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Breaking the Barrier to Slow Water Exchange Rates for Optimal Magnetic Resonance Detection of paraCEST Agents W. Shirangi Fernando,† André F. Martins,† Piyu Zhao,† Yunkou Wu,‡ Garry E. Kiefer,†,§ Carlos Platas-Iglesias,∥ and A. Dean Sherry*,‡,† †

Department of Chemistry, University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United States Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States § Macrocyclics, Incorporated, Dallas, Texas 75235, United States ∥ Grupo QUICOOR, Centro de Investigaciones Científicas Avanzadas (CICA) and Departamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira, Rúa da Fraga 10, 15008 A Coruña, Spain ‡

S Supporting Information *

ABSTRACT: EuDOTA-tetraamide complexes as paraCEST agents offer an attractive platform for designing biological sensors and responsive agents. The early versions of these agents showed low sensitivity at temperature and power levels suitable for in vivo applications partly due to non-optimal water exchange rates. Here we report two new EuDOTA derivatives having glutamyl-phosphonate side arms that display the slowest water exchange rates of any other paraCEST agent reported so far. The advantages of such systems are demonstrated experimentally both in vitro and in vivo and DFT calculations were performed to help understand the physical-chemical reasons for this interesting behavior.

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INTRODUCTION Magnetic resonance imaging (MRI) is arguably the most important diagnostic tool available to clinicians today. The most widely used MRI contrast agents (CA) in clinical medicine today are gadolinium-based T1 agents.1,2 Although new Gd-based agents continue to be reported, identification of T1 agents that respond to specific biological events by a change in r1 relaxivity has some limitations, so development of newer types of responsive CAs for MRI remains a high priority.3,4 For instance, a new generation of MRI contrast agents based upon a chemical exchange saturation transfer (CEST) mechanism has been shown to offer potential advantages over Gd-based T1 agents as biosensors.5−9 To create CEST contrast, a frequencyselective presaturation pulse is applied to one pool of protons that is in slow-to-intermediate exchange with bulk water protons. The exchange of those spins from one pool of exchangeable protons with the bulk water protons pool alters the image contrast by reducing the magnetic resonance (MR) signal intensity of the tissue water. A general requirement for CEST is that the frequency difference between the two pools of protons, Δω, must be ≥kex (rate of exchange between the pools).10,11 The CEST signal is readily influenced by physiological parameters such as pH, temperature, or the presence of other metabolites, so CEST provides an attractive platform for development of biologically responsive MR agents by modulation of either the proton exchange rate constant (kex) or the frequency difference (Δω).4 Since CEST agents must be © XXXX American Chemical Society

activated with a RF pulse, image contrast can be turned on and off at will. As typical of other contrast agents, the CEST intensity also depends on agent concentration, but newer ratiometric imaging methods allow for the possibility of making the signal independent of agent concentration.6,12,13 One could in principle administer multiple types of paraCEST agents simultaneously and activate them separately by use of different presaturation frequencies. There are many advantages of CEST agents over conventional T1-based agents, and even though several in vitro proofof-principle examples have been reported, reports of successful in vivo applications have so far been limited for many reasons including (1) non-optimal water or proton exchange rates, (2) interference from the inherent MT signal of tissues, (3) limitations in the amount of RF power that can be used to activate such agents, and (4) the low inherent sensitivity of CEST.14 One goal of our lab over the past few years is to create highly shifted exchange sites (large Δω) with much slower proton or water molecule exchange rates so the agent can be activated using relatively low-power RF pulses.14−17 We have shown that the polarity of the ligand side chains in EuDOTAtetraamide complexes (DOTA = 1,4,7,10-tetraaza cyclododecane-1,4,7,10-tetraacetic acid) can dramatically alter the bound water lifetimes in such complexes. Typically, the Eu(III)Received: December 14, 2015

A

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Figure 1. Structures of Eu(III) complexes investigated here.

