An Fe complex for ¹⁹F magnetic resonance-based reversible redox sensing and multicolor imaging

Abstract

We report a first-in-class responsive, pentafluorosulfanyl (–SF₅)-tagged ¹⁹F MRI agent capable of reversibly detecting reducing environments via an Fe^II/III redox couple. In the Fe^III form, the agent displays no ¹⁹F MR signal due to paramagnetic relaxation enhancement-induced signal broadening; however, upon rapid reduction to Fe^II with one equivalent of cysteine, the agent displays a robust ¹⁹F signal. Successive oxidation and reduction studies validate the reversibility of the agent. The –SF₅ tag in this agent enables ‘multicolor imaging’ in conjunction with sensors containing alternative fluorinated tags and this was demonstrated via simultaneous monitoring of the ¹⁹F MR signal of this –SF₅ agent and a hypoxia-responsive agent containing a –CF₃ group.

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Introduction

Antioxidants, or reducing agents, play a pivotal role in maintaining biological redox balance and counteracting oxidative stress. In a biological setting, common antioxidants include ascorbic acid, which reduces nitrite ions to nitric oxide in the stomach and protects agains reactive oxygen species (ROS); vitamin E, which scavenges free radicals formed during metabolism; and thiol-containing molecules. These thiols, including cysteine and glutathione, help maintain redox balance through their ability to be reversibly oxidized to form disulfides. Reductive stress is a condition in which the cellular redox balance is shifted to a more reducing state. For example, reducing environments are associated with cellular hypoxia, impair signalling functions, promote mitochondrial dysfunction, increase apoptosis, and decrease cell survival.^1,2 Reductive stress is associated with a number of pathologies, including muscular dystrophy, cancer, Alzheimer's disease, rheumatoid arthritis, and alcohol abuse.^3,4

Many techniques currently exist to detect biological reductants and reducing environments, including positron emission tomography (PET)^5,6 and fluorescence imaging.^7,8 While these are powerful techniques, PET requires the use of ionizing radiation and fluorescence imaging has limited depth penetration for in vivo imaging.⁹ Thus, alternative techniques that can be readily applied and minimize the use of radioactive materials for in vivo sensing are needed. Traditionally, ¹H magnetic resonance imaging (MRI) is used to obtain anatomical and physiological images with high temporal resolution and depth penetration without the use of ionizing radiation; however, abundant endogenous ¹H signals deriving from water and lipids results in low imaging specificity. As an alternative, fluorine (¹⁹F) MRI provides the same excellent depth penetration, and importantly, higher specificity as there is no detectable endogenous fluorine present in the body. Additionally, fluorine-19 is 100% isotopically abundant, and contains high MR receptivity that nears that of ¹H (83% of ¹H).¹⁰ In addition to its generally favorable magnetic resonance properties, the ¹⁹F nucleus can display MR signals over a broad range of chemical shifts (>350 ppm).¹¹ A major advantage of molecular MR imaging agents is the exquisite ability to design selective analyte-responsive agents. However, this is partially counter-balanced by high limits of detection on the order of 10⁻⁴ M or more, which is a barrier to the application of small molecule agents. Regardless, elaborating new mechanisms for MR-based sensing with fluorinated small molecules will inform the development of MR sensors with much greater fluorine density (polymers and other macromolecules, nanoparticles) that provide the fluorine concentrations applicable to in vivo detection.

A number of transition metals and lanthanides have been used for redox sensing using magnetic resonance techniques including Eu^II/III, Mn^II/III, Fe^II/III, Co^II/III, and Cu^I/II.^12–37 In order to monitor dynamic redox events in biology, a reversible sensing agent is desirable and this requires a ligand that can effectively cage and sequester a metal ion in two oxidation states. Interest in the use of Fe^II/III has increased due to the superior biocompatibility of iron relative to other redox active metals and the ability to achieve redox reversibility in biological contexts.^10,30–35 From a magnetic resonance perspective, the differences in electronic relaxation times of the different oxidation states lead to modulation of MR properties, with high spin Fe^III complexes exhibiting substantial ¹⁹F MR signal attenuation due to strong paramagnetic relaxation enhancement (PRE) and Fe^II complexes exhibiting robust and detectable ¹⁹F MR signals due to weak PRE. To the best of our knowledge, while Fe complexes have been reported for ¹⁹F MRI applications,^38–45 this is the first demonstration of a reversible, redox-responsive Fe complex for ¹⁹F MR biosensing.

