Synthesis, Characterization, and Handling of EuII-Containing Complexes for Molecular Imaging Applications

Considerable research effort has focused on the in vivo use of responsive imaging probes that change imaging properties upon reacting with oxygen because hypoxia is relevant to diagnosing, treating, and monitoring diseases. One promising class of compounds for oxygen-responsive imaging is EuII-containing complexes because the EuII/III redox couple enables imaging with multiple modalities including magnetic resonance and photoacoustic imaging. The use of EuII requires care in handling to avoid unintended oxidation during synthesis and characterization. This review describes recent advances in the field of imaging agents based on discrete EuII-containing complexes with specific focus on the synthesis, characterization, and handling of aqueous EuII-containing complexes.


SYNTHESIS OF Eu II -CONTAINING COMPLEXES
In this section, we describe procedures for the preparation of Eu II -containing complexes using the ligands depicted in Figure 2. These procedures are divided into two general strategies (Figure 3): (1) chemical or electrochemical reduction of Eu III -containing complexes or mixtures of Eu III salts and ligands and (2) metalation of ligands with Eu II salts. Depending on the route used to generate Eu II -containing complexes, different techniques are appropriate to evaluate the identity and purity of the resulting complexes. These characterization techniques and strategies for effectively handling solutions of Eu II -containing complexes for analyses are described in the handling section of this article.

Reduction of Eu III to Produce Eu II -containing Complexes
In the process of forming Eu II -containing complexes, Eu III is often reduced using electrochemical or chemical methods. Transient formation of detectable amounts of Eu II -containing complexes can be achieved using cyclic voltammetry, and isolable Eu II -containing complexes can be obtained through electrochemical or chemical reduction of either Eu III -containing complexes or mixtures of Eu III with ligands. The most favorable route to form a Eu II -containing complex should be determined based upon the ligand and the type of analysis that is needed.

Transient Formation of Eu II -Containing Complexes From Cyclic Voltammetry
To obtain information about the reduction and oxidation potentials of Eu II -containing complexes, several research groups have formed Eu II -containing complexes transiently using cyclic voltammetry. A description of air-free electrochemical techniques used for cyclic voltammetry or bulk electrolysis to form Eu II -containing complexes is described in the handling section of this review. When reducing Eu III to form Eu II , two routes are commonly taken: the corresponding Eu III -containing complexes are synthesized and purified before electrolysis, such as in the case of Eu-containing complexes of 8, 20, 21, 25, 26, 27, 31-34, and 39 (Gansow et al., 1977;Yee et al., 1980Yee et al., , 1983Burai et al., 2003;Vanek et al., 2016;Basal et al., 2017a;Burnett et al., 2017). Alternatively, Eu III salts-such as Eu(OTf) 3 , EuCl 3 , or Eu(NO 3 ) 3 -are dissolved in the presence of ligands, enabling the formation of complexes upon electrolysis of Eu III to Eu II , such in the case of Eu-containing complexes of 1-8, 10, 11, 13-15, 20-23, 28-30, and 35-38 (Yee et al., 1980(Yee et al., , 1983Sabbatini et al., 1984;Burai et al., 2002Burai et al., , 2003Botta et al., 2003;Gamage et al., 2010;Gál et al., 2013;Regueiro-Figueroa et al., 2015). In these experiments, cyclic voltammetry peaks that are different than the peaks for Eu II/III aqua or the ligand (if the ligand is redox active in the potentials spanned by the voltammogram) are attributed to the formation of Eu II/III -containing complexes (Figure 4).
For the selection of an appropriate route, consideration of ligand structure and solubility is necessary. For example, ligands that readily form complexes with Eu II upon reduction from Eu III , like cryptands 8, 20, and 21, produce the same electrochemical profiles whether starting with a mixture of Eu III and ligand or a Eu II -containing complex (Yee et al., 1980(Yee et al., , 1983. When solubility differences exist between ligands and their corresponding complexes, such as if the ligand is not soluble but the complex is, then one must ensure complexation prior to CV analysis. If the complex is insoluble in aqueous media, then organic solvents can be employed with the caveat that the measured standard electrode potential might not be reflect the potential under aqueous conditions.

