Two fluorinated thulium complexes as molecular temperature sensors in MR applications

Abstract

¹⁹F-based magnetic resonance is a powerful tool to overcome several difficulties of standard ¹H MR. We present the syntheses and characterization (including cell viability and stability tests) of two Tm³⁺ complexes. Both complexes allow the detection of temperature (ΔC_T = −0.2319 ppm K⁻¹ and −0.2122 ppm K⁻¹) without a reference compound.

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MR thermometry enables the detection of non-invasive temperature distributions within extended objects.¹ Temperature plays an important role in various biochemical processes, where it can fluctuate within a range of 1–5 K over the course of a day.² Therefore, medical applications such as discrimination of healthy and abnormal tissue or progressing of diseases are also of interest.^2a,3 In heat treatments such as hypothermia, monitoring local temperature helps prevent damage to healthy tissue.^3,4

There are some MR parameters of water that are temperature-sensitive, including T₁ and T₂ relaxation times and proton resonance frequency (PRF).^2a,3,4b The PRF is currently the most widely used method for temperature detection and imaging. It involves measuring the resonance frequencies of water protons using a gradient-echo-based pulse sequence and calculating the phase coefficient from the measured phase shift as a function of temperature.^3,5 However, when measuring the water protons using this technique, only a low temperature sensitivity of 0.01 ppm K⁻¹ is measured.^4b,5b–d,6

Therefore, the development of substances with highly temperature-sensitive MR signals is necessary. For this purpose, paramagnetic lanthanoid^1b,6,7 (with C_T-values up to 1.45 ppm K⁻¹)^7f and transition metal⁸ complexes are coming into focus. The lanthanoid complexes TmDOTMA⁻ and TmDOTP⁻ are probably the most promising examples, with temperature sensitivities of 0.57 ppm K⁻¹ and 1.0 ppm K⁻¹, respectively.^1b,6,7 However, TmDOTP⁻ has a high affinity towards Ca²⁺-ions and is strongly affected by pH value.^1b,7c–e,9 Tsitovich et al. synthesized some Fe- and Co-complexes with temperature sensitivities up to 0.52 ppm K⁻¹.⁸

These MR probes show a significant advantage in comparison to the PRF method, but are limited by the inherent Curie temperature dependence of chemical shift in paramagnetic complexes.³¹⁹F NMR and MRI are gaining more and more interest for diagnostic studies due to the fact that they have some benefits compared to other nuclei. There is only one natural isotope of fluorine (¹⁹F). Additionally, it has a similar gyromagnetic ratio close to ¹H. The most important advantage is the absence of fluorine signals in the body, which makes detection of ¹⁹F MR probes without background signals possible.^2a,3,10

To the best of our knowledge, the list of molecular paramagnetic ¹⁹F MR temperature sensors is limited to only a few numbers of compounds.^3,11 Other examples include perfluorocarbons or organofluorine compounds, but they are limited in their temperature sensitivity.^2a,12 Lee et al. and Li et al. recently published a study in which they examined the temperature sensitivity of organofluorine compounds. The synthesized compounds contained several fluorine cores and use the difference between the strongest shifting signals while increasing temperature to determine the temperature sensitivity.^2a,13 These compounds had a temperature sensitivity of 0.0195 ppm K⁻¹, which is almost two times higher than that of PFCs.^2a,13

The spin-crossover complexes of Thorarinsdottir et al. showed much greater potential than ¹⁹F MR thermosensitive probes. These results showed a temperature sensitivity of up to 0.45 ppm K⁻¹ in FBS (0.67 ppm K⁻¹ in MeCN-d₃) and, to our knowledge, are the highest values for ¹⁹F.³ In this paper, we present the synthesis of two Tm³⁺ complexes (TmL1 and TmL2, see Fig. 1) that have high temperature sensitivity. Both complexes possess two CF₃ groups with different chemical shifts. Accordingly, the temperature is determined by the difference between the two signals, and no added reference substance is required.

