a case study – detection of saxitoxinkelep.web.elte.hu/ea/pmr8ea.pdf · saxitoxin • harmful...
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Saxitoxin
• One of the most toxic non‐protein substances
• 0.2 mg is lethal to an average weight human
• Produced by certain algae species (dinoflagellates, cyanobacteria)
• The toxin accumulates in shellfish (mussels, clams and scallops) Saxitoxin, STX
Saxitoxin• Harmful Algal Blooms• The toxin accumulates in shellfish• Paralytic shellfish poisoning (PSP)• Blocks neuronal sodium channels and mostly causes respiratory failure (artifical respiration is essential)
• Military interest (1000 times more toxic than nerve gas sarin)
• Suicide capsules (U2 pilot Francis G. Powers )• In 1970 ”all” stx was destroyed
Saxitoxin
• Harmful Algal Blooms – e.g. red tides
...and the waters that were in the river were turned to blood. And the fish that were in the river died; and the river stank and the Egyptians could not drink of the water of the river... Exodus 7: 20‐21
• Official test: mouse ”assay” (LD50 = 8 μg/kg)
• A more sensitive and friendly assay is needed
pH dependency of PET sensors
2 4 6 8 10
compound 8
Fluo
resc
ence
, A. U
.
pH
compound 11 • pKa of comaryl sensors are: 5.8 and 6.0
• pKa of anthracy sensor: 8.0
Presence of cations
-7 -6 -5 -4 -3 -2 -1
0.6
0.8
1.0
1.2
-7 -6 -5 -4 -3 -2 -1
0.6
0.8
1.0
1.2
-7 -6 -5 -4 -3 -2 -1
0.6
0.8
1.0
1.2
Na+
F/F 0
K+
Log[Mn+]
Ca2+
Presence of STX
• 1 is the most water soluble and gives the largest signal
• 2 binds STX the strongest
• 1:1 complex is assumed
Kele et al. Tetahedron Lett. 2002, 43, 4413-4416.
Sensor fabrication
• Sensory layer on a support (physically adsorbed, chemically bound)
• Surface properties should be characterized first (photophysical and sensing properties)
• Surface chemistry (Langmuir and Langmuir-Blodgett films)
• Air / water interface using sensor amphiphile
OO
O
ONO
O ONH
O
hydrobhobic tale hydrophylic head
Langmuir film• An amphiphil monolayer at the air/water interface
• The surface presure – area curve shows where it forms a stable compact film
Surface pressure – area curve of sensor amphiphile
0 20 40 60 80 100 120 140 160 180 2000
10
20
30
40
50
60
π, m
N/m
Area, Å2/molecule
Limiting molecular area: 38 Å2/molecule
Fluorescence is quenched when fluorphores are compressed
350 400 450 500 550
0
1000
2000
3000
4000
5000
30 mN/m
0 mN/m
Fluo
resc
ence
inte
nsity
, cps
Wavelength, nm
• Fluorescence intensity increased until 15 mN/m
• Peptidolipid as two-dimensional solvent
C17H35CO-Gly-Gly-Ala-Gly-NH2
Langmuir-Blodgett film detects STX
400 450 5000
5000
10000
15000
20000
25000
LB(ODAC)+ STX LB(ODAC)
LB(PL/ODAC 100:1)+ STX LB(PL/ODAC 100:1)
Fluo
resc
ence
Inte
nsity
, cps
Wavelength, nmKele et al. Langmuir 2002, 18, 8523-8526.
Covalent attachment of sensor film onto support – Self assembling monolayer
(SAM)
• No leaching from thesupport
• Reusable sensor
400 450 500 5500
5000
10000 quartz modified quartz
Fluo
resc
ence
inte
nsity
, cps
Wavelength, nm
Fluorescence of modified quartz slide
Detection of STX with coumaryl-crown modified quartz slide
350 400 450 500 5500
2000
4000
6000
8000
10000
12000
14000
Fluo
resc
ence
inte
nsity
, cps
Wavelength, nm
Buffer
1.0 x 10-5 M STX
2.5 x 10-5
5.0 x 10-5
7.5 x 10-5
1.0 x 10-4
Kele et al. Chem. Commun. 2006, 14, 1494-1496.
