colloquium by ruben e. a. musson department of bio-organic synthesis
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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%).
18
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