Influence of Calcium-induced Aggregation on the Sensitivity of. Aminobis(methylenephosphonate)-Containing Potential MRI Contrast.

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1 Supporting Information for Influence of Calcium-induced Aggregation on the Sensitivity of Aminobis(methylenephosphonate)-Containing Potential MRI Contrast Agents Jörg Henig, Ilgar Mamedov, Petra Fouskova, Éva Tóth, Nikos K. Logothetis, Goran Angelovski, * and Hermann A. Mayer * Institut für Anorganische Chemie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 7076 Tübingen, Germany, Abteilung für Physiologie kognitiver Prozesse, Max-Planck-Institut für biologische Kybernetik, Spemannstrasse 38, 7076 Tübingen, Germany, Centre de Biophysique Moléculaire, CNRS rue Charles Sadron, 4507 Orléans, Cedex, France and Division of Imaging Science and Biomedical Engineering, The University of Manchester, Manchester M3 9PT, U. K. Table S. Relaxometric Ca + titration of GdL at 5 C, ph 7.3 and 9.4 T [Gd 3+ ] (mm) [Ca + ] (mm) [Ca + ]/[SCA] T (s) r (mm - s - )

2 Table S. Relaxometric Ca + titration of GdL at 5 C, ph 7.3 and 9.4 T [Gd 3+ ] (mm) [Ca + ] (mm) [Ca + ]/[SCA] T (s) r (mm - s - ) Table S3. Relaxometric Ca + titration of GdL 3 at 5 C, ph 7.3 and 9.4 T [Gd 3+ ] (mm) [Ca + ] (mm) [Ca + ]/[SCA] T (s) r (mm - s - ) Table S4. Relaxometric Ca + titration of GdL 4 at 5 C, ph 7.3 and 9.4 T [Gd 3+ ] (mm) [Ca + ] (mm) [Ca + ]/[SCA] T (s) r (mm - s - )

3 Figure S. 3 P{ H} NMR spectra of EuL at 98 K. *Impurity from the ligand synthesis, being hardly visible in solutions of the free ligand, but more intense in these spectra as it experiences only a weak paramagnetic influence. Figure S. 3 P{ H} NMR spectra of EuL at different temperatures. *Impurity from the ligand synthesis (see above). 348 K 38 K ppm 98 K Figure S3. 3 P{ H} NMR spectra of EuL at 98 K in the absence of Ca + ions (a) and in the presence of three equiv. Ca + ions (b). *Impurity from the ligand synthesis. a) b) * * ppm ppm 3

4 Figure S4. Luminescence emission spectra of EuL (5 mm) in the presence and absence of 3 equiv. Ca + (5 C, ph 7.3, HEPES). 5x0 5 EuL EuL + Ca + 4x0 5 Intensity [a.u.] 3x0 5 x0 5 x λ [nm] Figure S5. Luminescence emission spectra of EuL 3 (5 mm) in the presence and absence of 3 equiv. Ca + (5 C, ph 7.3, HEPES). 4x0 5 EuL 3 EuL 3 + Ca + Intensity [a.u.] 3x0 5 x0 5 x λ [nm] 4

5 Table S5. Emission lifetimes and estimated q values of EuL in Ca + -free solution and upon addition of 3 equiv. Ca + (5 o C, HEPES, ph 7.3) τ HO (ms) τ DO (ms) q EuL 50 mm mm mm mm EuL + 3 equiv. Ca + 50 mm mm mm mm Figure S6. Estimated q values of EuL in Ca + -free solution and upon addition of 3 equiv. Ca + (5 o C, HEPES, ph 7.3) Apparent q value EuL EuL + 3 equiv Ca EuL [mm] 5

6 Table S6. Emission lifetimes and estimated q values of EuL in Ca + -free solution and upon addition of 3 equiv. Ca + (5 o C, HEPES, ph 7.3) τ HO (ms) τ DO (ms) q EuL 40 mm mm mm mm EuL + 3 equiv. Ca + 40 mm mm mm mm Figure S7. Estimated q values of EuL in Ca + -free solution and upon addition of 3 equiv. Ca + (5 o C, HEPES, ph 7.3)..0 Apparent q value EuL EuL + 3 equiv Ca EuL [mm] 6

7 Table S7. Emission lifetimes and estimated q values of EuL 3 in Ca + -free solution and upon addition of 3 equiv. Ca + (5 o C, HEPES, ph 7.3) τ HO (ms) τ DO (ms) q EuL 3 3 mm mm mm mm EuL equiv. Ca + 3 mm mm mm mm Figure S8. Estimated q values of EuL 3 in Ca + -free solution and upon addition of 3 equiv. Ca + (5 o C, HEPES, ph 7.3).6.4 EuL 3 EuL equiv Ca +. Apparent q value EuL 3 [mm] 7

