1 68% 50% H 2 L C2 Chapter 5 Selected applications

1

1)SOCl2, CH2Cl2, DMF

2)

NO2NO2

NH

NH

N

O

O

O

NO2 NO2

N NN

O

O

O

O

O

O

O

O

O

O

O

NEtO

O

OEt

O

OH

NEtO OEt

O

OC7H15O3

O

1) NaOH EtOH H2ODIAD

OO

OOH

Ph3P, THF, reflux 12h

2) HCl NHO OEt

O

OC7H15O3

O69%

56%

Fe, EtOH, HCl

NaOH, EtOH

2)

1)

68%

50%

O

OO

NN N

N

N

HOO

N

OHO

O

O

OO

O

H2LC2

Chapter 5 Selected applications

2

2 + 3 (LC2)2-

H2O

?

neutral complex

Complex formation

0.0

0.2

0.4

0.6

0.8

1.0

144214441446

[M+MeCN]2+/2

[Tb2(LC2)3]

C129H144N18O36Tb2

ESI-MS

Simulated


3

0.22

0.44

0.66

2.83.23.64.04.44.85.25.66.06.46.87.27.6 2.83.23.64.04.44.85.25.66.06.46.87.27.6

/ ppm

Har (LC2)2- OCH3

O(CH2)2

Hbenz Hpy Hpy CH2

bridgeOCH3

NCH3

NCH3O(CH2)2

CH2

bridge

R = 2

NMR titration ofH2LC2 in D2O(pD = 7.8)

By

Lu(ClO4)3

R = [LuIII]t/[H2LC2]t


4

0.0

0.1

0.2

0.3

0.4

0.5

Ab

sorb

an

ce

225 250 275 300 325 350 375 400

/ nm

LC2

[Eu2(LC2)3]

UV-vis titration, in water, pH 7.4, = 0.1 M, 25 oC

Addition of Eu3+


5

UV-vis titration, in water, pH 7.4, H2 LC2

10-5 M: > 95 % [Eu2(LC2)3]10-4 M: > 98 %

La Eu Lu

LC2

log 13 18.8(2) 18.1(2) 18.7(3)

log 23 24.9(4) 25.5(4) 26.3(4)

log 21 11.7(3) 11.8(5) 12.4(2)


6

LC2

[Eu2(LC2)3]

[Eu2(LC2)]

[Eu(LC2)3]

R =[EuIII]t/[H2LC2]t

0

20

40

60

80

100%

sp

ecie

s

Ligand speciation in water, pH 7.4, ctot(lig) = 10-4 M

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

R

[H2LC3]t = 10-4 M 95 % helicate 4 % 1:3 < 1 % 2:1

4.510-4 M 97 % 2 % < 0.5 %

0.67


7

24 20 16 1244 4036 32 28

/ 103 cm-1~Photophysical properties

H2LC2

250 300 350 400 450 500 550 600 650 700 750 800 850 / nm

[Eu2(LC2)3]5D0

653

24

1

J = 0

7FJ

[Tb2(LC2)3]

5J = 6

32

4

1-0

5D47FJ

1 3


8579 580 581

580 600 620 640 660 680 700 / nm

5D0 7FJ

J =

3

4

21

0

10 K

High resolution emission spectrum, [Eu2(LC2)3] = 10-4 M

146 cm-1

580 600590

AE

Pseudo-D3 symmetryAt 295 K: 580.25 nm 17 234 cm-1

calc. 17 231 cm-1

B20

-600 cm-1

31 cm-1

Small distortion


9

Ln (H2O)/s (D2O)/s q Q LnL / %

Nd 0.21 ± 0.02 1.28 ± 0.01 0.1 0.031 ±0.006

Sm 30.4 ± 0.4 163 ± 3 n.a. 0.38 ± 0.06

Eu 2430 ± 90 4380 ± 40 -0.1 21 ± 2

Tb 650 ± 20 940 ± 20 (-0.2)77K 11 ± 2

Dy 0.16 ± 0.01 0.26 ± 0.1 n.a. n.a.

Yb 4.40 ± 0.07 49.3 ± 0.8 0.0 0.15 ± 0.03

Photophysical properties

[Eu2(LC2)3]Distorted D3 symmetry

No water in the inner coordination sphere


10

Stability of the [Eu2(LC2)3] helicate vsligand exchange and trans-metalation

Luminescence intensity:

edta 100 eqs, 2 days, no loss

dtpa 100 eqs, 1 day, 10 % loss

L-ascorbate 100 eqs, 4 days, 10 % loss

citrate 100 eqs, 4 days, no loss

zinc 10 eqs, 1 day, 10 % loss

100 eqs, 1 day, 15 % loss

pH no effect down to pH = 3

pH 2, 1 day, 15 % loss


11

Cell viability:

[Eu2(LC2)3]

WST-1 test after 24 h incubation

90 %


12

0 M0M 25M 25M

50M 50M 125M 125M

250M250M 500M500M

10m

0M0M 25M 25M

50M 50M 125M 125M

250M250M 500M500M

10m

HeLa cells incubated 7 h at 37 oC with [Eu2(LC2)3]exc = 330 nm, exposure time 60 s, x40


13


Concentration dependence: HeLa cells incubated7 h at 37 oC

12

15

18

21

24

27in

ten

sit

y (

a.u

.)

0 100 200 300 400 5009

c / M

[Eu2(LC2)3]

14


Cells incubated5 h at 37 oCon plastic with

500 M [Eu2(LC2)3]

Hacat

Hela

MCF-7

Hacat

Hela

MCF-7

10 m

exc = 330 nmexposure time 60 sx40

Imaging othercell lines

15


ex = 330 nm (BP 80 nm), exposure time 60 s, lens x40

HeLa cells incubated 7h (Tb) or 24 h Sm) at 37 oC

[Tb2(LC2)3]

Imaging with other lanthanides

[Sm2(LC2)3]

Q = 0.38 % !

