Laszlo Jicsinszky

Improving the Trapping of Superoxide Radical with a β-Cyclodextrin– 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) Conjugate

During the last decade, the detection of superoxide radical in biological systems continued to receive increasing attention. The superoxide radical is positioned upstream in many free radical cascades leading to reactive oxygen species (ROS). These species are assumed to act as mediators in various physiological and pathological processes, such as signal transduction, aging, inflammatoryand age-related diseases. The search for reliable tools to study ROS in biological systems is important not only for unraveling their complex contributions but also for the development of diagnostic and therapeutic applications. In recent years, hydroethidine derivatives and other fluorescent probes have been developed successfully as ROS probes. However, the complexity of the dye chemistry in biological systems could lead potentially to misinterpretations concerning the ROS implication, and so there is a need to develop complementary techniques. In the late 70s, the coupled spin trapping/EPR technique (ST/EPR) has been developed for the detection and characterization of free radicals. This technique is based on the trapping of free radicals by diamagnetic spin traps leading to the formation of persistent spin adducts exhibiting EPR patterns characteristic of the radical trapped. A large number of studies using the ST/EPR technique has been devoted to the characterization of superoxide generated by various chemical and biological systems. Until the late 90s, almost all these studies reported the use of the wellknown 5,5-dimethyl-pyrroline-N-oxide (DMPO) as spin trap. However, in biological systems, the short half-lifetime ( 1 min) of the DMPO–superoxide spin adduct and its spontaneous decomposition to the DMPO–hydroxyl spin adduct severely limited the characterization of superoxide by ST/EPR. In the meantime, spin traps such as 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) and (Mito-DEPMPO), which exhibit improved performances compared with those of DMPO, have been developed. However, in biological systems the characterization of superoxide by ST/EPR is still hampered by the readily reduction of the spin adducts to EPR silent compounds by bioreductants and the relatively low trapping rate of superoxide radical (from 0.1 to 85 mol 1 L s , depending of the spin trap) compared with its disproportionation rate (3 10 mol 1 L s 1 at pH 7.3) and its reaction with SOD enzymes (5 10 mol 1 L s ). A few years ago, we showed that the addition of six equivalents of b-cyclodextrin derivatives during spin-trapping experiments resulted in an improved superoxide radical detection, due to the increase of the half-life time of the corresponding spin adduct and to its partial protection to reduction by ascorbate and glutathione/glutathione peroxidase. These results were attributed to the selective inclusion of the superoxide spin adduct (Kspinadduct > Knitrone) preventing further interactions with its immediate surrounding. Aiming at expanding these features to biological systems, we and others recently turned our attention to cyclodextrin functionalized nitrone spin traps. In the recent [a] Dr. M. Hardy, Dr. D. Bardelang, Dr. H. Karoui, Dr. J.-P. Finet, Dr. O. Ouari, Prof. P. Tordo UMR 6264, Laboratoire Chimie Provence, Equipe SREP Universit s d’Aix-Marseille 1, 2, 3 et CNRS Avenue Escadrille Normandie Niemen 13397 Marseille Cedex 20 (France) Fax: (+33) 491-288-758 E-mail : paul.tordo@univ-provence.fr olivier.ouari@univ-provence.fr [b] Prof. A. Rockenbauer Chemical Research Center, Institute for Structural Chemistry 1525 Budapest, PO Box 17 (Hungary) [c] Dr. L. Jicsinszky Cyclolab Ltd, 1525 Budapest, PO Box 435 (Hungary) [d] R. Rosas Spectropole, Universit Paul C zanne Centre Scientifique de Saint-J r me 13397 Marseille cedex 20 (France) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901342.

Symmetry Requirements for Effective Blocking of Pore-Forming Toxins: Comparative Study with α-, β-, and γ-Cyclodextrin Derivatives

ABSTRACT We compared the abilities of structurally related cationic cyclodextrins to inhibit Bacillus anthracis lethal toxin and Staphylococcus aureus α-hemolysin. We found that both β- and γ-cyclodextrin derivatives effectively inhibited anthrax toxin action by blocking the transmembrane oligomeric pores formed by the protective antigen (PA) subunit of the toxin, whereas α-cyclodextrins were ineffective. In contrast, α-hemolysin was selectively blocked only by β-cyclodextrin derivatives, demonstrating that both symmetry and size of the inhibitor and the pore are important.

Recent Applications of Cyclodextrins as Food Additives and in Food Processing

Nowadays the application of cyclodextrin-assisted molecular encapsulation in foods offers many advantages. Cyclodextrins, their derivatives and their cross-linked polymers can all improve the quality of food in storage, remove specific components and stabilize and increase the presence of components that are important for a healthy diet. The application of cyclodextrins and their complexes in packaging materials can help not only transport of previously nontransportable foods, but may also prevent, or at least decelerate, the spread of microbial infections. The number of publications, particularly analytical papers, on this matter is constantly increasing. Although the application of modern analytical methods and equipment allows for the quantitation of previously subjectively characterized parameters, bio-sensory methods are still important. The application of cyclodextrins in the nutraceutical industry has many advantages; however, some side effects connected with the inclusion complexation ability of these carbohydrates should lead scientists to study cases on an individual basis. Recent developments in the major fields of cyclodextrin related food research are herein summarized.