bound water lifetime, τM = 1/kex, correlates with the polarity of the ligand side chains as follows: phosphonates ≥ carboxylates ≫ alkyl groups ≥ simple amides.18 Our working hypothesis based on these observations is that the favorable hydrogenbonding network formed by carboxylate or phosphonate groups with the metal ion-bound water stabilizes the inner-sphere and second-sphere water molecules (surrounding the metal ion chelate), while inclusion of hydrophobic groups hinder access of outer-sphere water molecules to the inner-sphere metal ionbound water molecule.4,14,19 Thus, any increase in hydrophobicity in the amide side-chain groups would repel outersphere water molecules and hence reduce the rate of molecular exchange with the single lanthanide ion-bound water molecule. The optimal bound water lifetime (τm) for paraCEST agents lies in the range of 10−4−10−2 s, depending upon the strength of the B1 field applied during the presaturation period.14 The first paraCEST agents reported were simple EuDOTAtetraamide complexes that exhibited sufficiently slow water exchange kinetics (τM ≈ 10−4 s) to satisfy the CEST requirement (Δω ≥ kex) but too rapid to yield a maximum CEST signal.14 Here we report the design and synthesis of two novel glutamyl-methylene phosphonate ligands (Figure 1) designed to minimize water exchange based on the principles described above. Characterization of these Eu complexes by high-resolution NMR and CEST demonstrated that these complexes display the slowest water exchange kinetics reported so far (τM values ranging from 500 to 700 μs at room temperature). These unusually long bound water lifetimes allow one to activate these complexes using much weaker B1 fields. Furthermore, temperature-dependent studies show that a maximum CEST signal is observed near the physiological temperature of 37 °C. This demonstrates that ligand design principles can be used to optimize the sensitivity of paraCEST complexes for in vivo applications.

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animal monitoring system. The respiration rate was monitored and maintained at a rate of 15−20 breaths/min. MR images were acquired using an Agilent 9.4 T (400 MHz) animal scanner equipped with a 38 mm quad-birdcage volume coil. Images were acquired using a steady state gradient echo pulse sequence (GEMS) with centric k-space encoding preceded by a rectangular saturation pulse (10 μT of 5 s duration). A 200 μL amount of a stock solution of agent was administered via a tail vein catheter to achieve an injection dose of 0.4 mmol/kg. CEST spectra were acquired over the frequency range from 60 to −60 ppm in 24 steps. The water saturation shift referencing (WASSR) method20 was used to correct the B0 magnetic field inhomogeneity, where a total of 101 images with presaturation offset (step = 0.2 ppm, power =2 μT, and duration = 5 s) varied from 10 to −10 ppm were acquired to create the B0 maps. Other acquisition parameters are TR = 5.2 s, TE = 1.69 ms, average = 1, FOV = 3.0 cm × 3.0 cm, matrix = 64 × 128, slice thickness = 2 mm, and flip angle = 20°. CEST Data Analysis. CEST images were processed using selfwritten scripts in MATLAB (The Mathworks, Inc., Natick, MA, USA). The intensity of the water signal in the WASSR experiments was interpolated by a smoothing spline algorithm to identify the true center of the water frequency in each pixel. This yielded a B0 map across the entire FOV, which was used to correct the frequency of the CEST spectra in each pixel in the imaging experiment. The resulting corrected CEST spectra were then fitted to a sum of one Gaussian line shape and two Lorentzian line shapes representing the tissue MT signal and the bound and bulk water resonances, respectively. The noise filter (CEST < 1%) was applied to remove all voxels containing insufficient signal to accurately define the CEST spectrum in that voxel. Methods for Evaluation of τM. Water residence life times were estimated using three different methods.21 1. Fitting the CEST Spectra to the Bloch Equations Modified for Exchange. The experimentally obtained CEST spectra at different B1 powers were fitted to the Bloch equations for a three-pool exchange system (bulk water, water coordinated with the Ln3+, and the amide proton) using a MATLAB program. The T1 of solvent water was measured using a standard inversion recovery without the presaturation pulse, while T2 was determined with the CPMG pulse sequence or estimated from the bulk water line width. The following parameters were considered in the fitting algorithm: presaturation pulse power (B1 in Hz), static magnetic field (B0), presaturation time, T1 and T2 of bulk water, proton concentration in each pool, and resonance frequencies of bulk water, bound water, and amide protons. 2. Omega Plots . Mz/(Mo − Mz) versus 1/ω2 (ω = saturation power in radians per second) are plotted using the steady state intensity of bulk water after a 10 s presaturation pulse was applied using B1 values ranging from 50 to 400 Hz. The x-axis intercept equals 1/k2, from which the bound water lifetime, τM, could be calculated.21 3. Classical NMR Line Width Measurements. Classical NMR line width measurements were used for slow water exchange systems where bound water resonance was visible by 1H NMR. The line width at half height of the Eu-bound and bulk water resonances are measured as a function of temperature and fitted to a classical two-site exchange model.7

