Spin-crossover and high-spin iron(ii) complexes as chemical shift 19F magnetic resonance thermometers

The potential utility of paramagnetic transition metal complexes as chemical shift 19F magnetic resonance (MR) thermometers is demonstrated.


Introduction
The noninvasive measurement of temperature in vivo represents a growing area of research, largely due to its utility in medical applications such as low-temperature hyperthermia, 1,2 high-temperature thermal ablation, 1,2 and the treatment of heart arrhythmias. 3 Here, thermometry may be used to discriminate normal from abnormal tissue, and also to ensure that thermal treatments are localized to prevent damage to healthy tissue. 1,2,4 Magnetic resonance spectroscopy (MRS) and imaging (MRI) are particularly well-suited toward this end, owing to their use of non-ionizing radiation and ability to deeply penetrate tissue. 1,5 Indeed, a number of temperaturesensitive MR parameters of water, including T 1 and T 2 relaxation times, proton resonance frequency (PRF), diffusion coefficient, and proton density, can be used to monitor tissue temperature. 1,4,6 Currently, methods based on water PRF shi are the most widely used for imaging temperature in clinical studies due to their high-resolution and independence on tissue type. 7 However, these techniques suffer from a low temperature sensitivity of ca. À0.01 ppm per C, and their ability to accurately determine absolute temperature is limited. 1,7,8 In order to overcome sensitivity limitations, paramagnetic lanthanide 9 and transition metal complexes 10 that function as MRS probes have been developed for thermometry. These complexes feature paramagnetically shied proton resonances, thus minimizing the interference from background signal in biological tissue. In particular, proton resonances of Tm 3+ , Tb 3+ , Dy 3+ and Yb 3+ complexes have been shown to exhibit temperature sensitivities of up to 1.8 ppm per C, 9q and have been employed for temperature mapping in vitro and in vivo. 9 Additionally, transition metal MRS probes have been shown to exhibit similar sensitivity 10 and may alleviate toxicity concerns associated with lanthanides. 11 While paramagnetic MRS probes offer signicant improvements in sensitivity over PRF thermometry, they are nevertheless limited to the inherent Curie temperature dependence of chemical shi in paramagnetic compounds. 12 Alternatively, one can employ a strategy of tuning a physical parameter that itself depends on temperature and governs chemical shi. Since both contact (through-bond) and dipolar (through-space) hyperne shi scale as S(S + 1), where S represents the electronic spin state, variation of S as a function of temperature can result in dramatic changes in chemical shi. 12 As such, an ideal temperature-responsive chemical shi probe might feature a value of S that changes with temperature. Spin-crossover Fe II complexes that undergo a thermally-induced electronic spin transition from a low-spin, S ¼ 0 ground state to a high-spin, S ¼ 2 excited state satisfy just such a criterion. Moreover, the ligand eld in spin-crossover complexes can be chemically modulated to precisely tune the crossover temperature (T 1/2 ), dened as the temperature at which the low-spin and high-spin states are equally populated, 13 to near 37 C. Indeed, the utility of spin-crossover in MR thermometry has been demonstrated through T * 2 modulation in Fe II -based nanoparticles 14 and through paramagnetic chemical exchange saturation transfer (PARACEST) in molecular Fe II complexes. 15 While the vast majority of MRS thermometry probes exploit changes in the chemical shi of 1 H NMR resonances, the employment of 19 F MR offers several key advantages. First, the 19 F nucleus features a 100% natural abundance, a nuclear spin of I ¼ 1 / 2 , and a gyromagnetic ratio and sensitivity close to that of 1 H. 16 Moreover, the near absence of endogenous uorine signals in the body, the large spectral window of 19 F resonances, and the remarkable sensitivity of 19 F chemical shi to the local environment, give rise to NMR spectra with minimal peak overlap. 17 Indeed, it has been demonstrated that 19 F chemical shis of transition metal porphyrin complexes are highly sensitive to their solution electronic structure, in particular to oxidation state and spin state. 18 In addition, lanthanide-based 19 F chemical shi probes for monitoring pH have been reported. 19 However, despite the potential of S as a tunable parameter to increase the temperature sensitivity of 19 F MR chemical shi, to our knowledge no paramagnetic 19 F MR thermometers have been reported. In fact, diamagnetic per-uorocarbons represent the only examples of 19 F MR thermometry, but the application of these compounds is limited by the small temperature dependence of their 19 F chemical shis that affords a maximum sensitivity of only 0.012 ppm per C. 20 Given the advantages of 19 F over 1 H MR, in conjunction with the temperature sensitivity of 1 H MR chemical shis of our previously reported spin-crossover Fe II PARACEST probes 15 and the high-spin Fe II 1 H MR shi probes reported by Morrow and coworkers, 10 we sought to develop uorine-substituted spincrossover and high-spin Fe II complexes for chemical shi 19 F MR thermometry. Herein, we report a series of complexes that feature new symmetrically and asymmetrically-substituted 1,4,7-triazacyclononane (tacn) derivatives with uorinated 2picolyl donors. The potential utility of spin-crossover and highspin Fe II complexes as chemical shi 19 F MR thermometers is demonstrated through detailed analysis of their temperaturedependent spectroscopic and magnetic properties. Furthermore, these compounds exhibit excellent stability in a physiological environment, as revealed by variable-temperature 19 F NMR spectra recorded in fetal bovine serum (FBS). To our knowledge, this work provides the rst examples of paramagnetic chemical shi 19 F MR thermometers.

