Enhancing r1 Relaxivity in GdDOTA-Monoamide Complexes through Polar Group-Mediated Ordering of Second-Sphere Water Molecules.

This study was designed to test whether the single appended phosphonate group in GdDOTA-1AmP is sufficient for catalyzing the exchange of proton from the single inner-sphere water-exchanging molecule. Unlike the other phosphonate derivatives in this series, GdDOTA-1AmP showed a surprisingly smooth increase in r1 relaxivity from 3.0 to 6.3 mM-1 s-1 at 20 MHz as the pH was lowered from 9 to 2.5. In comparison to the bis-, tris-, and tetrakis-phosphonate analogues, which all show a biphasic dependence of r1 with changes in pH, the unique r1 versus pH characteristics of GdDOTA-1AmP are shown to closely parallel deprotonation of the single appended phosphonate group. Although the tissue biodistribution and clearance rates of GdDOTA-1AmP are more favorable than the other more highly charged phosphonate derivatives, the pH dependency of r1 is substantially reduced at magnetic fields typically used for small animal imaging (7 and 9.4T), so the attractiveness of this new molecule for quantitative imaging of tissue pH is diminished. However, this study provides some new insights into the feasibility of designing pH-responsive MRI contrast agents based upon fundamental acid-base prototropic mechanisms.

A Responsive Magnetic Resonance Imaging Contrast Agent for Detection of Excess Copper(II) in the Liver In Vivo.

The design, synthesis, and properties of a new gadolinium-based copper-responsive magnetic resonance imaging (MRI) contrast agent is presented. The sensor (GdL1) has high selectivity for copper ions and exhibits a 43% increase in r1 relaxivity (20 MHz) upon binding to 1 equiv of Cu2+ in aqueous buffer. Interestingly, in the presence of physiological levels of human serum albumin (HSA), the r1 relaxivity is amplified further up to 270%. Additional spectroscopic and X-ray absorption spectroscopy (XAS) studies show that Cu2+ is coordinated by two carboxylic acid groups and the single amine group on an appended side chain of GdL1 and forms a ternary complex with HSA (GdL1-Cu2+-HSA). T1-weighted in vivo imaging demonstrates that GdL1 can detect basal, endogenous labile copper(II) ions in living mice. This offers a unique opportunity to explore the role of copper ions in the development and progression of neurological diseases such as Wilson's disease.

Imaging Insulin Secretion from Mouse Pancreas by MRI Is Improved by Use of a Zinc-Responsive MRI Sensor with Lower Affinity for Zn2+ Ions.

It has been demonstrated that divalent zinc ions packaged with insulin in ß-cell granules can be detected by MRI during glucose-stimulated insulin secretion using a gadolinium-based Zn2+-sensitive agent. This study was designed to evaluate whether a simpler agent design having single Zn2+-sensing moieties but with variable Zn2+ binding affinities might also detect insulin secretion from the pancreas. Using an implanted MR-compatible window designed to hold the pancreas in a fixed position for imaging, we now demonstrate that focally intense "hot spots" can be detected in the tail of the pancreas using these agents after administration of glucose to stimulate insulin secretion. Histological staining of the same tissue verified that the hot spots identified by imaging correspond to clusters of islets, perhaps reflecting first-responder islets that are most responsive to a sudden increase in glucose. A comparison of images obtained when using a high-affinity Zn2+ sensor versus a lower-affinity sensor showed that the lower-affinity sensors produced the best image contrast. An equilibrium model that considers all possible complexes formed between Zn2+, the GdL sensor, and HSA predicts that a GdL sensor with lower affinity for Zn2+ generates a lower background signal from endogenous Zn2+ prior to glucose-stimulated insulin secretion (GSIS) and that the weaker binding affinity agent is more responsive to a further increase in Zn2+ concentration near ß-cells after GSIS. These model predictions are consistent with the in vivo imaging observations.

Enantiomeric Recognition of d- and l-Lactate by CEST with the Aid of a Paramagnetic Shift Reagent.

A previous report demonstrated that EuDO3A could be used as an NMR shift reagent for imaging extracellular lactate produced by cancer cells using CEST imaging. In this work, a series of heptadentate macrocyclic YbDO3A-trisamide complexes with δ-chiral carbons in the three pendant side-arms were examined as shift reagents for lactate detection. High resolution 1H NMR spectra and DFT calculations provided evidence for the formation of stereoselective lactate·YbDO3A-trisamide complexes each with a different CEST signature. This stereoselectivity allowed discrimination of d- versus l-lactate by both high-resolution NMR and CEST. This work demonstrates that lanthanide-based paramagnetic shift reagents can be designed to detect important metabolites by CEST MRI selectively.

