A Direct C11 Alkylation Strategy on the Saxitoxin Core: A Synthesis of (+)-11-Saxitoxinethanoic Acid.

The bis-guanidinium ion family of natural products are revered for their utility in the study of ion channel physiology. While many congeners have been isolated with various oxidation and sulfation patterns, only two members of this family have been isolated bearing a carbon-carbon bond at C11, namely 11-saxitoxinethanoic acid and zetekitoxin AB. Herein we described a synthetic platform capable of efficiently targeting (+)-saxitoxin and 11-saxitoxinethanoic acid with an embedded C11 carbon-carbon bond. We demonstrate that this strategy enables direct enolate coupling in both an inter- and intramolecular fashion to create the C11-C15 carbon-carbon bond.

Synthetic Approaches to Zetekitoxin AB, a Potent Voltage-Gated Sodium Channel Inhibitor.

Voltage-gated sodium channels (NaVs) are membrane proteins that are involved in the generation and propagation of action potentials in neurons. Recently, the structure of a complex made of a tetrodotoxin-sensitive (TTX-s) NaV subtype with saxitoxin (STX), a shellfish toxin, was determined. STX potently inhibits TTX-s NaV, and is used as a biological tool to investigate the function of NaVs. More than 50 analogs of STX have been isolated from nature. Among them, zetekitoxin AB (ZTX) has a distinctive chemical structure, and is the most potent inhibitor of NaVs, including tetrodotoxin-resistant (TTX-r) NaV. Despite intensive synthetic studies, total synthesis of ZTX has not yet been achieved. Here, we review recent efforts directed toward the total synthesis of ZTX, including syntheses of 11-saxitoxinethanoic acid (SEA), which is considered a useful synthetic model for ZTX, since it contains a key carbon-carbon bond at the C11 position.

Divergent Synthesis of Natural Derivatives of (+)-Saxitoxin Including 11-Saxitoxinethanoic Acid.

The bis-guanidinium toxins are a collection of natural products that display nanomolar potency against select isoforms of eukaryotic voltage-gated Na+ ion channels. We describe a synthetic strategy that enables access to four of these poisons, namely 11-saxitoxinethanoic acid, C13-acetoxy saxitoxin, decarbamoyl saxitoxin, and saxitoxin. Highlights of this work include an unusual Mislow-Evans rearrangement and a late-stage Stille ketene acetal coupling. The IC50 value of 11-saxitoxinethanoic acid was measured against rat NaV 1.4, and found to be 17.0 nm, similar to those of the sulfated toxins gonyautoxin II and III.

Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles.

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.

Synthesis of the Paralytic Shellfish Poisons (+)-Gonyautoxin 2, (+)-Gonyautoxin 3, and (+)-11,11-Dihydroxysaxitoxin.

The paralytic shellfish poisons are a collection of guanidine-containing natural products that are biosynthesized by prokaryote and eukaryote marine organisms. These compounds bind and inhibit isoforms of the mammalian voltage-gated Na(+) ion channel at concentrations ranging from 10(-11) to 10(-5) M. Here, we describe the de novo synthesis of three paralytic shellfish poisons, gonyautoxin 2, gonyautoxin 3, and 11,11-dihydroxysaxitoxin. Key steps include a diastereoselective Pictet-Spengler reaction and an intramolecular amination of an N-guanidyl pyrrole by a sulfonyl guanidine. The IC50's of GTX 2, GTX 3, and 11,11-dhSTX have been measured against rat NaV1.4, and are found to be 22 nM, 15 nM, and 2.2 µM, respectively.

Saxitoxin.

The paralytic agent (+)-saxitoxin (STX), most commonly associated with oceanic red tides and shellfish poisoning, is a potent inhibitor of electrical conduction in cells. Its nefarious effects result from inhibition of voltage-gated sodium channels (Na(V)s), the obligatory proteins responsible for the initiation and propagation of action potentials. In the annals of ion channel research, the identification and characterization of Na(V)s trace to the availability of STX and an allied guanidinium derivative, tetrodotoxin. The mystique of STX is expressed in both its function and form, as this uniquely compact dication boasts more heteroatoms than carbon centers. This Review highlights both the chemistry and chemical biology of this fascinating natural product, and offers a perspective as to how molecular design and synthesis may be used to explore Na(V) structure and function.