Fig. 1 Molecular structures of the synthesized complexes.

Both complexes were synthesized according to a previously published method.¹⁴ First, the tri- or bis-alkylated DO3A-tBu or DO2A-tBu and the bromoacetamide 2 were synthesized according to a literature procedure.¹⁵ The free amine(s) of DO3A-tBu and DO2A-tBu were substituted, following a slightly modified procedure from literature.¹⁴ The compounds were solved in CHCl₃ and Na₂CO₃ was added, with stirring taking place over a period of 7 days. Deprotection can be carried out in two different ways: one way involves deprotection with TFA and the other involves a hydrolysis of the esters using formic acid. Due to difficulties in removing residues of TFA, hydrolysis with formic acid was the method of choice. Both complexes L1 and L2 were obtained in high yields (89% respectively 83%).

L2 had a higher hydrophobicity than L1, the usage of different complexation methods was necessary. Complexation with TmCl₃ of L1 was carried out in water at 45 °C, stirred for one day, while complexation of L2 was carried out in MeOH at room temperature, stirred for three days. The purification step of the complexation started with precipitation of Tm(OH)₃ at pH 10. After filtration of the salt, the solution was neutralized and the solvent was evaporated. The resulting solid was suspended in EtOH and centrifuged to remove NaCl, which yielded the pure complexes (92% for TmL1 and 85% for TmL2). Starting from DO3A-tBu or DO2A-tBu, both complexes were obtained with an overall yield of 75% for TmL1 and 34% for TmL2 (see Fig. 2).

Fig. 2 Synthesis and reagents of L1 and L2. (a) Bromoacetyl bromide (2 eq.), K₂CO₃ (2 eq.), DCM, 30 min at 273.15 K (0 °C) to 90 min at rt, quant., (b) DO3A-tBu (0.94 eq.), Na₂CO₃ (2.2 eq.), CHCl₃, 7 d, 55 °C, 92%, (c) DO2A-tBu (0.42 eq.), Na₂CO₃ (2.2 eq.), CHCl₃, 7 d, 55 °C, 35%, (d) formic acid, 48 h, 111 °C, 89% for L1 and 83% for L2.

Stability experiments were performed with TmL1, here, the complex was dissolved in D₂O and two equivalents of ZnCl₂ were added. Control of the pH-value showed a slight decrease after 48 h from 3.35 (before the addition of ZnCl₂) to 2.83. The stability was verified by recording ¹⁹F NMR spectra after five minutes, thirty minutes, seven hours and twenty-four hours. For a long-term study, the probe was measured again after six months, and no transmetalation was observed. Additionally, the mixture was heated to 323.15 K and no changes in ¹⁹F NMR spectrum were observable. Finally, TmL1 showed a high stability towards Zn²⁺-ions (see Fig. 3).

Fig. 3 ¹⁹F NMR spectra of TmL1 after addition of two equivalents of ZnCl₂ at different times: 1 : ¹⁹F NMR spectrum before addition, 2 : ¹⁹F NMR spectrum after 5 min, 3 : ¹⁹F NMR spectrum after 30 min, 4 : ¹⁹F NMR spectrum after 7 h, 5 : ¹⁹F NMR spectrum after 24 h and 6 : ¹⁹F NMR spectrum after 6 months.

A solution of 0.1 mmol mL⁻¹ of the complexes in 0.5 mL D₂O was used in the experiments. The temperature range used was between 298.15 K and 323.15 K. The chemical shift changes of the two fluorine signals and the difference between these were determined. The difference between two fluorine signals allows the determination of the absolute temperature without the need for an internal or external reference. Complex TmL1 showed a decrease in the difference between the chemical shifts of both CF₃-groups with rising temperature (see Fig. 4).

Fig. 4 Variable-temperature ¹⁹F NMR spectra of TmL1 in D₂O. The temperature range was chosen as 298 K to 323 K. 1: T = 298 K, 2: T = 303 K, 3: T = 308 K, 4: T = 313 K, 5: T = 318 K and 6: T = 323 K.

