A case study – detection of saxitoxin

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

Saxitoxin detection by PET sensors

• aza-18-crown 6 ethers

• coumaryl and anthracylsignaling units

Saxitoxin detection by PET sensors

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 curveof stearic acid

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

Mixed monolayers – restored fluorescence

• Fluorescence intensity increased until 15 mN/m

• Peptidolipid as two-dimensional solvent

C17H35CO-Gly-Gly-Ala-Gly-NH2

Transfer of the monolayer onto a solid support – Langmuir‐Blodgett films

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

Epifluorescence microscopy of quartz slides

Modified Unmodified

Imaging ellipsometry of modified quartz slide

C

~72% Surface coverage

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)

Further elaboration of conformational effects

Receptor

Amplifier

Signaling unit

O OMeO

O

O

O

O

On

N

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.

Summary

-Conformational freedom / rigidity can have a profound effect on PET

- There are cases where PET is dependent on :

- purely direct coordination

- purely on conformation

- both

O OMeO

O

O

O

O

O

N