Protease sensors for bioimaging

Optical imaging of specific molecular targets and pathways in vivo has recently become possible through continued developments in imaging equipment, reconstruction algorithms, and more importantly the availability of imaging reporter molecules. These reporter molecules encompass photoproteins expressed in vivo and exogenously administered probes detectable by fluorescence and/or bioluminescence imaging. One particularly enticing aspect of optical imaging is the ability to design activatible probes with inherent amplification. This review summarizes our experience in developing novel near-infrared fluorescent (NIRF) imaging agents that report on protease activities. These agents are designed to be biocompatible, highly activatible, and able to produce bright NIRF following protease cleavage.     

Surface modification of highly dispersed rubber fillers and pigments by titanate proadhesive and hydrophobic compounds

A method of modifying silicate and carbonate fillers with titanate coupling agents and proadhesive agents was worked out. The modification aimed at hydrophobization of filler surface by introduction to the surface of functional groups with chemical affinity to polymers. Optimum amounts of modifying substances and appropriate conditions for performing the modification process were established. The obtained fillers showed a high degree of hydrophobicity.The modified fillers were applied in rubber mixtures based on butadienestyrene or natural rubber, in polyurethanes, in PVC, and as pigments in oil dyes of high corossive resistance.     

An investigation of heparinase immobilization

A systematic investigation of the parameters that affect the efficiency of immobilizing heparinase onto cyanogen bromide activated crosslinked 8% agarose beads was conducted. Two experimental measures, the “fraction bound” and the “fraction retained,” were used to monitor the coupling efficiency. The fraction bound is the portion of the total initial enzyme that is bound to the agarose gel. The fraction retained is the fraction of bound enzyme that is active. The product of the two measures indicates the coupling efficiency. The activity of the immobilized heparinase was measured under conditions free of both internal and external mass transfer limitations, and thus, the fraction retained represents the true immobilized enzyme activity. Increasing the degree of activation of the beads results in an increase in the fraction bound, the fraction retained, and consequently, the coupling efficiency. As the ratio of enzyme solution to gel volume increases from 1.5 to 2.2, the fraction bound remains constant but the fraction retained decreases (heparinase concentration; 0.15 mg/mL and degree of activation; 9.5 μmol of cyanate esters/g of gel). At volume ratios greater than 2.2, both the fraction bound and the fraction retained decline continuously. Changing the heparinase concentration in the coupling solution changes the coupling efficiency in a manner similar to that of the volume ratio change. When heparin is added during the coupling process, the fraction bound declines as the heparin concentration increases, whereas the fraction retained increases up to a heparin concentration of 12 mg/mL and decreases thereafter. When arginine, lysine, and glycine are used to block the unreacted cyanate ester groups after the coupling process, the immobilized heparinase shows different pH optima of 6.5, 6.9, and 7.2, respectively. Based upon these findings, a protocol to optimize heparinase immobilization is developed.     

pH-sensitive modulation of the second hydration sphere in lanthanide(III) tetraamide-DOTA complexes: a novel approach to smart MR contrast media

The lanthanide(III) complexes of three tetraamide DOTA bearing pyridyl, phenolic and hydroxypyridyl substituents have been studied by NMR, luminescence and cyclic voltammetry. The relaxivity profiles of the gadolinium complexes of the pyridyl and phenolic ligands were flat and essentially the same between pH 2 and 8. The hydroxypyridyl ligand, however, exhibited two regions of enhanced relaxivity. The small relaxivity enhancement (25 %) at lower pH (pH 2-4) has been attributed to an increase in the prototropic exchange of the coordinated water molecule while the slightly larger enhancement (84 %) at higher pH (pH 6-9) reflects deprotonation of the ligand amide protons. Deprotonation of the amides results in the formation of an intramolecular acid-base pair interaction with the phenolic protons and this, in turn, causes a highly organized second hydration sphere to come into effect, thereby increasing the relaxivity. The water relaxivity of the Gd(3+)-hydroxypyridyl complex is further enhanced upon binding to serum albumin.     

Synthesis, binding affinity, and relaxivity of target-specific MRI contrast agents

Although magnetic resonance imaging (MRI) is one of the most important imaging modalities of the central nervous system (CNS), one of the main drawbacks of MRI is its limited specificity. This can potentially be partially alleviated by target-specific contrast agents. In the present paper we describe a simple high yield synthesis of two such gadolinium-based spiperone targeted MRI contrast agents, 1a and 1b. The R₁ relaxivities of 1a and 1b were evaluated and found to be 5.94 and 8.31 mM⁻¹ s⁻¹, respectively at 9.4T, while their R₂ relaxivities at the same magnetic field were found to be 18.05 and 22.60 mM⁻¹ s⁻¹, respectively. In addition and very importantly compound 1a, which is a gadolinium-based, spiperone-targeted MRI contrast agent, was found to preserve some of the spiperone affinity toward the dopamine D2 receptor. Compounds 1a and 1b thus represent potential agents for in vitro dopamine receptor imaging using MRI in experimental models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New Cisplatin Analogues—On the Way to Better Antitumor Agents