Most small molecule ¹⁹F MRI agents utilize –CF₃ containing moieties as ¹⁹F tags; however, the large ppm range that is available for the ¹⁹F nucleus opens up opportunities to design imaging agents with chemical shift values that can be differentiated via chemical shift-specific magnetic resonance imaging, enabling ‘multicolor’ (or chemical shift-selective) imaging of multiple species containing distinct ¹⁹F frequencies within the same specimen. The pentafluorosulfanyl (–SF₅) moiety is promising in this respect due to its biostability⁴⁶ and distinct chemical shift relative to –CF₃ (∼+60 ppm for –SF₅, ∼−70 ppm for –CF₃). One disadvantage of an –SF₅ tag is the presence of inequivalent fluorine atoms that reduce signal density and result in doublet (equatorial fluorine atoms) and quintet (axial fluorine atom) signals. Regardless, the value of the –SF₅ tag has been successfully demonstrated in drug tracking studies,⁴⁷ though it has never been incorporated for biosensing applications as will be described.

Herein, we report the first Fe-based redox responsive ¹⁹F MRI agent, Fe^IIIDO3ASF₅ (Scheme 1), capable of reversibly detecting reducing environments. This agent contains a novel –SF₅ tag that has potential to be used simultaneously alongside a –CF₃ containing agent for multicolor ¹⁹F MR imaging.¹⁴

Scheme 1 Design strategy for reversible redox responsive ¹⁹F MRI probe exploiting Fe^II/III redox chemistry. The initial “off” ¹⁹F signal “turns-on” following reduction of Fe^III to Fe^II by cysteine and “turns-off” following oxidation by H₂O₂. An –SF₅ tag provides a means to simultaneously image these agents with –CF₃ and other ¹⁹F-tagged species.

Results and discussion

DO3ASF₅ was synthesized from ^tBuDO3A, chloroacetyl chloride, and 4-(pentafluorosulfanyl)aniline in good yield (48%) following tert-butyl ester deprotection (Scheme S1†). The ligand was complexed with anhydrous FeCl₃ to obtain Fe^IIIDO3ASF₅ (75%) or with aqueous Fe(BF₄)₂ to obtain Fe^IIDO3ASF₅ (64%). DO3ASF₅ and Fe^II/IIIDO3ASF₅ were purified using C18 reverse-phase chromatography. Full details are provided in the ESI.† Previous reports have shown that Fe^IIIDO3A complexes form neutral seven-coordinate complexes derived from three carboxylate and four nitrogen donors.⁴⁸ We propose that Fe^IIIDO3ASF₅ forms a similar neutral structure due to its low solubility in water and the one proton difference seen by mass spectrometry between Fe^IIIDO3ASF₅ and Fe^IIDO3ASF₅. These observations are reinforced by a remarkable increase in water solubility of Fe^IIDO3ASF₅, which would have a water solubilizing free carboxylic acid and only two carboxylate donors directly bound to the metal. The possibility of the amide group also coordinating cannot be ruled out since seven- and eight-coordinate Fe^II complexes have been reported.^49–51

