Fluorinated Paramagnetic Chelates as Potential Multi-chromic ¹⁹F Tracer Agents

Abstract

A class of potential multi-chromic ¹⁹F imaging tracers is made by pairing metal ions with a fluorinated chelator. All fluorinated metal chelates emit a single ¹⁹F signal. Paramagnetic metal ions shifted the ¹⁹F signal frequency and made the ¹⁹F relaxation rates insensitive toward local chemical environment.

¹H and ¹⁹F are respectively the most and 2^nd most sensitive stable nuclei for magnetic resonance spectroscopy and imaging (MRS/I), each with a nuclear spin of ½. Although ¹H MRI has wide clinical usage, ¹⁹F MRI, is hardly used in medicine due to lack of suitable ¹⁹F imaging agents. Recently, there is a renewed interest in ¹⁹F MRI due to the need to track therapeutic agents (genes, cells, etc.) and to monitor biochemical reactions in vivo non-invasively.^1–10 Compared with ¹H, the ¹⁹F signal has negligible endogenous background and a much wider frequency range (~ 400 ppm) and thus is ideally suited for tracking objects and monitoring reactions in vivo. For various biomedical applications, it is highly desirable to monitor several objects simultaneously with ¹⁹F MRS/I. This requires a set of ¹⁹F imaging tracers, each emitting the ¹⁹F signal as a specific radio frequency.

One way to achieve ¹⁹F multi-chromicity is to use different fluorinated compounds that have distinct ¹⁹F chemical shifts.¹¹ This is essentially a “one synthesis, one compound, one frequency” approach, which is synthetically inefficient. Here, we develop a “one synthesis, one set of compounds, multiple frequencies” approach, which is synthetically much more efficient. This is achieved by synthesizing a fluorinated chelator FC (Fig. 1) and then pairing FC with various paramagnetic metal ions (M^x+). Paramagnetic metal ions have unpaired electrons whose magnetic moments can greatly influence nearby nuclear spins.^12–14 FC contains nine chemically identical fluorine atoms as the ¹⁹F signal emitter while the paramagnetic ion acts as the ¹⁹F signal modulator. This approach has several advantages. First, by synthesizing just one fluorinated chelator, multiple ¹⁹F signal frequencies can be generated. Second, paramagnetic ions can shorten the relaxation times of nearby nuclei. Shorter ¹⁹F relaxation times can lead to higher signal/noise ratio.^15–17 Third, paramagnetic ions can dominate the ¹⁹F relaxation times of fluorinated metal chelates and thus reduce their dependency on the local environment. Fourth, as a result of their chemical similarity, different FC-M^x+ will cause similar perturbation to the biodistribution of the various objects that they label.

Fig.1.

Structure of a fluorinated chelator FC

With these ideas in mind, a convenient synthesis of FC on a 5-g scale was developed (Supplementary Information). A set of di-and tri-valent metal ions, was easily chelated by FC. All these metal ions can bind to the macrocyclic chelator in FC with high affinity, with Bi³⁺ being the strongest and Ca²⁺ the weakest.¹⁸ The chelates were purified by HPLC using fluorinated columns. FC and all the metal chelates have excellent aqueous solubility due to low fluorine content (21.4% for FC). In contrast, two fluorinated chelators we previously synthesized had low aqueous solubility due to higher fluorine contents (39.6 & 45.9%).¹⁹