Imaging Extracellular Lactate In Vitro and In Vivo Using CEST MRI and a Paramagnetic Shift Reagent.

Overproduction of lactate is a hallmark of cancer, yet a method to quantitatively measure lactate production by cancer cells is not straight-forward. Chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) can potentially be used to image lactate but the small difference in chemical shift of the lactate -OH proton and water proton resonances make it challenging. Like other spectroscopic methods, CEST MRI cannot discriminate intracellular lactate from extracellular lactate. Herein, we demonstrate a relatively simple way to shift the lactate -OH proton resonance far away from water by addition of the paramagnetic shift reagent, EuDO3A, while retaining the CEST properties of lactate itself. The potential of the method was demonstrated by imaging extracellular lactate excreted from lung cancer cells in tissue culture without interference from other components in the culture media and by imaging excess lactate excreted into the bladder of a mouse.

Protonation of carboxyl groups in EuDOTA-tetraamide complexes results in catalytic prototropic exchange and quenching of the CEST signal.

The CEST properties of EuDOTA-tetraamide complexes bearing pendant carboxylate and carboxyl ethyl esters were measured as a function of pH. The CEST signal from the Eu3+-bound water molecule decreased in intensity between pH 8.5 and 4.5 while the proton exchange rates (kex) increased over this same pH range. In comparison, the CEST signal in the corresponding carboxyl ester derivatives was nearly constant. Both observations are consistent with stepwise protonation of the four carboxylic acid groups over this same pH range. This indicates that negative charges on the carboxyl groups above pH 6 facilitate the formation of a strong hydrogen-bonding network in the coordination second sphere above the single Eu3+-bound water molecule, thereby decreasing prototropic exchange of protons on the bound water molecule with bulk water protons. The percentage of square antiprismatic versus twisted square antiprism coordination isomers also decreased as the appended carboxylic acid groups were positioned further away from the amide. The net effect of lowering the pH was an overall increase in kex and a quenching of the CEST signal.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

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 r2ex 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 T2ex 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 pKa 's are much higher than the observed CEST or T2ex effects. This demonstrated the utility of using appended acidic/basic groups to catalyze prototropic exchange for imaging tissue pH by MRI.

The Relationship between NMR Chemical Shifts of Thermally Polarized and Hyperpolarized 89 Y Complexes and Their Solution Structures.

Recently developed dynamic nuclear polarization (DNP) technology offers the potential of increasing the NMR sensitivity of even rare nuclei for biological imaging applications. Hyperpolarized 89 Y is an ideal candidate because of its narrow NMR linewidth, favorable spin quantum number (I=1/2 ), and long longitudinal relaxation times (T1 ). Strong NMR signals were detected in hyperpolarized 89 Y samples of a variety of yttrium complexes. A dataset of 89 Y NMR data composed of 23 complexes with polyaminocarboxylate ligands was obtained using hyperpolarized 89 Y measurements or 1 H,89 Y-HMQC spectroscopy. These data were used to derive an empirical equation that describes the correlation between the 89 Y chemical shift and the chemical structure of the complexes. This empirical correlation serves as a guide for the design of 89 Y sensors. Relativistic (DKH2) DFT calculations were found to predict the experimental 89 Y chemical shifts to a rather good accuracy.

Unexpected Changes in the Population of Coordination Isomers for the Lanthanide Ion Complexes of DOTMA-Tetraglycinate.

Lanthanide complexes with DOTA-tetraglycinate (DOTA-(gly)4) heavily favor the square antiprismatic (SAP) coordination isomer in aqueous solution, a structural feature that has made them useful as water-based paraCEST agents. In an effort to create amide-based paraCEST agents with rapid water exchange rates, we prepared the analogous tetraglycinate complexes with DOTMA, a ligand known to favor the twisted square antiprismatic (TSAP) coordination structures. Unexpectedly, NMR investigations show that the LnDOTMA-(gly)4 complexes, like the LnDOTA-(gly)4 complexes, also favor the SAP isomers in solution. This observation led to density functional theory (DFT) calculations in order to identify the energy terms that favor the SAP structures in lanthanide complexes formed with macrocyclic DOTA- and DOTMA-tetraamide ligands. The DFT calculations revealed that, regardless the nature of the ligand, the TSAP isomers present more negative hydration energies than the SAP counterparts. The extent to which the TSAP isomer is stabilized varies, however, depending on the ligand structure, resulting in different isomeric populations in solution.

