1 / 30

colloquium by Ruben E. A. Musson Department of Bio-organic Synthesis

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.

kyle
Download Presentation

colloquium by Ruben E. A. Musson Department of Bio-organic Synthesis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

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

  3. 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. Rodrigue et al., Am. J. Trop. Med. Hyg.1990, 42, 267-271

  4. 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 EJ Schantz, Environm. Lett. 1975, 9, 225-237

  5. 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)

  6. saxitoxin: toxicity • most toxic non-protein poison known • potential for use in chemical warfare (1ooo more toxic than Sarin)

  7. 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

  8. 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 Kao et al., Arch. Int. Pharmacodyn. 1967, 165, 438-450

  9. structural model of STX-binding to a Na-channel Penzotti et al., Biophys. J. 1998, 75, 2647-2657

  10. 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.

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

  12. Kishi synthesis (I) Kishi et al., JACS1977, 99, 2818-2819

  13. mechanism of the condensation

  14. Kishi synthesis (II) Kishi et al., JACS1977, 99, 2818-2819

  15. Jacobi synthesis (I) Jacobi et al., JACS1984, 106, 5594-5598

  16. Jacobi synthesis (II) Jacobi et al., JACS1984, 106, 5594-5598

  17. 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

  18. 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%).

  19. 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

  20. detection of saxitoxin Mouse bioassay is the current benchmark technique. Detection limit is 40 mg of STX / 100 g of shellfish. For both economic and ethical reasons, an alternative is desired. New approaches to detection include insect bioassay tissue biosensors molecular pharmacology neurophysiology whole-cell bioassay HPLC/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. The fluorophore is switched on and off by intramolecular photoinduced electron transfer (PET). de Silva et al., Chem. Rev.1997, 97, 1515-1566

  22. examples of known chemosensors de Silva et al., PNAS1999, 96, 8336-8337

  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, 5461-5465 Gawley et al., JACS2002, 124, 13448-13453

  24. detection of saxitoxin: chemosensors Emission spectrum of 22 (R=H): Gawley et al., JACS2002, 124, 13448-13453

  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., JACS2002, 124, 13448-13453

  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., JACS2002, 124, 13448-13453

  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. • Coumarins generally show good spectral features: • large Stokes shifts (70-100 nm) • high quantum yields Kele et al., Tetrahedron Lett.2002, 43, 4413-4416

  28. detection of saxitoxin: coumaryl crown based chemosensors Synthesis: Kele et al., Tetrahedron Lett.2002, 43, 4413-4416

  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+/Ca2+ in aqueous solution has no influence on the fluorescence intensities Kele et al., Tetrahedron Lett.2002, 43, 4413-4416

  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

More Related