Producing Eu II -Containing Complexes via Electrochemical Reduction of Eu III
Beyond the transient reduction of Eu III on the surface of electrodes during cyclic voltammetry, Eu III can be reduced on an isolable scale electrochemically using bulk electrolysis in oxygen-free solvent under an atmosphere of inert gas. Reduction via bulk electrolysis involves holding a sufficiently negative potential to reduce Eu III to Eu II . The electrochemical potential used to reduce a Eu III -containing complex is often 0.1-0.5 V more negative than the E 1/2 of the target complex (Burai et al., 2002(Burai et al., , 2003Botta et al., 2003); however, the reduction potential of the ligand functional groups should be considered before selecting this technique to avoid the possibility of reducing redox-active ligands. Bulk electrolysis was used to obtain Eu IIcontaining complexes of 1-8, 22-25, and 31 (Sabbatini et al., 1982(Sabbatini et al., , 1984Burai et al., 2000Burai et al., , 2002Christoffers and Starynowicz, 2008). In these studies, solutions containing both Eu III salts and ligands were held at the appropriate potential, typically in a two-compartment glass cell with a fritted glass separator with sparging of inert gas (Bard and Faulkner, 2000). The resulting Eu II -containing complexes can be used for further analysis of molecular-imaging-relevant properties, including UV-visible and luminescence spectroscopy, relaxivity measurements, 17 O-NMR spectroscopy, and NMRD measurements (Sabbatini et al., 1984;Burai et al., 2000Burai et al., , 2002Christoffers and Starynowicz, 2008). Bulk electrolysis of a solution of metal and ligand can provide enough material to obtain crystals for X-ray diffraction: for example, bulk electrolysis of Eu III to Eu II in the presence of ligand 24 followed by slow evaporation or cooling under inert atmosphere resulted in crystals of Eu II 24 (Christoffers and Starynowicz, 2008). Bulk electrolysis to produce isolable Eu II -containing complexes is appropriate when the potential needed to reduce a Eu III -containing complex to a Eu II -containing complex does not overlap with the redox-activity of the ligand, when the desired Eu II salt is unavailable, or when the standard potential or pH of the complex in solution is incompatible with chemical reductants.

Chemical Reduction of Eu III -Containing Complexes to Form Eu II -Containing Complexes
In addition to bulk electrolysis, chemical reductants are used to generate Eu II -containing complexes. Depending on the standard potential of the Eu III -containing complex to be reduced, different reducing agents will be appropriate. For example, the reduction potential of Zn (Zn II + 2e − → Zn 0 ) is −0.960 V vs Ag/AgCl (saturated KCl) (Bard and Faulkner, 2000); therefore, complexes that have standard electrode potentials more positive than −0.960 V vs Ag/AgCl should, thermodynamically, be reduced by Zn 0 . Eu III -containing complexes were reduced using Zn 0 to form Eu II 26, Eu II 27, and Eu II 34 (Ekanger et al., 2016a;Basal et al., 2017a,b). In these studies, the Eu III -containing complexes were dissolved in water in the presence of zinc metal dust, and the pH was adjusted between 4 and 6.5 to expose Zn 0 , resulting in the reduction of Eu III to Eu II . To date, only amalgamated Zn and Zn 0 have been used to chemically reduce Eu III -containing complexes to Eu II -containing complexes in water (McCoy, 1935;Ekanger et al., 2016a;Basal et al., 2017a,b). However, other chemical reductants, which have been used to reduce other Ln III ions to Ln II ions (Teprovich et al., 2008;MacDonald et al., 2013;Fieser et al., 2015), could be used if the low pH required for the use of zinc metal is undesirable or if the standard electrode potential of the Eu-containing complex is more negative than that of Zn 0 .

