Total synthesis of ciguatoxin CTX3C: a venture into the problems of ciguatera seafood poisoning

Hirama, Masahiro

doi:10.1002/tcr.20049

Cited by 57 publications

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“…More than 50,000 people suffer from ciguatera annually, making it one of the most common sources of food poisoning (1)(2)(3)(4). The disease is characterized by gastrointestinal, neurological, and cardiovascular disturbances that often persist for months or years, and in severe cases, paralysis, coma, and death may occur (1). Ciguatoxins exert their effects by binding to voltage-sensitive sodium ion channels, causing persistent activation of the channels (1).…”

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“…The disease is characterized by gastrointestinal, neurological, and cardiovascular disturbances that often persist for months or years, and in severe cases, paralysis, coma, and death may occur (1). Ciguatoxins exert their effects by binding to voltage-sensitive sodium ion channels, causing persistent activation of the channels (1). Ciguatoxin and its congener, CTX3C, are structurally classified as ladder-like polyethers ( Fig.…”

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Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings

Tsumoto

Yokota

Tanaka

et al. 2008

Journal of Biological Chemistry

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To address how proteins recognize polyether toxin compounds, we focused on the interaction between the ABC ring compound of ciguatoxin 3C and its specific antibody, 1C49. Surface plasmon resonance analyses indicated that Escherichia coliexpressed variable domain fragments (Fv) of 1C49 had the high affinity constants and slow dissociation constants typical of antigen-antibody interactions. Linear van't Hoff analyses suggested that the interaction is enthalpy-driven. We resolved the crystal structure of 1C49 Fv bound to ABC ring compound of ciguatoxin 3C at a resolution of 1.7 Å . The binding pocket of the antibody had many aromatic rings and bound the antigen by shape complementarity typical of hapten-antibody interactions. Three hydrogen bonds and many van der Waals interactions were present. We mutated several residues of the antibody to Ala, and we used surface plasmon resonance to analyze the interactions between the mutated antibodies and the antigen. This analysis identified Tyr-91 and Trp-96 in the light chain as hot spots for the interaction, and other residues made incremental contributions by conferring enthalpic advantages and reducing the dissociation rate constant. Systematic mutation of Tyr-91 indicated that CH-and -interactions between the aromatic ring at this site and the antigen made substantial contributions to the association, and van der Waals interactions inhibited dissociation, suggesting that aromaticity and bulkiness are critical for the specific recognition of polyether compounds by proteins.Ciguatera is a form of food poisoning caused by the ingestion of reef fish that have accumulated trace amounts of ciguatoxins of dinoflagellate origin via the food chain. More than 50,000 people suffer from ciguatera annually, making it one of the most common sources of food poisoning (1-4). The disease is characterized by gastrointestinal, neurological, and cardiovascular disturbances that often persist for months or years, and in severe cases, paralysis, coma, and death may occur (1). Ciguatoxins exert their effects by binding to voltage-sensitive sodium ion channels, causing persistent activation of the channels (1). Ciguatoxin and its congener, CTX3C, are structurally classified as ladder-like polyethers ( Fig. 1) (5, 6). A major obstacle to avoiding the disease is that ciguateric fish look, taste, and smell the same as uncontaminated fish. In addition, neither cooking nor freezing detoxifies the heat-stable ciguatoxins. Despite the seriousness of ciguatera, there is currently no rapid and reliable method for detecting these toxins at fisheries. The traditional method is a mouse bioassay of lipid extracts (7). Several additional methods for detecting ciguatoxins have recently been developed, including assays based on cytotoxicity (8), radioligand binding (8, 9), high performance chromatography (10), mass spectrometry (11-13), and an antibody-based immunoassay (14 -16). Among these, the antibody-based immunoassay is attractive, because it is accurate, sensitive, easily performed, and portable. Dur...

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Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings

Tsumoto

Yokota

Tanaka

et al. 2008

Journal of Biological Chemistry

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“…Ciguatoxins are a family of ladder-like polycyclic ethers that causes ciguatera seafood poisoning in tropical and subtropical regions; more than 50,000 people suffer annually from this type of food poisoning (1)(2)(3)(4)(5)(6)(7). CTX3C, which contains 13 ether rings, is probably the best known the member of polycyclic ether family.…”

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“…10C9 IgG is prepared by immunization of mice with a carrier protein (keyhole limpet hemocyanin (KLH) 3 ) conjugated to the ABCDE-ring of CTX3C. 10C9 IgG binds not only to CTX3C-ABCDE but also to full-length CTX3C (Fig.…”

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How Protein Recognizes Ladder-like Polycyclic Ethers