8 Table S8. Emission lifetimes and estimated q values of EuL 4 in Ca + -free solution and upon addition of 3 equiv. Ca + (5 o C, HEPES, ph 7.3) τ HO (ms) τ DO (ms) q EuL 4 50 mm mm mm mm EuL equiv. Ca + 50 mm mm mm mm Figure S9. Estimated q values of EuL 4 in Ca + -free solution and upon addition of 3 equiv. Ca + (5 o C, HEPES, ph 7.3)..0 EuL 4 EuL equiv Ca + Apparent q value EuL 4 [mm] 8

9 Figure S0. Potentiometric titration curve of GdL. 0 8 ph base equivalents Figure S. Potentiometric titration curve of GdL. 0 8 ph base equivalents 9

10 Figure S. Potentiometric titration curve of GdL ph base equivalents Figure S3. Potentiometric titration curve of GdL ph base equivalents 0

11 Figure S4: H-/T of GdL 4 in the presence of 3 equivalents of Ca + as a function of the temperature, (.5 mm, 500 MHz). Figure S5: 7 O T values as a function of temperature for GdL 4, (c = 50 mm, B =.75 T).

12 Equations used for the analysis of the 7 O NMR data From the measured 7 O NMR relaxation rates of the paramagnetic solutions, /T, /T, and of the reference, /T A, /T A, one can calculate the reduced relaxation rates, /T r, /T r, which may be written as in Equations ()-(), where P m is the molar fraction of bound water, /T m, /T m are the relaxation rates of the bound water and ω m is the chemical shift difference between bound and bulk water. = = Tr Pm T TA T m +τ m () = = Tr Pm T TA τ m T - - m + τ m Tm ( τ m + Tm ) + ω ω m m () ω m is determined by the hyperfine or scalar coupling constant, A/h, according to Equation (3), where B represents the magnetic field, S is the electron spin and g L is the isotropic Landé g factor. ω m = g L µ BS ( S+ ) B A 3k T h B (3) The 7 O longitudinal relaxation rates are given by Equation (4), where γ S is the electron and γ I is the nuclear gyromagnetic ratio (γ S =.76 0 rad s - T -, γ I = rad s - T - ), r is the effective distance between the electron charge and the 7 O nucleus, I is the nuclear spin (5/ for 7 O), χ is the quadrupolar coupling constant and η is an asymmetry parameter: µ 0 h I S d = γ γ S( S+ ) 6 6 τ d+ 4 τ m 5 4π rgdo + ωsτ d T 3π + 0 I + 3 χ (+ η / 3) τ I (I ) RO (4) where:

13 τ = τ + τ + di m RO T i e i =, (5) The τ RO overall rotational correlation time is assumed to have simple exponential temperature dependence with E R activation energy as follows: 98 ER τ RO = τ RO exp (6) R T 98.5 In the transverse relaxation the scalar contribution, /T sc, is the most important [Eq. (7)]. /τ s is the sum of the exchange rate constant and the electron spin relaxation rate. T T m sc 3 = + τ s τ m S( S+ ) A = τ h T e s (7) (8) The inverse binding time (or exchange rate, k ex ) of water molecules in the inner sphere is assumed to obey the Eyring equation [Eq. (9)], where S and H are the entropy and enthalpy of activation for the exchange, and k ex 98 is the exchange rate at 98.5 K. τ m = k ex kbt = h S exp R H RT 98 k ex T H = exp 98.5 R 98.5 T (9) The electron spin relaxation rate, /T e has been fitted to a simple exponential equation: 98 E / T e = / Te exp (0) R T 98.5 T 3

14 7 O NMR spectroscopy on GdL, GdL and GdL 3 For complexes GdL (Figure S6), GdL and GdL 3 (Figure S7), the reduced 7 O transverse relaxation rates (/T r ) decrease with increasing temperature in the whole temperature range investigated, indicating that they are in the fast exchange region. For GdL and GdL 3, the experimentally measured paramagnetic 7 O chemical shifts were considerably smaller than what would be expected for a Gd 3+ complex with the given q value, therefore, they have not been included in the final fitting. Figure S6. Variable temperature reduced 7 O transverse ( ) and longitudinal ( ) relaxation rates and chemical shifts ( ) for GdL. B =.75 T. The lines correspond to the least-squares fit as explained in the text.. Figure S7. Variable temperature reduced 7 O transverse ( ) and longitudinal ( ) relaxation rates for GdL (a) and GdL 3 (b). B =.75 T. The lines correspond to the least-squares fit as explained in the text.. Even though.7 has been obtained for EuL from the luminescence lifetime measurements and the possibility of the existence of some dehydrated species with non coordinated phosphonate groups cannot be excluded from the luminescence and 3 P NMR data, the hydration number of GdL was fixed to one in this analysis, as correlates better with the obtained values of the reduced chemical shifts ω r. For GdL and GdL 3 the increased formation of aggregates at 50 mm concentration leads to a reduction of inner sphere 4