The signal at −34.5 ppm (298.15 K) shifted to −37.5 ppm (323.15 K) at higher temperatures, resulting in a C_T-value of −0.1058 ppm K⁻¹. The other signal shifted from −78.7 ppm to −75.6 ppm, resulting in a C_T-value of 0.1261 ppm K⁻¹. This change in the difference between both signals resulted in a temperature coefficient ΔC_T of −0.2319 ppm K⁻¹, which is over 20 times higher than the temperature coefficient of water (C_T = 0.01 ppm K⁻¹).^6b In the following step, the influence of Zn²⁺-ions on the temperature sensitivity of TmL1 was examined. A slight increase to C_T = −0.2370 ppm K⁻¹ was observed. We assumed that changes in the C_T-value of ± 0.005 ppm K⁻¹ were measurement errors. Thus, the presence of Zn²⁺-ions does not affect the temperature sensitivity.

Complex TmL2 showed a slightly lower overall temperature sensitivity compared to TmL1 (see ESI†). The CF₃-groups displayed C_T-values of 0.0904 ppm K⁻¹ and −0.1218 ppm K⁻¹, respectively. This resulted in a ΔC_T between both signals of −0.2122 ppm K⁻¹. Structural differences, such as the positive charge or higher hydrophobicity of TmL2, are suggested to have no significant influence on the sensitivity.

The work of Pujales-Paradela et al. proved that these types of complexes can be used for paraCEST (paramagnetic Chemical Exchange Saturation Transfer) imaging and fluorine imaging.¹⁴ In addition to their high temperature sensitivity, the synthesis of two multifunctional contrast agents was achieved.

Compared to the results of Thorarinsdottir et al., a lower temperature sensitivity (−0.2319 ppm K⁻¹vs. 0.52 ppm K⁻¹) was obtained.³ However, the use of two different CF₃-groups for the determination of the temperature enables the calculation of the absolute temperature by determining the difference between the two signals. Also, the usage of one CF₃-group gives these complexes an advantage for future imaging experiments. Furthermore, these complexes can be used as multifunctional contrast agents.

After conducting successful ¹⁹F VT NMR measurements, the toxicity of the synthesized complexes TmL1 and TmL2 was evaluated using fibroblasts (L929) in cell culture experiments. The method used is described in the experimental section. The complexes were dissolved in a cell culture medium at concentrations of 0.19 mM for TmL1 and 0.24 mM for TmL2 and added to the cells. The cell viability was determined after 24 h and 48 h of incubation, and the results were compared to a control sample containing only the cells and the cell culture medium. Both complexes showed no significant toxicity (Fig. 5). Complex TmL1 had a cell viability of 88.56% after 24 h and 87% after 48 h, while complex TmL2 had slightly lower viability at 85.5% and 79.5%, respectively. These results are comparable to the control samples (91.25% and 85.56%, respectively) and indicate that the complexes can be used in future in in vivo experiments (Fig. 5).

Fig. 5 Results of the viability tests after an incubation time of 24 h and 48 h.

We presented the synthesis of two multifunctional complexes, TmL1 and TmL2, with a high ¹⁹F MR signal temperature sensitivity and no significant toxicity towards fibroblasts (L929). TmL1 had a slightly higher temperature sensitivity than TmL2, with a ΔC_T of -02319 ppm K⁻¹ (and -0.2122 ppm K⁻¹), which is over 20 times higher than the temperature coefficient of water. The presence of Zn²⁺-ions did not affect the temperature sensitivity of TmL1. Based on the previously published complexes,¹⁴ these complexes could be used for paraCEST imaging and ¹⁹F imaging. Furthermore, the two different CF₃-groups in each complex enables the determination of the absolute temperature without an internal or external reference. The promising complexes TmL1 and TmL2 will be examined in future MR imaging experiments.

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3cc02724d

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