The clinical success of cisplatin (cis -diamminedichloroplatinum(II )) in antitumor chemotherapy has encouraged an all-out search for analogues with lower toxicity, improved therapeutic index and increased activity. Literally thousands of analogues, obtained by replacement of the ammine- and chloro-ligands by other amines and anionic ligands, respectively, have been systematically screened for activity in experimental tumor models. Some of these analogues have been selected for clinical evaluation, but only very few of them appear to be promising antitumor agents. More recently, cisplatin analogues have been designed and synthesized on the basis of, inter alia, the following considerations: 1) platinum complexes with carrier molecules as ligands should prove useful for achieving increasing drug concentration in tumor tissues; 2) platinum complexes with chemotherapeutic agents as ligands could afford polyfunctional drugs with synergistic action; 3) complexes containing more than one platinum atom might be more effective than complexes containing only one platinum atom; 4) platinum complexes could be used as sensitizers in radiation therapy. In this paper, we shall give a brief account of the “traditional” analogues, and then critically discuss what we believe could be the new trends in the design of cisplatin analogues.     

Preparation of 5-diaminomethylenebarbiturates by barbituric acid addition to carbodiimides

Through NMR spectroscopic monitoring of barbituric acid addition to carbodiimide, a general synthetic procedure for the preparation of 5-diaminomethylenebarbiturates (DABA) was developed. This procedure is very simple and applicable to the preparation of large quantities of DABA derivatives. Through an X-ray structural study of one of the DABA derivatives, it was established that these compounds have an ylide-type structure with strong charge separation within the molecule.     

Obtainment of chelating agents through the enzymatic oxidation of lignins by phenol oxidase

Oxidation of lignin obtained from acetosolv and ethanol/water pulping of sugarcane bagasse was performed by phenol oxidases: tyrosinase (TYR) and laccase (LAC), to increase the number of carbonyl and hydroxyl groups in lignin, and to improve its chelating capacity. The chelating properties of the original and oxidized lignins were compared by monitoring the amount of Cu²⁺ bound to lignin by gel permeation chromatography. The Acetosolv lignin oxidized with TYR was 16.8% and with LAC 21% higher than that of the original lignin. For ethanol/water lignin oxidized with TYR was 17.2% and with LAC 18% higher than that of the original lignin.     

Impregnated silica-based layers in thin layer chromatography

Abstract

Impregnated thin-layer chromatography (TLC) layers based on silica gel are presented. Impregnating agents such as metal cations, inorganic ions, chelating agents, chiral selectors, surfactants, ion-pairing reagents, and ionic liquids are discussed. The role of impregnated TLC layers in medicinal chemistry is highlighted. The historical overview of TLC separations on physically coated layers is given and some future prospects ahead of this technique are discussed.     

A bifunctional platinum(II) complex capable of intercalation and hydrogen-bonding interactions with DNA: binding studies and cytotoxicity

The interactions of [Pt(CNN)(4-dpt)]PF(6), (1; 4-dpt=2,4-diamino-6-(4-pyridyl)-1,3,5-triazine, HCNN=6-phenyl-2,2'-bipyridine) with double-stranded DNA, poly(dA-dT)(2), and poly(dG-dC)(2) were examined by spectroscopic, electrophoretic, and hydrodynamic methods. The spectroscopic data were analyzed with McGhee, van't Hoff, and Gibbs-Helmholtz equations. In a comparative study, [Pt(CNN)(py)]PF(6) (2; py=pyridine) was prepared and the nature of its binding towards DNA was investigated [preliminary results: ChemBioChem 2003, 4, 62-68]. For reactions with calf thymus DNA at 20 degrees C, the intrinsic binding constants for 1 and 2 are (4.6+/-0.2)x10(5) and (2.3+/-0.3)x10(4) mol(-1) dm(3), respectively. Results of DNA-binding reactions revealed that 1 and 2 preferentially bind to the AT sequence of duplex DNA. Intercalation is the preferred binding mode for 2, whereas both intercalation and minor-groove binding are observed for 1. Complex 1 is cytotoxic against a number of carcinoma cell lines, including KB-3-1, CNE-3, and HepG2, and remains potent against multidrug- or cisplatin-resistant KB-V-1 and CNE1 cell lines, for which the resistance ratios are 1.6 and 1.5, respectively. Importantly, 1 is almost an order of magnitude less toxic to the normal cell line CCD-19Lu (IC(50)=176+/-1.7 microM) and it selectively induced apoptosis leading to cancer cell death with less than 5 % detectable necrosis.