Oxidative Conversion of a Europium(II)‐Based T1 Agent into a Europium(III)‐Based paraCEST Agent that can be Detected In Vivo by Magnetic Resonance Imaging

The Eu^II complex of 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) tetra(glycinate) has a higher reduction potential than most Eu^II chelates reported to date. The reduced Eu^II form acts as an efficient water proton T₁ relaxation reagent, while the Eu^III form acts as a water‐based chemical exchange saturation transfer (CEST) agent. The complex has extremely fast water exchange rate. Oxidation to the corresponding Eu^III complex yields a well‐defined signal from the paraCEST agent. The time course of oxidation was studied in vitro and in vivo by T₁‐weighted and CEST imaging.     

Six,Seven or Eight Coordinate FeII,CoII or NiII Complexes of Amide‐Appended Tetraazamacrocycles for ParaCEST Thermometry

Fe^II, Co^II and Ni^II complexes of two tetraazamacrocycles (1,4,8,11‐tetrakis(carbamoylmethyl)‐1,4,8,11‐tetraazacyclotetradecane ( L1 ) and 1,4,7,10‐tetrakis(carbamoylmethyl)‐1,4,7,10‐tetraazacyclododecane ( L2 ) show promise as paraCEST agents for registration of temperature (paraCEST=paramagnetic chemical exchange saturation transfer). The Fe^II, Co^II and Ni^II complexes of L1 show up to four CEST peaks shifted ≤112 ppm, whereas analogous complexes of L2 show only a single CEST peak at ≤69 ppm. Comparison of the temperature coefficients (C_T) of the CEST peaks of [Co( L2 )]²⁺, [Fe( L2 )]²⁺, [Ni( L1 )]²⁺ and [Co( L1 )]²⁺ showed that a CEST peak of [Co( L1 )]²⁺ gave the largest C_T (?0.66 ppm ^oC^?1 at 4.7 T). NMR spectral and CEST properties of these complexes correspond to coordination complex symmetry as shown by structural data. The [Ni( L1 )]²⁺ and [Co( L1 )]²⁺ complexes have a six‐coordinate metal ion bound to the 1‐, 4‐amide oxygen atoms and four nitrogen atoms of the tetraazamacrocycle. The [Fe( L2 )]²⁺ complex has an unusual eight‐coordinate Fe^II bound to four amide oxygen atoms and four macrocyclic nitrogen atoms. For [Co( L2 )]²⁺, one structure has seven‐coordinate Co^II with three bound amide pendents and a second structure has a six‐coordinate Co^II with two bound amide pendents.     

Chemical Exchange Saturation Transfer (CEST) Agents: Quantum Chemistry and MRI

Diamagnetic chemical exchange saturation transfer (CEST) contrast agents offer an alternative to Gd³⁺‐based contrast agents for MRI. They are characterized by containing protons that can rapidly exchange with water and it is advantageous to have these protons resonate in a spectral window that is far removed from water. Herein, we report the first results of DFT calculations of the ¹H nuclear magnetic shieldings in 41 CEST agents, finding that the experimental shifts can be well predicted (R²=0.882). We tested a subset of compounds with the best MRI properties for toxicity and for activity as uncouplers, then obtained mice kidney CEST MRI images for three of the most promising leads finding 16 (2,4‐dihydroxybenzoic acid) to be one of the most promising CEST MRI contrast agents to date. Overall, the results are of interest since they show that ¹H NMR shifts for CEST agents—charged species—can be well predicted, and that several leads have low toxicity and yield good in vivo MR images.     

Lanthanide‐Based T2ex and CEST Complexes Provide Insights into the Design of pH Sensitive MRI Agents

The CEST and T₁ /T₂ relaxation properties of a series of Eu³⁺ and Dy³⁺ DOTA‐tetraamide complexes with four appended primary amine groups are measured as a function of pH. The CEST signals in the Eu³⁺ complexes show a strong CEST signal after the pH was reduced from 8 to 5. The opposite trend was observed for the Dy³⁺ complexes where the r_2ex of bulk water protons increased dramatically from ca. 1.5 mm ⁻¹ s⁻¹ to 13 mm ⁻¹ s⁻¹ between pH 5 and 9 while r₁ remained unchanged. A fit of the CEST data (Eu³⁺ complexes) to Bloch theory and the T_2ex data (Dy³⁺ complexes) to Swift–Connick theory provided the proton‐exchange rates as a function of pH. These data showed that the four amine groups contribute significantly to proton‐catalyzed exchange of the Ln³⁺‐bound water protons even though their pK _a’s are much higher than the observed CEST or T_2ex effects. This demonstrated the utility of using appended acidic/basic groups to catalyze prototropic exchange for imaging tissue pH by MRI.     

