19F PARASHIFT Probes for Magnetic Resonance Detection of H2O2

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Article 19

F PARASHIFT Probes for Magnetic Resonance Detection of HO and Peroxidase Activity 2

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Meng Yu, Bailey S. Bouley, Da Xie, José S. Enriquez, and Emily L. Que J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05685 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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Journal of the American Chemical Society

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F PARASHIFT Probes for Magnetic Resonance Detection of H2O2 and Peroxidase Activity Meng Yu, Bailey S. Bouley, Da Xie, José S. Enriquez, Emily L. Que* Department of Chemistry, University of Texas at Austin, Austin, Texas 78712-1224, United States. ABSTRACT: Elevated levels of reactive oxygen species and peroxidase expression are often associated with inflammation and inflammatory diseases. We developed two novel Co(II) complexes that can be used to detect oxidative activity associated with inflammation using 19F magnetic resonance imaging (MRI). These agents display a large change in 19F chemical shift upon oxidation from Co(II) to Co(III), facilitating selective visualization of both species using chemical shift and relaxation time selective pulse sequences. This large chemical shift change is attributed to a large magnetic anisotropy in the high spin Co(II) complexes. Importantly, the differing reactivity of the two agents allows for detection of either H2O2 production and/or the activity of peroxidase enzymes, providing two useful platforms for 19F MR hot spot imaging of oxidative events associated with biological inflammation.

INTRODUCTION Oxidative activity in biological systems is essential and associated with several diverse physiological processes.1-5 In particular, inflammation accompanying the defensive immune response is often accompanied by increased concentrations of reactive oxygen species.6 Further, elevated expression of oxidative enzymes including peroxidases is observed in atherosclerosis, cancer, cystic fibrosis, Alzheimer’s, and other diseases involving tissue inflammation.7,8 In order to monitor oxidative activity in vivo, magnetic resonance imaging is an attractive modality due to its good penetration depth and spatial resolution, and lack of use of ionizing radiation. To this end, a number of groups have reported oxidation-responsive probes for use in 1H-based magnetic resonance imaging (MRI) including probes based off of Gd3+, Eu2+, Mn2+, and Co2+.9-16 While 1H MRI provides great sensitivity and high-resolution 3D images, the intrinsic large and heterogeneous background associated with this imaging modality complicates the interpretation of 1H MR images taken with exogenous bioresponsive probes. 19 F MRI provides a promising alternative to 1H MRI as all detected 19F signal derives from exogenous probes. In this way, 19F signals are detected as ‘hot spots’ that highlight areas of interest against a zero background signal and whose signal is directly proportional to the 19F concentration. 19F MRI with nanoformulations has been employed in vivo with fruitful outcomes.17-22 However, only a few 19F MR agents have been reported that are responsive to different redox environments via toggling between diamagnetic and paramagnetic states in response to either reduction or oxidation at the metal center.23-25 These agents rely on paramagnetic relaxation enhancement (PRE) to modulate 19F signal that requires close proximity of fluorines to the metal center in order to maximize the

relaxation effect and quench the 19F signal in the paramagnetic oxidation state. However, the chemical shift does not alter significantly upon change in oxidation state in these probes, which limits the ability to detect and differentiate both the oxidized and reduced states. For biological redox mapping, an ideal probe would display a large change in chemical shift (ΔδF) following oxidation or reduction. Distinct, detectable signals for both the oxidized and reduced states would then enable quantification since probes based solely on signal intensity change require a second reference.26 Diamagnetic 19F chemical shift probes have been widely explored and they typically function through the change of chemical environment around the 19F nuclei; however, these probes generally suffer from low sensitivity due to their limited fluorine density or small chemical shift change (∆δF ≤ 2 ppm).27-29 While small chemical shift changes are readily distinguished by NMR, differentiation of these by MRI is practically more challenging due to greater signal broadening and lower spectral resolution in typical scanners. Thus, producing sensors with large changes in chemical shift in response to analyte is desirable to able to distinguish both species in imaging experiments. Another commonly employed strategy to induce changes in chemical shift is incorporating metal ions with large magnetic anisotropy in order to further enhance the chemical shift through pseudocontact shift (PCS) interactions. PCS is highly dependent on the nature of metals as well as the relative orientation between nuclei of interest and the magnetic dipolar axis of the metal ion. While lanthanide ions are most commonly used to induce large changes in chemical shift,26,30,31 most are redox inert in the biological redox regime, with Eu(II) systems being an exception if chelated by the appropriate ligands.25,32 Co(II)-based systems are intriguing alternatives con-

