Tuning Phenols with Intra‐Molecular Bond Shifted HYdrogens (IM‐SHY) as diaCEST MRI Contrast Agents

The optimal exchange properties for chemical exchange saturation transfer (CEST) contrast agents on 3 T clinical scanners were characterized using continuous wave saturation transfer, and it was demonstrated that the exchangeable protons in phenols can be tuned to reach these criteria through proper ring substitution. Systematic modification allows the chemical shift of the exchangeable protons to be positioned between 4.8 to 12 ppm from water and enables adjustment of the proton exchange rate to maximize CEST contrast at these shifts. In particular, 44 hydrogen‐bonded phenols are investigated for their potential as CEST MRI contrast agents and the stereoelectronic effects on their CEST properties are summarized. Furthermore, a pair of compounds, 2,5‐dihydroxyterephthalic acid and 4,6‐dihydroxyisophthalic acid, were identified which produce the highest sensitivity through incorporating two exchangeable protons per ring.     

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.     

Enzyme‐Responsive LipoCEST Agents: Assessment of MMP‐2 Activity by Measuring the Intra‐liposomal Water 1H NMR Shift

Mobile proton‐containing solutes can be detected by MRI by the chemical exchange saturation transfer (CEST) method. CEST sensitivity is dramatically enhanced by using, as exchanging protons, the water molecules confined inside liposomes, shifted by a paramagnetic shift reagent. The chemical shift of the intraliposomal water resonance (δ^IL) is affected by the overall shape of the supramolecular system. δ^IL of a spherical LipoCEST acts as a sensitive reporter of the distribution of streptavidin proteins anchored at the liposome surface by biotinylated phospholipids. This finding prompted the design of a MMP‐2 responsive LipoCEST agent as the streptavidin moieties can be released from the liposome surfaces when a properly tailored enzyme‐cleavable peptide is inserted on the phospholipids before the terminal biotin residues. δ^IL reports on the overall changes in the supramolecular architecture associated to the cleavage carried out by MMP‐2.     

Acid-catalyzed proton exchange as a novel approach for relaxivity enhancement in Gd-HPDO3A-like complexes

A current challenge in medical diagnostics is how to obtain high MRI relaxation enhancement using Gd^III-based contrast agents (CAs) containing the minimum concentration of Gd^III ions. We report that in GdHPDO3A-like complexes a primary amide group located in close proximity to the coordinated hydroxyl group can provide a strong relaxivity enhancement at slightly acidic pH. A maximum relaxivity of r₁ = 9.8 mM⁻¹ s⁻¹ (20 MHz, 298 K) at acidic pH was achieved, which is more than double that of clinically approved MRI contrast agents under identical conditions. This effect was found to strongly depend on the number of amide protons, i.e. it decreases with a secondary amide group and almost completely vanishes with a tertiary amide. This relaxivity enhancement is attributed to an acid-catalyzed proton exchange process between the metal-coordinated OH group, the amide protons and second sphere water molecules. The mechanism and kinetics of the corresponding H⁺ assisted exchange process are discussed in detail and a novel simultaneous double-site proton exchange mechanism is proposed. Furthermore, ¹H and ¹⁷O NMR relaxometry, Chemical Exchange Saturation Transfer (CEST) on the corresponding Eu^III complexes, and thermodynamic and kinetic studies are reported. These highlight the optimal physico-chemical properties required to achieve high relaxivity with this series of Gd^III-complexes. Thus, proton exchange provides an important opportunity to enhance the relaxivity of contrast agents, providing that labile protons close to the paramagnetic center can contribute.

A novel GdHPDO3A-like complex featuring primary amide side chain induces extraordinary high relaxivity by virtue of a simultaneous double-site proton exchange mechanism under slight acidic conditions.     

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.     

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.     

Monitoring enzyme activity using a diamagnetic chemical exchange saturation transfer magnetic resonance imaging contrast agent

Chemical exchange saturation transfer (CEST) is a new approach for generating magnetic resonance imaging (MRI) contrast that allows monitoring of protein properties in vivo. In this method, a radiofrequency pulse is used to saturate the magnetization of specific protons on a target molecule, which is then transferred to water protons via chemical exchange and detected using MRI. One advantage of CEST imaging is that the magnetizations of different protons can be specifically saturated at different resonance frequencies. This enables the detection of multiple targets simultaneously in living tissue. We present here a CEST MRI approach for detecting the activity of cytosine deaminase (CDase), an enzyme that catalyzes the deamination of cytosine to uracil. Our findings suggest that metabolism of two substrates of the enzyme, cytosine and 5-fluorocytosine (5FC), can be detected using saturation pulses targeted specifically to protons at +2 ppm and +2.4 ppm (with respect to water), respectively. Indeed, after deamination by recombinant CDase, the CEST contrast disappears. In addition, expression of the enzyme in three different cell lines exhibiting different expression levels of CDase shows good agreement with the CDase activity measured with CEST MRI. Consequently, CDase activity was imaged with high-resolution CEST MRI. These data demonstrate the ability to detect enzyme activity based on proton exchange. Consequently, CEST MRI has the potential to follow the kinetics of multiple enzymes in real time in living tissue.     