METHODS

CEST Measurements in Vitro. CEST spectra were recorded by applying a frequency-selective presaturation pulse for a variable period of time (determined experimentally) over a range of frequencies (typically ±100 ppm) followed by a single sampling pulse to measure the intensity of the water signal at equilibrium (Mz). The data are presented as a plot of Mz/M0, where M0 is the water intensity in the absence of an applied pulse, against saturation frequency (in ppm). The applied B1 field (in Hz) was calibrated prior to collection of the CEST spectra by measuring the 360° pulse width for bulk water protons as a function of transmitter power level. CEST Imaging in Vivo. All animal studies were performed in compliance with guidelines set by the UT Southwestern Institutional Animal Care and Use Committee (IACUC). SCID mice were anesthetized using 2% isofluorane in air, and body temperature was monitored with a rectal probe and regulated at 30.0 ± 0.1 °C using the B

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exchange in the TSAP isomers is ∼50 faster than corresponding SAP isomers.28 Note also that the chemical shift of the H4 protons in these complexes (∼17 ppm) is located somewhat upfield compared to most other EuDOTA-tetraamide complexes (typically 24−36 ppm).4 This also indicates that water exchange must be unusually slow in these complexes because the chemical shift of the H4 proton has been found to be proportional to water exchange rates.29 It should be noted that although TSAP isomer was not detected by NMR, a small amount of TSAP isomer is likely present in solution since the interconversion process of these isomers (arm rotation or ring flip) still occurs even when the concentration of the other isomers is too low to be detected.30,31 Another feature characteristic of very slow water exchange systems is the appearance of Eu-bound water resonance. Interestingly, in both EuL1 and EuL2, the bound water NMR peaks were observed in pure water (Figure 2), not in a less polar solvent such as acetonitrile, which is commonly done to restrict the rate of water exchange.30 The CEST peak that reflects exchange of the Eu(III)-bound water molecule of EuL1 (Figure 3) and EuL2 (Supporting

Computational Studies. All calculations were performed employing density functional theory (DFT) within the hybrid meta-GGA approximation using the TPSSh exchange-correlation functional22 and Gaussian 09 package (Revision D.01).23 The TPSSh functional was selected on the basis of previous studies showing that it provides more accurate geometries of Ln3+ complexes than the popular B3LYP functional,24 as well as accurate 17O Aiso values of the coordinated water molecules for different Gd3+ complexes with polyaminocarboxylate ligands.25 Geometry optimizations of the [Ln(L)(H2O)]3+ systems (Ln = Eu or Yb) were performed in aqueous solution by using the large-core relativistic effective core potential (LCRECP) of Dolg et al., the related [5s4p3d]-GTO valence basis set for the lanthanides,26 and the standard 6-31G(d) basis set for C, H, N, O, and P atoms. For the sake of computational simplicity, the ethyl groups on the appended phosphonates in L1 were replaced by methyl groups (more information available in the Supporting Information). Synthesis and Preparation of Ligands and Complexes. See Supporting Information.

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RESULTS AND DISCUSSION Two new EuDOTA-tetraamide complexes, EuL1 and EuL2, were designed to test our hypothesis that ligand side chains containing a combination of charged polar groups (glutamyl) plus phosphonate ester groups would have a synergistic impact in reducing the rate of water exchange between the innersphere of the Eu(III) ion and bulk water.18,19,21 The synthetic details are found in the Supporting Information. High-resolution 1H NMR studies of EuL1 and EuL2 showed that these complexes exist largely as SAP coordination isomers in both D2O and H2O as evidenced by the single, highly shifted H4 cyclen protons (Figure 2). In all EuDOTA-tetraamide

Figure 3. CEST spectra of 20 mM EuL1 at pH 7, 20 μT, at different temperatures, and sat. time = 5 s.