Syntheses and structures
With the goal to prepare air-and water-stable complexes, tacnbased ligands bearing three pendent pyridyl groups offer an ideal platform, as these hexadentate scaffolds have been shown to afford highly-stable Fe II complexes. 10,21 In addition, the ligand eld can be readily tuned to obtain spin-crossover complexes within a physiologically relevant temperature range by chemical modulation of the electronic and steric properties of the pyridyl donors. 21e, 22 Toward this end, we sought to synthesize related ligands that support Fe II complexes in selected spin states through controlled introduction of methyl groups into the 6-position of the pyridyl groups, which serves to weaken the ligand eld by virtue of steric crowding at the Fe II center. In addition, in order to enable utilization of these compounds in 19 F MRS thermometry, we installed uorine substituents onto the 3-positions of the pyridyl groups.
The preparation of ligands L x (x ¼ 1-3; see Fig. 1) was carried out through a ve-step synthesis involving stepwise addition of 2-picolyl derivatives to the tacn backbone via reductive amination of the corresponding 2-pyridinecarboxaldehydes with tacn precursors (see Experimental section and Scheme S1 †). Through judicious selection of the aldehyde reagent in each step, this synthetic route enabled the preparation of both symmetric and asymmetric tri-functionalized tacn-based ligands, appended with one or two types of 2-picolyl donors. Metalation of the ligands with Fe II and Zn II was effected through reaction of equimolar amounts of L x and the corresponding divalent metal ion in CH 3 CN. Subsequent diffusion of Et 2 O into a concentrated CH 3 CN or CH 3 Single-crystal X-ray diffraction analysis for 1a$0.5CH 3 CN, 1b, 2a, 2b, and 3a, was carried out at 100 K (see Table S1 †). Compound 1a$0.5CH 3 CN crystallized in the triclinic space group P 1, and features two [Fe(L 1 )] 2+ cations in the asymmetric unit. Compound 1b crystallized in the monoclinic space group Pc, with the asymmetric unit comprised of two [Zn(L 1 )] 2+ cations. In contrast to the metal complexes of asymmetric L 1 , compounds 2a and 2b are isostructural and crystallized in the cubic space group F 43c, with one third of the [M(L 2 )] 2+ (M ¼ Fe, Zn) cation in the asymmetric unit. In these two structures, the M II metal center resides on a site of crystallographic three-fold symmetry. Finally, the asymmetric unit of the crystal structure of 3a, which crystallized in the trigonal space group P3, features one-third of three unique [Fe(L 3 )] 2+ cations, with the remainder of each complex related through a crystallographic three-fold axis (see Fig. S1 †).