Cyclic voltammetry of 1 mM Fe^IIIDO3ASF₅ in 0.1 M KCl was conducted to characterize the complex's ability to switch between the Fe^III and Fe^II oxidation states. Fe^IIIDO3ASF₅ displayed a half wave potential, E_1/2, at 389 mV vs. normal hydrogen electrode (NHE, Fig. 1A) with an anodic peak potential, E_pa, at 358 mV vs. NHE and a cathodic peak potential, E_pc, at 420 mV vs. NHE, consistent with a reversible redox process. This potential is similar to an Fe^IIIDOTA peptide conjugate reported in the literature (E_1/2 = 396 mV vs. NHE).⁵² With an E_1/2 of 389 mV, both the ferric and ferrous oxidation states of FeDO3ASF₅ should be biologically accessible, as this potential is near that of the one electron reduction of H₂O₂ at pH 7 (380 mV vs. NHE).⁵³ Compared to reversible redox responsive agents for ¹H MRI (FePyC3A E_1/2 = 230 mV vs. NHE,³¹ MnHBET E_1/2 = 356 mV vs. NHE),²⁸¹H/¹⁹F MRI (MnHTFBED E_1/2 = 250 mV vs. NHE),²⁹ and CEST (CoTPT, E_1/2 = −107 mV vs. NHE),²¹Fe^IIIDO3ASF₅ has a slightly higher potential, indicating our agent favors the reduced state more than these previously reported agents. The cyclic voltammograms of Fe^IIIDO3ASF₅ were similar when the scan speed varied from 50–400 mV s⁻¹, demonstrating the complex is stable and exhibits reversible redox behavior within the given scan speed range.

Fig. 1 (A) Cyclic voltammograms of 1 mM Fe^IIIDO3ASF₅ in 0.1 M KCl recorded at various scan speeds (50–400 mV s⁻¹). (B) UV-vis absorption spectra of 100 μM Fe^IIIDO3ASF₅ in pH 7.4 buffer (black), 100 μM of Fe^IIDO3ASF₅ in pH 7.4 buffer (gray), and 100 μM Fe^IIDO3ASF₅ in pH 7.4 buffer generated in situ from Fe^IIIDO3ASF₅ with one equivalent of cysteine (green). (C) UV-vis absorption spectra of 25 μM Fe^IIIDO3ASF₅ in deoxygenated pH 7.4 buffer (black) with five equivalents cysteine after five minutes (blue), five equivalents DTT after five minutes (green), and five equivalents of GSH after ten minutes (red). (D) The natural log of the change of absorbance at 310 nm vs. time of the reduction of 25 μM Fe^IIIDO3ASF₅ with 150–350 μM of cysteine. Slopes of linear regression fits correspond to the pseudo first-order reaction rates, k_obs. (E) Plot of k_obsvs. concentration of cysteine, slope of linear regression corresponds to second order rate constant. 5 mM HEPES (pH 7.4) with 10 mM KNO₃ was used for all UV-vis experiments.

The absorption spectrum for Fe^IIIDO3ASF₅ displays a characteristic charge-transfer band (extinction coefficient, ε = 3280 M⁻¹ cm⁻¹) at 310 nm (Fig. 1B, black trace). The Fe^IIDO3ASF₅ complex displays a lower absorbance at 310 nm relative to the Fe^III complex (Fig. 1B, gray trace). Therefore, we performed an initial screen of different reducing agents and monitored the Fe^III to Fe^II change using UV-vis spectroscopy. Cysteine, dithiothreitol (DTT), and glutathione (GSH) were selected since they are either commonly used in biological assays (DTT) or because they are biologically relevant agents (cysteine and GSH) that have been shown to reduce other metal MRI agents.^16,54 The initial screening of the three reducing agents showed that DTT and cysteine caused the disappearance of the absorbance peak at 310 nm within 5 minutes. This disappearance is assigned to the reduction of the Fe^III complex to Fe^II since the resulting absorbance matches the spectrum of Fe^IIDO3ASF₅ (Fig. 1B and C). In contrast, when 5 equivalents of GSH was used, the reduction was incomplete after 10 minutes (Fig. 1C). However, full reduction was observed when the GSH concentration was increased to 25 equivalents (Fig. S1†). Notably, Fe^IIIDO3ASF₅ was reduced within five minutes by just one equivalent of cysteine (green trace, Fig. 1B), an improvement over our previously reported macrocyclic Cu^II-based sensor that required three equivalents of cysteine for full reduction.¹⁶

Given the promising reduction results and the biological relevance of cysteine, the kinetics of cysteine-mediated reduction were measured via UV-vis (Fig. S2†). The measurements were determined under pseudo-first order conditions with excess (6–14-fold) cysteine in HEPES buffer (pH 7.4) (Fig. 1D). The reduction is first-order in cysteine and gives a second-order rate constant of k = 74 M⁻¹ s⁻¹ (Fig. 1E). This rate constant is ∼50-fold faster than the redox activated ¹H agent, FePyC3A. This is consistent with Fe^IIIDO3ASF₅ having a higher E_1/2 (389 mV compared to 230 mV vs. NHE).³¹

