Synthesis of MRI contrast agents derived from DOTAM-Gly-l-Phe-OH incorporating a disulfide bridge: Conjugation to a cell penetrating peptide and preparation of a dimeric agent

A cell penetrating peptide conjugate and dimeric PARACEST MRI contrast agents, based on the DOTAM-Gly-l-Phe-OH scaffold have been prepared in moderate yields using diethyl azodicarboxylate (DEAD) or iodine-mediated disulfide bridge formation as a key step. Magnetic (PARACEST) properties of these agents have been evaluated.     

A robust and convergent synthesis of dipeptide-DOTAM conjugates as chelators for lanthanide ions: new PARACEST MRI agents

A generally applicable synthetic approach to dipeptide-DOTAM conjugates has been developed which is based on the peralkylation of 1,4,7,10-tetraazacyclododecane (cyclen) with protected N-iodoacetyl dipeptides. Standardized procedures were used for the alkylation, metalation, and purification of the resultant lanthanide complexes. Using this approach, we have been able to rapidly and reliably prepare and screen five different ligands each with up to six lanthanide ions. This preliminary investigation has identified several paramagnetic compounds with strong chemical exchange saturation transfer (PARACEST) properties in water at physiological temperature and pH. Extension of the synthetic approach to a wide variety of amino acids is possible.     

Method for enhancing cell penetration of Gd3+-based MRI contrast agents by conjugation with hydrophobic fluorescent dyes

Gadolinium ion (Gd(3+)) complexes are commonly used as magnetic resonance imaging (MRI) contrast agents to enhance signals in T(1)-weighted MR images. Recently, several methods to achieve cell-permeation of Gd(3+) complexes have been reported, but more general and efficient methodology is needed. In this report, we describe a novel method to achieve cell permeation of Gd(3+) complexes by using hydrophobic fluorescent dyes as a cell-permeability-enhancing unit. We synthesized Gd(3+) complexes conjugated with boron dipyrromethene (BDP-Gd) and Cy7 dye (Cy7-Gd), and showed that these conjugates can be introduced efficiently into cells. To examine the relationship between cell permeability and dye structure, we further synthesized a series of Cy7-Gd derivatives. On the basis of MR imaging, flow cytometry, and ICP-MS analysis of cells loaded with Cy7-Gd derivatives, highly hydrophobic and nonanionic dyes were effective for enhancing cell permeation of Gd(3+) complexes. Furthermore, the behavior of these Cy7-Gd derivatives was examined in mice. Thus, conjugation of hydrophobic fluorescent dyes appears to be an effective approach to improve the cell permeability of Gd(3+) complexes, and should be applicable for further development of Gd(3+)-based MRI contrast agents.     

Lanthanide(III) complexes of pyridine-tetraacetic acid-glycoconjugates: synthesis and luminescence studies of mono and divalent derivatives

A potent lanthanide chelate, fulfilling the requirements for the development of MRI contrast agents or luminescent probes, was armed with alkyne groups. We then implemented a click methodology to graft the bifunctional ligand to azide-containing glucoside and maltoside scaffolds. The resulting hydrophilic glycoconjugates retained the ligand binding capacity for Eu(3+) or Tb(3+) ion as evidenced by the number of bound water molecules to the lanthanide ion. Divalent Eu(3+) and Tb(3+) complexes were shown to double the brightness of the emitted fluorescent signal compared to its monovalent derivatives. Designing multivalent lanthanide luminescent probes would enable the fluorescent signal of labeled biomolecules to be enhanced.     

Cyclen-based phenylboronate ligands and their Eu3+ complexes for sensing glucose by MRI

Novel cyclen-based phenylboronate ligands and their corresponding Eu(3+) complexes have been examined as glucose sensors using chemical exchange saturation transfer (CEST) MR imaging for detection. Two isomeric bis-phenylboronate complexes, Eu(4) and Eu(10), and a mono-phenylboronate complex, Eu(12), had been prepared and characterized by UV and circular dichroism spectroscopy, mass spectrometry, and CEST imaging. Both the free ligands and their Eu(3+) complexes bind to simple sugars, but their selectivity and binding affinities vary with sugar structure. Interestingly, the free ligands, 4 and 10, are selective for fructose over glucose, but this selectivity order switches in the respective Eu(3+) complexes. Of the complexes examined, Eu(4) shows the highest selectivity and binding affinity for glucose (2275 +/- 266 M(-)(1) at pH 10.2 and 339 +/- 29 M(-)(1) at pH 7). Glucose acts as a "capping"moiety in the Eu(4).glucose binary complex and modulates water exchange between a single Eu(3+)-bound water molecule and bulk water, an effect that can be detected by CEST imaging. Thus, Eu(4) represents a new class of metabolite-specific imaging agents that may allow mapping of metabolites by MRI of the bulk water signal.     