Europium(III) macrocyclic complexes with alcohol pendant groups as chemical exchange saturation transfer agents

Paramagnetic lanthanide(III) complexes that contain hyperfine-shifted exchangeable protons offer considerable advantages over diamagnetic molecules as chemical exchange saturation transfer (CEST) agents for MRI. As part of a program to investigate avenues to improve the sensitivity of such agents, the CEST characteristics of europium(III) macrocyclic complexes having appended hydroxyethyl groups were investigated. The CEST spectrum of the asymmetrical complex, EuCNPHC3+, shows five distinct peaks for each magnetically nonequivalent exchangeable proton in the molecule. The CEST spectra of this complex were fitted to NMR Bloch theory to yield exchange rates between each of six exchanging proton pools (five on the agent plus bulk water). Exchange between the Eu3+-bound hydroxyl protons and bulk water protons was slow in dry acetonitrile but accelerated incrementally upon stepwise addition of water. In pure water, exchange was too fast to observe a CEST effect. The utility of this class of europium(III) complex for CEST imaging applications is ultimately limited by the small chemical shifts induced by the hydroxyl-appended ligands of this type and the resulting small Deltaomega values for the exchangeable hydroxyl protons.     

N-Aryl Amides as Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agents

Chemical exchange saturation transfer (CEST) MRI has recently emerged as a versatile molecular imaging approach in which diamagnetic compounds can be utilized to generate an MRI signal. To expand the scope of CEST MRI applications, herein, we systematically investigated the CEST properties of N-aryl amides with different N-aromatic substitution, revealing their chemical shifts (4.6–5.8 ppm) and exchange rates (up to thousands s⁻¹) are favorable to be used as CEST agents as compared to alkyl amides. As the first proof-of-concept study, we used CEST MRI to detect the enzymatic metabolism of the drug acebutolol directly by its intrinsic CEST signal without any chemical labeling. Our study implies that N-aryl amides may enable the label-free CEST MRI detection of the metabolism of many N-aryl amide-containing drugs and a variety of enzymes that act on N-aryl amides, greatly expanding the scope of CEST MR molecular imaging.     

Detection of Sulfatase Enzyme Activity with a CatalyCEST MRI Contrast Agent

A chemical exchange saturation transfer (CEST) MRI contrast agent has been developed that detects sulfatase enzyme activity. The agent produces a CEST signal at δ=5.0 ppm before enzyme activity, and a second CEST signal appears at δ=9.0 ppm after the enzyme cleaves a sulfate group from the agent. The comparison of the two signals improved detection of sulfatase activity.     

A pH‐Responsive MRI Agent that Can Be Activated Beyond the Tissue Magnetization Transfer Window

A terbium‐based complex that displays a water exchange CEST resonance well outside the normal magnetization transfer (MT) frequency range of tissues provides a direct readout of pH values by MRI. Deprotonation of the phenolic proton in this complex results in a frequency shift of 56 ppm in a bound water molecule exchange peak between pH 5 and 8. This allows direct imaging of pH without prior knowledge of the agent concentration and with essentially no interference from the tissue MT signal.     