In the cationic complex of each compound, the M II center resides in a distorted octahedral coordination environment, comprised of three facially bound tertiary amine nitrogen atoms from the tacn backbone and three picolyl nitrogen atoms (see Fig. 2). Examination of bond distances associated with the Fe II cations reveals the spin state of these complexes in the solidstate at 100 K (see Table 1). The mean Fe-N bond distances for 1a$0.5CH 3 CN and 3a fall in the ranges 1.974(2)-2.088(2) and 1.969(3)-1.999(3)Å, respectively, indicative of low-spin Fe II . 15,22,23 In 1a$0.5CH 3 CN, the Fe-N Me-pyr bond lengths of 2.085(2) and 2.090(2)Å are signicantly longer than the Fe-N F-pyr bond distances of 1.970(2)-1.978(2)Å, due to the steric effects imposed by the methyl substituent on one of the picolyl groups. 22 In contrast, the average Fe-N MeF-pyr and Fe-N tacn bond distances for 2a of 2.224(2) and 2.230(2)Å, respectively, are substantially longer and are characteristic of high-spin Fe II . 22,23a-c,24 Finally, the mean Zn-N bond distances of 2.196 (3) and 2.212(2)Å for 1b, and 2b, respectively, are consistent with reported distances for Zn II ions in similar coordination environments. 25 The presence of uoro and methyl substituents on the 2-picolyl pendent groups of ligands L 1-3 leads to a distortion from octahedral coordination at the metal centers. This deviation from perfect octahedral geometry can be quantied through the octahedral distortion parameter S, dened as the sum of the absolute deviations of the 12 cis-oriented N-M-N angles from 90 . 26 Analysis of the Fe II centers in 1a$0.5CH 3 CN, 2a, and 3a gives values of S ¼ 72.4(3), 134.8(3), and 59.9(4) , respectively. The much larger value for 2a than for 1a$0.5CH 3 CN and 3a reects the signicant steric crowding in 2a and further corroborates the high-spin and low-spin assignments of these complexes. 27 The larger distortion of the [Fe(L 1 )] 2+ cation in 1a$0.5CH 3 CN relative to [Fe(L 3 )] 2+ in 3a is attributed to presence of one vs. zero picolyl methyl substituents, respectively. The coordination environment of the Fe II complex in 2a and its isostructural Zn II analogue in 2b are similar, where 2b is slightly less distorted than 2a, evident from a smaller S value of 127.7(2) . In contrast, the difference between the structures of 1a$0.5CH 3 CN and 1b is substantial. Upon moving from Fe to Zn, the mean N tacn -M-N tacn angle decreases by 7.1%, from 85.07(6) to 79.1(2) , and the mean trans N tacn -M-N pyr angles decrease by 10.7 (N Me-pyr ), and 10.2% (N F-pyr ), respectively. Finally, a more than two-fold increase in S is observed for 1b relative to 1a$0.5CH 3 CN. These differences reect a much greater degree of distortion at the Zn II center in 1b than at the Fe II center in 1a$0.5CH 3 CN, which likely stems from increased coordination exibility at the d 10 Zn II ion due to lack of ligand eld stabilization, and the larger six-coordinate ionic radius of Zn II (0.88Å) compared to low-spin Fe II (0.75Å). 27a Compounds 1a$0.5CH 3 CN, 1b, 2a, 2b, and 3a feature intramolecular M/F distances in the range 5.094(2)-5.277(2)Å. The shortest M/F distances are observed between the 3-uoro-2picolyl pendent groups and the Fe II centers in compounds 1a$0.5CH 3 CN and 3a, with slightly longer M/F distances of 5. 26-5.28Å in compounds 1b, 2a, and 2b. The longer Zn/F distance in 1b, compared to the corresponding Fe/F distance in 1a$0.5CH 3 CN, can be attributed to the longer Zn-N bond distances relative to Fe. In the case of compounds 2a and 2b, the presence of bulky 3-uoro-6-methyl-2-picolyl groups increase the M/F distances relative to 1a$0.5CH 3 CN and 3a. Importantly, the M/F distances of 1a and 2a are within the optimal range of 4.5-7.5Å to balance the benets of paramagnetic hyperne shi with the decrease in sensitivity due to spectral broadening, 19d,e which demonstrates the potential of these complexes as candidates for 19 F chemical shi MR probes.