Information) remained relatively sharp at all B1 fields up to 20 μT and at temperatures up to 321 K. Both observations are consistent with complexes exhibiting unusually slow water exchange kinetics. The Eu(III)−water residence lifetimes in these complexes were measured using two to three different well-established methods. Each method has certain limitations, but each reported reasonably similar τM values for each complex. Those values are summarized in Table 1. The most striking observation from these data was that EuL1 and EuL2 display the longest bound water lifetimes of any EuDOTA-tetraamide complexes reported to date when measured in pure water.19,21,32 Most paraCEST agents display maximum CEST intensities at or below room temperature (∼298 K) and typically show a decrease in CEST at more physiologically relevant temperatures (310 K).32 This has been one of the limitations in translating potential agents for use in vivo. Interestingly, it was found that both EuL1 and EuL2 showed an increase in CEST as the temperature was changed from 298 to 310 K when using B1 = 20 μT, similar CEST intensities at 298 and 310 K using B1

Figure 2. 1H NMR spectra of 30 mM EuL1 in water at pH 7 (top) and 14 mM EuL2 in water at pH 7 (bottom). Note that the chemical shifts are reported relative to the water resonance at 0 ppm to be consistent with the CEST spectra. The inset in the top spectrum shows the Eubound water resonance at different temperatures. The resonance near 17 ppm (*) reflects the axial H4 protons in a SAP isomer.

complexes, the H4 proton resonances are typically observed between 24 and 36 ppm for the SAP isomers and between 5 and 12 ppm for the TSAP isomers.27 In these complexes, a single H4 resonance was found near 22 ppm (when residual water peak is at 4.7 ppm) for both complexes (Figure 2); no resonances were observed in the 5−12 ppm region, indicating that these Eu3+ complexes adopt the SAP configuration exclusively, the coordination isomer that typically exhibits the slowest water exchange kinetics. It has been reported that water C

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Table 1. Eu-Bound Water Residence Lifetimes (τM) Measured for EuL1 and EuL2 compared with Similar Previously Reported Complexes τM (μs) estimated using different methods (in H2O at 298 K) Eu complex EuL1 EuL2 EuDOTA-(asp)419 EuDOTA-(asp-OEt2)419 EuDOTA-(gly OEt)421 EuDOTA-(gly)421 a

3-pool fitting-Bloch equations 735 538 266 446 210 160

± ± ± ± ± ±

15/272 ± 20b 24 23a 33 6 30/52 ± 3b

omega plot 688 ± 482 ± 223a 440a 247 ± 156 ±

temperature-dependent line width measurements

10/214 ± 15b 4

24 10

670 ± 20 n.d. n.d. n.d. n.d. n.d.

Average lifetime of multiple species. bMeasurements at 310 K.

the most widely studied paraCEST agents, EuDOTA-(gly)4−, has a bound water lifetime τM of 156 μs at 298 K.32,14 As shown in Figure 4a, this agent would be predicted to have a maximum CEST signal only when using B1 ≈ 23 μT. The current data show EuL1 and EuL2 have near-optimal τM values for in vivo CEST imaging using clinically acceptable B1 levels (5−10 μT).14 Although EuL1 displays a somewhat longer τM than EuL2, the CEST properties of EuL2 were found to be equally favorable (Supporting Information). It is known that the bulkiness and polarity of the side chain plays an important role in controlling the τM value. Mani et. al reported that introduction of hydrophobic bulky groups on the amide side chains of EuDOTA-tetraamide increases the steric bulk around the bound water site and increased the rate of water exchange.27 Introduction of even bulkier groups such as −CCH(CH2CH2CH3)2 and CCH(CH3)(CH2CH3) resulted in a further decrease in τM not by sterics but rather by a switch in geometric preference away from SAP toward TSAP in these complexes. Interestingly, although we introduced much bulkier glutamyl-phosphonate groups here, both EuL1 and EuL2 remained largely in the SAP geometry while the TSAP geometric isomer was undetectable by NMR spectroscopy. This suggests that the SAP geometry is likely governed by the four charged glutamate carboxyl groups closest to the Eu(III) combined with the added benefit of the somewhat more hydrophobic phosphonate ester moieties further away from the Eu(III) ion. This favorable combination yielded the record slow water exchange rates seen in both complexes. In an effort to further assess the molecular details that led to the unusually slow water exchange characteristics of these complexes in solution, high-resolution 1H NMR (1D and 2D), DFT calculations, and a lanthanide-induced shift (LIS) analysis were performed.4 The 1H NMR shifts observed for Yb(III) complexes are largely dipolar in origin (δdip ij ), so for axially symmetric complexes such as these, the shifts can be described by eq 1, where Cj is the Bleaney’s constant33 characteristic of the Ln3+ ion, A02⟨r2⟩ is a ligand field coefficient, r is the distance from the observed nucleus to the Yb3+ ion, and θ is the angle defined by the observed nucleus and the principal axis of the magnetic susceptibility tensor.