500 M

250 M 250 M

10 m

16


Co-localization with labelled LDL or transferrin

[Eu2(LC2)3] 500 M LDL 15 g/mL, incubation 0.5 h transferrin 50 g/mL, incubation 2 h

merge365 nm/10 s

10 m

10 m

[Eu2(LC2)3]

[Eu2(LC2)3]

10 m

10 m

[Eu2(LC2)3]

[Eu2(LC2)3]

470 nm/1 s

LDL

Transferrin

LDL

Transferrin

17


Uptake mechanism: endocytosis?

0 M0 0 M 125 125 125 M

0

10

20

30

40Intensity (a.u.)

12501250c / mM

0 M 125 M0 M 125 M0 125

4 oC

37 oC

HeLa cellsIncubated with

[Eu2(LC2)3]

4 oC37 oC

18


Concentration:0.18-0.30 MConcentration:0.18-0.30 M

5

6

7Log

I

5

6

7Log

I

5

6

7Log

I

-11 -10 -9 -8 -7Log c

-11 -10 -9 -8 -7Log c

[Eu2(LC2)3]

500 cells

[Eu2(LC2)3]

500

1.9 10-9M

Volume:200 L

Cell volume:

2600-4200m3

Actual concentration of the helicate in HeLa cellsincubated with a 50 M solution

19


580 600 620 640 660 680 700 720

/ nm

Does the [Eu2(LC2)3] helicate survives in the cells?

03

24

1

5D07FJ

J =

In solution0.015 mM

0.5 mM inHela cells

Eu(5D0) lifetime

2.4 0.1 ms

2.4 0.1 ms

1.7 0.3 ms

In culturemedium0.5 mM

20


500 550 600 650 700

/ nm

[Tb2(LC2)3]

10-4 M

in cellulo

579 580 581 / nm

[Eu2(LC2)3] 10-4 Maq.

5D0 7F0

cellular

21


Time-resolved microscopy

ChopperLamp

Electronics

Data treatment

Chopper

Wallac Signifier® (Nikon Eclipse E600 microscope)

22



TRD, Time-delay 100 s

Bright field

TRD, No time-delay

HeLa cells incubated with 500 M[Eu2(LC2)3] 5 h at 37 oC (in RPMI-1640)Lens x40

23


20 m


HeLa cells incubated 6 h at 37 oC with [Eu2(LC2)3] 100 M (in RPMI-1640). Lens x100

TR mode, delay 100 s

24



0 20 40 60 80 100 120 140

0

1

2

3

4 conventional microscopy TRF microscopy

I-I0 / I0 (a.u.)

c /M

HeLa cells incubated with [Eu2(LC2)3] 5 h at 37 oC(in RPMI-1640). Lens x40

25MSc: f-Elements, Prof. J.-C. Bünzli, 2008

5.9 Tracing biomolecular interactions

Molecular interactions between biomolecules are keymechanisms in living cells.

Moreover, high throughput screening strategies are beingdeveloped in which pharmaceutical industry is testingas many compounds as possible (from combinatorial chemis-try) on molecular targets.

Henceforth the need for developing adequate analyticaltechniques able to work in the microliter range.

Homogeneous immunoassays based on LnIII luminescence(cf. § 2.7) are ideal in this respect.Technically, a luminescence resonance energy transfer (LRET) is used.



TRACE® technology(Time Resolved Amplified Cryptate Emission)

a) Choosing the right chelate

Key parameters are stability and dissociation kinetics

HO2CN N N

HO2C

CO2H

CO2H

CO2H

dtpa logK = 19-23Gdiss

# = 10-50 kJmol-1

NR

NR

N

N

N

N

O

O

N

H2N

H

H2N

H

N

R

N

N

R

bipyridine cryptandGdiss

# = 100-120 kJmol-1

[Eu(tbp)]

R = COOH



The quantum yield is relatively low: Q = 2 %, mainlybecause water is co-ordinated in the firstco-ordination sphere and because of a PET process.Water can be expelled by fluoride ions: Q = 7 %.PET process (leading to the reduction into EuII) canbe minimized by decreasing the cavity size, sincethe ionic radius of EuII is larger (1.30 Å) comparedto EuIII (1.12 Å, CN = 9).

NR

NR

N

N

R

N

N

R

N

N

O

H2N

H



b) Choosing the energy acceptor

550 750650

Allophycocyanin (105 kDa phycobiliprotein)High absorption coefficient, Q = 70 %R0 = 95 Å (distance for 50 % energy transfer fromthe Eu cryptate)

665 nm



allophycocyanin

Measuringwindow

time

337 nm 665 nm(a)

time

337 nm 665 nm(b)

time

337 nm 665 nm(c)

Eu chelate



time

337 nm

665 nm

time

337 nm 665 nm (c)

(d)

Signals (a) and (b) are time-discriminated and signals(c) and (d) are ratioed

H. Bazin et al., Rev. Mol. Biotechnol. 2002, 82, 233



c) Application: cell surface detection of membrane protein

D. Maurel et al. Anal. Biochem. 2004, 329, 253

cellmembraneR2R1 R2R1

no LRETLRET

Idea: prove that the GABAB receptor is a heterodimer



Kinetic of association showing the saturation of thebinding sites after 8 hours

0 4 8 24

em

issio

n/

a.u

.

t / h

D. Maurel et al. Anal. Biochem. 2004, 329, 253


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1 68% 50% H 2 L C2 Chapter 5 Selected applications