A 18F-labeled saxitoxin derivative for in vivo PET-MR imaging of voltage-gated sodium channel expression following nerve injury.

Both chronic and neuropathic pain conditions are associated with increased expression of certain voltage-gated sodium ion channel (NaV) isoforms in peripheral sensory neurons. A method for noninvasive imaging of these channels could represent a powerful tool for investigating aberrant expression of NaV and its role in pain pathogenesis. Herein, we describe the synthesis and evaluation of a positron emission tomography (PET) radiotracer targeting NaVs, the design of which is based on the potent, NaV-selective inhibitor saxitoxin. Both autoradiography analysis of sciatic nerves excised from injured rats as well as whole animal PET-MR imaging demonstrate that a systemically administered [(18)F]-labeled saxitoxin derivative concentrates at the site of nerve injury, consistent with upregulated sodium channel expression following axotomy. This type of PET agent has potential use for serial monitoring of channel expression levels at injured nerves throughout wound healing and/or following drug treatment. Such information may be correlated with pain behavioral analyses to help shed light on the complex molecular processes that underlie pain sensation.

Synthesis of saxitoxin derivatives bearing guanidine and urea groups at C13 and evaluation of their inhibitory activity on voltage-gated sodium channels.

Here, we describe the synthesis of the first C13-N-substituted STX derivatives 4, 5, and 6 bearing a guanidine, a urea group, and an acetamide, respectively, via the fully protected saxitoxinol derivative 8. These compounds are of interest because a previous docking study of saxitoxin (STX) with voltage-gated sodium channels (NaVCh) suggested that the C13 carbamoyl group of STX interacts with residue E403 in the pore region of NaVCh. In a cell-based assay with Neuro-2a cells, the NaVCh-inhibitory activities of 4 and 5 were more than 20- to 50-fold weaker than that of decarbamoyl-STX (3), which is 10-fold less potent than STX. On the other hand, 6 was 1000 times less potent than 3. The electrostatic analysis of C13 in STX and its analogs 4-6 using EON calculations suggested that the NaVCh-inhibitory activity of these derivatives is influenced by both the hydrophilicity and the charge balance of the substituent at C13.

A novel method for preparing complete antigens of gonyautoxin 2,3 and their feature of immunogenicity.

In this paper, a novel method was proposed to prepare artificial antigens of gonyaulax parlaytic shellfish toxin 2 and 3 (GTX2,3). An intermediate GTX2,3-aldehyde was first synthesized by activating the NH2 group of the 2nd and 8th amino acid residues with three different aldehydes and two artificial complete antigens GTX2,3-aldehyde-bovine serum albumin (BSA) and GTX2,3-aldehyde- keyhole limpet hemocyanin (KLH) were then prepared by cross-linking the intermediate with BSA or KLH. The successful preparation of the two complete antigens was confirmed by UV spectral scanning, HPLC, production of antibodies with titer of 1.28 × 104 from mice immunized with the two complete antigens, indirect ELISA and Western-blot. In conclusion, the synthesized complete antigens have strong immunogenicity, which provides a solid foundation for preparing GTX2,3 monoclonal antibody and rapid detection kit.

A stereocontrolled synthesis of (+)-saxitoxin.

A concise stereoselective total synthesis of (+)-saxitoxin is described. A silver(I)-initiated hydroamination cascade constructs the bicyclic guanidinium ion core from a alkynyl bisguanidine. This sequence creates two C-N bonds, one C-O bond, and three rings and forms a single stereoisomer in a single synthetic transformation. This process enabled us to complete the synthesis of (+)-saxitoxin in 14 steps from N-Boc-l-serine methyl ester.