= 9.4 μT, and a smaller CEST intensity at 310 K when B1 = 4.7 μT (Figure 4b−d). These data are easily explained by inspection of the simulated curves in Figure 4a, where it is shown that the maximum CEST intensity depends upon both temperature (exchange rate) and B1 field. In comparison, one of

δijdip =

Cjμ2B ⎡ A 20r 2(3 cos2 θ − 1) ⎤ ⎥ ⎢ 60k 2T 2 ⎣ r3 ⎦

(1)

Since the LIS values for Yb3+ are much larger than Eu3+ and have negligible contact contributions,34−37 structure elucidation of YbL1 was first attempted before analogous calculations were also performed for EuL1 and TbL1. It was assumed that the ligands contribute eight donor atoms to the Ln(III) ion and the coordination sphere also contained a single water molecule.

Figure 4. (a) Simulated curves at 298 K showing the effect of τM on CEST intensity (Mz/Mo); color curves are for different applied B1. (b− d) CEST spectra of EuL1 at 298 and 310 K collected with three B1 values: (b) 20, (c) 9.4, and (d) 4.7 μT. D

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The DFT-derived structure was used to calculate LIS values for each proton in the complex, and these were compared with the experimental LIS values measured for the YbL1 complex by 1 H NMR (Figure 6). The furthest downfield resonance at 80.7

The structure of YbL1 obtained by DFT (Figure 5a and Supporting Information) had average Yb−Namine and Yb−

Figure 6. 1H NMR spectrum of YbL1 in D2O.

ppm can be assigned by comparison to other Yb3+ cyclen-based complexes to the axial protons of cyclen that are pointing to the side of the macrocycle opposite to the amide pendant arms (a(ax)). The chemical shift of this resonance is typical of YbDOTA-like complexes with SAP coordination.40,41 The remaining macrocyclic-backbone ethylene proton resonances were easily assigned by the spin-coupling network displayed in a 1H−1H COSY NMR spectrum (Supporting Information). According to eq 1, the Yb3+-induced 1H paramagnetic shifts in Yb-1 are expected to be proportional to the geometric factor (3 cos2 θ − 1)/r3, which can be estimated for each nucleus from the structure obtained with DFT calculations. These geometrical factors were obtained by assuming that the principal magnetic axis coincides with the Yb−Owater vector. A plot of the experimental paramagnetic shifts (δpara i ) versus the geometric term, (3 cos2 θ − 1)/r3, obtained from DFT calculations was linear (R2 > 0.998) with a slope of −2542 ± 65 ppm × Å3 (Figure 7).

Figure 5. (a) DFT-derived structure of the YbL1 complex optimized in aqueous solution at the TPSSh/LCRECP/6-31G(d) level. The blue cone shows the dipolar field induced by the Yb3+ ion. (b) Computed TPSSh/LCRECP/6-31G(d) electrostatic potential (Hartree) for the Yb-2 complex on the molecular surface defined by the 0.001 electrons· bohr−3 contour of the electronic density. (Top) Lateral view. (Bottom) Views along the pseudo C4 symmetry axis.