Tanaka

Tsumuraya

et al. 2008

Journal of Biological Chemistry

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Ciguatoxins are a family of marine toxins composed of transfused polycyclic ethers. It has not yet been clarified at the atomic level on the pathogenic mechanism of these toxins or the interaction between a polycyclic ether compounds and a protein.Using the crystal structures of anti-ciguatoxin antibody 10C9 Fab in ligand-free form and in complexes with ABCD-ring (CTX3C-ABCD) and ABCDE-ring (CTX3C-ABCDE) fragments of the antigen CTX3C at resolutions of 2.6, 2.4, and 2.3 Å , respectively, we elucidated the mechanism of the interaction between the polycyclic ethers and the antibody. 10C9 Fab has an extraordinarily large and deep binding pocket at the center of the variable region, where CTX3C-ABCD or CTX3C-ABCDE binds longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab adjusts to the antigen fragments by means of rotational motion in the variable region. In addition, the antigen fragment lacking the E-ring induces a large motion in the constant region. Consequently, the thermostability of 10C9 Fab is enhanced by 10°C upon complexation with CTX3C-ABCDE but not with CTX3C-ABCD. The crystal structures presented in this study also show that 10C9 Fab recoginition of CTX3C antigens requires molecular rearrangements over the entire antibody structure. These results further expand the fundamental understanding of the mechanism by which ladder-like polycyclic ethers are recognized and may be useful for the design of novel therapeutic agents by antibodies, marine toxins, or new diagnostic reagents for the detection and targeting of members of the polycyclic ether family.Ciguatoxins are a family of ladder-like polycyclic ethers that causes ciguatera seafood poisoning in tropical and subtropical regions; more than 50,000 people suffer annually from this type of food poisoning (1-7). CTX3C, which contains 13 ether rings, is probably the best known the member of polycyclic ether family. Although CTX3C is known to bind to voltage-sensitive sodium channels, like other polycyclic ether toxins (8 -10), the precise pathogenic mechanism remains to be elucidated, and methods for the treatment of ciguatera poisoning have not yet been developed (5).Oguri et al. (11) established a direct sandwich enzyme-linked immunosorbent assay using two distinct monoclonal antibodies (10C9 and 3D11) to detect CTX3C at the parts per billion level with no cross-reactivity against other related marine toxins. 10C9 IgG is prepared by immunization of mice with a carrier protein (keyhole limpet hemocyanin (KLH) 3 ) conjugated to the ABCDE-ring of CTX3C. 10C9 IgG binds not only to CTX3C-ABCDE but also to full-length CTX3C (Fig. 1), with dissociation constants of 0.8 and 2.8 nM, respectively. Because the skeletons of polycyclic ethers are so rigid and because the compounds are so large (12-15) (they extend beyond the binding surface of the paratope of the antibody (200 -400 Å 2 ) (16 -21)), how the antibody recognizes the polycyclic ethers within the limited areas of the antigen-bindin...

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“…In 1989, Hirama's group initiated collaborative investigations with the Yasumoto's group with the aim of elucidating the absolute configuration of ciguatoxins. 7 …”

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Bioorganic Studies Utilizing Rationally Designed Synthetic Molecules: Absolute Configuration of Ciguatoxin and Development of Immunoassay Systems

Oguri¹

2007

Bulletin of the Chemical Society of Japan

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Ciguatoxins are the major causative toxins in ciguatera seafood poisoning. The limited availability of ciguatoxins has precluded structural elucidation and development of a reliable and specific immunoassay for detecting the toxins in contaminated fish. To seek a solution for the longstanding problem of ciguatera, we addressed the synthetic challenge by utilizing rationally designed model compounds of ciguatoxins. The C2 configuration and entire absolute configuration of ciguatoxin were successfully elucidated with minutest amounts of natural toxins, using an approach which combined partial structure synthesis, microscale chemical transformations, and the CD exciton chirality method. On the basis of the absolute configuration, the partial structures of ciguatoxins were designed and synthesized as haptens for the preparation of anti-ciguatoxin antibodies. Monoclonal antibodies (mAbs) against both ends of ciguatoxin CTX3C were prepared by immunization of mice with protein conjugates of synthetic haptens of the ABCDE-ring and the IJKLM-ring, in place of the natural toxin. Haptenic groups with surface areas larger than 400 Å 2 were required to produce mAbs, which could bind strongly to CTX3C itself. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was shown to detect CTX3C at the ppb level with no cross-reactivity against other related marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin. In order to make the sandwich immunoassay protocol a general method for detecting other ciguatoxins congeners, the preparation of mAbs for the left-end of ciguatoxin was investigated. Expeditious synthesis of the left end of ciguatoxin with installation of the 3-butene-1,2-diol side-chain of the A-ring as well as surface plasmon resonance (SPR) analysis of the antibody-hapten interactions are also described.

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Total synthesis of ciguatoxin CTX3C: a venture into the problems of ciguatera seafood poisoning

Cited by 57 publications

References 59 publications

Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings

Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings

How Protein Recognizes Ladder-like Polycyclic Ethers

Bioorganic Studies Utilizing Rationally Designed Synthetic Molecules: Absolute Configuration of Ciguatoxin and Development of Immunoassay Systems

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