CoII Complexes as Liposomal CEST Agents

Three paramagnetic Co^II macrocyclic complexes containing 2‐hydroxypropyl pendant groups, 1,1′,1′′,1′′′‐(1,4,8,11‐tetraazacyclotetradecane‐1,4,8,11‐tetrayl)tetrakis‐ (propan‐2‐ol) ([Co(L1)]²⁺, 1,1′‐(4,11‐dibenzyl‐1,4,8,11‐tetraazacyclotetradecane‐1,8‐diyl)bis(propan‐2‐ol) ([Co(L2)]²⁺), and 1,1′‐(4,11‐dibenzyl‐1,4,8,11‐tetraazacyclotetradecane‐1,8‐diyl)bis(octadecan‐2‐ol) ([Co(L3)]²⁺) were synthesized to prepare transition metal liposomal chemical exchange saturation transfer (lipoCEST) agents. In solution, ([Co(L1)]²⁺) forms two isomers as shown by ¹H NMR spectroscopy. X‐ray crystallographic studies show one isomer with 1,8‐pendants in cis‐configuration and a second isomer with 1,4‐pendants in trans‐configuration. The [Co(L2)]²⁺ complex has 1,8‐pendants in a cis‐configuration. Remarkably, the paramagnetic‐induced shift of water ¹H NMR resonances in the presence of the [Co(L1)]²⁺ complex is as large as that observed for one of the most effective Ln^III water proton shift agents. Incorporation of [Co(L1)]²⁺ into the liposome aqueous core, followed by dialysis against a solution of 300 mOsm L^?1 produces a CEST peak at 3.5 ppm. Incorporation of the amphiphilic [Co(L3)]²⁺ complex into the liposome bilayer produces a more highly shifted CEST peak at ?13 ppm. Taken together, these data demonstrate the feasibility of preparing Co^II lipoCEST agents.     

Modulation of water exchange in europium(III) DOTA-tetraamide complexes via electronic substituent effects

The chemical exchange saturation transfer (CEST) efficiency for a series Eu3+-based tetraamide complexes bearing p-substituents on a single coordinating pendant arm is highly sensitive to water exchange rates. The CEST effect increases in the order Me < MeO < F approximately CO2tBu < CN < H. These results show that CEST contrast can be modulated by changes in electron density at a single ligating atom, and this forms the basis of creating imaging agents that respond to chemical oxidation and reduction.     

On‐bead combinatorial synthesis and imaging of europium(III)‐based paraCEST agents aids in identification of chemical features that enhance CEST sensitivity

The rate of water exchange between the inner sphere of a paramagnetic ion and bulk water is an important parameter in determining the magnitude of the chemical exchange saturation transfer signal from paramagnetic CEST agents (paraCEST). This is governed by various geometric, steric and ligand field factors created by macrocyclic ligands surrounding the paramagnetic metal ion. Our previous on‐bead combinatorial studies of di‐peptoid–europium(III)–1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA)–tetraamide complexes revealed that negatively charged groups in the immediate vicinity of the metal center strongly enhances the CEST signal. Here, we report a solid phase synthesis and on‐bead imaging of 76 new DOTA derivatives that are developed by coupling with a single residue onto each of the three arms of a DOTA–tetraamide scaffold attached to resin beads. This single residue predominantly carries negatively charged groups blended with various physico‐chemical characteristics. We found that non‐bulky negatively charged groups are best suited at the immediate vicinity of the metal ion, while positive, bulky and halogen containing moieties suppress the CEST signal. Copyright © 2017 John Wiley & Sons, Ltd.     

A paramagnetic CEST agent for imaging glucose by MRI

The europium(III) complex of a DOTA-tetraamide ligand (DOTA = 1,4,7,10-tetraazacyclododecane-N,N',N' ',N' '-tetraacetic acids) containing two phenyl boronate pendent arms binds glucose reversibly with an association constant of 383 M-1 at pH 7. Glucose binding results in slowing of water exchange between a single Eu(III)-bound water molecule and bulk water, and this can be imaged by MRI using chemical exchange saturation transfer (CEST) imaging sequence. This metabolite-responsive paramagnetic CEST agent responds to changes in glucose over the physiologically important range (0-20 mM), and thus it offers the possibility of high-sensitivity MR imaging glucose in tissues using bulk water protons as antenna.     