Contrast agents for MRI: 30+ years and where are we going?

Thirty years ago, Schering filed the first patent application for a contrast agent for magnetic resonance imaging (MRI) covering the forefather of the gadolinium contrast agents and still the most widely used gadolinium probe: gadolinium(III) diethylenetriaminepentaacetate (Magnevist). To date, 11 contrast agents have been approved by the US Food and Drug Administration for intravenous use. Coordination chemists have done a great deal to move the field forward. Our understanding of lanthanide chemistry now makes possible the design of complexes with long rotational correlation times, fast or slow water-exchange rates, high thermodynamic stabilities, and kinetic inertness, leading to sensitive and nontoxic contrast agents. Chemists did not stop there. The last few decades has seen the development of novel classes of probes that yield contrast through different mechanisms, such as paramagnetic chemical exchange saturation transfer agents. Thirty years since the first patent, chemists are still leading the way. The development of high-sensitivity contrast agents for high magnetic fields, safe probes for patients with kidney disorders, and multimodal, targeted, and responsive agents demonstrates that the field of contrast agents for MRI still has much to offer.     

Crystalline and water soluble CR(4+) and CR(5+:) model compounds for chromium toxicity studies

We discuss two complexes of Cr(4+) for their possible utility as models for Cr toxicity studies. They are Cr(dien)(O2)2(.)H2O (dien = diethylenetriamine) and Cr(NH3)3(O2)2, which have been recently characterized by x-ray diffraction and magnetic techniques. We present their optical and infrared absorption spectra as quick aids in their identification procedure. We also summarize the general properties of some well-characterized Cr(5+) compounds. All of these compounds are water soluble with the Cr(5+) samples being fairly stable in basic solutions. They can all be prepared as pure crystals with a shelf life of over 2 years when stored in a refrigerator.     

Preparation,Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and purification methods.     

Magnetic Circular Dichroism of Porphyrin Lanthanide M3+ Complexes

Lanthanide complexes exhibit interesting spectroscopic properties yielding many applications as imaging probes, natural chirality amplifiers, and therapeutic agents. However, many properties are not fully understood yet. Therefore, we applied magnetic circular dichroism (MCD) spectroscopy, which provides enhanced information about the underlying electronic structure to a series of lanthanide compounds. The metals in the M³⁺ state included Y, La, Eu, Tb, Dy, Ho, Er, Tm, Yb, and Lu; the spectra were collected for selected tetraphenylporphin (TPP) and octaethylporphin (OEP) complexes in chloroform. While the MCD and UV‐VIS absorption spectra were dominated by the porphyrin signal, metal binding significantly modulated them. MCD spectroscopy was found to be better suited to discriminate between various species than absorption spectroscopy alone. The main features and trends in the lanthanide series observed in MCD and absorption spectra of the complexes could be interpreted at the Density Functional Theory (DFT) level, with effective core potentials on metal nuclei. The sum over state (SOS) method was used for simulation of the MCD intensities. The combination of the spectroscopy and quantum‐chemical computations is important for understanding the interactions of the metals with the organic compounds. Chirality 26:655–662, 2014. © 2014 Wiley Periodicals, Inc.     

New magnetic resonance contrast agents as biochemical reporters

Magnetic resonance imaging is a noninvasive, volume rendering diagnostic technique that uses lanthanide complexes to enhance proton relaxation. Magnetic resonance imaging is not limited by light scattering as optical microscopic techniques are, and allows imaging of whole animals. Clinical contrast agents are nonspecific and report solely on anatomy, whereas contrast agents that can be activated can be tailored to report on the physiological status or metabolic activity of biological systems. These new classes of magnetic resonance contrast agents represent a substantial leap in the type of information that can be derived from imaging experiments, and are the focus of this review.     

Increasing the luminescence of lanthanide complexes.

This review compares the chemical and physical properties of lanthanide ion complexes and of other narrow-emitting species that can be used as labels for cytometry. A series of luminescent lanthanide ion macrocyclic complexes, Quantum Dyes, which do not release or exchange their central lanthanide ion, do accept energy transfer from ligands, and are capable of covalent binding to macromolecules, including proteins and nucleic acids, is described and their properties are discussed.Two methods are described for increasing the luminescence intensity of lanthanide ion complexes, which intrinsically is not as high as that of standard fluorophores or quantum dots. One method consists of adding a complex of a second lanthanide ion in a micellar solution (columinescence); the other method produces dry preparations by evaporation of a homogeneous solution containing an added complex of a second lanthanide ion or an excess of an unbound antenna ligand. Both methods involve the Resonance Energy Transfer Enhanced Luminescence, RETEL, effect as the mechanism for the luminescence enhancement.     

