colloquium by Ruben E. A. Musson Department of Bio-organic Synthesis Faculty of Mathematics and Natural Sciences Leiden University

overview introduction to saxitoxin: biochemistry and clinical toxicology synthesis of saxitoxin novel methods of saxitoxin detection concluding remarks 2

selected cases of saxitoxin poisoning 1987: Mass die-offs among whales and other sea-life near Cape Cod. The cause of these deaths was initially blamed on pollution. 1987: Outbreak in Guatemala. Of 187 people affected following ingestion of clam soup, 26 died. 2000: Mysterious death of an East-Timorese after eating a tropical crab. 2002: 13 cases reported in Florida. Nowadays annually >300 fatalities around the world. 3 Rodrigue et al., Am. J. Trop. Med. Hyg. 1990, 42,

saxitoxin (STX): overview elaborated by dinoflagellates (planktonic algae) accumulates in shellfish (mussels, clams, oysters) feeding on these algae during “red tides” highly poisonous; causes PSP (paralytic shellfish poisoning) essential structural features: two guanidino moieties and a hydrated ketone 4 EJ Schantz, Environm. Lett. 1975, 9,

other toxins found in shellfish brevetoxins (causing NSP: neurotoxic shellfish poisoning) ciguatoxins (causing CFP: ciguatera fish poisoning) domoic acid (causing ASP: amnesic shellfish poisoning) okadaic acid and derivatives (causing DSP: diarrheic shellfish poisoning) 5

saxitoxin: toxicity most toxic non-protein poison known potential for use in chemical warfare (1ooo  more toxic than Sarin) 6 poison LD 50 (mice,  g/kg) botulinum toxin D tetanus toxin0.001 diphtheria toxin0.1 shigella dysenteriae toxin (verotoxin)1.3 ricin2.7 saxitoxin8 tetrodotoxin8 crotoxin82 cholera toxin250 aflatoxins

saxitoxin: toxicity tetrodotoxin (TTX) has the same mechanism of action as saxitoxin but its structure boasts only one guanidino moiety TTX is found in pufferfish (fugu) TTX is therapeutically used in pain control 7

saxitoxin: mechanism of action STX is rapidly absorbed through the GI-tract and excreted. Site of action: voltage-gated Na-channels of nerve cells the guanidino groups of STX bind to carboxylate sidechains near the mouth of a Na-channel that normally guide hydrated sodium ions into the channel upon coordinating, the remainder of the molecule plugs the channel, thereby blocking sodium influx normal membrane polarization/depolarization processes cannot take place: nerve pulses cannot pass anymore, resulting in paralysis 8 Kao et al., Arch. Int. Pharmacodyn. 1967, 165,

structural model of STX-binding to a Na-channel 9 Penzotti et al., Biophys. J. 1998, 75,

treatment of saxitoxin poisoning artificial respiration gut decontamination (gastric lavage (?), activated charcoal) monitoring of blood pressure and pH no antidote known When supportive treatment is applied in time, recovery from PSP usually is complete. 10

synthesis First total synthesis of saxitoxin was reported in 1977 by Yoshito Kishi. Second total synthesis was reported in 1984 by Peter Jacobi. 11

Kishi synthesis (I) 12 Kishi et al., JACS 1977, 99,

mechanism of the condensation 13

Kishi synthesis (II) 14 Kishi et al., JACS 1977, 99,

Jacobi synthesis (I) 15 Jacobi et al., JACS 1984, 106,

Jacobi synthesis (II) 16 Jacobi et al., JACS 1984, 106,

Kishi vs. Jacobi (I) Kishi: key step is the condensation of a vinylogous carbamate with silicon tetraisothiocyanate and benzyloxyacetaldehyde. Jacobi: key step is the intramolecular 1,3-dipolar cycloaddition of a highly reactive azomethine imine. final steps of both syntheses are identical: protective group manipulations 17

