A case study detection of saxitoxin

Transcription

1 A case study detection of saxitoxin

2 Saxitoxin ne 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

3 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

4 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: fficial test: mouse assay (LD 50 = 8 μg/kg) A more sensitive and friendly assay is needed

5 Saxitoxin detection by PET sensors aza-18-crown 6 ethers coumaryl and anthracyl signaling units

6 Saxitoxin detection by PET sensors

7 ph dependency of PET sensors Fluorescence, A. U. compound 8 compound 11 pka of comaryl sensors are: 5.8 and 6.0 pka of anthracy sensor: ph

8 Presence of cations 1.2 a K F/F Ca Log[M n+ ]

9 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,

10 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 H hydrobhobic tale hydrophylic head

11 Langmuir film An amphiphil monolayer at the air/water interface The surface presure area curve shows where it forms a stable compact film

12 Surface pressure area curve of stearic acid

13 Surface pressure area curve of sensor amphiphile π, m/m Area, Å 2 /molecule Limiting molecular area: 38 Å 2 /molecule

14 Fluorescence is quenched when fluorphores are compressed 5000 Fluorescence intensity, cps m/m 30 m/m Wavelength, nm

15 Mixed monolayers restored fluorescence

16 Peptidolipid as two-dimensional solvent C 17 H 35 C-Gly-Gly-Ala-Gly-H 2 Fluorescence intensity increased until 15 m/m

17 Transfer of the monolayer onto a solid support Langmuir Blodgett films

18 Langmuir-Blodgett film detects STX Fluorescence Intensity, cps LB (DAC) + STX LB (DAC) LB (PL/DAC 100:1) + STX LB (PL/DAC 100:1) Wavelength, nm Kele et al. Langmuir 2002, 18,

19 Covalent attachment of sensor film onto support Self assembling monolayer (SAM) o leaching from the support Reusable sensor

20 Fluorescence of modified quartz slide quartz modified quartz Fluorescence intensity, cps Wavelength, nm

21 Epifluorescence microscopy of quartz slides Modified Unmodified

22 Imaging ellipsometry of modified quartz slide C ~72% Surface coverage

23 Detection of STX with coumaryl-crown modified quartz slide Fluorescence intensity, cps Buffer 1.0 x 10-5 M STX 2.5 x x x x Wavelength, nm Kele et al. Chem. Commun. 2006, 14,

24 Afterparty Docking experiments revealed that the guanidino function is not at the right orientation to H-bond with the quencher nly the 7th lowest energy conformation showed close proximity of this two groups Why does it work?

25 The effect of conformational mobility on PET Efficiency of PET in case of different membered rings Cl H n n Me n=0-5 Me

26 PET efficiency = protonation induced FE Me FE, I/I M sensor 10-3 M HBF 4 MeC Ring size

27 Electronic effects Me 200 Effect of substituents Fluoreszcencia erősítés (I/I 0 ) Me Me - o electronic effect (dimethoxy derivative) - Large conformational effect (rigid acetonide) 0

28 Further elaboration of conformational effects Amplifier n Receptor Me Signaling unit

29 Effect of protonation 10-6 M sensor 10-3 M HBF 4 MeC Me Fluoreszcencia erősítés (I/I 0 ) Me Me 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

30 Stoichiometry of the Crownneopentyl-ammonium complex 1 H MR H H Me Δδ trans * x crown etherr t BuCH 2 H 3 Cl 4 only 1:1 complex was formed x crown ether Job-plot

31 Stability of different complexes H H Me CH 2 Cl 2 : CDCl 3 : CD 3 D 90 : 9 : A transz hidrogének eltolódás-változásai a két ligandumra t BuCH 2 H 3 Cl 4 neopentil BuH 3 Cl 4 butil Δδ max H trans logk K butyl 0,074 ppm 3,30 ± 0, neopentyl 0,114 ppm 3,00 ± 0,

32 0.03 Δδ trans * x crown etherr Me H H + BuH 3 Cl Xcrown ether Job-plot

33 MR studies Binding constants butyl 6.00 (1) 3.30 (3) neopentyl 5.23 (8) 3.00 (8) Changes in chemical shifts (Δδ) H H transh H H a b CH 2 CH 2a CH 2b CH trans nbu np

34 Systems elaborated - Sensors Me Me Me Me Guests BuH 3 Cl 4 CyH 3 Cl 4 i PrH 3 Cl 4 t BuH 3 Cl 4 t BuCH 2 H 3 Cl 4 size H-donor ability

35 acidity size ButilH 3 + Cl 4-36 CiklohexilH Cl 4 IzopropilH Cl 4 Fluoreszcencia erõsítés (I / I 0 ) tbutilh Cl 4 eopentilh Cl 4 0 szenzorok Kele et al. Angew. Chem., Intl. Ed. 2006, 45(16),

36 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 Me