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.     

A Structural Basis for 129Xe Hyper-CEST Signal in TEM-1 β-Lactamase

Genetically encoded (GE) contrast agents detectable by magnetic resonance imaging (MRI) enable non-invasive visualization of gene expression and cell proliferation at virtually unlimited penetration depths. Using hyperpolarized ¹²⁹Xe in combination with chemical exchange saturation transfer, an MR contrast approach known as hyper-CEST, enables ultrasensitive protein detection and biomolecular imaging. GE MRI contrast agents developed to date include nanoscale proteinaceous gas vesicles as well as the monomeric bacterial proteins TEM-1 β-lactamase (bla) and maltose binding protein (MBP). To improve understanding of hyper-CEST NMR with proteins, structural and computational studies were performed to further characterize the Xe-bla interaction. X-ray crystallography validated the location of a high-occupancy Xe binding site predicted by MD simulations, and mutagenesis experiments confirmed this Xe site as the origin of the observed CEST contrast. Structural studies and MD simulations with representative bla mutants offered additional insight regarding the relationship between local protein structure and CEST contrast.     

19F MRI Probes for Multimodal Imaging**

Magnetic resonance imaging (MRI) is one of the most powerful imaging tools today, capable of displaying superior soft-tissue contrast. This review discusses developments in the field of ¹⁹F MRI multimodal probes in combination with optical fluorescence imaging (OFI), ¹H MRI, chemical exchange saturation transfer (CEST) MRI, ultrasonography (USG), X-ray computed tomography (CT), single photon emission tomography (SPECT), positron emission tomography (PET), and photoacoustic imaging (PAI). In each case, multimodal ¹⁹F MRI probes compensate for the deficiency of individual techniques and offer improved sensitivity or accuracy of detection over unimodal counterparts. Strategies for designing ¹⁹F MRI multimodal probes are described with respect to their structure, physicochemical properties, biocompatibility, and the quality of images.     

Osmotically Shrunken LIPOCEST Agents: An Innovative Class of Magnetic Resonance Imaging Contrast Media Based on Chemical Exchange Saturation Transfer

The peculiar properties of osmotically shrunken liposomes acting as magnetic resonance imaging–chemical exchange saturation transfer (MRI–CEST) contrast agents have been investigated. Attention has been primarily devoted to assessing the contribution arising from encapsulated and incorporated paramagnetic lanthanide(III)‐based shift reagents in determining the chemical shift of the intraliposomal water protons, which is a relevant factor for generating the CEST contrast. It is demonstrated that a highly shifted resonance for the encapsulated water can be attained by increasing the percentage of the amphiphilic shift reagent incorporated in the liposome bilayer. It is also demonstrated that the shift contribution arising from the bulk magnetic susceptibility can be optimized through the modulation of the osmotic shrinkage. In terms of sensitivity, it is shown that the saturation transfer efficiency can be significantly improved by increasing the size of the vesicle, thus allowing a high number of exchangeable protons to be saturated. In addition, the role played by the intensity of the saturating radiofrequency field has also been highlighted.     

Carbon Dots as a New Class of Diamagnetic Chemical Exchange Saturation Transfer (diaCEST) MRI Contrast Agents

While carbon dots (C‐dots) have been extensively investigated pertaining to their fluorescent, phosphorescent, electrochemiluminescent, optoelectronic, and catalytic features, their inherent chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) properties are unknown. By virtue of their hydrophilicity and abundant exchangeable protons of hydroxyl, amine, and amide anchored on the surface, we report here that C‐dots can be adapted as effective diamagnetic CEST (diaCEST) MRI contrast agents. As a proof‐of‐concept demonstration, human glioma cells were labeled with liposomes with or without encapsulated C‐dots and implanted in mouse brain. In vivo CEST MRI was able to clearly differentiate labeled cells from non‐labeled cells. The present findings may encourage new applications of C‐dots for in vivo imaging in deep tissues, which is currently not possible using conventional fluorescent (near‐infrared) C‐dots.     