Kishi vs. Jacobi (II) Kishi: construction of third ring by efficient cyclization reaction. Jacobi: conversion of a 5-membered ring to a 6-membered ring. Number of reaction steps (from commercially available material): Kishi (>18), Jacobi (17). Both: tight stereochemical control. Overall yields: Kishi (0.25%), Jacobi (0.5%). 18

detection of saxitoxin Why are quick methods of detection important? STX has been used in covert government operations and chemical warfare. Governments need to monitor shellfish beds for the presence of STX to prevent PSP outbreaks. Rapid diagnosis of PSP victims improves survival rates. Main problems: small amounts numerous variations in composition most family-members are labile towards alkaline and oxidative conditions and therefore hard to purify 19

detection of saxitoxin Mouse bioassay is the current benchmark technique. Detection limit is 40  g of STX / 100 g of shellfish. For both economic and ethical reasons, an alternative is desired. 20 New approaches to detection include insect bioassaytissue biosensors molecular pharmacologyneurophysiology whole-cell bioassayHPLC/MS HPLC with postcolumn oxidation of the C4-C12 bond

21 detection of saxitoxin: chemosensors Fluorescence signaling has several advantages: high detection sensitivity on-off switchability high spatial and temporal resolution “Catch-and-tell” approach: combining a receptor and a fluorophore. de Silva et al., Chem. Rev. 1997, 97, The fluorophore is switched on and off by intramolecular photoinduced electron transfer (PET).

22 examples of known chemosensors de Silva et al., PNAS 1999, 96,

23 detection of saxitoxin: chemosensors STX is a good candidate for fluorescence sensing by quenching of PET: inorganic and organic cations can be detected by fluorescence sensing guanidinium ions are known to bind to crown ethers large number (11) of potential hydrogen-bond donors Gawley et al., Tetrahedron Lett. 1999, 40, Gawley et al., JACS 2002, 124,

24 detection of saxitoxin: chemosensors Emission spectrum of 22 (R=H): Gawley et al., JACS 2002, 124,

25 detection of saxitoxin: chemosensors Control substances used to assess selectivity of 21 towards saxitoxin: arginine and guanidine.HCl adenine o-bromophenol Solvent: ethanol/water mixture [ammonium phosphate pH 7.1] in water, this sensor is insensitive to metal ions elimination of the possibility of simple proton-transfer enhancing fluorescence None of these compounds showed any evidence of binding. Gawley et al., JACS 2002, 124,

26 detection of saxitoxin: chemosensors The exact way of binding is still somewhat enigmatic. Attempts to grow crystals of a crown-STX complex have failed. Monte Carlo docking searches: lowest-energy structures possessed hydrogen bonds between the C-8 guanidinium and the crown ether oxygens. How is the benzylic nitrogen involved? Gawley et al., JACS 2002, 124,

27 detection of saxitoxin: coumaryl crown based chemosensors Optical fiber based fluorescence sensor detecting STX requires a monolayer of fluorophore molecules covalently bound on the fiber surface. Anthracylmethyl-aza-crowns not suitable: fluorescence quenching due to aggegrate formation. Kele et al., Tetrahedron Lett. 2002, 43, Coumarins generally show good spectral features: large Stokes shifts ( nm) high quantum yields

28 detection of saxitoxin: coumaryl crown based chemosensors Synthesis: Kele et al., Tetrahedron Lett. 2002, 43,

29 detection of saxitoxin: coumaryl crown based chemosensors Results: absorption maximum at 323 nm; emission maximum at 419 nm excellent response to saxitoxin no pH dependency reflected in fluorescence intensity fluorescence quenching only when benzylic nitrogen is unprotonated addition of Na + /K + /Ca 2+ in aqueous solution has no influence on the fluorescence intensities Kele et al., Tetrahedron Lett. 2002, 43,

30 concluding remarks Despite its high toxicity, saxitoxin is the object of medical interest; therefore, its synthesis continues to be an intriguing goal. Use in chemical warfare: In 1970, President Nixon ordered the CIA to destroy its entire stock of saxitoxin, painstakingly collected over several years, as part of the US commitment in accordance with the United Nations agreement on biological weapons. However, in 1975 William Colby, the CIA Director, revealed to Congress that they still possessed over 10 grammes of the material in downtown Washington. Luckily, this supply of saxitoxin was eventually distributed to scientists and medical researchers under the auspices of the National Institutes of Health (NIH). Neil Edwards University of Sussex