Complex Formation by Direct Mixing of Eu II Salts and Ligands
Another technique to synthesize Eu II -containing complexes is mixing Eu II halide salts with ligands. Eu II chloride, bromide, and iodide salts are available commercially. When mixing Eu II and ligands, often a slight excess of a Eu II halide salt (1.1-1.2 equivalents) is mixed with a water-soluble ligand (1 equivalent) in water. Complexes tend to be easier to purify from an excess of Eu II relative to an excess of ligand: the addition of phosphate buffer precipitates excess Eu II from solution as phosphate salts that can be removed with a small (0.2 micrometer) hydrophilic filter to yield a buffered solution of Eu II -containing complex (Garcia and Allen, 2012b). This technique was used to synthesize Eu II -containing complexes of 8-11 and 17-19 (Zucchi et al., 2010;Garcia and Allen, 2012b;Garcia et al., 2012;Ekanger et al., 2014Ekanger et al., , 2016bEkanger et al., , 2017Lenora et al., 2017).
When a ligand is not water-soluble but the resulting complex is, aqueous solutions of Eu II -containing complexes can be prepared by mixing Eu II salts with ligands in an organic solvent and then separating the resulting complex from the organic solvent. Purification by precipitation or crystallization results in solids that are soluble in water for imaging. For an example of purification by precipitation, Eu II 16 was synthesized in tetrahydrofuran: EuI 2 and 16 are soluble in tetrahydrofuran, but Eu II 16 is not, enabling isolation of Eu II 16 by precipitation (Kuda-Wedagedara et al., 2015). For an example of purification by crystallization, crystals were grown of cryptates Eu II 4, Eu II 8, Eu II 10, Eu II 11, Eu II 12, and Eu II 16 from slow evaporation of a mixture of ligand and Eu II halide in acetone, methanol, or methanol/tetrahydrofuran (Burai et al., 2000;Kuda-Wedagedara et al., 2015;Jin et al., 2016;Lenora et al., 2017).