A new class of cationic cyclodextrins: synthesis and chemico-physical properties

A new class of positively charged triazole-bridged β-cyclodextrin (β-CD) derivatives have been synthesized in excellent yields (over 70%) through a copper-catalyzed azide-alkyne cycloaddition (CuAAC), starting from 6I-azido-6I-deoxy-β-CD or its permethylated derivative and a series of 1-butyl-3-alkynylimidazolium salts. The regioselective azide/alkyne [3 + 2] cycloaddition was efficiently carried out using metallic copper as the catalyst, affording very clean products. Easy access to such hybrid molecules that combine the properties of CDs and ionic liquids, may pave the way to a number of new applications in analytical chemistry and catalysis.

Enabling technologies and green processes in cyclodextrin chemistry

Summary The design of efficient synthetic green strategies for the selective modification of cyclodextrins (CDs) is still a challenging task. Outstanding results have been achieved in recent years by means of so-called enabling technologies, such as microwaves, ultrasound and ball mills, that have become irreplaceable tools in the synthesis of CD derivatives. Several examples of sonochemical selective modification of native α-, β- and γ-CDs have been reported including heterogeneous phase Pd- and Cu-catalysed hydrogenations and couplings. Microwave irradiation has emerged as the technique of choice for the production of highly substituted CD derivatives, CD grafted materials and polymers. Mechanochemical methods have successfully furnished greener, solvent-free syntheses and efficient complexation, while flow microreactors may well improve the repeatability and optimization of critical synthetic protocols.

Structural equilibrium in new nitroxide-capped cyclodextrins: CW and pulse EPR study.

Design of the new spin-labeled cyclodextrins can significantly extend the functionality of nitroxides. A series of new complexes based on fully methylated cyclodextrin (TRIMEB) covalently bound to the piperidine, pyrroline, pyrrolidine, and pH-sensitive imidazoline type nitroxides has been synthesized and studied using pulse and continuous wave electron paramagnetic resonance (EPR). The influence of the radical and linker properties on the structure of complexes formed has been investigated. Using the electron spin echo envelope modulation technique, we have analyzed quantitatively the accessibility of radicals to solvent molecules in studied complexes depending on the structure and length of the linkers. In all studied systems we observed different types of equilibria between conformations with radical fragment being outside the TRIMEB cavity and radical fragment capping the cavity of TRIMEB. The observed guest-induced shift of equilibrium toward the complex with radical capping TRIMEB cavity was explained by a change of macrocyclic configuration of TRIMEB. Complex with the -NH-CO- linker has been found most perspective for the applications requiring close location of nitroxide to the inclusion complex of TRIMEB. Using continuous wave EPR, we have shown that the pH-sensitive radical covalently bound to TRIMEB maintains its pH-sensitivity, but this complexation does not reduce radical reduction rate in the reaction with ascorbic acid.

Efficient mechanochemical synthesis of regioselective persubstituted cyclodextrins

A number of per-6-substituted cyclodextrin derivative syntheses have been effectively carried out in a planetary ball mill under solvent-free conditions. The preparation of Bridion® and important per-6-amino/thiocyclodextrin intermediates without polar aprotic solvents, a source of byproducts and persistent impurities, could be performed. Isolation and purification processes could also be simplified. Considerably lower alkylthiol/halide ratio were necessary to reach the complete reaction in comparison with thiourea or azide reactions. While the presented mechanochemical syntheses were carried out on the millimolar scale, they are easily scalable.

Influence of the milling parameters on the nucleophilic substitution reaction of activated β-cyclodextrins

The present work focuses on the mechanochemical preparation of industrially important β-cyclodextrin (CD) derivatives. Activated CDs have been reacted with nitrogen and sulfur nucleophiles using a planetary mill equipped with stainless steel, zirconia and glass milling tools of different sizes. It is shown that the milling frequency and the number as well as the size of the milling balls have an effect on the nucleophilic reaction.

Synthesis of Randomly Substituted Anionic Cyclodextrins in Ball Milling

A number of influencing factors mean that the random substitution of cyclodextrins (CD) in solution is difficult to reproduce. Reaction assembly in mechanochemistry reduces the number of these factors. However, lack of water can improve the reaction outcomes by minimizing the reagent’s hydrolysis. High-energy ball milling is an efficient, green and simple method for one-step reactions and usually reduces degradation and byproduct formation. Anionic CD derivatives have successfully been synthesized in the solid state, using a planetary ball mill. Comparison with solution reactions, the solvent-free conditions strongly reduced the reagent hydrolysis and resulted in products of higher degree of substitution (DS) with more homogeneous DS distribution. The synthesis of anionic CD derivatives can be effectively performed under mechanochemical activation without significant changes to the substitution pattern but the DS distributions were considerably different from the products of solution syntheses.

Inhibition of Clostridium perfringens epsilon toxin by β-cyclodextrin derivatives

Clostridium perfringens epsilon toxin (ETX) is considered as one of the most dangerous potential biological weapons. The goal of this work was to identify inhibitors of ETX using a novel approach for the inactivation of pore-forming toxins. The approach is based on the blocking of the target pore with molecules having the same symmetry as the pore itself. About 200 various β-cyclodextrin derivatives were screened for inhibitors of ETX activity using a colorimetric cell viability assay. Several compounds with dose-dependent activities at low micromolar concentrations have been identified. The same compounds were also able to inhibit lethal toxin of Bacillus anthracis.