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

(L C2 ) 2- H2OH2O ? neutral complex Complex formation [M+MeCN] 2+ /2 [Tb 2 (L C2 ) 3 ] C 129 H 144 N 18 O 36 Tb 2 ESI-MS Simulated Chapter 5 Selected applications

 / ppm H ar (L C2 ) 2- OCH 3 O(CH 2 ) 2 H benz H py CH 2 bridge OCH 3 NCH 3 O(CH 2 ) 2 CH 2 bridge R = 2 NMR titration of H 2 L C2 in D 2 O (pD = 7.8) By Lu(ClO 4 ) 3 R = [Lu III ] t /[H 2 L C2 ] t Chapter 5 Selected applications

Absorbance / nm L C2 [Eu 2 (L C2 ) 3 ] UV-vis titration, in water, pH 7.4,  = 0.1 M, 25 o C Addition of Eu 3+ Chapter 5 Selected applications

5 UV-vis titration, in water, pH 7.4, H 2 L C M: > 95 % [Eu 2 (L C2 ) 3 ] M: > 98 % LaEuLu L C2 log  (2)18.1(2)18.7(3) log  (4)25.5(4)26.3(4) log  (3)11.8(5)12.4(2) Chapter 5 Selected applications

6 L C2 [Eu 2 (L C2 ) 3 ] [Eu 2 (L C2 )] [Eu(L C2 ) 3 ] R = [Eu III ] t /[H 2 L C2 ] t % species Ligand speciation in water, pH 7.4, c tot (lig) = M R [H 2 L C3 ] t = M95 % helicate 4 % 1:3 < 1 % 2:1 4.5  M97 % 2 % < 0.5 % 0.67 Chapter 5 Selected applications

/ 10 3 cm -1 ~ Photophysical properties H 2 L C / nm [Eu 2 (L C2 ) 3 ] 5D05D J = 0 7FJ7FJ [Tb 2 (L C2 ) 3 ] 5 J = D45D4 7FJ7FJ 1   3   Chapter 5 Selected applications

/ nm 5 D 0  7 F J J = K High resolution emission spectrum, [Eu 2 (L C2 ) 3 ] = M 146 cm A E Pseudo-D 3 symmetry At 295 K: nm cm -1 calc cm -1 B 2 0  -600 cm cm -1 Small distortion Chapter 5 Selected applications

9 Ln  (H 2 O)/  s  (D 2 O)/  sqQ Ln L / % Nd0.21 ± ± ±0.006 Sm30.4 ± ± 3 n.a.0.38 ± 0.06 Eu2430 ± ± ± 2 Tb650 ± ± 20 (-0.2) 77K 11 ± 2 Dy0.16 ± ± 0.1 n.a. Yb4.40 ± ± ± 0.03 Photophysical properties [Eu 2 (L C2 ) 3 ]Distorted D 3 symmetry No water in the inner coordination sphere Chapter 5 Selected applications

10 Stability of the [Eu 2 (L C2 ) 3 ] helicate vs ligand 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: [Eu 2 (L C2 ) 3 ] WST-1 test after 24 h incubation 90 % Chapter 5 Selected applications

12 HeLa cells incubated 7 h at 37 o C with [Eu 2 (L C2 ) 3 ] exc = 330 nm, exposure time 60 s, x40 Chapter 5 Selected applications

13 Chapter 5 Selected applications Concentration dependence: HeLa cells incubated 7 h at 37 o C intensity (a.u.) c /  M [Eu 2 (L C2 ) 3 ]

14 Chapter 5 Selected applications Cells incubated 5 h at 37 o C on plastic with 500  M [Eu 2 (L C2 ) 3 ] Hacat Hela MCF-7 Hacat Hela MCF-7 10  m exc = 330 nm exposure time 60 s x40 Imaging other cell 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 o C [Tb 2 (L C2 ) 3 ] Imaging with other lanthanides [Sm 2 (L C2 ) 3 ] Q = 0.38 % ! 500  M 250  M 10  m

16 Chapter 5 Selected applications Co-localization with labelled LDL or transferrin [Eu 2 (L C2 ) 3 ] 500  M LDL 15  g/mL, incubation 0.5 h transferrin 50  g/mL, incubation 2 h merge365 nm/10 s 10  m  m [Eu 2 (L C2 ) 3 ] [Eu 2 (L C2 ) 3 ] 10  m  m [Eu 2 (L C2 ) 3 ] [Eu 2 (L C2 ) 3 ] 470 nm/1 s LDL Transferrin LDL Transferrin