Directly Functionalized Cucurbit[7]uril as a Biosensor for the Selective Detection of Protein Interactions by 129Xe hyperCEST NMR

Advancement of hyperpolarized ¹²⁹Xe MRI technology toward clinical settings demonstrates the considerable interest in this modality for diagnostic imaging. The number of contrast agents, termed biosensors, for ¹²⁹Xe MRI that respond to specific biological targets, has grown and diversified. Directly functionalized xenon-carrying macrocycles, such as the large family of cryptophane-based biosensors, are good for localization-based imaging and provide contrast before and after binding events occur. Noncovalently functionalized constructs, such as cucurbituril- and cyclodextrin-based biosensors, benefit from commercial availability and optimal exchange dynamics for CEST imaging. In this work, we report the first directly functionalized cucurbituril used as a xenon biosensor. Biotinylated cucurbit[7]uril (btCB7) gives rise to a ¹²⁹Xe hyperCEST response at the unusual shift of δ=28 ppm when bound to its protein target with substantial CEST contrast. We posit that the observed chemical shift is due to the deformation of btCB7 upon binding to avidin, caused by proximity to the protein surface. Conformational searches and molecular dynamics (MD) simulations support this hypothesis. This construct combines the strengths of both families of biosensors, enables a multitude of biological targets through avidin conjugation, and demonstrates the advantages of functionalized cucurbituril-based biosensors.     

Detecting Enzyme Activities with Exogenous MRI Contrast Agents

This review focuses on exogenous magnetic resonance imaging (MRI) contrast agents that are responsive to enzyme activity. Enzymes can catalyze a change in water access, rotational tumbling time, the proximity of a ¹⁹F‐labeled ligand, the aggregation state, the proton chemical‐exchange rate between the agent and water, or the chemical shift of ¹⁹F, ³¹P, ¹³C or a labile ¹H of an agent, all of which can be used to detect enzyme activity. The variety of agents attests to the creativity in developing enzyme‐responsive MRI contrast agents.     

A conjugated polymer-Gd (III) complex as pH sensitive contrast agent in magnetic resonance imaging

A novel pH-responsive contrast agent (PFP-aa/Gd) for magnetic resonance imaging (MRI) was prepared by binding Gd(III) to a water-soluble conjugated polyfluorene with pendant carboxylate and amine moieties. The PFP-aa is a good chelator for Gd³⁺ and the PFP-aa/Gd complex has good stability. As the pH changes from 10.0 to 4.0, both the carboxylate and amine are protonated, thus PFP-aa exhibits positive charges and forms tight aggregation, which reduces molecular tumbling and accelerates the exchange of bound water leading to the increase of relaxivity R ₁. More importantly, the R ₁ increases by about eight fold as the pH changes from 8.0 to 6.0, which makes PFP-aa/Gd suitable as a potential marker of the pH below physiological level. In comparison to other contrast agents, the unique sensitivity of the water relaxivity of PFP-aa/Gd indicates that this complex could be used in MRI experiments to monitor physiological pH change.     

Lanthanide(III) Complexes of Tris(amide) PCTA Derivatives as Potential Bimodal Magnetic Resonance and Optical Imaging Agents

Lanthanide complexes of two tris(amide) derivatives of PCTA were synthesized and characterized. The relaxometric and luminescence properties of their lanthanide complexes were investigated as bimodal magnetic resonance (MR) and optical imaging agents. Luminescence studies show that one of the Tb^III complexes dimerizes in solution at low millimolar concentrations, whereas the other may have a higher than expected coordination number in solution. The corresponding Gd^III complexes display unusually high T₁ relaxivities and enhanced kinetic inertness compared to GdPCTA. These features suggest that these new chelates may be suitable for in vivo applications. The fast water‐exchange rates observed for these complexes make them unsuitable as paramagnetic chemical exchange saturation transfer (PARACEST) agents.     

Towards an Improved Design of MRI Contrast Agents: Synthesis and Relaxometric Characterisation of Gd-HPDO3A Analogues

The properties of Ln^III-HPDO3A complexes as relaxation enhancers and paraCEST agents are essentially related to the hydroxylpropyl moiety. A series of three HPDO3A derivatives, with small modifications to the hydroxyl arm, were herein investigated to understand how heightened control can be gained over the parameters involved in the design of these agents. A full ¹H and ¹⁷O-NMR relaxometric analysis was conducted and demonstrated that increasing the length of the OH group from the lanthanide centre significantly enhanced the water exchange rate of the gadolinium complex, but with a subsequent reduction in kinetic stability. Alternatively, the introduction of an additional methyl group, which increased the steric bulk around the OH moiety, resulted in the formation of almost exclusively the TSAP isomer (95 %) as identified by ¹H-NMR of the europium complex. The gadolinium analogue of this complex also exhibited a very fast water exchange rate, but with no detectable loss of kinetic stability. This complex therefore demonstrates a notable improvement over Gd-HPDO3A.     