Oamide distances of 2.66 and 2.30 Å, respectively. These distances are reasonably close to those typically observed in the solid state for Yb3+ complexes with DOTA-tetraamide ligands (Yb−Namine ≈ 2.60 Å and Yb−Oamide ≈ 2.33 Å).15,38,39 The inner-sphere water molecule had a Yb−Owater distance of 2.488 Å. Solid state structures of complexes with DOTA-tetraamide show quite variable Yb−Owater distances, most falling in the range of 2.33−2.44 Å.15,38,39 The somewhat long calculated Yb−Owater distance in YbL1 is related to the limitations of polarizable continuum solvation models to account for hydrogen-bonding interaction involving the coordinated water molecule and second-sphere water molecules.25 The geometry around the Yb3+ ion was square antiprism (SAP) with N−C−C−N and N−C−C−O dihedral angles of −54.4° and 32.4°. Analogous calculations performed for EuL1 provided a very similar geometry with somewhat longer bond distances as a consequence of the larger ionic radius of the metal ion (Eu−Namine = 2.68 Å, Eu−Oamide = 2.42 Å, and Eu−Owater = 2.52 Å).

Figure 7. Plot of the Yb3+-induced paramagnetic shifts in (YbL1) versus the geometric factors obtained from DFT calculations. The diamagnetic contribution was assumed to be 3.0 ppm for all nuclei. Negative paramagnetic shifts correspond to shifts to higher field. The solid line represents the linear fit of the data with R2 > 0.998. E

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Inorganic Chemistry The excellent linear correlation between the experimental and the calculated shifts using the axial symmetry model described by eq 1 confirms that the 1H paramagnetic shifts in Yb-1 are largely pseudocontact in origin and that the DFTcalculated structure reasonably reflects the average dynamic structure of the complex in water. Given that the Bleaney coefficients of Yb3+ and Tb3+ are 22 and −86, respectively,33 the NMR spectrum of the Tb3+ complex would be expected to display paramagnetic shifts ca. 4 times larger than the Yb3+ complex but in the opposite direction. Several other proton resonances were easily identified in the 1H NMR spectrum of TbL1 (Supporting Information), and those data also agreed well with the structure predicted by the DFT calculations. On the basis of these combined DFT/NMR data, it was readily apparent that the pendent carboxyl groups of the glutamyl residues are positioned axially relative to the 4-fold symmetry axis of the complex while the phosphonate ester groups occupy equatorial positions. This is confirmed by the negative paramagnetic 31P NMR shift observed for YbL1 (−2.22 ppm with respect to the 31P resonance for the Lu3+ complex at 23.78 ppm), which correlates very well with the geometric factor obtained from the DFT structure (Figure 7). The negative paramagnetic shift indicates that the phosphonate groups lie beyond the magic angle of 54.7°. The DFT results provide some important insights into the slow water exchange properties of EuL1. As expected, the electrostatic potential studies carried out for YbL1 demonstrate (Figure 5b) that the most negative electrostatic potential on the molecular surface is located at the negatively charged oxygen atoms of the carboxylate groups, while the hydrophobic cyclen unit presents a slightly positive electrostatic potential.42,43 Thus, the water molecule is located in a hydrophobic pocket of neutral electrostatic potential, and this likely minimizes local hydrogen-bonding between the coordinated water molecule and nearby functional groups, resulting in slower water exchange.44 Furthermore, the bulky hydrophobic phosphonate ethyl ester groups on the periphery of the complex likely restrict the conformational movement of the glutamyl side chains, thereby reducing the solvent-accessible surface around the bound water.45 In the case of EuL2, the single ethyl ester group on each phosphonate group results in a somewhat faster water exchange and hence a decrease in CEST intensity relative to that seen for an equivalent amount of EuL1. This is likely related to the lower rigidity of the EuL2 complex resulting from the presence of phosphonate monoester compared to EuL1. Nevertheless, the water exchange rate measured for EuL2 is still lower than those measured in most previous complexes (Table 1), so the hydrophobicity of the pocket containing the coordinated water molecule together with a reduced conformational freedom seems to be the key factor in reducing water exchange rates. Only a few in vivo studies have been successfully reported on water exchange-based paraCEST complexes because most introduce too much line broadening to be detected.46−48 However, as shown in Figure 8, the complex with the lowest overall charge (EuL1) was readily detected in CEST spectra of kidneys of healthy SCID mice after injection of a single 0.4 mmol/kg dose. This amount of agent had no measurable impact on heart rate or developed pressure, and the mice fully recovered at the conclusion of each imaging experiment. The CEST intensity produced by EuL1 at 8 min after injection ranged from 6% to 14% across the entire kidney. On the basis of CEST versus [EuL1] calibration curves measured in plasma