CHARACTERIZATION FOR IDENTITY AND PURITY IN AQUEOUS MEDIA
Depending the route chosen to form Eu II -containing complexes, different techniques for the characterization of identity and purity of Eu II -containing complexes must be used (Figure 5). Assessment of identity of Eu II -containing complexes includes evidence for the oxidation state of Eu, coordination environment, and metal-to-ligand ratio. Assessment of the purity of Eu IIcontaining complexes includes the detection of Eu II or Eu III , ligand, reactants, or byproducts.
When Eu II -containing complexes are generated in situ via cyclic voltammetry, purity with respect to excess Eu II aqua can be assessed by observation of a peak for Eu II/III aqua . The minimum detectable concentration of europium by cyclic voltammetry, and hence the boundary of usefulness for this technique, is influenced by multiple experimental parameters including concentration of supporting electrolyte, the identity of the buffer and solvent, and choice of reference electrode (Bard and Faulkner, 2000). With these parameters in mind, minimum detectable concentrations can be determined experimentally (Harris, 2003). Unlike Eu II/III , if a ligand is not redox-active, cyclic voltammetry does not provide evidence for the presence of uncomplexed ligand. Therefore, the usefulness of cyclic voltammetry for detection of excess ligand is situationally dependent.
Regarding the identity of the complex that is formed during the course of cyclic voltammetry, formation of a Eu-containing complex can be validated by comparing the standard electrode potential of the new complex with the standard electrode potential of a sample of the Eu-containing complex (Tables 1, 2). For example, Eu II -containing complexes of 8, 20, and 21 were synthesized and found to produce the same E 1/2 whether starting with a mixture of Eu III and ligand or an alreadysynthesized Eu II -containing complex (Yee et al., 1980(Yee et al., , 1983. However, the standard electrode potential is influenced by the same experimental parameters that are listed for consideration of purity using cyclic voltammetry; therefore, care must be taken to note experimental parameters when comparing standard electrode potentials. In the case where Eu III -containing complexes are reduced chemically with zinc, such as 2Eu22Cl 3 + Zn 0 → 2Eu22Cl 2 + ZnCl 2 , a combination of spectroscopic techniques can be used to provide evidence of the oxidation state and degree of purity. For evidence that Zn II was removed from solution postreduction, the concentration of Zn II (down to parts-per-billion levels) can be monitored with inductively coupled plasmamass spectrometry (Ekanger et al., 2016a). For evidence of the reduction of Eu III , a lack of overlap of the excitation bands of Eu II -and Eu III -containing species enables monitoring of the presence of Eu III (down to micromolar levels) by luminescence spectroscopy when excitation is performed with a Eu III -specific wavelength (Ekanger et al., 2016a;Basal et al., 2017a). Evidence for the generation of Eu II is obtained using electron paramagnetic resonance (EPR) spectroscopy. In its ground state, Eu III has no net magnetic moment (Cullity and Graham, 2009) despite having six unpaired electrons. The magnetic moment (µ eff ) of lanthanides is calculated using the total angular momentum (J), unlike the magnetic moments of 3d n transition metals that take into account the number of unpaired electrons (Cotton, 2006;Layfield et al., 2015). This difference is due to the quenching of orbital angular momentum by ligands for 3d orbitals but not for the shielded 4f orbitals. Therefore, the Eu III ground state would not be expected to be observed in EPR spectroscopy (Abragam and Bleaney, 1970). However, Eu II is paramagnetic and characterized by a signal in EPR spectroscopy with a g factor of ∼1.99 (Abragam and Bleaney, 1970;Caravan et al., 1999). Also, Eu II -containing complexes can be colored yellow or orange and give rise to broad and relatively intense UV-visible absorptions and emissions that are distinct from the corresponding Eu IIIcontaining species (Burai et al., 2003;Kuda-Wedagedara et al., 2015;Ekanger et al., 2016a). For example, a combination of spectroscopic techniques were used to monitor the formation of Eu II -containing complexes Eu22, Eu26, and Eu34 (Ekanger et al., 2016a;Basal et al., 2017a).
In the case where the formation of a complex was achieved by mixing EuCl 2 with ligands, evidence of 1:1 complex formation in solution was determined by measuring the change in relaxivity as a function of Eu II -to-ligand ratio, a technique known as proton relaxation enhancement (Lauffer, 1987;Lenora et al., 2017). Another solution-phase technique to monitor complex formation is a Job plot where both ligand and metal ratios are varied, and a unique property of the complex, such as a complex-specific emission, is monitored (Renny et al., 2013;Kuda-Wedagedara et al., 2015). The choice of spectral feature to monitor in a Job plot is complex-dependent. Typically, Eu IIbased emission is largely quenched in aqueous media due to the abundance of OH oscillators (Jiang et al., 1998); therefore, luminescence spectroscopy is not suitable to characterize the formation of every Eu II -containing complex. Other features to monitor as a function of metal-to-ligand ratio include complexspecific absorbance peaks, relaxivity, or cyclic voltammetry peaks. In the case where single crystals of a Eu II -containing complex are obtained, X-ray diffraction combined with elemental analysis provides information regarding identity and purity. X-ray diffraction provides information about the oxidation state and coordination environment of the Eu II ion in the solid state, including bond distances and number and identity of counter ions (Zucchi et al., 2010;Kuda-Wedagedara et al., 2015;Jin et al., 2016;Basal et al., 2017a;Lenora et al., 2017). Elemental analysis provides information about elemental composition as an indication of purity. However, it is important to note that for molecular-imaging applications, solution-phase characterization is often more important than solid-phase analysis of solids because solid-state properties do not necessarily [a] converted to V vs. Ag/AgCl by subtracting 0.044 V from the saturated calomel values (Bard and Faulkner, 2000) or by adding |E pa(Ag/AgCl) -E pa(ferrocene/ferrocenium) | to the reported E 1/2 values vs. ferrocene/ferrocenium (Gamage et al., 2010); [b] average of the anodic and cathodic peak potentials; [c] Lenora et al., 2017).