15 water, which was indicated by a drop in q to 0.7 in the concentration-dependent q measurements. However, as the equation applied is not optimized for these systems, the reduction can also be due to a change in the second coordination sphere and the number of inner sphere water molecules might still be one. Therefore, for GdL and GdL 3 two fits of the 7 O NMR data were performed assuming q = 0.7 or (Figure S7 shows the fit with q = ).The experimental data have been fitted to the Solomon-Bloembergen-Morgan theory of paramagnetic relaxation. In the fitting procedure, r GdO has been fixed to the common value of.50 Å. 5 The quadrupolar coupling constant χ(+η /3) / has been set to the value for pure water, 7.58 MHz. For GdL and GdL 3, the scalar coupling constant A/ħ, was fixed to -3.6 MHz, the value obtained for GdL. The electron spin relaxation rate /T e, has been fitted to a simple exponential function. Since it has a negligible contribution to the transverse relaxation rates, the fit of parameters related to the zero-field splitting mechanism, as usually done, has very low confidence. The following parameters have been adjusted: the water exchange rate k 98 ex, or the activation entropy S, the activation enthalpy for water exchange H, the rotational correlation time τ 98 RO, and its activation energy E R, as well as the electron spin relaxation rate at 98 K /T 98 e (Table S9) and its activation energy E T. When E T was fixed to kj/mol, /T 98 e was calculated between (.-.3) 0 7 s -, otherwise small negative values were obtained. Table S9. Parameters obtained from the fitting of the 7 O NMR data at.75 T. GdL GdL GdL 3 GdDOTA b q a k 98 ex / 0 6 s - 70±3 60±5 0±5 90±5 30±0 4. H / kj mol - 7.± ± ± ± ± S / J mol - K - 0± -3± -36± -4± -44± A/h / 0 6 rad s ± a -3.6 a -3.6 a -3.6 a -3.7 τ 98 RO / ps 90±0 390±0 540±0 380±0 550±0 90 E R / kj mol - 7.8±0.9 7.± ± ± ±.0 7 a fixed in the fit. b Ref.: Powell, D. H.; NiDhubhghaill, O. M.; Pubanz, D.; Helm, L.; Lebedev, Y. S.; Schlaepfer, W.; Merbach, A. E., J. Am. Chem. Soc. 996, 8, Note that by assuming q = for GdL we calculate k ex 98 = s -. 5

16 Table S0. Variable temperature reduced longitudinal and transverse 7 O relaxation rates and chemical shifts of GdL aqueous solution. c Gd = mol/kg. B =.75 T T (K) Shift T/ms(ref) T/ms(ref) (ref)/ppm T/ms T/ms ln(/tr) Shift/ppm ln(/tr) ωr /Hz E E E E E E E E E+05 Table S. Variable temperature reduced longitudinal and transverse 7 O relaxation rates and chemical shifts of GdL aqueous solution. c Gd = mol/kg. B =.75 T T (K) Shift T/ms(ref) T/ms(ref) (ref)/ppm T/ms T/ms ln(/tr) Shift/ppm ln(/tr) ωr /Hz E E E E E E E E+05 6

17 Table S. Variable temperature reduced longitudinal and transverse 7 O relaxation rates and chemical shifts of GdL 3 aqueous solution. c Gd = mol/kg. B =.75 T T (K) Shift T/ms(ref) T/ms(ref) (ref)/ppm T/ms T/ms ln(/tr) Shift/ppm ln(/tr) ωr /Hz E E E E E E E E+05 Table S3. Variable temperature reduced longitudinal and transverse 7 O relaxation rates and chemical shifts of GdL 4 aqueous solution. c Gd = mol/kg. B =.75 T T (K) Shift T/ms(ref) T/ms(ref) (ref)/ppm T/ms T/ms ln(/tr) Shift/ppm ln(/tr) ωr /Hz E E E E E E E E E+05 7

18 Table S4. Variable temperature reduced longitudinal and transverse 7 O relaxation rates and chemical shifts of GdL aqueous solution. c Gd = mol/kg; in the presence of three equivalents of calcium; B =.75 T T (K) T/ms T/ms Shift/ppm T/ms(ref) T/ms(ref) Shift (ref)/ppm ln(/tr) ln(/tr) ωr /Hz E E E E+04 8

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