UV-vis spectroscopy
To probe the solution electronic structures of the cationic complexes in 1a, 1b, 2a, 2b, and 3a, UV-vis absorption spectra were collected for crystalline samples in CH 3 CN solution. The spectrum of 1a obtained at 25 C exhibits an intense band at 264 nm (3 ¼ 10 700 M À1 cm À1 ), in addition to a weaker broad band at 424 nm (3 ¼ 2800 M À1 cm À1 ) with a high-energy shoulder (see Fig. 3 and S2 †). Based on literature precedent of Fe II complexes in similar ligand environments, we assign these absorption bands as ligand-centered p-p* and metal-ligand charge transfer (MLCT) transitions, respectively. 22,28 The UV-vis spectrum of 2a at 25 C is dominated by the intense p-p* band (l max ¼ 273 nm, 3 max ¼ 11 100 M À1 cm À1 ), and an additional broad feature of low intensity between 320 and 460 nm (l max ¼ 375 nm) corresponds to a MLCT transition (see Fig. 3, lower, and S3 †). The weak intensity and the small temperature dependence between À35 and 65 C for the latter band (3 max ¼ 1000 vs. 700 M À1 cm À1 , respectively) are characteristic of high-spin Fe II . 28c, 29 Compound 3a is also relatively insensitive to temperature changes and at 25 C displays a similar ligand-centered p-p* transition at 261 nm, but with a more intense MLCT band at Turquoise, orange, green, blue and gray spheres represent Zn, Fe, F, N and C atoms, respectively; H atoms are omitted for clarity. 436 nm (3 max ¼ 10 600 M À1 cm À1 ), and as such is indicative of low-spin Fe II (see Fig. 3, lower, and S4 †). 22,30 The variabletemperature UV-vis spectra of the Zn II compounds 1b and 2b in CH 3 CN each exhibits a single intense band with l max ¼ 268 and 278 nm, respectively (see Fig. S5 and S6 †), consistent with ligand-centered p-p* transitions. 31 The absorption spectra of 1a demonstrate remarkable temperature dependence between À35 and 65 C (see Fig. 3 , upper). While the position of the p-p* band is relatively invariant to temperature, 3 max decreases signicantly from 14 800 to 8400 M À1 cm À1 upon warming, as has been observed for related pyridyl complexes. 32 At À35 C, the MLCT band exhibits a l max value of 439 nm (3 max ¼ 5500 M À1 cm À1 ) with a shoulder at ca. 385 nm. Upon warming, the MLCT bands broaden and decrease in intensity, resulting in a single peak with l max ¼ 385 nm (3 max ¼ 1600 M À1 cm À1 ) at 65 C that corresponds to ca. 3.5-fold reduction in intensity from the À35 C spectrum. This temperature dependence of the spectra is indicative of a thermally-induced spin state transition. 22,33 Indeed, approximating a metal complex of O h symmetry, the intensity of the MLCT band is directly correlated to the number of electrons in t 2g orbitals. 32c,d As such, moving from low-spin Fe II (t 6 2g ) to high-spin Fe II (t 4 2g e 2 g ) with increasing temperature results in a weaker absorption. Moreover, the presence of three isosbestic points at 222, 273, and 302 nm suggests an equilibrium between two spin states for the Fe II centers in 1a.
The temperature-dependent spin state of Fe II in 1a in CH 3 CN can be further examined by comparing the UV-vis spectra of 1a with the corresponding spectra of the high-spin compound 2a and the low-spin compound 3a (see Fig. 3, lower). At lower temperature, the spectrum of 1a strongly resembles that of 3a (see Fig. S7 †), whereas at higher temperature the broad spectrum resembles that of 2a (see Fig. S8 †). These temperature-dependent spectral changes demonstrate the thermally-induced spin-crossover of 1a in CH 3 CN solution from primary population of a lowspin state at À35 C to a high-spin state at 65 C.
With an eye toward employing these complexes in MR thermometry, UV-vis spectra were collected for aqueous solutions of compounds 1a, 1b, 2a, 2b, and 3a at ambient  (8) 79.39 (7) (2) a N Me-pyr corresponds to a N atom on a 6-methyl-2-picolyl group. b N F-pyr corresponds to a N atom on a 3-uoro-2-picolyl group. c N MeF-pyr corresponds to a N atom on a 3-uoro-6-methyl-2-picolyl group. d Octahedral distortion parameter (S) ¼ sum of the absolute deviations from 90 of the 12 cis angles in the MN 6 coordination sphere. e Data obtained from Zn1 due to severe crystallographic disorder associated with Zn2. temperature. All compounds show similar characteristics in H 2 O as in CH 3 CN, giving comparable values of l max and 3 max (see Fig. S9-S13 †). Nevertheless, the spectrum of 1a in H 2 O reveals some key differences from the spectrum obtained in CH 3 CN at 25 C. The absorption maximum of the MLCT band is shied to a longer wavelength in H 2 O (l max ¼ 436 nm), and the intensity of this band compared to the intensity of the analogous band for 3a in the same solvent is considerably greater in H 2 O than in CH 3 CN (H 2 O: 3 max,3a /3 max,1a ¼ 1.5; CH 3 CN: 3 max,3a / 3 max,1a ¼ 3.8). These observations indicate that moving from CH 3 CN to H 2 O serves to stabilize the low-spin state of [Fe(L 1 )] 2+ , leading to a higher T 1/2 . Similar trends have been reported for other spin-crossover Fe II complexes and stem from the donor strength of the two solvents. 34 Importantly, 1a exhibits remarkable water and air stability, as the absorption spectra of this compound in deoxygenated water and aer four weeks in oxygenated water are identical (see Fig. S9 †).