17 Chapter 5 Selected applications Uptake mechanism: endocytosis? 0  M0  0  M 125    M Intensity (a.u.) c / mM 0  M125  M0  M  M 0   4 o C 37 o C HeLa cells Incubated with [Eu 2 (L C2 ) 3 ] 4 o C 37 o C

18 Chapter 5 Selected applications Concentration:  M Concentration:  M Logc Logc [Eu 2 (L C2 ) 3 ] 500 cells [Eu 2 (L C2 ) 3 ] M  Volume: 200  L Cell volume:  m 3 Actual concentration of the helicate in HeLa cells incubated with a 50  M solution

19 Chapter 5 Selected applications / nm Does the [Eu 2 (L C2 ) 3 ] helicate survives in the cells? D07FJ5D07FJ J = In solution mM 0.5 mM in Hela cells Eu( 5 D 0 ) lifetime 2.4  0.1 ms 2.4  0.1 ms 1.7  0.3 ms In culture medium 0.5 mM

20 Chapter 5 Selected applications / nm [Tb 2 (L C2 ) 3 ] M in cellulo / nm [Eu 2 (L C2 ) 3 ]10 -4 M aq. 5D0  7F05D0  7F0 cellular

21 Chapter 5 Selected applications Time-resolved microscopy Chopper Lamp 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 [Eu 2 (L C2 ) 3 ] 5 h at 37 o C (in RPMI-1640) Lens x40

23 Chapter 5 Selected applications 20  m Time-resolved microscopy HeLa cells incubated 6 h at 37 o C with [Eu 2 (L C2 ) 3 ] 100  M (in RPMI-1640). Lens x100 TR mode, delay 100  s

24 Chapter 5 Selected applications Time-resolved microscopy c /  M HeLa cells incubated with [Eu 2 (L C2 ) 3 ] 5 h at 37 o C (in RPMI-1640). Lens x40

25 MSc: f-Elements, Prof. J.-C. Bünzli, Tracing biomolecular interactions Molecular interactions between biomolecules are key mechanisms in living cells. Moreover, high throughput screening strategies are being developed in which pharmaceutical industry is testing as many compounds as possible (from combinatorial chemis- try) on molecular targets. Henceforth the need for developing adequate analytical techniques able to work in the microliter range. Homogeneous immunoassays based on Ln III luminescence (cf. § 2.7) are ideal in this respect. Technically, a luminescence resonance energy transfer (LRET) is used. Chapter 5 Selected applications

26 MSc: 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 dtpa logK =  G diss # = kJmol -1 bipyridine cryptand  G diss # = kJmol -1 [Eu(tbp)] R = COOH Chapter 5 Selected applications

27 MSc: f-Elements, Prof. J.-C. Bünzli, 2008 The quantum yield is relatively low: Q = 2 %, mainly because water is co-ordinated in the first co-ordination sphere and because of a PET process. Water can be expelled by fluoride ions: Q = 7 %. PET process (leading to the reduction into Eu II ) can be minimized by decreasing the cavity size, since the ionic radius of Eu II is larger (1.30 Å) compared to Eu III (1.12 Å, CN = 9). Chapter 5 Selected applications

28 MSc: f-Elements, Prof. J.-C. Bünzli, 2008 b) Choosing the energy acceptor Allophycocyanin (105 kDa phycobiliprotein) High absorption coefficient, Q = 70 % R 0 = 95 Å (distance for 50 % energy transfer from the Eu cryptate) 665 nm Chapter 5 Selected applications

29 MSc: f-Elements, Prof. J.-C. Bünzli, 2008 allophycocyanin Measuring window time 337 nm 665 nm (a) time 337 nm 665 nm (b) time 337 nm 665 nm (c) Eu chelate Chapter 5 Selected applications

30 MSc: 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

31 MSc: f-Elements, Prof. J.-C. Bünzli, 2008 c) Application: cell surface detection of membrane protein D. Maurel et al. Anal. Biochem. 2004, 329, 253 cell membrane R2 R1 R2 R1 no LRET LRET Idea: prove that the GABA B receptor is a heterodimer Chapter 5 Selected applications

32 MSc: f-Elements, Prof. J.-C. Bünzli, 2008 Kinetic of association showing the saturation of the binding sites after 8 hours  emission/a.u. t / h D. Maurel et al. Anal. Biochem. 2004, 329, 253 Chapter 5 Selected applications