Exceptionally Inert Lanthanide(III) PARACEST MRI Contrast Agents Based on an 18‐Membered Macrocyclic Platform

We report a macrocyclic ligand based on a 3,6,10,13‐tetraaza‐1,8(2,6)‐dipyridinacyclotetradecaphane platform containing four hydroxyethyl pendant arms (L¹) that forms extraordinary inert complexes with Ln³⁺ ions. The [EuL¹]³⁺ complex does not undergo dissociation in 1 M HCl over a period of months at room temperature. Furthermore, high concentrations of phosphate and Zn²⁺ ions at room temperature do not provoke metal‐complex dissociation. The X‐ray crystal structures of six Ln³⁺ complexes reveal ten coordination of the ligand to the metal ions through the six nitrogen atoms of the macrocycle and the four oxygen atoms of the hydroxyethyl pendant arms. The analysis of the Yb³⁺‐ and Pr³⁺‐induced paramagnetic ¹H NMR shifts show that the solid‐state structures are retained in aqueous solution. The intensity of the ¹H NMR signal of bulk water can be modulated by saturation of the signals of the hydroxy protons of Pr³⁺, Eu³⁺, and Yb³⁺ complexes following chemical‐exchange saturation transfer (CEST). The ability of these complexes to provide large CEST effects at 25 and 37 °C and pH 7.4 was confirmed by using CEST magnetic resonance imaging experiments.     

A pH-Sensitive Zwitterionic Iron Complex Probe with High Biocompatibility for Tumor-Specific Magnetic Resonance Imaging

Accurate diagnosis of tumor characteristics, including its location and boundary, is of immense value to subsequent therapy. Activatable magnetic resonance imaging (MRI) contrast agents that respond to tumor-specific microenvironments, such as the redox state, pH, and enzyme activity, enable better mapping of tumor tissue. However, the practical application of most reported activatable agents is hampered by problems including potential toxicity, inefficient elimination, and slow activation. In this study, we developed a zwitterionic iron complex (Fe-ZDS) as a positive MRI contrast agent for tumor-specific imaging. Fe-ZDS could dissociate in weakly acidic solution rapidly, accompanied by clear longitudinal relaxivity (r₁) enhancement, which enabled the complex to act as a pH-sensitive contrast agent for tumor-specific MR imaging. In vivo experiments showed that Fe-ZDS rapidly enhanced the tumor-to-normal contrast ratio by >40 %, which assisted in distinguishing the tumor boundary. Furthermore, Fe-ZDS circulated freely in the bloodstream and was excreted relatively safely via kidneys owing to its zwitterionic nature. Therefore, Fe-ZDS is an ideal candidate for a tumor-specific MRI contrast agent and holds considerable potential for clinical translation.     

Hexameric MnII Dendrimer as MRI Contrast Agent

A Mn^II chelating dendrimer was prepared as a contrast agent for MRI applications. The dendrimer comprises six tyrosine‐derived [Mn(EDTA)(H₂O)]^2? moieties coupled to a cyclotriphosphazene core. Variable temperature ¹⁷O NMR spectroscopy revealed a single water co‐ligand per Mn^II that undergoes fast water exchange (k_ex=(3.0±0.1)×10⁸ s^?1 at 37 °C). The 37 °C per Mn^II relaxivity ranged from 8.2 to 3.8 mM ^?1 s^?1 from 0.47 to 11.7 T, and is sixfold higher on a per molecule basis. From this field dependence a rotational correlation time was estimated as 0.45(±0.02) ns. The imaging and pharmacokinetic properties of the dendrimer were compared to clinically used [Gd(DTPA)(H₂O)]^2? in mice at 4.7 T. On first pass, the higher per ion relaxivity of the dendrimer resulted in twofold greater blood signal than for [Gd(DTPA)(H₂O)]^2?. Blood clearance was fast and elimination occurred through both the renal and hepatobiliary routes. This Mn^II containing dendrimer represents a potential alternative to Gd‐based contrast agents, especially in patients with chronic kidney disease where the use of current Gd‐based agents may be contraindicated.