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sidering the ability of Co(II) to induce large changes in 1H

prolonged stirring of 2 in basic aerated water (pH = 8) followed by purification. The slow oxidation rate of 1 together with purification difficulty prevented isolation of pure oxidized product of 1.

Scheme 1. CoII-based 19F MR probes for bio-oxidation. chemical shifts,33-35 as well as its ability to undergo oxidation to Co(III) in a biologically relevant redox regime. Further, the relatively short electronic relaxation times of Co(II) complexes (T1e = 5×10-12-10-13 s for five-/six- coordinating Co(II) complexes) will produce robust 19F resonances that have minimal signal broadening while still possessing enhanced relaxation rates via PRE. Thus, here we report two novel Co(II)-based redox-responsive 19F MR probes that function by modulating both chemical shift and relaxation properties in response to H2O2 or peroxidase activity (Scheme 1). In the Co(II) state, the magnetic anisotropy and paramagnetism of the complex results in a highly shifted 19F resonance that possesses a very short longitudinal (T1) relaxation time. Upon oxidation to Co(III), these effects are abrogated, which results in a large 19F chemical shift difference between the two oxidation states (∆δF) (Scheme 1) and a lengthened T1 relaxation time. These changes can be exploited to differentiate the two states by both NMR and MRI and be used to monitor the activity of two enzymes involved in oxidative metabolism. RESULTS AND DISCUSSION Synthesis. Both ligands are hexadentate with a TACN (1,4,7-triazacyclononane) heteromacrocycle as the structural core. The fluorine substituents were installed on one side arm via amide coupling between an amine containing the fluorine tag and a carboxylate group on the chelate core. Carboxylate groups and pyrazole rings were selected to complete the remaining coordination sites with two main considerations: (1) hydrophilic moieties are highly preferred since compounds with high fluorine content generally suffer from poor water solubility; (2) the ligand environment must be able to stabilize both Co(II) and Co(III) oxidation states in aqueous environment. Indeed, aqueous stable Co(III) complexes with TACN appended with three carboxylate groups or three pyrazole rings have been reported in literature.16,36 The synthesis of ligand L1 (Scheme S1) is straightforward whereas the synthesis of ligand L2 (Scheme S2) requires protection of TACN with Boc first followed by substitution and amide coupling. Both complexes were obtained through direct complexation between a Co(II) salt and ligands followed by purification using reversed phase chromatography. Co(III) complex 3 can be obtained by

Figure 1. ORTEP representation of 2 and 3. Hydrogen atoms, solvent molecules and counter-anions are omitted for clarity. All thermal ellipsoids are drawn at 50% probability level. Crystal structures contain the Λ(λλλ) isomer exclusively. (Grey: carbon, blue: nitrogen, purple: cobalt, red: oxygen, green: fluorine).

