Structure-redox-relaxivity relationships for redox responsive manganese-based magnetic resonance imaging probes.

A library of 10 Mn-containing complexes capable of switching reversibly between the Mn(II) and Mn(III) oxidation states was prepared and evaluated for potential usage as MRI reporters of tissue redox activity. We synthesized N-(2-hydroxybenzyl)-N,N',N'-ethylenediaminetriacetic acid (HBET) and N-(2-hydroxybenzyl-N,N',N'-trans-1,2-cyclohexylenediaminetriacetic acid (CyHBET) ligands functionalized (-H, -OMe, -NO2) at the 5-position of the aromatic ring. The Mn(II) complexes of all ligands and the Mn(III) complexes of the 5-H and 5-NO2 functionalized ligands were synthesized and isolated, but the Mn(III) complexes with the 5-OMe functionalized ligands were unstable. (1)H relaxivity of the 10 isolable complexes was measured at pH 7.4 and 37 °C, 1.4 T. Thermodynamic stability, pH-dependent complex speciation, hydration state, water exchange kinetics of the Mn(II) complexes, and pseudo-first order reduction kinetics of the Mn(III) complexes were studied using a combination of pH-potentiometry, UV-vis spectroscopy, and (1)H and (17)O NMR measurements. The effects of ligand structural and electronic modifications on the Mn(II/III) redox couple were studied by cyclic voltammetry. The Mn(II) complexes are potent relaxation agents as compared to the corresponding Mn(III) species with [Mn(II)(CyHBET)(H2O)](2-) exhibiting a 7.5-fold higher relaxivity (3.3 mM(-1) s(-1)) than the oxidized form (0.4 mM(-1) s(-1)). At pH 7.4, Mn(II) exists as a mixture of fully deprotonated (ML) and monoprotonated (HML) complexes and Mn(II) complex stability decreases as the ligands become more electron-releasing (pMn for 10 µM [Mn(II)(CyHBET-R')(H2O)](2-) decreases from 7.6 to 6.2 as R' goes from -NO2 to -OMe, respectively). HML speciation increases as the electron-releasing nature of the phenolato-O donor increases. The presence of a water coligand is maintained upon conversion from HML to ML, but the water exchange rate of ML is faster by up to 2 orders of magnitude (k(ex)(310) for H[Mn(II)(CyHBET)(H2O)](-) and [Mn(II)(CyHBET)(H2O)](2-) are 1.2 × 10(8) and 1.0 × 10(10) s(-1), respectively). The Mn(II/III) redox potential can be tuned over a range of 0.30 V (E(1/2) = 0.27-0.57 V) through electronic modifications to the 5-substituent of the aromatic ligand component. However, care must be taken in tuning the ligand electronics to avoid Mn(III)-ligand autoredox. Taken together, these results serve to establish criteria for optimizing Mn(III) versus Mn(II) relaxivity differentials, complex stability, and Mn(II/III) redox potential.

Activation and retention: a magnetic resonance probe for the detection of acute thrombosis.

Blood-clot formation that results in the complete occlusion of a blood vessel (thrombosis) often leads to serious life-threatening events, such as strokes and heart attacks. As the composition of a thrombus changes as it matures, new imaging methods that are capable of distinguishing new clots from old clots may yield important diagnostic and prognostic information. To address this need, an activatable magnetic resonance (MR) probe that is responsive to a key biochemical process associated with recently formed clots has been developed.

Redox-activated manganese-based MR contrast agent.

Here we report a simple Mn coordination complex with utility as a redox-sensitive MR probe. The HBET ligand stabilizes both the Mn(2+) and Mn(3+) oxidation states. In the presence of glutathione (GSH), low relaxivity Mn(III)-HBET is converted to high relaxivity Mn(II)-HBET with a 3-fold increase in relaxivity, and concomitant increase in MR signal. Alternately, hydrogen peroxide can convert Mn(II)-HBET to Mn(III)-HBET with a reduction in MR signal.

Fibrin-targeted PET probes for the detection of thrombi.