Lanthanide binding and IgG affinity construct: Potential applications in solution NMR, MRI, and luminescence microscopy

Paramagnetic lanthanide ions when bound to proteins offer great potential for structural investigations that utilize solution nuclear magnetic resonance spectroscopy, magnetic resonance imaging, or optical microscopy. However, many proteins do not have native metal ion binding sites and engineering a chimeric protein to bind an ion while retaining affinity for a protein of interest represents a significant challenge. Here we report the characterization of an immunoglobulin G-binding protein redesigned to include a lanthanide binding motif in place of a loop between two helices (Z-L2LBT). It was shown to bind Tb(3+) with 130 nM affinity. Ions such as Dy(3+) , Yb(3+) , and Ce(3+) produce paramagnetic effects on NMR spectra and the utility of these effects is illustrated by their use in determining a structural model of the metal-complexed Z-L2LBT protein and a preliminary characterization of the dynamic distribution of IgG Fc glycan positions. Furthermore, this designed protein is demonstrated to be a novel IgG-binding reagent for magnetic resonance imaging (Z-L2LBT:Gd(3+) complex) and luminescence microscopy (Z-L2LBT: Tb(3+) complex).     

Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.

We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca(2+) activation of the transport process. In this study, yeast membranes, expressing wild type and mutant Ca(2+)-ATPases, were reacted with Cr small middle dotATP and tested for their ability to occlude (45)Ca(2+) by HPLC analysis, after cation resin and C(12)E(8) treatment. We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca(2+) to the same extent as wild type ATPase. Using NMR and mass spectrometry we have analyzed the conformational properties of the synthetic L6-7 loop and demonstrated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum. All three aspartate Asp(813)/Asp(815)/Asp(818) were required to coordinate the trivalent lanthanide ion. Overall these observations suggest a dual function of the loop: in addition to mediating contact between the intramembranous Ca(2+)-binding sites and the cytosolic phosphorylation site (Zhang, Z., Lewis, D., Sumbilla, C., Inesi G., and Toyoshima, C. (2001) J. Biol. Chem. 276, 15232-15239), the L6-7 loop, in a preceding step, participates in the formation of an entrance port, before subsequent high affinity binding of Ca(2+) inside the membrane.     

Near-infrared Yb(3+) vibronic sideband spectroscopy: application to Ca(2+)-binding proteins.

We have used near-infrared (NIR) vibronic fluorescence spectroscopy to study the vibrational structure of ligands associated with model complexes of the lanthanide Yb(3+). This technique exploits the similar binding properties of the lanthanide Yb(3+) to probe Ca(2+)-binding sites in proteins. The (NIR) fluorescence of complexed Yb(3+) exhibits, in addition to main 0-0 (2F5/2----2F7/2) electronic transition of Yb(3+), weak vibronic sidebands which provide infrared-like, local vibrational spectra of the chelates (inner sphere ligands) of Yb(3+). A similar approach has been used for the lanthanide Gd(3+) (MacGregor, R.B., Jr (1989) Arch. Biochem. Biophys. 274, 312-316) which fluoresces in the UV and which is usually complicated by amino-acid residues fluorescing in the same spectral region. In this same spectral region, other complications in studying photosynthetic membranes occur in the form of the excitation wavelength being actinic, promoting photodegradation of the membranes, as well as the reabsorption of Gd(3+) fluorescence. NIR excitation and fluorescence detection of Yb(3+) avoid these problems when studying photosynthetic membranes. A preliminary study has been conducted here on rat muscle parvalbumin.     

Lanthanide-binding tags with unnatural amino acids: sensitizing Tb3+ and Eu3+ luminescence at longer wavelengths

Lanthanide-binding tags (LBTs) are small, genetically encoded, versatile protein fusion partners that selectively bind lanthanide ions with high affinity. The LBT motif features a strategically positioned tryptophan residue that sensitizes Tb3+ luminescence upon excitation at 280 nm. Herein, we describe the preparation of new LBT peptides that incorporate unnatural amino acids in place of tryptophan, and which sensitize both Tb3+ and Eu3+ luminescence at lower energies. We also report the semisynthesis of proteins tagged with these new LBTs using native chemical ligation. This expands the scope of LBTs and will enable their wider use in luminescence applications.     

