colloquiumby

Ruben E. A. Musson

Department of Bio-organic SynthesisFaculty of Mathematics and Natural Sciences

Leiden University

overviewoverview

• introduction to saxitoxin: biochemistry and clinical toxicology

• synthesis of saxitoxin• novel methods of saxitoxin detection• concluding remarks

2

selected cases of saxitoxin poisoningselected 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, 267-271

saxitoxin (STX): overviewsaxitoxin (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, 225-237

N

NH

HN

NH

+H2N

O NH2

O

NH2+

HO

HO

H

82

12

6

other toxins found in shellfishother 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: toxicitysaxitoxin: toxicity

• most toxic non-protein poison known• potential for use in chemical warfare (1ooo more toxic than

Sarin)

6

poison LD50 (mice, g/kg)botulinum toxin D 0.0004tetanus toxin 0.001diphtheria toxin 0.1shigella dysenteriae toxin (verotoxin) 1.3ricin 2.7saxitoxin 8tetrodotoxin 8crotoxin 82cholera toxin 250aflatoxins 2 - 1500

saxitoxin: toxicitysaxitoxin: 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 actionsaxitoxin: 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, 438-450

structural model of STX-binding to a Na-channelstructural model of STX-binding to a Na-channel

9

Penzotti et al., Biophys. J. 1998, 75, 2647-2657

treatment of saxitoxin poisoningtreatment 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

synthesissynthesis

• 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)Kishi synthesis (I)

12

Kishi et al., JACS 1977, 99, 2818-2819

N

O

O

O

O

OMe

1. HO(CH2)3OH / TsOH in toluene, 2. hydrazine hydratein MeOH, , 74%

O

OHN

X

1P2S5 / benzene2: X=O

3: X=S

O

OHN

MeOOC

HN

N

Y

Y

S

CH2OBn

X

1. NH2NH2.H2O in MeOH; 2. NOCl in DCM, -50 oC; 3. NH3 in benzene, 75%5: X=COOMe; Y=O6: X=NHCONH2; Y=O7: X=NHCONH2; Y=S

4

1. CH3C(O)CHBrCO2CH3 / NaHCO3 in DCM, 2. KOH in MeOH, 50%

BnOCH2C(O)H / Si(NCS)4in benzene, 75%

HS(CH2)3SH / BF3.OEt2 in MeCN, 63%

mechanism of the condensationmechanism of the condensation

13

O

OHN

COOMe

S

C

NO

OBn

N

NS

HO OBn

COOMe

O

O

HN

NS

COOMe

O

OH

OBnSiX3

Kishi synthesis (II)Kishi synthesis (II)

14

Kishi et al., JACS 1977, 99, 2818-2819

AcOH/TFA, 50 oC, 50%

1. Et3O+BF4- / NaHCO3 in DCM2. EtCO2NH4, , 33%

8: X=S; Y=O

9: X=Y=NH

HN

N

S

S

S

CH2OBn

NHCONH2

10: Z=S(CH2)3S11: Z=OH

ClSO2NCO in HCOOH, 5 oC, 50%

1. NBS in MeCN2. MeOH, , 30%

HN

NX

CH2OBn

NH

HN

S

S

Y

H

7

HN

NHN

CH2OH

NH

HN

Z

Z

NH

H

HN

NHN

CH2OC(O)NH2

NH

HN

OH

OH

NH

H

BCl3 in DCM, 0 oC,75%

d,l-saxitoxin

Jacobi synthesis (I)Jacobi synthesis (I)

15

Jacobi et al., JACS 1984, 106, 5594-5598

S S

NH

HN

NH

O

O

NH

Ph

HN

NH

ON

N

CO2Me

Ph

OS

S

HN

NH

O

N

N

H R

Ph

SS

O

13: R=CO2Me14: R=CH2OH ()

1. NaOMe in MeOH2. NaBH4 in MeOH, 72%

1,3-dipolar cycloaddition

MeOC(OH)HCO2Me /BF3.Et2O, 65-75%

HN

NH

O

N

N

H

SS

15: X=Bn16: X=C(S)OPh

1. Pd / AcOH / HCOOH2. ClC(S)OPh, 80%

OH

X BH3.Me2S, 98%

12

Jacobi synthesis (II)Jacobi synthesis (II)

16

Jacobi et al., JACS 1984, 106, 5594-5598

17: R=H18: R=OAc Ac2O / pyr

HN

NH

O

N

N

H

SS

OH

S

OPh

HN

NH

O

NH

NH

H

SS

OH

S

OPh

HN

NH

O

H

Na in NH3-78 oC

N

NH

SS

OR

S

75%

19: Z=S(CH2)3S20: Z=OH

ClSO2NCO in HCOOH, 5 oC

1. NBS in MeCN2. MeOH,

NH

N NH

CH2OAc

NH

HN

Z

Z

HN

H

NH

N NH

CH2OC(O)NH2

NH

HN

HO

HO

HN

H

d,l-saxitoxin

1. Et3O+BF4- / KHCO3 in DCM2. EtCO2NH4, , 48%

16

Kishi vs. Jacobi (I)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 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%).

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detection of saxitoxindetection 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 saxitoxindetection 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 bioassay tissue biosensors

molecular pharmacologyneurophysiology

whole-cell bioassay HPLC/MSHPLC with postcolumn oxidation of the C4-C12

bond

21

detection of saxitoxin: chemosensorsdetection 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, 1515-1566

The fluorophore is switched on and off by intramolecular photoinduced electron transfer (PET).

22

examples of known chemosensorsexamples of known chemosensors

de Silva et al., PNAS 1999, 96, 8336-8337

23

detection of saxitoxin: chemosensorsdetection 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, 5461-5465Gawley et al., JACS 2002, 124, 13448-13453

N

O

O

O

O

O

CH2

N

O

O

N

O

O

CH2

R

21 22

24

detection of saxitoxin: chemosensorsdetection of saxitoxin: chemosensors

Emission spectrum of 22 (R=H):

Gawley et al., JACS 2002, 124, 13448-13453

N

O

O

N

O

O

CH2

R

22

25

detection of saxitoxin: chemosensorsdetection 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, 13448-13453

26

detection of saxitoxin: chemosensorsdetection 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, 13448-13453

N

NH

HN

NH

+H2N

O NH2

O

NH2+

HO

HO

H

82

12

6

27

detection of saxitoxin: coumaryl crown based detection of saxitoxin: coumaryl crown based chemosensorschemosensors

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, 4413-4416

Coumarins generally show good spectral features:• large Stokes shifts (70-100 nm)• high quantum yields

28

detection of saxitoxin: coumaryl crown based detection of saxitoxin: coumaryl crown based chemosensorschemosensors

Synthesis:

Kele et al., Tetrahedron Lett. 2002, 43, 4413-4416

Ac2O/pyr (66%)

26

H2SO4 (25%)

1-aza-18-crown-6Et3N in MeCN, (30%)

H2N OH OHNH

O

O O

Cl

NH

O

ClOEt

O O

23O ONH

O

OO

O

ONO

24

25

29

detection of saxitoxin: coumaryl crown based detection of saxitoxin: coumaryl crown based chemosensorschemosensors

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 remarksconcluding 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 EdwardsUniversity of Sussex