Solid State Structures. We obtained solid-state structural information for pyrazole-containing complexes 2 and 3 (full structural details are presented in the Supporting Information). Green, long block-shape crystals of 2 were obtained by slow vapor diffusion of Et2O into the MeCN solution of 2 at room temperature (Figure 1). The Co(II) center is six-coordinate with three amine nitrogen donors from the TACN backbone, two pyrazole nitrogen atoms and one oxygen donor from the amide pendent arm. The coordination geometry is best viewed as an unusual, slightly distorted trigonal prism with three TACN nitrogen atoms defining one trigonal plane and the pyrazole nitrogen atoms and amide oxygen atom defining the other plane. The nitrogen atoms on two pyrazole rings are protonated. The Co–L bond distances are consistent with a hexacoordinate high-spin Co(II) complex (Table S1). The oxidation product of 2, complex 3, was crystallized as red, long needles by slow vapor diffusion of Et2O into a MeCN solution of 3 at -20 ºC (Figure 1). Shorter Co– L bond distances (Table S1), anion count, as well as the 1 H/19F NMR spectra (Figure S18, S19) support the assignment of a low-spin Co(III) complex. The coordination geometry is best described as slightly distorted octahedral with two pyrazole nitrogen atoms bound in cis-α conformation. Note that in this structure, both pyrazole rings are deprotonated given the absence of counteranions within the unit cell. Different from 2, the Co(III) metal center is bound to the deprotonated amide nitrogen atom (N8) instead of the oxygen. This is not entirely unexpected given that trivalent Co(III) ion is much more effective in promoting amide nitrogen deprotonation than its divalent form and strongly favors nitrogen donors.37 In addition, the IR spectrum of 3 displays an intense feature at 1586 cm-1 that corroborates the presence of a deprotonated amide moiety (Figure S1).37 Solution behavior. Complexes 1 and 2 display excellent solubility in 100% aqueous solution. Despite the


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Journal of the American Chemical Society presence of the hydrophobic perfluoro-tBu moiety, both 1 and 2 are soluble in water up to at least 30 mM, allowing all experiments to be performed in 100% aqueous solution. Complexes 1 and 2 possess similar magnetic moments in aqueous solution (Evans’ method38, 4.6(±0.1) µB and 4.8(±0.1) µB, respectively). The large values suggest significant contribution from orbital angular momentum to the net magnetic moments in these two high spin (S = 3/2) Co(II) complexes. At 25 °C, the 1H NMR spectra of 1 and 2 in D2O are both significantly broadened and peaks are observed across a wide range between 0 and 250 ppm (Figure S16, S17). 2 displays one relatively sharp feature at 77 ppm that we assigned to the aromatic protons of pyrazoles – these peaks are not present in the spectrum of complex 1. At elevated temperature (50 °C), new broad resonances appear from for both complexes that are consistent with generation of different isomeric forms and reveals the fluxional nature of the ligand backbone (Figure S16, S17). The 1H NMR spectrum of Co(III) complex 3, conversely, displays sharp resonances in the diamagnetic region between 0-10 ppm (Figure S18). The aliphatic and pyrazole protons are split, likely due to the coordination of Co(III) and isomerization.

Figure 2. Variable-temperature magnetic susceptibility data for aqueous solutions of 1 (square) and 2 (triangle) determined by the Evans’ method using a 11.7 T NMR spectrometer.

Importantly for 19F MR-based sensing, the 19F NMR spectra of the Co(II) and Co(III) complexes display signals that are distinct both in terms of relaxation parameters and chemical shift. The 19F NMR spectra of 1 and 2 contain sharp and robust 19F signals at -80.4 ppm and -79.6 ppm respectively with FWHM (full width at half maximum) values determined to be 9 Hz and 12 Hz. At 9.4 T, the 19F T1 and T2 relaxation times were measured to be 43 ms and 39 ms for 1, 48 ms and 42 ms for 2 in buffered solution, demonstrating significant relaxation enhancement by the paramagnetic centers. The R2/R1 values for both of these complexes are close to unity (~1.1) which can minimize the signal loss in MRI due to T2 reduction; this is also consistent with the observed sharp NMR signals without significant line broadening.39 Minimal changes in 19 F chemical shift were observed in solutions of both complexes in the presence of fetal bovine serum (FBS) (Figure S20). Although no significant changes in T1 were

