoxins match the model, and relatively smaller but biologi-
cally active LSPs, such as brevenal (pentacyclic) and gambierol
(octacyclic), possess somewhat longer side chains than other
LSPs. It would be correlated to the concept of hydrophobic
matching when the LSPs bind to the target proteins in the lipid
bilayer membranes. Although the concept of hydrophobic
matching is still controversial on the interaction of LSPs and
TM proteins, it might be a clue to understanding the mode of
action of naturally occurring LSPs and also a guiding principle
to design ALPs possessing potent affinities with TM proteins.
Besides these studies for elucidating the molecular mechanism
underlying the powerful toxicity of natural LSPs, the charac-
teristic property that some ALPs such as ALP7B and ALP10A
interact, dissociate, or precipitate TM proteins can be utilized
to fish out membrane-integral proteins from cell lysates in
proteomics and related areas.
Although there are few detailed proposals on the molecular
recognition between the TM proteins and the LSPs to date, it
has been suggested that (i) van der Waals interaction including
CH/π interaction53 might be an important factor because of the
hydrophobic nature of the TM domain, and (ii) hydrogen
bonding between skeletal oxygen atoms of LSPs and the OH/
NH groups of the R-helical peptides stabilizes their complexation
(51) (a) Jensen, M. Ø.; Mouritsen, O. G. Biochim. Biophys. Acta 2004,
1666, 205–226. (b) Orza´ez, M.; Lukovic, D.; Abad, C.; Pe´rez-Paya´,
E.; Mingarro, I. FEBS Lett. 2005, 579, 1633–1638.
(52) (a) Sengupta, D.; Meinhold, L.; Langosch, D.; Ullmann, G. M.; Smith,
J. C. Proteins 2005, 58, 913–922. (b) Sengupta, D.; Behera, R. N.;
Smith, J. C.; Ullmann, G. M. Structure 2005, 13, 849–855.
(53) Nishio, M.; Hirota, M.; Umezawa, Y. CH/π Interaction: EVidence,
Nature, and Consequences; Wiley-VCH: New York, 1998.
9
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A R T I C L E S
Torikai et al.
because the distance between the neighboring skeletal oxygen
atoms on the same side of the LSPs is consistent with the helix
pitch (ca. 5 Å).26 On the other hand, a glycine zipper is expected
to be one of the candidates for the interacting motifs because
of the common structural feature of GpA and integrins.28 The
glycine zipper motif is abundantly found in homooligomeric
domains of channel complexes: potassium channel pore-lining
helices (KcsA),54 mechanosensitive channel of large conduc-
tance (MscL),55 vacuolating toxin anion selective channel
(VacA),56 mechanosensitive channel of small conductance
(MscS),57 and also in homooligomeric domains of G-protein-
coupled receptors (GPCRs) such as R-factor receptors,58 ErbB,59
and CCK4.60 In the glycine zipper motif, weak hydrogen bonds
between the CR-H of glycine and a carbonyl oxygen
(CR-H· · ·O)61 were suggested to play an important role in
stabilizing the dimeric structure; therefore, the CR-H of glycine
could be also expected to be a hydrogen bond donor toward
skeletal oxygen atoms of LSPs. In addition to the weak hydrogen
bonding, dipole-dipole interactions between weakly polarized
functional groups presented in the hydrophobic environment
would be an important factor of the molecular recognition
between LSPs and TM proteins.
ethers) were systematically synthesized on the basis of the
convergent strategy via R-cyano ethers. The interaction with
transmembrane (TM) proteins, glycophorin A (GpA) and
integrin R1ꢀ1, were evaluated by SDS-PAGE and partly by SPR.
The heptacyclic ether (ALP7B) exhibited notable activity to
induce dissociation of GpA dimers to monomers in the presence
of 2% SDS. The KD value between ALP7B and a peptide
composed of 29 residues corresponding to TM domain of GpA
(GpA-TM) was determined to be 48 µM by SPR. The decacyclic
ether (ALP10A) exhibited an intriguing phenomenon to induce
precipitation of GpA dose-dependently under the low concentra-
tion of SDS (0.03%). ALP10A also induced precipitation of
integrin R1ꢀ1 which is also known to form heterodimers in
the lipid bilayer membranes. These results can be related to the
concept of hydrophobic matching; that is, the length of the
hydrophobic region including the side chains of ALP7B and
ALP10A is ca. 25 Å, which matches the lengths of the
hydrophobic region of R-helical TM proteins. Preparation of
the focused library of ALPs possessing different ring size and
functional groups and evaluation of their biological activities
and the interaction with various TM proteins are in progress in
our laboratory.
Acknowledgment. We are grateful to Prof. Yasuhiro Uozumi,
Dr. Hiroaki Sasagawa, and Ms. Michiko Nakano of the Institute
for Molecular Science for measurement of 920 MHz 1H NMR
spectra. We thank Mr. Seiji Adachi in our department for invaluable
discussions and Ryota Mouri, Futoshi Hasegawa, Hiroaki Minato,
and Tomoyoshi Imaizumi in our laboratory for preliminary
experimentation. This work was supported by Grant-in-Aid for
Scientific Research on Priority Areas (No. 16073211) from MEXT,
for Exploratory Research (No. 15655033), and for Scientific
Research (S) (No. 18101010) from JSPS, Japan.
Conclusion
In conclusion, aritificial ladder-shaped polyethers (ALPs)
composed of an iterative 6/7/6 ring system with different
molecular lengths (tetracyclic, heptacyclic, and decacyclic
(54) Doyle, D. A.; Cabral, J. M.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.;
Cohen, S. L.; Chait, B. T.; MacKinnon, R. Science 1998, 280, 69–77.
(55) Chang, G.; Spencer, R. H.; Lee, A. T.; Barclay, M. T.; Rees, D. C.
Science 1998, 282, 2220–2226.
(56) Kim, S.; Chamberlain, A. K.; Bowie, J. U. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 5988–5991.
(57) Bass, R. B.; Strop, P.; Barclay, M.; Rees, D. C. Science 2002, 298,
1582–1587.
Supporting Information Available: Experimental section
including the synthesis of the ALPs and copies of the NMR
spectra of new compounds, SDS-PAGE, and SPR analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(58) Overton, M. C.; Chinault, S. L.; Blumer, K. J. J. Biol. Chem. 2003,
278, 49369–49377.
(59) Gerber, D.; Sal-Man, N.; Shai, Y. J. Biol. Chem. 2004, 279, 21177–
21182.
(60) Kobus, F. J.; Fleming, K. G. Biochemistry 2005, 44, 1464–1470.
(61) Senes, A.; Ubarretxena-Belandia, I.; Engelman, D. M. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 9056–9061.
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