Figure 8. (a) Proton density image of mouse after a bolus injection of 0.4 mmol/kg EuL1. (b) CEST spectrum from the ROI (yellow circle in a) shows a distinct water exchange peak near 42 ppm characteristic of the agent as it was filtered through the kidney. (c) B0 map used to correct small variations in the frequency of the water exchange peak in each pixel. CEST images (intensity differences between images collected after presaturation at ±42 ppm) of (d) the entire crosssection of the mouse at 8 min post injection and (e) the kidneys only. B1 = 10 μT.

in vitro, this corresponds to about 10−15 mM agent in kidney at the 8 min time point. In conclusion, this study illustrates that it is possible to use chemical design principles to create new paraCEST complexes with water exchange kinetics optimized to yield the largest CEST signal at 37 °C. This was made possible by combining the properties of glutamyl (charged) and phosphonate ester (hydrophobic) groups on each amide side chain. This design feature offers opportunities to create new types of responsive paraCEST agents that can be detected in vivo. The proper balance between polarity and hydrophobicity and the rigidity introduced by the presence of bulky side chains appears to be the key factor in optimizing the water exchange kinetics for CEST detection of these agents by MRI. High-resolution 1H NMR revealed that the most predominant geometry for Eu3+, Tb3+, and Yb3+ complexes is the square antiprism (SAP) structure. This correlated well with standard DFT modeling and LIS analyses of the high-resolution NMR spectra. The slow water exchange features of EuL1 and EuL2 allow the use of weaker B1 saturation pulses to activate CEST, and the in vivo experiments demonstrated that the bound water CEST signals of EuL1 and EuL2 are easily detected in mice at physiological temperatures. In comparison, the bound water signal from EuDOTA-(gly)4, a complex that displays a nearly 4-fold faster water exchange rate,48 cannot be detected in kidney images after injection at the same dosage level used here. From a general perspective, translation of paraCEST agents from in vitro observations to in vivo applications has not been fully realized even after many years of basic chemical research on optimizing their sensitivity. The agents described herein have water exchange rates nearly optimized for CEST in vitro, yet even these remain difficult to detect in vivo. The reasons for this are not fully understood, but given that many processes in biology and physiology involve proton exchange, it is likely that CEST agents behave much differently in vivo than they do in vitro. Understanding these added factors that alter the properties of CEST agents in vivo will require further detailed imaging experiments. F

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02850. Organic synthesis methods, NMR spectra for L1/L2 synthesis steps, HPLC separation of L1/L2, TbL1 LIS values, full ref 23, and Cartesian coordinates obtained with DFT calculations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; sherry@utdallas. edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge partial financial support for this work from the National Institutes of Health (CA-115531, EB-01598, EB-00482), Harold C. Simmons Cancer Center through an NCI Cancer Center Support Grant, 1P30 CA142543, and the Robert A. Welch Foundation (AT-584).

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ABBREVIATIONS CEST, chemical exchange saturation transfer; paraCEST, paramagnetic CEST; LIS, lanthanide-induced shifts; TLC, thin layer chromatography; SAP, square antiprism; TSAP, twisted square antiprism

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REFERENCES

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DOI: 10.1021/acs.inorgchem.5b02850 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry (47) Wang, X.; Wu, Y.; Soesbe, T. C.; Yu, J.; Zhao, P.; Kiefer, G. E.; Sherry, A. D. Angew. Chem., Int. Ed. 2015, 54, 8662−8664. (48) Wu, Y.; Zhang, S.; Soesbe, T. C.; Yu, J.; Vinogradov, E.; Lenkinski, R. E.; Sherry, A. D. Magn. Reson. Med. 2015, DOI: 10.1002/ mrm.25844.

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DOI: 10.1021/acs.inorgchem.5b02850 Inorg. Chem. XXXX, XXX, XXX−XXX