measured under these conditions (48 ms for 1, 46 ms for 2), T2 values were shortened to 15 ms and 7 ms, respectively. This is likely due to hydrophobic interactions between protein and the –OC(CF3)3 moiety. Remarkably, upon oxidation of 1 and 2, their 19F NMR resonances undergo a near 10 ppm downfield shift. The 19F NMR spectrum of 3 displays a sharp resonance (FWHM = 3 Hz) at 70.5 ppm with T1 and T2 relaxation times of 1.05 s and 0.64 s respectively (at 9.4 T), consistent with a diamagnetic Co(III) complex. While we weren’t able to isolate the oxidized product of 1 in pure form, the crude 19F NMR spectrum of this species displays a dominant peak at -70.6 ppm that we assign to the Co(III) species, and this peak has a FWHM of 2 Hz, similar to 3. Compared to the 19F NMR spectra of L1 and L2, both Co(II) complexes show approximately 10 ppm 19F chemical shift change which is comparable to some lanthanidebased 19F chemical shift agents.26,30 This is noteworthy in that both Co(II) complexes likely have relatively long metal-fluorine distances based on the solid state structure of 2 (7-8 Å), where pseudocontact shift (PCS) or throughspace interactions usually governs. For first-row transition metals, this effect diminishes rapidly over distance.40 Based on the crystal structure of 2, we hypothesize that the unusual trigonal prismatic geometry contributes to a larger magnetic anisotropy than the common octahedral geometry and that results in greater chemical shift. Trigonal prismatic geometry has been reported to result in a high negative value of magnetic anisotropy (D) and possible SMM (single-molecule magnet) behavior in d7 ions41-43 and a series of Co(II) complexes were recently reported by Morrow that exhibit larger 1H chemical shifts in complexes with trigonal prismatic geometry vs. normal octahedral geometry.33 To support this hypothesis, we measured the temperature dependence of ߯MT for 1 and 2, where a gradual decrease of ߯MT as the temperature increases was observed for both complexes (Figure 2). In the absence of magnetic anisotropy or exchange coupling, the ߯MT of a transition metal ion should remain constant versus temperature due to Curie behavior. The temperature dependence of ߯MT for 1 and 2 is a good indicator of magnetic anisotropy in these complexes.41,43,44 DFT calculations for d-electron configurations and energy profiles of 2 also meet with the prerequisites for high magnetic anisotropy. (Figure S5) Oxidative reactivity. The redox properties of the two Co(II) complexes were first examined by cyclic voltammetry (CV) in 0.1 M phosphate buffer (pH 7.2). Both complexes display a quasi-reversible feature around 213 mV and 154 mV (vs. NHE) for 1 and 2, respectively (Figure S2). These redox features were assigned to the Co(II)/(III) redox couple. It is worth noting that replacing one carboxylate or pyrazole ring with the amide pendant arm shifts the E1/2 values cathodically by approximately 200 mV compared to the Co(II) TACN complexes with three carboxylate or three pyrazole arms demonstrating the high sensitivity of Co(II)/(III) redox potential to ligand environment.16,36 This is beneficial in that Co(II) complexes with negative E1/2 are normally air sensitive and unable to differentiate reactive oxidative species from O2. The sta-



bility of 1 and 2 in air was assessed by UV-vis and 19F NMR in aerated aqueous solution. Indeed, no change in the UV/vis spectra were observed after 20 h after dissolving 1 and 2 in aerated H2O (Figure S3, S4). Further analysis of the solution using 19F NMR reveals no or negligible oxidation of the Co(II) complexes (Figure S3, S4). Although 1 and 2 are air stable in neutral aqueous solution, they react readily with H2O2 and convert into Co(III) complexes. Both Co(III) complexes display charge transfer bands around 500 nm where 1 and 2 have negligible absorption around this region. Under pseudo firstorder conditions at 37 °C, the reaction rates for 1 and 2 were determined to be 3.15×10-4 s-1 and 1.75×10-3 s-1, respectively (Figure 3A, 3B). The reaction rate for 2 is ~5–6 times faster than 1 and exceeds that of a Co(II) complex previously reported by our group under the same conditions (7.3×10-4 s-1, Figure S9).23 2 is ~75% oxidized within just 10 min based on UV-vis whereas only ~20% conversion was achieved for 1 in the same amount of time. This demonstrates the superior reactivity of 2 towards H2O2 over 1. To gain further mechanistic insight into the oxidation reaction with H2O2, we performed 17O NMR experiments on 1 and 2 (Figure S10) in D2O doped with H217O. The peak width at half maximum (∆ν1/2)of 17O exhibits no concentration dependence for both complexes which suggests little or no inner-sphere interaction between Co(II) and bulk water since T2 relaxation of H217O is almost exclusively due to inner-sphere contributions.45 This suggests that oxidation by H2O2 does not occur via an inner-sphere