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 Frequency-Selective pH-Responsive paraCEST Agent

Paramagnetic chemical exchange saturation transfer (paraCEST) agents are well-suited for imaging tissue pH because the basis of CEST, chemical exchange, is inherently sensitive to pH. Several previous pH-sensitive paraCEST agents were based on an exchanging Ln³⁺-bound water molecule as the CEST antenna but this design often added additional line-broadening to the bulk water signal due to T₂ exchange. We report herein a pH-sensitive paraCEST agent that lacks an inner-sphere water molecule but contains one Ln-bound −OH group for CEST activation. The Yb³⁺ complex, Yb( 1 ), displayed a single, highly shifted CEST peak originating from the exchangeable Yb-OH proton, the frequency of which changed over the biologically relevant pH range. CEST images of phantoms ranging in pH from 6 to 8 demonstrate the potential of this agent for imaging pH. Initial rodent imaging studies showed that Gd( 1 ) remains in the vascular system much longer than anticipated but is cleared slowly via renal filtration.     

Iron(II) PARACEST MRI contrast agents

The first examples of Fe(II) PARACEST magnetic resonance contrast agents are reported (PARACEST = paramagnetic chemical exchange saturation transfer). The iron(II) complexes contain a macrocyclic ligand, either 1,4,7-tris(carbamoylmethyl)-1,4,7-triazacyclononane (L1) or 1,4,7-tris[(5-amino-6-methyl-2-pyridyl)methyl]-1,4,7-triazacyclononane (L2). The macrocycles bind Fe(II) in aqueous solution with formation constants of log K = 13.5 and 19.2, respectively, and maintain the Fe(II) state in the presence of air. These complexes each contain six exchangeable protons for CEST which are amide protons in [Fe(L1)](2+) or amino protons in [Fe(L2)](2+). The CEST peak for the [Fe(L1)](2+) amide protons is at 69 ppm downfield of the bulk water resonance whereas the CEST peak for the [Fe(L2)](2+) amine protons is at 6 ppm downfield of bulk water. CEST imaging using a MRI scanner shows that the CEST effect can be observed in solutions containing low millimolar concentrations of complex at neutral pH, 100 mM NaCl, 20 mM buffer at 25 °C or 37 °C.     

Rotaxane Probes for the Detection of Hydrogen Peroxide by 129Xe HyperCEST NMR Spectroscopy

The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in ¹H MRI are impaired by the high abundance of protons in the body. ¹²⁹Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional ¹H MRI because of its ability to report specific chemical environments. To date, the scope of responsive ¹²⁹Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane‐based ¹²⁹Xe hyperCEST NMR contrast agent that can be turned on in response to H₂O₂, which is upregulated in several disease states. Added H₂O₂ was detected by ¹²⁹Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H₂O₂ produced by HEK 293T cells activated with tumor necrosis factor.     

Multilamellar LipoCEST Agents Obtained from Osmotic Shrinkage of Paramagnetically Loaded Giant Unilamellar Vescicles (GUVs)

Moving from nano‐ to micro‐systems may not just be a matter of scale, but it might imply changes in the properties of the systems that can open new routes for the development of efficient MRI contrast agents. This is the case reported in the present paper, where giant liposomes (giant unilamellar vesicles, GUVs) loaded with Ln^III complexes have been studied as chemical exchange saturation transfer (CEST) MRI contrast agents. The comparison between nanosized liposomes (small unilamellar vesicles, SUVs) and GUVs sharing the same formulation led to differences that could not be accounted for only in terms of the increase in size (from 100–150 nm to 1–2 μm). Upon osmotic shrinkage, GUVs yielded a saturation‐transfer effect three order of magnitude higher than SUVs consistent with the increase in vesicles volume. Confocal microscopy showed that the shrinkage of GUVs resulted in multilamellar particles whereas SUVs are known to yield asymmetrical, discoidal shape.     

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.     

High Exchange Rate Complexes of 129Xe with Water-Soluble Pillar[5]arenes for Adjustable Magnetization Transfer MRI

Macrocyclic host structures for generating transiently bound ¹²⁹Xe have been used in various ultra-sensitive NMR and MRI applications for molecular sensing of biochemical analytes. They are based on hyperpolarized nuclei chemical exchange saturation transfer (Hyper-CEST). Here, we tested a set of water-soluble pillar[5]arenes with different counterions in order to compare their potential contrast agent abilities with that of cryptophane-A (CrA), the most widely used host for such purposes. The exchange of Xe with such compounds was found to be sensitive to the type of ions present in solution and can be used for switchable magnetization transfer (MT) contrast that arises from off-resonant pre-saturation. We demonstrate that the adjustable MT magnitude depends on the interplay of saturation parameters and found that the optimum MT contrast surpasses the CrA CEST performance at moderate saturation power. Since modification of such water-soluble pillar[5]arenes is straightforward, these compounds can be considered a promising platform for designing various sensors that may complement the field of Xe HyperCEST-based biosensing MRI.     

Live‐cell MRI with Xenon Hyper‐CEST Biosensors Targeted to Metabolically Labeled Cell‐Surface Glycans

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper‐CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper‐CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live‐cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell‐surface glycans at nanomolar concentrations.