Breaking the Barrier to Slow Water Exchange Rates for Optimal Magnetic Resonance Detection of paraCEST Agents.

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.

Amplifying the sensitivity of zinc(II) responsive MRI contrast agents by altering water exchange rates.

Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI contrast agent, GdDOTA-diBPEN, new structural targets were rationally designed to increase the rate of water exchange to improve MRI detection sensitivity. These new sensors exhibit fine-tuned water exchange properties and, depending on the individual structure, demonstrate significantly improved longitudinal relaxivities (r1). Two sensors in particular demonstrate optimized parameters and, therefore, show exceptionally high longitudinal relaxivities of about 50 mM(-1) s(-1) upon binding to Zn(II) and human serum albumin (HSA). This value demonstrates a 3-fold increase in r1 compared to that displayed by the original sensor, GdDOTA-diBPEN. In addition, this study provides important insights into the interplay between structural modifications, water exchange rate, and kinetic stability properties of the sensors. The new high relaxivity agents were used to successfully image Zn(II) release from the mouse pancreas in vivo during glucose stimulated insulin secretion.

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.

The stereochemistry of amide side chains containing carboxyl groups influences water exchange rates in EuDOTA-tetraamide complexes.

Many Eu(III) complexes formed with DOTA-tetraamide ligands (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) have sufficiently slow water exchange kinetics to meet the slow-to-intermediate condition required to serve as chemical exchange saturation transfer (CEST) contrast agents for MRI. This class of MRI contrast agents offers an attractive platform for creating biological sensors because water exchange is exquisitely sensitive to subtle ligand stereochemistry and electronic effects. Introduction of carboxyl groups or carboxyl ethyl ester groups on the amide substituents has been shown to slow water exchange in these complexes, but less is known about the orientation or position of these side-chain groups relative to the inner-sphere Eu(III)-bound water molecule. In this study, a series of Eu(III) complexes having one or more carboxyl groups or carboxyl esters at the δ-position of the pendant amide side chains were prepared. Initial attempts to prepare optically pure EuDOTA-[(S)-Asp]4 resulted in a chemically pure ligand consisting of a mixture of stereochemical isomers. This was traced to racemization of (S)-aspartate diethyl ester during the synthetic procedure. Nevertheless, NMR studies of the Eu(III) complexes of this mixture revealed that each isomer had a different water exchange rate, differing by a factor of 2 or more. A second controlled synthesis and CEST study of EuDOTA-[(S)-Asp]4 and cis-EuDOTA-[(S)-Asp]2[(R)-Asp]2 confirmed that the water exchange rates in these diastereomeric complexes are controlled by the axial versus equatorial orientation of the carboxyl groups on the amide side chains. These observations provide new insights toward the development of even more slowly water exchanging systems which will be necessary for practical in vivo applications.

Strategies for labeling proteins with PARACEST agents.

Reactive surface lysine groups on the monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4 and 10.1 chelates were added per 3G4 molecule and between 5.6 and 5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53µs for the non-conjugated chelate to 65-77µs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73-75µs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein.

A responsive europium(III) chelate that provides a direct readout of pH by MRI.

A europium(III) DO3A-tris(amide) complex is reported for imaging pH by MRI using ratiometric chemical exchange saturation transfer (CEST) principles. Deprotonation of a single phenolic proton between pH 6 and 7.6 results in an ∼5 ppm shift in the water exchange CEST peak that is easily detected by MRI. Collection of two CEST images at two slightly different activation frequencies provides a direct readout of solution pH without the need of a concentration marker.

Polymeric PARACEST MRI contrast agents as potential reporters for gene therapy.