Afterparty
• Docking experimentsrevealed that the guanidinofunction is not at the right orientation to H-bond withthe quencher N
• Only the 7th lowest energyconformation showed closeproximity of this twogroups
• Why does it work?
O OMeO
N
n=0-5O OMeO
ClHN n
n
N N N N N N
The effect of conformational mobility on PET
• Efficiency of PET in case of different membered rings
PET efficiency = protonation induced FE
0
50
100
150
200
876543
FE,
I/I0
Ring size
O O
N
MeO
10-6 M sensor10-3M HBF4MeCN
0
50
100
150
200
O OMeO
N
OMeMeO
NOO
N
Effect of substituents
- No electronic effect (dimethoxy derivative)
- Large conformational effect (rigid acetonide)
Electronic effects Fl
uore
szce
ncia
erő
síté
s (I/
I 0)
Effect of protonation
N
OO
O
OO
N
OMeMeO
O O
O
O
O
O
N
OO
O
OON
O OMeO
• the 18-crown moiety distorts the 5 membered ring – less eficient PET
• the 15-crown has larger signaling potential (small changes in conformation results in lage increase
10-6 M sensor10-3M HBF4MeCN
Fluo
resz
cenc
ia e
rősí
tés
(I/I 0)
Stoichiometry of the Crown-neopentyl-ammonium complex
Job-plot
+ tBuCH2NH3ClO4
only 1:1 complex was formed
O OMeO
NO
OO
OO
H
H
1H NMR
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0 0.2 0.4 0.6 0.8 1x
Δδtra
ns*
x cr
own
ethe
rr
crown ether
Stability of different complexes
Δδmax Htrans logK K
butyl 4 000
neopentyl 1 000
CH2Cl2 : CDCl3 : CD3OD 90 : 9 : 1
0,074 ppm 3,30 ± 0,02
0,114 ppm 3,00 ± 0,02
A transz hidrogének eltolódás-változásai a két ligandumra
4.064.074.084.094.104.114.124.134.144.154.16
0.0 2.0 4.0 6.0 8.0
neopentil
butil
tBuCH2NH3ClO4
BuNH3ClO4
O OMeO
NO
OO
OO
H
H
+ BuNH3ClO4
O OMeO
OO
OO
O
NHH
0
0.005
0.01
0.015
0.02
0.025
0.03
0.00 0.50 1.00Xcrown ether
Δδtra
ns*
x cr
own
ethe
rr
Job-plot
N
O O
O
O O
HH
NO
O
O
OO
transH
HaHb
CH2 CH2a CH2b CHtrans
nBu 0.052 0.069 0.096 0.077
nP 0.061 0.096 0.13 0.116
butyl 6.00 (1) 3.30 (3)
neopentyl 5.23 (8) 3.00 (8)
N
O O
O
O O
NO
O
O
OO
Binding constants
Changes in chemical shifts (Δδ)
NMR studies
O OMeO
NO
OO
OO
O OMeO
N
O
O O
O
O O O OMeO
OO
O
O
O
N
O OMeO
ON
O
OO
Systems elaborated
BuNH3ClO4 CyNH3ClO4iPrNH3ClO4
tBuNH3ClO4tBuCH2NH3ClO4
size
H-donor ability
- Sensors
Guests
sizeacidity
0
5
12
24
36
szenzorok
Fluo
resz
cenc
ia e
rõsí
tés
(I / I
0) ButilNH3
+ClO4-
CiklohexilNH3+ClO4
-
IzopropilNH3+ClO4
-
tButilNH3+ClO4
-
NeopentilNH3+ClO4
-
N
O
OO
O
O
N
O
O
O O
O
O
NO
OO
O
O
OO
O
NO
Kele et al. Angew. Chem., Intl. Ed. 2006, 45(16), 2565-2567.