HANDLING Eu II -CONTAINING SAMPLES TO PREVENT OXIDATION
Preventing Eu II -containing complexes from oxidizing over the course of analyses is critical for the collection of accurate data: Eu III and Eu II have different properties, and misinterpretation of experimental results can occur if care is not taken to prevent unintentional oxidation of Eu II . Rigorous techniques must be used in the synthesis and handling of Eu II -containing complexes. This section describes apparatuses and techniques that were successfully used to study Eu II -containing complexes (Figures 6, 7). Cyclic voltammetry or bulk electrolysis performed either inside or outside of a glovebox should use solvents that have been degassed (under reduced pressure, for example, on a Schlenck line) or well-sparged (≥5 min of vigorous bubbling with an inert gas for volumes of ∼3 mL in a capped vessel that contains a vent needle). To ensure that there is no detectable dissolved oxygen in solution, cyclic voltammetry of degassed solvents should not show peaks for O 2 (Green, 2018). If outside of a glovebox, the inert-gas source should be retracted to the head-space of the vessel once sparging is complete prior to cyclic voltammetry or bulk electrolysis. Inside the glovebox, there is no need for an inert gas line in the headspace because the atmosphere is O 2 -free. If outside of a glovebox, transfer of solutions of Eu II from bulk electrolysis for crystal growth or other analyses must be performed using air-free techniques (Shriver and Drezdzon, 1986).
For other routes to synthesize and handle Eu II -containing complexes in aqueous media, air-free handling is achieved using Shlenck techniques, a wet (water allowed but no molecular oxygen) glovebox, or a combination of both. During the synthesis of Eu II -containing complexes in a glovebox, the glovebox atmosphere must not be contaminated with oxygen. Either a commercial oxygen sensor or chemical indicator can be used to monitor the atmosphere. One suitable chemical indicator for this purpose is dicyclopentadienyltitanium(IV) dichloride (Ti IV Cp 2 Cl 2 ) (Burgmayer, 1998). When the titanium metallocene is dissolved in acetonitrile in the presence of zinc metal, a deep blue solution is obtained. An aliquot of this solution FIGURE 6 | Diagram describing techniques that can be used to analyze Eu II -containing complexes with air-free conditions. Frontiers in Chemistry | www.frontiersin.org is filtered through celite or a hydrophilic filter and diluted with acetonitrile to yield a diffuse, blue solution caused by the presence of Ti III Cp 2 (NCCH 3 ) 2 . If the solution remains blue upon evaporation, Ti III Cp 2 (NCCH 3 ) 2 is unoxidized, indicating that the atmosphere is good (<5 ppm O 2 ) (Shriver and Drezdzon, 1986). A color change to green is caused by formation of a dimeric species or some oxidation of Ti III to Ti IV . A color change to yellow is indicative of near-complete oxidation to Ti IV Cp 2 (NCCH 3 ) 2 . Either green or yellow suggests a bad atmosphere with respect to O 2 , and steps to address the quality of the atmosphere should be taken prior to working with Eu II . If the indicator persists as green or yellow after refreshing the glovebox atmosphere by purging the glovebox with inert gas, then the oxygen-removing catalyst should be replaced or regenerated. Ideally, the atmosphere of a wet glovebox should be checked at least daily, and the atmosphere should be purged with inert gas before and after each use. All liquids to be used in a wet glovebox should be rigorously degassed before transport into the glovebox. Solids to be brought into a glovebox can be placed in an open vial and brought into the antechamber if the solids do not sublime at the temperature and pressure of the glovebox antechamber. To prevent loss of solid from bumping or the vial accidentally tipping, the top of the vial can be covered with tissue that is secured with a rubber band ( Figure 7I). If solids sublime at the temperature and pressure of the antechamber, then the solids should be placed under an inert atmosphere in a sealed container prior to being brought into the glovebox.