As expected, a frequency spread of 3708 Hz (7.89 ppm) was produced by pairing FC with different metal ions at the concentration of 50 mM (Fig. 2a). In all cases, there is a single un-split ¹⁹F signal. In a previous report on fluorinated lanthanide chelates, multiple ¹⁹F signals were observed.¹⁰ This difference is likely the result of the flexible linker between the fluorocarbons and the metal chelate. Diamagnetic ions (black bars), Bi³⁺, Hg²⁺, Y³⁺, Pb²⁺, La³⁺, Ca²⁺ and Cd²⁺, caused very little change in the ¹⁹F chemical shift relative to FC (empty bar). Among the paramagnetic ions (gray bars), Fe³⁺, Gd³⁺, Er³⁺, Dy³⁺, Ho³⁺ and Tb³⁺ caused significant ¹⁹F chemical shift change with Tb³⁺ being the most effective. Fig.3 plots the ¹⁹F spectra of FC-Tb³⁺, FC-Gd³⁺, FC-Y³⁺ and FC. Some metal ions, especially paramagnetic ions, also shorten the ¹⁹F relaxation times of the fluorinated metal chelates (Fig. 2b). Among the diamagnetic ions, only Bi³⁺ shortens ¹⁹F T₁ and T₂ significantly. The paramagnetic ions shorten the ¹⁹F relaxation times more effectively and can be classified into three groups. For Sm³⁺, Ce³⁺, Eu³⁺, Pr³⁺, Nd³⁺ and Yb³⁺, the relaxation time reduction is modest. In the case of Sm³⁺, ¹⁹F T₁ and T₂ actually increased slightly, as is the case with the diamagnetic Hg²⁺ and Y³⁺. For Dy³⁺, Tb³⁺, Ho³⁺and Er³⁺, the relaxation time reduction is dramatic, around 95%. For Gd³⁺, Fe³⁺, Ni³⁺ and Cu²⁺, the relaxation time reduction is overwhelming, around 99%. As expected, the most effective metal ion to shorten the ¹⁹F relaxation times is Gd³⁺, which reduces T₁ and T₂ to 4.2 ms and 1.7 ms in FC-Gd³⁺, respectively, from 1330 ms and 701 ms in FC.

Fig.2.

Effects of metal ions on the ¹⁹F signal frequency (a, upper) in terms of chemical shift in ppm unit; relaxation times (b, middle; left: T₁; right: T₂); and relaxation time reduction caused by O₂ (c, lower; left: T₁; right: T₂). Each sample contains 50 mM fluorinated metal chelate in phosphate-buffered saline (PBS), pH 7.0, 25°C. All samples are bubbled with a flow of pure N₂ (for a, b and c) or O₂ (for c) for 10 min at 0°C before measurement. The relaxation time reduction is defined as: [T_i(N₂)-T_i(O₂)]/T_i(N₂) ×100%, with i = 1 or 2. For experimental details, see Supplementary Information.

Fig.3.

¹⁹F NMR spectra of 50 mM of FC-Tb³⁺, FC-Gd³⁺, FC-Y³⁺, FC at pH7.0, 25°C in PBS saturated with N₂.

Then a study was carried out to see whether the intramolecular paramagnetic ion makes ¹⁹F T₁ and T₂ insensitive toward oxygen. Oxygen is paramagnetic and is known to reduce ¹⁹F relaxation times both in vitro²⁰ and in vivo.²¹ To test the effect of oxygen, ¹⁹F T₁ and T₂ were measured in solutions saturated with N₂ and O₂, respectively. It is found that, as expected, the paramagnetic oxygen resulted in dramatic reduction in ¹⁹F relaxation times in most cases (Fig. 2c). However, in the case of Tb³⁺, Ho³⁺, Dy³⁺, Er³⁺, Gd³⁺, Fe³⁺, Ni³⁺ and Cu²⁺, the relaxation time reduction caused by saturated oxygen is 5% or less. It is interesting to note that the diamagnetic Bi³⁺ also stabilizes T₁ and T₂ against oxygen.

Encouraged by this result, studies were carried out to verify whether the ¹⁹F signal frequency and relaxation times of paramagnetic fluorinated chelates have weaker dependency than those of diamagnetic fluorinated chelates on pH, temperature, serum protein and human sera. Here, the paramagnetic FC-Tb³⁺ and FC-Gd³⁺ are compared with the diamagnetic FC-Y³⁺ and FC. The results are summarized in Tables 1 and 2. First, pH, temperature, bovine serum albumin (BSA) and human sera produced little change in ¹⁹F chemical shift in both para- and diamagnetic compounds (Δδ < 1 ppm in all cases except for FC-Tb³⁺ in 10% BSA, where Δδ = 1.9 ppm). Second, pH, temperature, BSA and human sera produced much smaller change ¹⁹F relaxation times in the two paramagnetic compounds than in the two diamagnetic compounds. Thus, the intra-molecular paramagnetic ion indeed drastically shortens the ¹⁹F relaxation times and makes them rather insensitive toward environmental fluctuations.