Magnetic properties
To probe the magnetic properties of compounds 1a and 2a, variable-temperature magnetic susceptibility data were collected in the temperature range 5-60 C for aqueous solutions in a 9.4 T NMR spectrometer using the Evans method (see Fig. 4). 35 For 2a, c M T is constant over this temperature range, with an average value of c M T ¼ 3.63 cm 3 K mol À1 that corresponds to a high-spin, S ¼ 2 Fe II ion with g ¼ 2.20. In stark contrast, for 1a, c M T increases nearly linearly with increasing temperature, from a minimum value of 0.93 cm 3 K mol À1 at 5 C to a maximum value of 1.99 cm 3 K mol À1 at 60 C, indicative of thermally-induced spin-crossover. Note that the high-spin excited state contributes considerably to the overall magnetic moment of 1a at 5 C, as the observed value of c M T ¼ 0.93 cm 3 K mol À1 is signicantly higher than the theoretical value of 0 cm 3 K mol À1 for a solely populated S ¼ 0 ground state. Analogously, a mixture of low-spin and high-spin Fe II centers is present at 60 C, as evident from the signicant deviation of c M T ¼ 1.99 cm 3 K mol À1 from the average value of the high-spin analogue 2a.
Considering a value of c M T ¼ 0 cm 3 K mol À1 for a solely populated S ¼ 0 low-spin state and c M T ¼ 3.63 cm 3 K mol À1 for a solely populated S ¼ 2 high-spin state with g ¼ 2.20, the highspin molar fraction of Fe II centers in 1a was calculated as a function of temperature (see Fig. S14 †). A linear t to the data gives T 1/2 ¼ 325(1) K or 52(1) C. Moreover, the data were simulated using the regular solution model 36,37 to estimate thermodynamic parameters of DH ¼ 18.0(3) kJ mol À1 and DS ¼ 55.5(9) J K À1 mol À1 , which are similar in magnitude to related mononuclear spin-crossover Fe II complexes (see Fig. S15 †). 15,28c,36,38 To test our hypothesis that the low-spin state of [Fe(L 1 )] 2+ in 1a is stabilized in H 2 O relative to CH 3 CN, variable-temperature magnetic susceptibility data were collected for an acetonitrile solution of 1a, using the same procedure as described above (see Fig. S16 †). As observed in aqueous solution, c M T increases nearly linearly with increasing temperature, from 0.62 cm 3 K mol À1 at À42 C to 2.71 cm 3 K mol À1 at 60 C. Furthermore, a linear t to the data affords T 1/2 ¼ 17(1) C, which is 35 C lower than observed in H 2 O, and demonstrates the different donor strengths of the H 2 O and CH 3 CN (see Fig. S17 †).

Variable-temperature NMR spectroscopy
To further investigate the solution properties of compounds 1a, 1b, 2a, 2b, and 3a, variable-temperature 1 H NMR spectra were collected in CD 3 CN at selected temperatures. The 1 H NMR spectra of compounds 1b, 2b, and 3a resemble those of their respective ligands (see Fig. S18-S20 †) and show minimal changes in the temperature range 25-56 C, conrming diamagnetic electronic structures (see Fig. S21-S23 †). In contrast, the 1 H NMR spectra of 2a display nine paramagnetically shied resonances, consistent with time-averaged C 3 symmetry in CH 3 CN solution (see Fig. S24 †). At À1 C, these resonances span À18 to 225 ppm, typical for high-spin Fe II complexes. 10,12,21b,d,e,g,h,28c As the temperature is increased to 56 C, the peaks shi linearly toward the diamagnetic region. This Curie behavior (d f T À1 ) is characteristic of high-spin complexes and conrms that 2a remains S ¼ 2 over the entire temperature range. In contrast, the 1 H NMR resonances of 1a show anti-Curie behavior, shiing away from the diamagnetic region with increasing temperature (see Fig. S25 †). Specically, at À38 C, the proton resonances are dispersed between À2 and 13 ppm, barely beyond the diamagnetic region, suggesting primary population of an S ¼ 0 ground state. Increasing the temperature to 56 C results in an expansion of the chemical shi range to À25-150 ppm, indicative of thermal population of the high-spin excited state. An analogous trend is observed in the variable-temperature 1 H NMR spectra of 1a in D 2 O, though the resonances are broader and less shied than in CD 3 CN at analogous temperatures, giving a chemical shi range from À17 to 107 ppm at 56 C (see Fig. S26 †). These observations are consistent with the higher T 1/2 in H 2 O relative to CH 3 CN, as evident from solution magnetic measurements and UV-vis data.
