Transcript PowerPoint-presentatie

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
O
NH2
H
H
N
+
H2N
O
6
8
NH
2
N
H
HO
N
NH2+
12
HO
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)
poison
LD50 (mice, mg/kg)
botulinum toxin D
0.0004
tetanus toxin
0.001
diphtheria toxin
0.1
shigella dysenteriae toxin (verotoxin)
1.3
ricin
2.7
saxitoxin
8
tetrodotoxin
8
crotoxin
82
cholera toxin
250
aflatoxins
2 - 1500
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)
O
X
O
N
1
O
HN
OMe
O
O
1. HO(CH2)3OH / TsOH
in toluene, 
2. hydrazine hydrate
in MeOH, , 74%
O
2: X=O
3: X=S
P2S5 / benzene
1. CH3C(O)CHBrCO2CH3 /
NaHCO3 in DCM, 
2. KOH in MeOH, 50%
CH2OBn
MeOOC
X
HN
O
Y
S
HN
N
BnOCH2C(O)H / Si(NCS)4
in benzene, 75%
Y
5: X=COOMe; Y=O
6: X=NHCONH2; Y=O
7: X=NHCONH2; Y=S
O
4
1. NH2NH2.H2O in MeOH; 2. NOCl in DCM, -50 oC; 3. NH3 in benzene, 75%
HS(CH2)3SH / BF 3.OEt2 in MeCN, 63%
Kishi et al., JACS 1977, 99, 2818-2819
13
mechanism of the condensation
SiX3
OBn
N
C
O
HO
OBn
OBn
COOMe
COOMe
COOMe
HN
N
O
H
O
O
HN
S
S
S
N
N
O
O
O
14
Kishi synthesis (II)
CH2OBn
H
CH2OBn
NHCONH2
HN
HN
AcOH/TFA,
50 oC, 50%
S
S
N
7
8: X=S; Y=O
H
N
1. Et3O+ BF 4- /
NaHCO3 in DCM
2. EtCO2NH4, , 33%
Y
X
N
N
H
S
9: X=Y=NH
S
S
BCl3 in DCM, 0 oC,
75%
HN
CH2OC(O)NH2
H
H
N
CH2OH
H
HN
NH
HN
N
N
H
ClSO2NCO in HCOOH,
5 oC, 50%
NH
HN
OH
OH
d,l-saxitoxin
Kishi et al., JACS 1977, 99, 2818-2819
H
N
N
N
H
Z
Z
10: Z=S(CH2)3S
11: Z=OH
1. NBS in MeCN
2. MeOH, , 30%
15
Jacobi synthesis (I)
CO2Me
Ph
H
N
H
N
NH
N
Ph
O
O
N
H
NH
S
S
N
N
H
MeOC(OH)HCO2Me /
BF3.Et2O, 65-75%
O
O
12
S
S
1,3-dipolar cycloaddition
OH
H
H
N
H
N
O
N
N
H
X
BH3.Me2S, 98%
N
R
O
N
N
H
S
Ph
N
S
S
15: X=Bn
16: X=C(S)OPh
H
O
S
1. Pd / AcOH / HCOOH
2. ClC(S)OPh, 80%
Jacobi et al., JACS 1984, 106, 5594-5598
13: R=CO2Me
14: R=CH2OH ()
1. NaOMe in MeOH
2. NaBH4 in MeOH, 72%
16
Jacobi synthesis (II)
OH
H
H
N
N
N
H
S
Na in NH3
-78 oC
16
S
N
H
OPh
H
N
NH
O
N
S
H
H
N
S
O
OR
OH
NH
N
H
OPh
S
HO
CH2OAc
H
N
NH
N
NH
ClSO2NCO in HCOOH,
5 oC
HO
d,l-saxitoxin
Jacobi et al., JACS 1984, 106, 5594-5598
S
S
S
CH2OC(O)NH2
N
H
N
S
75%
H
HN
NH
O
17: R=H
18: R=OAc
H
N
H
H
Ac2O / pyr
1. Et3O+ BF 4- /
KHCO3 in DCM
2. EtCO2NH4, , 48%
NH
HN
Z
N
H
N
NH
Z
19: Z=S(CH2)3S
20: Z=OH
1. NBS in MeCN
2. MeOH, 
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., PNAS 1999, 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
R
O
N
O
O
O
O
O
O
O
O
21
N
N
CH2
CH2
22
Gawley et al., Tetrahedron Lett. 1999, 40, 5461-5465
Gawley et al., JACS 2002, 124, 13448-13453
24
detection of saxitoxin: chemosensors
R
N
Emission spectrum of 22 (R=H):
O
O
O
O
N
CH2
22
Gawley et al., JACS 2002, 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., JACS 2002, 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.
O
NH2
H
H
N
How is the benzylic nitrogen involved?
+
H2N
8
NH
2
N
H
HO
HO
Gawley et al., JACS 2002, 124, 13448-13453
O
6
N
12
NH2+
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:
O
25
Ac2O/pyr (66%)
H2 N
OH
N
H
24
OH
O
H2SO4 (25%)
Cl
OEt
O
O
O
N
O
Cl
O
O
O
N
H
O
O
O
23
1-aza-18-crown-6
Et3N in MeCN, (30%)
Kele et al., Tetrahedron Lett. 2002, 43, 4413-4416
N
H
O
26
O
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