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 mg/kg)

• A more sensitive and friendly assay is needed

Saxitoxin detection by PET sensors

• aza-18-crown 6 ethers

• coumaryl and anthracyl

signaling units

Saxitoxin detection by PET sensors

pH dependency of PET sensors

2 4 6 8 10

compound 8

Flu

ore

sce

nce

, 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. Tetrahedron 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

O

O

O

ON

O

O ONH

O

hydrobhobic talehydrophylic 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 80 100 120 140 160 180 200

0

10

20

30

40

50

60, 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

F

luo

resce

nce

in

ten

sity, cp

s

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

400 450 500

0

5000

10000

15000

20000

LB(PL/ODAC 100:1)

+ STX

LB(PL/ODAC 100:1)

Flu

ore

scen

ce

In

ten

sity, cp

s

Wavelength, nm

Kele et al. Langmuir 2002, 18, 8523-8526.

Covalent attachment of sensor film onto

support – Self assembling monolayer

(SAM)

• No leaching from the

support

• Reusable sensor

400 450 500 550

0

5000

10000 quartz

modified quartzF

luo

resce

nce

in

ten

sity, cp

s

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

350 400 450 500 550

0

2000

4000

6000

8000

10000

12000

14000

F

luo

resce

nce

in

ten

sity, cp

s

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 experiments

revealed that the guanidino

function is not at the right

orientation to H-bond with

the quencher N

• Only the 7th lowest energy

conformation showed close

proximity of this two

groups

• 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/

I 0

Ring size

O O

N

MeO

10-6 M sensor

10-3M HBF4

MeCN

0

50

100

150

200

5c5b5a

O OMeO

N

OMeMeO

NOO

N

Effect of substituents

- No electronic effect

(dimethoxy derivative)

- Large conformational

effect (rigid acetonide)

Electronic effects F

luo

reszce

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

O

O

N

OMeMeO

O O

O

O

O

O

N

O

O

O

OO

N

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 sensor

10-3M HBF4

MeCN

Flu

ore

szce

ncia

erő

síté

s (

I/I 0

)

Stoichiometry of the Crown-

neopentyl-ammonium complex

Job-plot

+ tBuCH2NH3ClO4

only 1:1 complex was formed

O OMeO

NO

O

O

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 1

x

Dd

tran

s*

x c

row

n e

ther

r

crown ether

Stability of different complexes

Ddmax 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

4.06

4.07

4.08

4.09

4.10

4.11

4.12

4.13

4.14

4.15

4.16

0.0 2.0 4.0 6.0 8.0

neopentil

butil

tBuCH2NH3ClO4

BuNH3ClO4

O OMeO

NO

O

O

OO

H

H

+ BuNH3ClO4

O OMeO

O

O

O

O

O

NH

H

0

0.005

0.01

0.015

0.02

0.025

0.03

0.00 0.50 1.00Xcrown ether

Dd

tran

s*

x c

row

n e

ther

r

Job-plot

N

O O

O

O O

H

H

N

OO

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

N

OO

O

OO

Binding constants logK

Changes in chemical shifts (Dd)

NMR studies

O OMeO

NO

O

O

OO

O OMeO

N

O

O O

O

O OO OMeO

O

O

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

Flu

ore

szce

ncia

erõ

síté

s (

I /

I 0)

ButilNH3

+ClO

4

-

CiklohexilNH3

+ClO

4

-

IzopropilNH3

+ClO

4

-

tButilNH3

+ClO

4

-

NeopentilNH3

+ClO

4

-

N

O

O

O

O

O

N

O

O

O O

O

O

N

O

OO

O

O

O

O

O

N

O

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