Table 1.

pH and temperature effects on the ¹⁹F chemical shift and relaxation times.²² All samples contain 50 mM fluorinated metal chelate in PBS after bubbled with a flow of pure N₂ for 10 min at 0°C before measurement. The changes are relative to the same sample at pH7, 25°C in PBS. For accuracies of T₁ and T₂ measurements, see Table S1 of Supplemental Information.

Changed parameters	Chelates	pH6	pH8	10°C	35°C	45°C
Δδ (ppm)	FC-Tb3+	0.2	0.2	0.6	−0.4	−0.8
	FC-Gd3+	0.1	0.6	0.5	−0.1	−0.1
	FC-Y3+	0	0	0	−0.1	−0.1
	FC	0	0	0.1	−0.1	−0.1
ΔT1 (ms)	FC-Tb3+	0.7	−0.9	−6.6	3.3	8.7
	FC-Gd3+	−0.3	−0.2	−0.1	−0.2	−0.3
	FC-Y3+	−83	−16	−462	239	619
	FC	101	110	−348	−15	50
ΔT2 (ms)	FC-Tb3+	4.8	5.1	−4.7	1.2	4.3
	FC-Gd3+	−0.6	−0.6	−0.3	−0.4	−0.4
	FC-Y3+	451	413	−63	748	728
	FC	89	125	−30	7	−82

Table 2.

The effect of serum proteins on the ¹⁹F chemical shift and relaxation times.²² All samples contain 50 mM fluorinated metal chelate in either 10% of BSA in PBS or human sera at 25°C. Samples were bubbled with a flow of pure N₂ or O₂ for 10 min a t 0°C before measurement. The changes are relative to the same sample at pH7, 25°C in PBS bubbled with pure N₂ for 10 min.

Changed parameters	Chelates	10% BSA in PBS	Human sera
			Under N2	Under O2
Δδ (ppm)	FC-Tb3+	1.9	0.1	0.1
	FC-Gd3+	0.8	0.3	0.3
	FC-Y3+	0	−0.1	0
	FC	0	0	0.1
ΔT1 (ms)	FC-Tb3+	−2.5	7.1	2.5
	FC-Gd3+	0.3	0	0.2
	FC-Y3+	−314	−303	−718
	FC	−219	−83	−490
ΔT2 (ms)	FC-Tb3+	−31.1	−29.8	−30.9
	FC-Gd3+	−1.1	−0.8	−1.0
	FC-Y3+	−644	−569	−593
	FC	−600	−492	−487

Conclusions

A “one synthesis, one set of compounds, multiple frequencies” approach has been developed for making fluorinated metal chelates. In such a fluorinated metal chelate, the fluorocarbon sphere is the ¹⁹F signal emitter while the metal chelate is the ¹⁹F signal modulator, which plays three roles: shifting ¹⁹F resonance frequency, which enables collecting ¹⁹F signals at different radio frequencies; reducing ¹⁹F relaxation times, which can improve the signal/noise ratio; dominating the ¹⁹F relaxation process, which makes the ¹⁹F signal insensitive toward environmental fluctuations. These features make fluorinated paramagnetic chelates potential tracer agents for multi-chromic ¹⁹F MRS/I.

The work presented here is just a proof of concept. Further studies, such as introducing more fluorine atoms to improve signal intensity, varying the ¹⁹F-M^x+ distance and the ¹⁹F/M^x+ ratio to generate larger ¹⁹F frequency shift and to further reduce the environment-dependency of relaxation times, are under active development.

A potential clinical application of multi-chromic ¹⁹F tracers is to use them to label different therapeutic agents, such as cells, antibodies and drug delivery vehicles. By labelling different objects with different fluorinated metal chelates, each object can be tracked at a specific radio frequency.