In order to determine the effect of spin state on 19 F resonances, and to assess these compounds as candidates for 19 F MRS thermometry, we collected variable-temperature 19 F NMR Fig. 4 Variable-temperature magnetic susceptibility data for aqueous solutions of 1a (purple) and 2a (red), obtained in a 9.4 T NMR spectrometer using the Evans method. Error bars represent standard deviations of the measurements. spectra for aqueous solutions of 1a and 2a from 4 to 61 C, using triuoroethanol (TFE) as an internal standard (see Experimental section, Fig. S27, and Table S2 †). To better understand how the temperature dependence of 19 F NMR chemical shis is affected by the electronic spin state, and to quantify the hyperne shis of the paramagnetic Fe II compounds 1a and 2a, their corresponding Zn II analogues, 1b and 2b, were employed as diamagnetic references (see Table 2). 18c Importantly, the chemical shis of the uorine resonances of Zn II compounds 1b and 2b are effectively invariant to temperature changes (see Fig. 5, S28, and S29 †).
At 4 C, the 19 F NMR spectrum of the high-spin compound 2a displays a single resonance at À59.4 ppm vs. CFCl 3 that is shied +67.3 ppm from its diamagnetic Zn II analogue 2b. As the temperature is raised to 61 C, the chemical shi of the paramagnetic signal shis upeld to À71.4 ppm, closer to the 19 F resonance of its diamagnetic analogue, as expected for Curie behavior (see Fig. S30 and S31, and Tables S3 and S4 †). The observation of a single signal for 2a further supports the C 3 symmetry of the [Fe(L 2 )] 2+ cation in solution, as suggested by 1 H NMR spectroscopy. Analysis of the temperature dependence of the 19 F NMR chemical shi reveals a linear temperature dependence over 4-61 C following the equation d ppm ¼ À0.21 Â T À 58.8, affording a temperature coefficient 39 of CT ¼ À0.21(1) ppm per C (see Fig. 5, and Table 2). Since linewidth has a signicant effect on the precision of MRS probes, the value |CT|/FWHM (FWHM ¼ full width at half maximum) is also a useful measure of probe sensitivity. At 40 C, the uorine resonance of 2a exhibits a FWHM of 868 Hz, giving a |CT|/ FWHM ¼ 0.11 per C.
The 19 F NMR spectrum of 1a obtained at 4 C exhibits two resonances of equal intensity at À99.3 and À102.1 ppm vs. CFCl 3 (see Fig. S32, and Table S3 †), suggesting that the two 3-uoro-2-picolyl arms of L 1 are inequivalent on the NMR timescale. These peaks are shied +23.1 and +20.3 ppm from the diamagnetic Zn II analogue 1b (see Fig. S33, and Table S4 †), which exhibits two overlapping resonances centered at À122.3 ppm (see Fig. S28 †). Increasing the temperature to 61 C results in a downeld shi of the resonances of 1a to +51.3 and +44.8 ppm from 1b, consistent with the anti-Curie behavior observed in the corresponding 1 H NMR spectra. The 19 F chemical shi of both resonances for 1a vary linearly between 4 and 61 C following the equations d ppm ¼ 0.52 Â T À 101.7 and d ppm ¼ 0.45 Â T À 104.2, providing temperature sensitivities of CT ¼ +0.52(1) and +0.45(1) ppm per C, respectively (see Fig. 5, and Table 2). Fluorine resonances with the narrowest linewidths are obtained at 20 C, but the peaks broaden signicantly above 55 C (FWHM > 500 Hz). At 40 C, the uorine resonances each shows a value of |CT|/FWHM ¼ 0.87 per C.