Total synthesis of (+)-decarbamoylsaxitoxin and (+)-gonyautoxin 3.

Facile construction of the complex saxitoxin (STX) skeleton is carried out by using a novel, conformationally controlled, guanidine cyclization process that relies on the use of neighboring group participation. The utility of this methodology is verified by its employment in syntheses of both natural and unnatural STX derivatives.

De novo synthesis of modified saxitoxins for sodium ion channel study.

Access to novel forms of (+)-saxitoxin (STX), a potent and selective inhibitor of voltage-gated Na(+) ion channels, has been made possible through de novo synthesis. Saxitoxin is believed to lodge in the outer mouth of the channel pore, thereby stoppering ion flux. Herein, we demonstrate that modification of the C13-carbamoyl unit can be accommodated in the binding site of the protein without significantly reducing ligand-receptor affinity. These discoveries have emboldened efforts to prepare photoaffinity-labeled and other unique forms of STX as pharmacological tools for interrogating both the molecular architecture and function of Na(+) channels. A synthetic plan that makes such compounds generally available is described.

Total synthesis of (-)- and (+)-decarbamoyloxysaxitoxin and (+)-saxitoxin.

Playing the sax: The enantioselective total syntheses of (-)- and (+)-decarbamoyloxysaxitoxin (doSTX) and (+)-saxitoxin (STX) are reported. A new methodology was developed for the synthesis of STXs, featuring discriminative reduction of the nitro group and N-O bond in nitroisoxazolidine. Enantioselective total syntheses of (-)- and (+)-decarbamoyloxysaxitoxin (doSTX) and (+)-saxitoxin (STX) were achieved. The characteristic spiro-fused cyclic guanidine structure of STX was constructed by oxidation at the C4 position with IBX via an alpha-iminium carbonyl intermediate and acid-promoted cyclization of guanidine at the C5 position. A second-generation methodology was developed for the synthesis of STX, featuring discriminative reduction of the nitro group and N-O bond in nitroisoxazolidine. This approach provides efficient access to the key diamine intermediate for STXs.

A stereoselective synthesis of (+)-gonyautoxin 3.

An asymmetric synthesis of the paralytic shellfish poison (PSP), (+)-gonyautoxin 3, is described. A unique, Rh-catalyzed amination reaction provides rapid access to the heteratom-rich, tricyclic core of the toxin, which is common to more than 30 related natural products. The completed route should facilitate the preparation of other naturally occurring PSPs and designed analogues thereof.

(+)-saxitoxin: a first and second generation stereoselective synthesis.

A stereoselective synthesis of the bis-guanidinium toxin (+)-saxitoxin (STX), the agent infamously associated with red tides and paralytic shellfish poisoning, is described. Our approach to this unique natural product advances through an unusual nine-membered ring guanidine intermediate 39 en route to the tricyclic skeleton that defines STX. The effectiveness of this strategy is notable, as only four steps are needed to transform 39 into the target molecule, including a four-electron alkene oxidation catalyzed by OsCl3. Construction of the critical monocyclic guanidine has been achieved through two channels, the first of which makes use of Rh-catalyzed C-H amination and highlights a novel class of heterocyclic N,O-acetals as iminium ion equivalents for crafting functionalized amines. A second route to 39 relies on a stereoselective acetylide dianion addition to a serine-based nitrone, thereby facilitating the preparation of STX in just 14 linear steps from commercial material.

A synthesis of (+)-saxitoxin.

An asymmetric synthesis of the bis-guanidinium poison, (+)-saxitoxin (STX), is described. Commencing from an N,O-acetal starting material made readily available through sulfamate ester C-H amination, the completed route to STX showcases the utility of oxathiazinane dioxide heterocycles for the assembly of polyfunctionalized amine derivatives. In the final preparative stages, an unusual nine-membered ring guanidine intermediate is oxidized selectively and made to undergo dehydrative cyclization to afford the tricyclic core of the natural product. Access to STX and related structures will provide unique pharmacological tools for the study of voltage-regulated Na+ ion channel proteins.