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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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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+
Chapter 5 Selected applications
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)
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
11
Cell viability:
[Eu2(LC2)3]
WST-1 test after 24 h incubation
90 %
Chapter 5 Selected applications
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
Chapter 5 Selected applications
13
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
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
Chapter 5 Selected applications
Time-resolved microscopy
ChopperLamp
Electronics
Data treatment
Chopper
Wallac Signifier® (Nikon Eclipse E600 microscope)
22
Chapter 5 Selected applications
Time-resolved microscopy
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
Chapter 5 Selected applications
20 m
Time-resolved microscopy
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
Chapter 5 Selected applications
Time-resolved microscopy
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.
Chapter 5 Selected applications
26MSc: f-Elements, Prof. J.-C. Bünzli, 2008
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
Chapter 5 Selected applications
27MSc: f-Elements, Prof. J.-C. Bünzli, 2008
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
Chapter 5 Selected applications
28MSc: f-Elements, Prof. J.-C. Bünzli, 2008
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
Chapter 5 Selected applications
29MSc: f-Elements, Prof. J.-C. Bünzli, 2008
allophycocyanin
Measuringwindow
time
337 nm 665 nm(a)
time
337 nm 665 nm(b)
time
337 nm 665 nm(c)
Eu chelate
Chapter 5 Selected applications
30MSc: f-Elements, Prof. J.-C. Bünzli, 2008
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
Chapter 5 Selected applications
31MSc: f-Elements, Prof. J.-C. Bünzli, 2008
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
Chapter 5 Selected applications