We further characterized these complexes by measuring their solution magnetic moments (μ_eff, Evans NMR method).^55,56Fe^IIIDO3ASF₅ exhibited a μ_eff of 5.7, consistent with a high spin, S = 5/2 Fe^III species. With this spin state, we expect strong PRE-induced signal attenuation of the –SF₅ tag in this Fe^III complex, resulting in the oxidized state being “off” in ¹⁹F MR measurements. Fe^IIDO3ASF₅ had a μ_eff of 4.5 consistent with a high spin S = 2 species.⁵⁷ While containing four unpaired electrons, the electronic relaxation times of high spin Fe^II species are typically very short, making them inefficient for PRE.¹⁰ Thus we expect to observe significantly less signal broadening for Fe^IIDO3ASF₅ compared to the Fe^III complex.

The ¹⁹F NMR spectrum of 1 mM Fe^IIIDO3ASF₅ in deoxygenated pH 7.4 HEPES (Fig. S3†) contained a severely broadened peak centered at +63.7 ppm with a very low signal-to-noise ratio (SNR). On the other hand, the ¹⁹F NMR spectrum of 1 mM Fe^IIDO3ASF₅ contained a doublet at +63.4 ppm (Fig. S4†), similar to ligand DO3ASF₅, which displayed a doublet at +63.2 ppm. The quintet of Fe^IIDO3ASF₅ was not visible even at concentrations as high as 2.5 mM, and therefore, our discussion will focus on the doublet. To confirm that the reduction can be monitored via¹⁹F NMR, 5 equivalents of cysteine were introduced to a sample of Fe^IIIDO3ASF₅, which caused the broadened signal to sharpen and increase in intensity, resulting in a doublet at +63.4 ppm, equivalent to that of Fe^IIDO3ASF₅ (Fig. S5†). The relaxation times for Fe^IIIDO3ASF₅ alone could not be measured due to the severely broadened nature of its ¹⁹F NMR peaks. Given this, the ¹⁹F longitudinal (T₁) and transverse (T₂) relaxation times of purified Fe^IIDO3ASF₅ and in situ generated Fe^IIDO3ASF₅ following reduction of Fe^IIIDO3ASF₅ were measured at room temperature. Fe^IIDO3ASF₅ had T₁ and T₂ values of 155 and 6.3 ms, respectively. The relaxation times measured for in situ generated Fe^IIDO3ASF₅ (Fe^IIIDO3ASF₅ + 5 equivalents DTT) were in strong agreement (T₁ = 152 ms, T₂ = 5.7 ms).

We next investigated the ability of our complex to reversibly detect environmental redox changes. A 0.5 mM sample of Fe^IIIDO3ASF₅ in deoxygenated HEPES was subjected to multiple reduction and oxidation cycles using cysteine and H₂O₂, respectively (Fig. 2). Initially, the 0.5 mM sampled displayed no ¹⁹F MR signal (Fig. 2B), exhibited an absorption spectrum consistent with the Fe^III species (Fig. 2A), and had the deprotonated Fe^IIIDO3ASF₅ mass (ESI⁺m/z = 659). After adding one equivalent of cysteine, a ¹⁹F doublet appeared at +63.4 ppm (Fig. 2B), the Fe^III was fully reduced to Fe^II according to UV-vis (Fig. 2A), and the protonated Fe^IIDO3ASF₅ mass was observed (ESI⁺m/z = 660). The same sample was reacted with 10 equivalents of H₂O₂, regenerating Fe^IIIDO3ASF₅ which caused a broadening in the ¹⁹F NMR signal (Fig. 2B), a reappearance of the Fe^III charge-transfer band (Fig. 2A), and a compound with an m/z of 659. This entire process was repeated multiple times with similar results. Oxidation from Fe^II to Fe^III with H₂O₂ occurs rapidly, with complete conversion occurring within 5 minutes in the presence of as low as one equivalent of H₂O₂ as monitored by UV-vis (Fig. S6†).