pathway since the Co(II) centers are unable to interact with external ligands such as H2O. An outer-sphere mechanism is thus likely involved. The reactions of 1 and 2 with H2O2 in HEPES buffer solution were then studied by NMR spectroscopy. Due to slower reaction rate, oxidation of 1 required large excess of H2O2. In the presence of H2O2, a sharp 19F peak emerged at -70.6 ppm (FWHM = 2 Hz) which we assigned to the generated Co(III) complex (Figure 3C). Simultaneously, the 19F signal of 1 at -80.2 ppm diminished, consistent with the consumption of the Co(II) species. With 5 eq. H2O2, only ~ 48% oxidation was achieved within an hour at 25 °C based upon NMR integration. The reaction of 2 with H2O2 is much more efficient. Similar to 1, oxidation of 2 results in a new resonance at -70.5 ppm (FWHM = 3 Hz) while the peak at -79.6 ppm decreases over time. In presence of 3 eq. H2O2, almost full oxidation (90%) was observed after 30 min (Figure 3D). The reaction rate is further enhanced at 37 °C where ~95% conversion of 2 is accomplished within 15 min (Figure S21). The chemical shift of the newly generated species is consistent with 3, synthesized using O2 as the oxidant at basic pH, indicating the formation of same Co(III) species (Figure S19). A minor peak at -70.4 ppm also appeared which can be assigned to compound 5 (Scheme S1), the side product from amide bond hydrolysis. Formation of 5 was confirmed by LC/MS (Figure S38). Nevertheless, we expect that the small chemical shift difference (~0.1 ppm) is insufficient to be distinguished by MRI to cause signal reduction.

Figure 3. (A) UV-vis spectra of 1 mM 1 in 50 mM HEPES buffer (pH 7.4, 0.1 M NaCl) after adding 10 mM H2O2 at 37 °C. The inset shows the change in absorbance at 520 nm and the curve fit of data in pseudo first-order kinetics; (B) UV-vis spectra of 1 mM 2 in 50 mM HEPES buffer (pH 7.4, 0.1 M NaCl) after adding 10 mM H2O2 at 37 °C. The inset shows the change in absorbance at 493 19 nm and the curve fit of data in pseudo first-order kinetics; (C) F NMR spectra of 1 mM 1 after reacting with 5 eq. and 10 eq. 19 H2O2 in 50 mM HEPES buffer (pH 7.4, 0.1 M NaCl) for 1 h at 25 °C; (D) F NMR spectra of 1 mM 2 after reacting with 2 eq. and 3 eq. H2O2 in 50 mM HEPES buffer (pH 7.4, 0.1 M NaCl) for 30 min at 25 °C; (E) UV-vis spectra of 1 mM 1 in presence of 32 mM glucose, 40 U/mL glucose oxidase and 85 U/mL horseradish peroxidase at 37 °C in PBS buffer; grey arrow denotes HRP absorbance bands and black arrow represents absorbance band for Co(III) complex. (F) Change in absorbance at 506 nm for Figure 3E (filled dots) and control in the absence of HRP (empty dots). Difference in initial absorbance is due to HRP.