Novel bimodal bifunctional ligands for radioimmunotherapy and targeted MRI

The structurally novel bifunctional ligands C-NETA and C-NE3TA, each possessing both acyclic and macrocyclic moieties, were prepared and evaluated as potential chelates for radioimmunotherapy (RIT) and targeted magnetic resonance imaging (MRI). Heptadentate C-NE3TA was fortuitously discovered during the preparation of C-NETA. An optimized synthetic method to C-NETA and C-NE3TA including purification of the polar and tailing reaction intermediates, tert-butyl C-NETA (2) and tert-butyl C-NE3TA (3) using semiprep HPLC was developed. The new Gd(III) complexes of C-NETA and C-NE3TA were prepared as contrast enhancement agents for use in targeted MRI. The T 1 relaxivity data indicate that Gd(C-NETA) and Gd(C-NE3TA) possess higher relaxivity than Gd(C-DOTA), a bifunctional version of a commercially available MRI contrast agent; Gd(DOTA). C-NETA and C-NE3TA were radiolabeled with (177)Lu, (90)Y, (203)Pb, (205/6)Bi, and (153)Gd; and in vitro stability of the radiolabeled corresponding complexes was assessed in human serum. The in vitro studies indicate that the evaluated radiolabeled complexes were stable in serum for 11 days with the exception being the (203)Pb complexes of C-NETA and C-NE3TA, which dissociated in serum. C-NETA and C-NE3TA radiolabeled (177)Lu, (90)Y, or (153)Gd complexes were further evaluated for in vivo stability in athymic mice and possess excellent or acceptable in vivo biodistribution profile. (205/6)Bi- C-NE3TA exhibited extremely rapid blood clearance and low radioactivity level at the normal organs, while (205/6)Bi- C-NETA displayed low radioactivity level in the blood and all of the organs except for the kidney where relatively high renal uptake of radioactivity is observed. C-NETA and C-NE3TA were further modified for conjugation to the monoclonal antibody Trastuzumab.     

Potential bile acid metabolites. 16. Synthesis of stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids

New synthetic routes to the four possible stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids (and their methyl esters), one of which (3 alpha,6 alpha 7 beta,12 alpha) is new, and some related compounds are described. In addition, the 5 alpha-epimer of the new acid was obtained. The final products were obtained in high purity for use as reference compounds in the analysis of bile acids in human biologic samples. The results of analysis of the prepared stereoisomers by proton and carbon 13 nuclear magnetic resonance spectroscopies are briefly discussed along with the thin-layer and gas-liquid chromatographic properties.     

Mononuclear metal complexes with piroxicam: synthesis, structure and biological activity

Piroxicam (=Hpir) is a non-steroidal anti-inflammatory and an anti-arthritic drug. VO(2+), Mn(2+), Fe(3+), MoO(2)(2+) and UO(2)(2+) complexes with deprotonated piroxicam have been prepared and characterized with the use of infrared, UV-Vis, nuclear magnetic resonance and electron paramagnetic resonance spectroscopies. The experimental data suggest that piroxicam acts as a deprotonated bidentate ligand in all complexes and is coordinated to the metal ion through the pyridine nitrogen and the amide oxygen. Molecular mechanics calculations in the gas state have been performed in order to propose a model for the Fe(3+), VO(2+) and MoO(2)(2+) complexes. Potential anticancer cytostatic and cytotoxic effects of piroxicam complexes with VO(2+), Mn(2+) and MoO(2)(2+) on human promyelocytic leukemia HL-60 cells have been investigated. Among all complexes, only VO(pir)(2)(H(2)O) clearly induces apoptosis after 24-h incubation, whereas piroxicam induces apoptosis after 57-h incubation.     

Polynuclear complexes of lanthanides with nickel and copper schiff bases as ligands

Polynuclear complexes of lanthanide(III) nitrates, thiocyanates, chlorides and perchlorates with N,N′- 1,3-propylen-bis(salicylideniminato)nickel(II) and N,N'?1,3-propylen-bis(salicylideniminato)copper(II) have been prepared and characterized by vibrational and electronic spectra, magnetic susceptibility, molar conductivity and thermal analysis measurements. Reliable information relating to the lanthanide- ligand bondings and geometries of the complexes have been inferred from infrared and electronic spectral data.     

In vivo visualizing of organs and tissues with liposomes

Abstract

The application of liposomes as carriers for imaging agents is considered. Liposomes loaded with the appropriate contrast agents have been shown to be suitable for gamma-, magnetic resonance (MR), computed tomography (CT) and ultrasound imaging. The methods are briefly described to prepare liposomes loaded with different contrast agents, as well as some data on their biodistribution. The application of contrast-loaded liposomes for liver/spleen, tumor, lymph nodes, infection and inflammation sites, myocardial infarction, and blood pool imaging is briefly reviewed together with some data available on the use of liposome for the ophtalmological imaging. New trends in the use of contrast-loaded liposomes are also considered, such as the application of long-circulating polymer-modified liposomes for imaging purposes and development of new lipid-coated liposome-like contrast agents.