Fig. 2 Repeated reduction and oxidation of Fe^IIIDO3ASF₅ analyzed via (A) UV-vis (0.1 mM complex, absorbance value at 310 nm) and (B) ¹⁹F NMR (0.5 mM) using cysteine (“Cys”) and H₂O₂ in 5 mM HEPES buffer (pH 7.4) with 10 mM KNO₃.

The kinetic and chemical stabilities of Fe^IIDO3ASF₅ and Fe^IIIDO3ASF₅ were then examined under multiple conditions. The stability of Fe^IIIDO3ASF₅ was studied in the presence of various biologically relevant metal salts, human serum, and different pH values using UV-vis spectroscopy or ¹⁹F NMR. The stability of Fe^IIIDO3ASF₅ (100 μM) in the presence of different metal salts (100 mM NaCl, 100 mM KCl, 10 mM CaCl₂, 10 mM MgCl₂, and 100 μM ZnCl₂) was monitored before and after incubation by UV-vis (Fig. S7†). The results showed that Fe^IIIDO3ASF₅ has high stability with no significant absorbance changes observed after 24 h of incubation at 37 °C. Given that Fe^IIIDO3ASF₅ has a E_1/2 = 389 mV vs. NHE, it may be sensitive to biological reductants while circulating through the body causing the complex to exist in the Fe^II state. If this is the case, the Fe^IIDO3ASF₅ complex will dominate, and no selective “turn-on” will be possible. To evaluate this possibility, we monitored Fe^IIIDO3ASF₅ in human serum (HS) by ¹⁹F NMR to estimate its redox sensitivity in that environment. No ¹⁹F NMR signal changes were observed for a solution of Fe^IIIDO3ASF₅ in 80% HS (20% D₂O) or 8% HS (20% D₂O) in 1X Minimum Essential Medium (MEM) after incubating at 37 °C for 24 h (Fig. S8 and S9†). Fe^IIDO3ASF₅ and Fe^IIIDO3ASF₅ were also stable under acidic (pH 6) conditions as evaluated by ¹⁹F NMR (Fig. S10–S12†). The stability of Fe^IIDO3ASF₅ under acidic conditions matched well with the in situ generated Fe^IIDO3ASF₅ (Fig. S11 and S12†). The stability of Fe^IIDO3ASF₅ with metal cations was determined with ¹⁹F NMR due to the low absorbance at wavelengths >300 nm of Fe^IIDO3ASF₅ (Fig. S13–S17†). Similar to the Fe^III complex, Fe^IIDO3ASF₅ showed good stability in the presence of abundant metal cations. The air stability of Fe^IIDO3ASF₅ was also evaluated in HEPES buffer by UV-vis. Upon exposure to atmospheric oxygen, only minimal absorbance changes were observed over 18 h (Fig. S18†) demonstrating that the complex is relatively stable to oxygen following reduction.

We measured the ¹H longitudinal relaxivity (r₁) and transverse relaxivity (r₂) of Fe^IIIDO3ASF₅ at 60 MHz to gauge the potential presence of an inner sphere water molecule in this complex. The r₁ and r₂ were found to be 0.37 mM⁻¹ s⁻¹ and 0.39 mM⁻¹ s⁻¹, respectively. The r₁ of Fe^IIIDO3ASF₅ is ∼3–22-fold less than many of the previously reported r₁ values for Fe(III) relaxation agents.⁵⁸ These lower values are likely because the metal center is coordinatively saturated by the macrocyclic ligand framework and contains no exchangeable protons, which leaves outer-sphere proton relaxation as the only possible mechanism.⁵⁹ This coordinatively saturated structure is consistent with the observed high kinetic stability of the complexes.