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Journal of the American Chemical Society The reaction was also carried out in fetal bovine serum and the same NMR changes were observed (Figure S22). Oxidation of 1 and 2 is reversible: following H2O2 oxidation, addition of Na2S2O4 regenerates the original Co(II) complexes which is supported by 19F NMR (Figure S23). Treatment with an excess of weaker reductant cysteine results in partial reduction (30-40%) of the Co(III) species (Figure S24); gluthathione and ascorbic acid did not reduce the Co(III) complexes. The reactivity of 1 and 2 towards other oxidants was studied. As expected, compound 1 displayed minimal or no reactivity with ClO-, TBHP, •OtBu, •OH and O2- (Figure S25). Similarly, 2 shows no oxidation in the presence ClO-, TBHP, •OtBu and •OH. However, a stronger oxidant such as O2- can lead to oxidation of 2 (Figure S26). Given that the oxidation of both complexes relies heavily on the redox potential of oxidants, it is reasonable that weaker oxidants such as ClOand TBHP are not as effective. Although radical species such as •OtBu and •OH are extremely reactive, they are prone to extract hydrogen atoms rapidly from the environment and lose reactivity.46 Although UV-vis and NMR studies demonstrate that the reaction between 2 and H2O2 is rapid in vitro, the reactivity may not be sufficient for in vivo applications. Therefore, we further explored the application of 1 and 2 as 19F MR probes for oxidative events within a biological context. Glucose oxidase (GOX) is an enzyme that catalyzes the oxidation of glucose by O2, generating gluconolactone and H2O2 (Figure 4C). Therefore, we investigated the application of 1 and 2 to detect H2O2 formed by glucose/glucose oxidase oxidation. As expected, complex 2 oxidized rapidly after incubation with glucose/glucose oxidase at 37 °C. More than 70% oxidation was achieved within 15 min based on 19F NMR (Figure S27). These results show that 2 is capable of detecting H2O2 generated in situ at lower concentration. On the contrary, complex 1 remained intact in the presence of glucose/glucose oxidase under the exact same conditions. Both 19F NMR and UV-vis indicated little or no reaction after incubating at 37 °C for 1 h (Figure S28). A plausible explanation may be that the kinetic studies were performed under pseudo first order conditions, namely, H2O2 is present at large excess amount. However, the reaction between glucose and GOX supplies the solution with H2O2 at a steady state instead of a sudden burst of H2O2 that overwhelms the Co(II) complexes. The reactivity of 1 thus may be much lower than it appears especially at low H2O2 concentrations. Peroxidase enzymes are capable of enhancing the reactivity of reactive oxygen species. High peroxidase activity is usually a sign of aberrant health conditions such as inflammation. Peroxidase activity of up to ~ 250000 units/mL has been observed in atherosclerotic lesions.47 The inertness of 1 towards H2O2 could enable the detection of oxidation mediated by peroxidase activity. Indeed, after the introduction of horseradish peroxidase (HRP), the reactivity is greatly enhanced. As shown in Figure 3E and 3F, in the presence of HRP (85 U/mL) and using glucose/GOX as a H2O2 source, the absorbance around 500 nm increases quickly attributing to the generation of

Co(III) complex. No absorbance change was observed without HRP, which further highlights the requirement of HRP for quick oxidation. Furthermore, 19F NMR spectroscopy was used to track the oxidation process which reveals the rapid conversion from Co(II) to Co(III) complex where ~80% conversion was achieved within 15 min; this well matches the UV-vis results (Figure S29). Overall, the intrinsic low reactivity of 1 renders it incompetent as a H2O2 probe for in vivo use but opens a new promising avenue for selectively detecting peroxidase mediated oxidative activity. 19 F MRI Studies. Given the distinct 19F NMR features of the reduced and oxidized species, we probed their potential as MRI imaging agents using a 7T preclinical MRI scanner, first conducting simple phantom experiments of compounds 1, 2 and 3. Due to the short 19F relaxation times of 1 and 2, 19F MR images were acquired using a RARE (Rapid Acquisition with Relaxation Enhancement) pulse sequence with a short repetition time (TR = 100 ms). Good linearity was obtained for both 1 and 2 when plotting the signal-to-noise ratio (SNR) versus concentration with goodness of fit of R2=0.995 and R2=0.994, respectively (Figure S31). Considering a detection SNR threshold of 3.5 and an acquisition time of 12.5 min, the limit of detection for 1 and 2 was determined to be 0.17 mM and 0.16 mM, respectively. These values are comparable to previously reported paramagnetic 19F MR probes.39,48 Under identical conditions, the SNRs of 1 and 2 are about twice the SNR of our previously reported Co(II) complex (Figure S32). Given that complex 3 has much longer 19F relaxation times, a RARE pulse sequence with a long repetition time (TR = 1500 ms) was employed instead. Within the same length of scan time, the limit of detection for 3 was measured to be 0.53 mM (Figure S31). It’s worth noting that 1 and 2 display about 4-fold of SNR increase comparing to 3 due to faster scan rate enabled by their shorter relaxation times due to PRE effect. The submillimolar detection limits indicate the good sensitivity of 1 and 2 as 19 F MR probes. The capability of using 19F MRI to monitor the oxidation process by tracking both Co(II)/(III) species was demonstrated through monitoring Co(II) and Co(III) signals following reaction with variable amounts of H2O2 (Figures 4A,B). Here 4 mM 1 was reacted with different equivalents of H2O2 for 1 h and 19F MR images were acquired. The low reactivity of 1 allows us to more readily control the oxidation and monitor the process. Because of the large 19F chemical shift difference (∆δF) between the Co(II)/(III) species, Co(II)/(III) images can be acquired separately by utilizing chemical shift selective excitation pulse sequence without any signal interference from the other species despite the compromise of some spatial resolution associated with this technique. This is superior compared to 19F MR probes relying only on relaxationtime difference as interference between short/long relaxation time species can hinder unambiguous differentiation. As we increased the concentration of H2O2 the SNR of 19F MR images corresponding to Co(II) species decreased (Figure 4A, top row). Meanwhile, the SNR of 19F MR