Solution-phase characterization of Eu II -containing samples, including NMR spectroscopy, MRI, and fluorescence or UVvisible absorbance spectroscopy, requires that samples be sealed to prevent oxygen contamination that would interfere with the integrity of the results. For NMR spectroscopy, J-young NMR tubes with Teflon seals or flame-sealed NMR tubes are appropriate for long term studies ( Figure 7G). Alternatively, NMR tubes capped with a plastic cap and sealed with paraffin wax or electrical tape suffice for studies that last a few hours (Figures 7E,F and Video S1). For samples that must be shipped, samples can be loaded into NMR tubes that are subsequently flame-sealed (Lenora et al., 2017). For MRI, tubes (for example, glass vials that have a 400 µL capacity) can be filled to the brim with solution (to avoid bubbles), capped, dipped in wax, and loaded into an apparatus that is then covered in paraffin wax ( Figure 7A) (Garcia and Allen, 2012b;Garcia et al., 2012;Basal et al., 2017a,b). For in-vivo injections, syringes with rubber-tip FIGURE 7 | Pictures of apparatuses included in the handling section of this manuscript: (A) empty tube holder (top) and tube holder with tubes that are covered in wax (bottom); (B) jar sealed with electrical tape that contains glass wool and syringes of Eu II -containing complexes packed under an inert atmosphere and (inset) a plastic 1 mL syringe with a rubber tip on the plunger that contains a solution of a Eu II -containing complex; (C) wax-sealed cuvette; (D) electrical-tape-sealed cuvette; (E) wax-sealed NMR tube; (F) electrical-tape-sealed NMR tube; (G) J-young NMR tube (left) and flame-sealed NMR tube (right); (H) electrical-tape-sealed vial containing a solid sample; and (I) glass vial that contains solid sample covered with a tissue that is secured with a rubber band. All scale bars represent 1 cm. plungers can be loaded with sample, packaged in a bottle that is under an atmosphere of N 2 or Ar, and sealed with electrical tape (Figure 7B; Basal et al., 2017a). Packed this way, the integrity of samples is sufficient for shipping with glass wool included in the bottle to minimize vibrations during shipping. For samples in cuvettes (for example, samples for emission or absorbance spectroscopy that must be removed from the glovebox), quartz cuvettes with Teflon caps can be sealed with paraffin wax ( Figure 7C) or electrical tape ( Figure 7D) (Kuda-Wedagedara et al., 2015;Ekanger et al., 2016a;Jin et al., 2016;Basal et al., 2017a). For samples that will undergo temperature changes (heating or cooling), we have observed that paraffin wax is more reliable than electrical tape.
To ensure that a technique or apparatus successfully seals Eu II from air over the course of an experiment, the relaxivity, UV-visible absorption, or luminescence spectra of the Eu IIcontaining complex can be measured immediately after sample preparation and after the analyses are complete, if the analyses are nondestructive. Another way to assess the air-free environment and handling of a Eu II -containing sample is to measure a spectral feature of the Eu II -containing complex as a function of time at different concentrations of europium. Time-dependent measurements can reveal the presence of oxidizing impurities (Burai et al., 2002). For example, the oxidation half-life (t 1/2 ), which is the time at which half of the complex has oxidized, was measured for Eu6 by monitoring the intensity of a complexspecific UV-visible absorbance peak as a function of time (Burai et al., 2002). For a 5 mM solution of Eu6, the t 1/2 was found to be 10 days. However, t 1/2 increased as a function of concentration of europium (>1 month for Eu6 at 100 mM), suggesting that the t 1/2 reported at 5 mM was influenced by the presence of oxidizing impurities such as O 2 . Oxygen can be avoided using the techniques described in this section.
If a Eu II -containing sample cannot be monitored to check the effectiveness of an air-free technique, then another way to assess if an apparatus is sealed from air is to monitor the color change of a solution of Ti III Cp 2 Cl 2 sealed in parallel to the sample to be measured (Burgmayer, 1998). The use of Ti III Cp 2 Cl 2 as an indicator provides information regarding the technique used to seal the solution from air. However, a limitation of this method is that it does not provide direct information about the Eu IIcontaining complex being analyzed.

CONCLUSIONS AND OUTLOOK
The unique and tuneable properties of Eu II make Eu II -containing complexes promising molecular imaging agents. The synthesis, characterization, and handling of Eu II require care with respect to the use of air-free techniques and characterization of oxidation states because Eu II and Eu III have different molecular imaging properties that confound results if the ions are inadvertently comingled within a sample. We expect that the techniques described in this review will guide the future synthesis, characterization, and handling of Eu II -containing complexes for molecular imaging.

AUTHOR CONTRIBUTIONS
LB and MA: Contributed to the manuscript and approved the final version.

ACKNOWLEDGMENTS
MA gratefully acknowledges support from the National Institutes of Health (R01EB013663), and LB was supported by a Rumble fellowship from Wayne State University.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2018.00065/full#supplementary-material Video S1 | Video demonstration of the sealing of an NMR sample with paraffin wax.