There is an ongoing effort to develop better methods for noninvasive detection and characterization of thrombi. Here we describe the synthesis and evaluation of three new fibrin-targeted positron emission tomography (PET) probes (FBP1, FBP2, FBP3). Three fibrin-specific peptides were conjugated as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-monoamides at the C- and N-termini and chelated with (64)CuCl2. Probes were prepared with a specific activity ranging from 10 to 130 µCi/nmol. Both the peptides and the probes exhibited nanomolar dissociation constants (Kd) for the soluble fibrin fragment DD(E), although the Cu-DOTA derivatization resulted in a 2-3 fold loss in affinity relative to the parent peptide. Biodistribution and imaging studies were performed in a rat model of carotid artery thrombosis. For FBP1 and FBP2 at 120 min post injection, the vessel containing the thrombus showed the highest concentration of radioactivity after the excretory organs, that is, the liver and kidneys. This was confirmed ex vivo by autoradiography, which showed >4-fold activity in the thrombus-containing artery compared to the contralateral artery. FBP3 showed much lower thrombus uptake, and the difference was traced to greater metabolism of this probe. Hybrid MR-PET imaging with FBP1 or FBP2 confirmed that these probes were effective for the detection of an arterial thrombus in this rat model. A thrombus was visible on PET images as a region of high activity that corresponded to a region of arterial occlusion identified by simultaneous MR angiography. FBP1 and FBP2 represent promising new probes for the molecular imaging of thrombi.

Discrete bimodal probes for thrombus imaging.

Here we report a generalizable solid/solution-phase strategy for the synthesis of discrete bimodal fibrin-targeted imaging probes. A fibrin-specific peptide was conjugated with two distinct imaging reporters at the C- and N-termini. In vitro studies demonstrated retention of fibrin affinity and specificity. Imaging studies showed that these probes could detect fibrin over a wide range of probe concentrations by optical, magnetic resonance, and positron emission tomography imaging.

Molecular MR imaging of liver fibrosis: a feasibility study using rat and mouse models.

BACKGROUND & AIMS: Liver biopsy, the current clinical gold standard for fibrosis assessment, is invasive and has sampling errors, and is not optimal for screening, monitoring, or clinical decision-making. Fibrosis is characterized by excessive accumulation of extracellular matrix proteins including type I collagen. We hypothesize that molecular magnetic resonance imaging (MRI) with a probe targeted to type I collagen could provide a direct and non-invasive method of fibrosis assessment. METHODS: Liver fibrosis was induced in rats with diethylnitrosamine and in mice with carbon tetrachloride. Animals were imaged prior to and immediately following i.v. administration of either collagen-targeted probe EP-3533 or non-targeted control Gd-DTPA. Magnetic resonance (MR) signal washout characteristics were evaluated from T1 maps and T1-weighted images. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for gadolinium and hydroxyproline. RESULTS: EP-3533-enhanced MR showed greater signal intensity on delayed imaging (normalized signal enhancement mice: control=0.39 ± 0.04, fibrotic=0.55 ± 0.03, p<0.01) and slower signal washout in the fibrotic liver compared to controls (liver t(1/2)=51.3 ± 3.6 vs. 42.0 ± 2.5 min, p<0.05 and 54.5 ± 1.9 vs. 44.1 ± 2.9 min, p<0.01 for fibrotic vs. controls in rat and mouse models, respectively). Gd-DTPA-enhanced MR could not distinguish fibrotic from control animals. EP-3533 gadolinium concentration in the liver showed strong positive correlations with hydroxyproline levels (r=0.74 (rats), r=0.77 (mice)) and with Ishak scoring (r=0.84 (rats), r=0.79 (mice)). CONCLUSIONS: Molecular MRI of liver fibrosis with a collagen-specific probe identifies fibrotic tissue in two rodent models of disease.

Development of a fluorogenic sensor for activated Cdc42.

Cdc42, a member of the Rho GTPase family, is a fundamental regulator of the actin cytoskeleton during cell migration. To generate a sensor for Cdc42 activation, we employed a multi-pronged approach, utilizing cysteine labeling and expressed protein ligation, to incorporate the environment sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) into the GTPase binding domain of the WASP protein. These constructs bind only the active, GTP-bound conformation of Cdc42 to produce a fluorescence signal. Studies with a panel of five sensor analogs revealed a derivative that exhibits a 32-fold increase in fluorescence intensity in the presence of activated Cdc42 compared to incubation with the inactive GDP-bound form of the protein. We demonstrate that this sensor can be exploited to monitor Cdc42 nucleotide exchange and GTPase activity in a continuous, fluorescence assay.

Monitoring protein interactions and dynamics with solvatochromic fluorophores.

Solvatochromic fluorophores possess emission properties that are sensitive to the nature of the local microenvironment. These dyes have been exploited in applications ranging from the study of protein structural dynamics to the detection of protein-binding interactions. Although the solvatochromic indole fluorophore of tryptophan has been utilized extensively for in vitro studies to advance our understanding of basic protein biochemistry, the emergence of new extrinsic synthetic dyes with improved properties, in conjunction with recent developments in site-selective methods to incorporate these chemical tools into proteins, now open the way for studies in more complex systems. Herein, we discuss recent technological advancements and their application in the design of powerful reporters, which serve critical roles in modern cell biology and assay development.