19

Figure 4. (A) F MR images of 4 mM 1 in HEPES buffer after reacting with 0, 2, 4, and 6 equivalents of H2O2 for 1 h at 25 19 °C(reactions were quenched with catalase before imaging); (B) Signal-to-noise ratios of the F MR images in Figure 4A; (C) (Left 19 column) F MR images of 2 mM 2 after incubating with 32 mM glucose and GOX (40 U/mL) in PBS buffer at 37 °C for 1 h; (Right 19 column) F MR images of 2 mM 1 after incubating with 32 mM glucose and GOX (40 U/mL) in the absence (top) / presence (bottom) of horseradish peroxidase (85 U/mL) in PBS buffer at 37 °C for 1 h. Detailed scanning parameters are provided in the supporting information.

images corresponding to Co(III) species increased (Figure 4A, bottom row). This was confirmed via quantification of the SNR (Figure 4B). Notably, the oxidation progress reflected by 19F MRI well matches the results from 19F NMR (Figure S30). Overall, the results here demonstrate that not only can we use chemical shift selective pulse to image both Co(II)/(III) species simultaneously with no interference but we can also track the oxidation process by using two channels with opposite signal response that would potentially provide additional diagnostic information. The concept of exploiting 1 and 2 for detecting H2O2 and peroxidase activity was next examined by 19F MRI (Figure 4C). Complete oxidation of 2 was observed using glucose/GOX through both Co(II) and Co(III) selective sequences (Figure 4C, left column). This is evident by the “turn-off” in the Co(II) selective 19F MR signal and concurrent “turn-on” in Co(III) selective channel. The capability of 1 for detecting horseradish peroxidase activity was demonstrated by 19F MRI as well. Without HRP, no Co(III) complex associated 19F MR signal was found (Figure 4C, right column, bottom) and 1 remains intact as shown by the Co(II) selective image (Figure 4C, right column, top). In the presence of HRP, full oxidation of 1 was achieved based on both channels. This is impressive given that the required peroxidase concentration is about 1000 fold lower than what is observed in vivo which further emphasized the potential of using 1 as a 19F MR probe for peroxidase activity. Taken together, the 19F MRI experiments vividly demonstrate the application of 1 and 2 for sensing H2O2 or peroxidase mediated oxidative activity in vitro through independent signal response of the two channels. The large chemical shift difference between the