Gene therapy is a potentially powerful treatment approach that targets molecular remedies for disease. Among other challenges it remains difficult to monitor gene delivery and its downstream metabolic consequences. Approaches to MRI gene reporters have been reported but few have the potential for translation beyond isolated cell systems. Herein, we report a polycationic polymer MRI contrast agent that binds to DNA in a ratio of one monomer unit per phosphate group of DNA. Significantly, this binding event diminishes the MR contrast signal from the agent itself potentially providing a platform for imaging delivery and release of a gene into cells and tissues. Importantly, we demonstrate here the proof of concept that a positively charged polymeric contrast agent can also act as a transfection agent, delivering the gene for encoding green fluorescent protein into cells. These observations provide support for the radical, new idea of creating a combined transfection/imaging agent for monitoring gene delivery in real time by MRI.

Effect of the regiochemistry of butyl amide substituents on the solution-state structures of lanthanide(III) DOTA-tetraamide complexes.

The coordination geometry adopted by the lanthanide complexes of DOTA-tetraamides is a critical factor in determining their water exchange kinetics. Controlling the water exchange kinetics of DOTA-tetraamide complexes, and by extension their coordination geometry, is of particular interest because of the potential application of this class of complex as PARACEST MRI contrast agents. To facilitate the maximum CEST effect at the lowest pre-saturation powers much slower exchange kinetics are required than are commonly observed with these types of chelates. Complexes that adopt the more slowly exchanging square antiprismatic coordination geometry are therefore preferred; however, the factors that govern which coordination geometry is preferred remain unclear. A series of DOTA-tetraamide complexes with butyl amide substituents in different regioisomeric configurations provides some insight into these factors. The population of each coordination geometry was found to vary substantially depending upon the regiochemistry of the butyl amide substituent. It was observed that the twisted square antiprism coordination geometry, usually favored in complexes with the larger lanthanide ions only, is also increasingly favored for certain DOTA-tetraamide complexes with the smaller lanthanides. This is in marked contrast to simple DOTA-tetraamide complexes such as DOTAM. The effect was more prevalent in complexes formed with more bulky and more electron donating amide butyl substituents. It is also associated with loss of an inner-sphere water molecule from the complexes of later lanthanides that adopt the twisted square antiprismatic geometry. The complexes with sec-butyl substituents are inherently more complicated because of the introduction of a stereochemical center into each pendant arm. Unlike chiral complexes with larger amide substituents there is no "locking" effect of the orientation of the pendant arms in these complexes and up to four diastereoisomeric coordination isomers can be observed.

Polymeric PARACEST agents for enhancing MRI contrast sensitivity.

Linear polymers of PARACEST agents were prepared by using classical free radical chain polymerization conditions. The Eu3+-polymers exhibited similar intermediate-to-slow water exchange and CEST characteristics as the Eu3+-monomers. This provided an avenue to lower the detection limit of these imaging agents substantially and makes them potentially useful as MRI sensors for molecular imaging.

A europium(III)-based PARACEST agent for sensing singlet oxygen by MRI.

A europium(III) DOTA-tetraamide complex was designed as a MRI sensor of singlet oxygen ((1)O2). The water soluble, thermodynamically stable complex reacts rapidly with (1)O2 to form an endoperoxide derivative that results in an ∼3 ppm shift in the position of the Eu(III)-bound water chemical exchange saturation transfer (CEST) peak. The potential of using this probe to detect accumulation of the endoperoxide derivative in biological media by ratiometric CEST imaging was demonstrated.

The pH sensitivity of -NH exchange in LnDOTA-tetraamide complexes varies with amide substituent.

The amide proton exchange rates in various lanthanide(III) DOTA-tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA-(gly)(4)(-) (Yb-1), YbDOTA-(NHCH(2)PO(3))(4 (5-) (Yb-2) and YbDOTA-(NHCH(2)PO(3)Et(2))(4)(3+) (Yb-3). The general shapes of the CEST vs pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb-1, Yb-2 and Yb-3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the -NH protons in Yb-1 and Yb-2 were similar (-17 ppm) while the -NH proton in Yb-3 was at -13 ppm. This shows that the crystal field produced by the amide oxygen donor atoms in Yb-3 is substantially weaker than that in the other two complexes. In an effort to expand the useful range of pH values that might be measured using these complexes as CEST agents, the shapes of the CEST vs pH curves were also determined for two thulium(III) complexes with much larger hyperfine shifted -NH proton resonances. The ratio of CEST from -NH exchange in Tm-1 compared with CEST from -NH exchange in Tm-3 was found to be linear over an extended pH range, from 6.3 to 7.4. This demonstrates a potential advantage of using mixtures of lanthanide(III) DOTA-tetraamides for mapping tissue pH by use of ratiometric CEST imaging.