The two 19 F NMR resonances of 1a exhibit 2.5-and 2.1-fold higher CT values than that of the high-spin 2a. Furthermore, the narrower linewidths of the resonances of 1a afford an 8-fold higher |CT|/FWHM value than 2a at 40 C. Remarkably, the two 19 F resonances of 1a represent 43-and 38-fold enhancement of temperature sensitivity compared to diamagnetic per-uorocarbons that have been employed for in vivo thermometry. 20 Despite the much narrower peak widths of the diamagnetic uorine resonances relative to those of 1a, the |CT|/FWHM value of 1a at 40 C is 2.9-fold higher owing to the strong temperature dependence of the chemical shi of its two resonances. These observations demonstrate that the use of spin-crossover complexes may provide an excellent strategy for improving the sensitivity of 19 F MR thermometers.
Furthermore, the separation between the two uorine resonances of 1a varies strongly with temperature, from 2.81 ppm at 4 C to 6.52 ppm at 61 C, following the linear relationship Dd ppm ¼ 0.069 Â T + 2.47 (see Fig. S34 †). This peak separation provides an internal method of correcting errors in the 19 F chemical shi that arise from complicating physiological effects, such as motion, magnetic susceptibility changes, and varying oxygen tension. 20 Overall, three temperature-dependent parameters of compound 1a can be followed for MR thermometry, namely the 19 F NMR chemical shis of two inequivalent uorine substituents, and the chemical shi difference between these signals.
To evaluate the efficacy of 1a and 2a in a physiological environment, 19 F NMR spectra were collected from 4 to 61 C on 13.4 and 15.0 mM solutions of 1a and 2a, respectively, in fetal bovine serum (FBS), using NaF as an internal standard (see Fig. S35 †). The 19 F NMR spectra in FBS are essentially identical to those recorded in H 2 O and provide the same CT values (see Fig. S36 and S37 and Tables 2 and S5 †). Plots of the temperature dependence of uorine chemical shis of compounds 1a and 2a in FBS are depicted in Fig. 6, where the chemical shis of the Fe II complexes have been referenced to the corresponding shis of Zn II analogues 1b and 2b in water (see Table S6 †). The linewidths for the resonance of 2a are similar in FBS and H 2 O, while 1a exhibits slightly narrower peaks in the high-temperature region (>30 C) in FBS compared to those in H 2 O, resulting in higher |CT|/FWHM values in FBS. Furthermore, both complexes remain intact while incubated with FBS for over 24 h, as evidenced by identical 19 F NMR spectra recorded at 25 C initially and aer 24 h (see Fig. S38 and S39 †). Taken together, these results demonstrate the stability of compounds 1a and 2a in a physiological environment and indicate that temperature measurements with +0.52(1) and À0.21(1) ppm per C sensitivity, respectively, can be achieved with these probes through chemical shi 19 F MR thermometry. Moreover, the excellent stability and favorable 19 F MR properties of 1a under physiological conditions suggest that this compound is a viable candidate for in vivo studies.
A comparison of the 19 F NMR properties of compounds 1a and 2a in CD 3 CN (see Fig. S40-S44 †), H 2 O and FBS is summarized in Table 2. The hyperne shi of the spin-crossover compound 1a is signicantly affected by the solvent, in contrast to high-spin 2a (see Tables S3 and S7 †). Along these lines, the resonances of 1a display a 1.3-fold higher temperature sensitivity in CD 3 CN than in H 2 O, which is consistent with a lower T 1/2 in CD 3 CN. These observations reect the pronounced effects of spin state on 19 F NMR chemical shi, as has been previously reported for transition metal porphyrin complexes. 18 Nevertheless, the results presented here provide a rare examination of spin state effects on 19 F NMR spectra across a series of metal complexes.

Conclusions
The foregoing results demonstrate the potential utility of paramagnetic Fe II complexes as chemical shi 19 F MR thermometers. Most importantly, we show that the sensitivity of 19 F MR thermometers can be improved by employing a temperature-dependent change in spin state, as illustrated in a series of Fe II complexes. To our knowledge, these complexes represent the rst examples of paramagnetic 19 F MR chemical shi agents proposed for thermometry applications. Future efforts will focus on in vitro and in vivo MRS thermometry experiments on these compounds and the synthesis of spin-crossover complexes with higher sensitivity by exploiting the chemical tunability of the tacn-based ligand scaffold.