Co(II) and Co(III) species allows convenient and accurate differentiation without the need for special designed sequence or additional imaging modalities such as 1H MRI. With these tools, 19F MRI could be used in conjunction with Magnetic Resonance Spectroscopy (MRS) in vivo and shed light on oxidative activity during metabolic processes. CONCLUSIONS A pair of water soluble, air stable, and redoxresponsive Co(II) complexes were synthesized and characterized. A ~10 ppm 19F chemical shift was observed for both Co(II) complexes which we attribute to their large magnetic anisotropy associated with their unique trigonal prismatic geometry. The appreciable chemical shift difference enables ready differentiation between Co(II)/(III) species by 19F MRI through chemical shift selective pulse sequences. It is noteworthy that complex 2 exhibits higher reactivity towards H2O2 than 1. Complex 1, however, readily oxidizes in the presence of H2O2 and HRP and thus can be utilized for imaging peroxidase mediated oxidation events. As a proof of concept, these two complimentary tools show promise for the development of hot spot in vivo imaging agents for inflammation. In order to accomplish this, next generation sensors will include incorporation of these probes into biocompatible carriers that will increase in vivo circulation time and enable targeting. We also note that diamagnetic Co(III) species formed following oxidation do not benefit from the PRE effect offered by Co(II) that enables higher 19F MR imaging sensitivity; Fe(II)/(III) systems could be employed to address this. Ongoing work is focused on further improving the reactivity, reversibility, sensitivity, and biological properties of these probes.


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Journal of the American Chemical Society (14) Rodríguez, E.; Nilges, M.; Weissleder, R.; Chen, J. W. J. Am. Chem. Soc. 2010, 132 (1), 168.

ASSOCIATED CONTENT

(15) Querol, M.; Chen, J. W.; Weissleder, R.; Bogdanov, A. Org. Lett. 2005, 7 (9), 1719.

Supporting Information Synthetic details, additional spectroscopic data, and results from DFT calculations. The Supporting Information is available free of charge on the ACS Publications website.

(16) Tsitovich, P. B.; Spernyak, J. A.; Morrow, J. R. Angew. Chem. Int. Ed. 2013, 52 (52), 13997. (17) Kislukhin, A. A.; Xu, H.; Adams, S. R.; Narsinh, K. H.; Tsien, R. Y.; Ahrens, E. T. Nat Mater 2016, 15 (6), 662. (18) Janjic, J. M.; Srinivas, M.; Kadayakkara, D. K. K.; Ahrens, E. T. J. Am. Chem. Soc. 2008, 130 (9), 2832.

AUTHOR INFORMATION Corresponding Author

(19) Ahrens, E. T.; Zhong, J. NMR Biomed 2013, 26 (7), 860.

* [email protected]

(20) Palekar, R. U.; Jallouk, A. P.; Lanza, G. M.; Pan, H.; Wickline, S. A. Nanomedicine (Lond) 2015, 10 (11), 1817.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources This work was funded by start-up funds from the University of Texas at Austin and by the Welch Foundation (F-1883).

Notes

(21) Partlow, K. C.; Chen, J.; Brant, J. A.; Neubauer, A. M.; Meyerrose, T. E.; Creer, M. H.; Nolta, J. A.; Caruthers, S. D.; Lanza, G. M.; Wickline, S. A. The FASEB Journal 2007, 21 (8), 1647. (22) Temme, S.; Grapentin, C.; Quast, C.; Jacoby, C.; Grandoch, M.; Ding, Z.; Owenier, C.; Mayenfels, F.; Fischer, J. W.; Schubert, R.; Schrader, J.; Flogel, Circulation 2015, 131, 1405−1414.

The authors declare no competing financial interest

(23) Yu, M.; Da Xie; Phan, K. P.; Enriquez, J. X. S.; Luci, J. J.; Que, E. L. Chem. Commun. 2016, 52, 13885.

ACKNOWLEDGMENT

(24) Xie, D.; King, T. L.; Banerjee, A.; Kohli, V.; Que, E. L. J. Am. Chem. Soc. 2016, 138 (9), 2937.

The authors thank Prof. M. J. Rose for use of his IR spectrometer, Prof. J, J. Luci for technical help with MRI experiments and Dr. V. Lynch for help with X-ray crystallography.

(25) Basal, L. A.; Bailey, M. D.; Romero, J.; Ali, M. M.; Kurenbekova, L.; Yustein, J.; Pautler, R. G.; Allen, M. J. Chemical Science 2017, 00, 1.

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19F PARASHIFT Probes for Magnetic Resonance Detection of H2O2

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