To validate our –SF₅ labelled agent's ability to “turn-on” and be monitored in concert with a –CF₃ labelled agent, Fe^IIIDO3ASF₅ and previously reported hypoxia sensor CuATSM-F₃ (Fig. S19†)¹⁴ were subjected to ¹⁹F MRI using a 7 T scanner. Samples were imaged both at the –SF₅ doublet frequency (282.588 MHz) and the –CF₃ frequency (282.550 MHz). As shown in Fig. 3, 3 mM Fe^IIIDO3ASF₅ only provides a signal at the –SF₅ frequency when reduced with one equivalent of cysteine (SNR = 25.38); no signal is seen in this sample at the –CF₃ frequency (SNR = 1.99). CuATSM-F₃ was imaged alongside Fe^IIIDO3ASF₅ to show the agents will only provide signals when reduced and imaged using their respective imaging frequency. Specifically, 5 mM CuATSM-F₃ displayed a signal when reduced with one equivalent of sodium dithionite (Na₂S₂O₄) using the –CF₃ frequency (SNR = 28.92) and no signal when imaged at the –SF₅ frequency (SNR = 1.26). We note that CuATSM-F₃ is unable to provide a “turn-on” response to cysteine, as more reducing conditions typically associated with severe hypoxia are needed to activate this agent (Fig. S20†). The reversibility of Fe^IIIDO3ASF₅ can also be monitored using MR imaging (Fig. S21†) as initially no signal is observed for 3 mM Fe^IIIDO3ASF₅ (SNR = 2.04), which then “turns-on” with one equivalent of cysteine (SNR = 25.38), and “turns-off” with hydrogen peroxide (SNR = 3.68). Finally, due to the shorter T₁ of Fe^IIDO3ASF₅, using a rapid acquisition with relaxation enhancement (RARE) pulse sequence we were able to triple the number of averages for the –SF₅ image compared to the –CF₃ image. This resulted in the Fe complex displaying a similar SNR when compared to reduced CuATSM-F₃ even though the fluorine concentration was smaller (12 mM ¹⁹F for Fe^IIIDO3ASF₅ and 15 mM ¹⁹F for CuATSM-F₃).

Fig. 3 ¹⁹F MRI phantoms of 5 mM CuATSM-F₃ in the presence and absence of Na₂S₂O₄ and 3 mM Fe^IIIDO3ASF₅ in the presence and absence of cysteine. Images were obtained at both the –CF₃ (282.550 MHz) and –SF₅ (282.588 MHz) frequencies. Acquisition time for all images was 15 minutes.

Conclusions

In conclusion, Fe^IIIDO3ASF₅ is a redox-responsive ¹⁹F MR agent that can undergo reversible redox chemistry and be detected using the distinct ¹⁹F frequency associated with the biostable –SF₅ moiety. Conversion from a high spin S = 5/2 Fe^III species to a high spin S = 2 Fe^II species following cysteine addition provides a mechanism for MR signal “turn-on”. Oxidation following addition of H₂O₂ provides a mechanism for signal “turn-off”. While these species are physiologically present at micromolar concentrations or below in the extracellular space, and thus may be difficult for detection by MRI, previous work in MRI-based redox imaging suggests a huge potential for monitoring redox environments associated with inflammation or hypoxia using MR imaging agents.³¹ In proof-of-concept experiments, we demonstrate that this complex can be imaged in concert with the –CF₃ tagged hypoxia-responsive agent CuATSM-F₃, opening up possibilities for multiplexed biosensing using ¹⁹F MR. Ongoing studies include investigating the effects of ligand structure on Fe redox reactivity and biosensing properties and further development of agents with unique tags for multicolor imaging.

Data availability

This data is archived in the Texas Data Repository, https://dataverse.tdl.org/dataverse/emilyque/.

Author contributions

RTK, RTR, and ELQ designed experiments. RTK, RTR, and DJC performed experiments and characterized compounds. RTK, RTR, DJC, and ELQ all contributed to data analysis and manuscript writing.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the National Science Foundation (1945401) (ELQ), the Welch Foundation (F-1883) (ELQ), a UT Austin Graduate Student Summer Fellowship (RTK), and a UT Austin Provost's Graduate Excellence Fellowship (DJC). We acknowledge the Biomedical Imaging Center at UT Austin for access to their facilities and Que Lab members for helpful discussions.

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: https://doi.org/10.1039/d2sc05222a

‡ These authors contributed equally to this work.

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