Konstantina Yannakopoulou

Synthesis, Characterization, and Remarkable Biological Properties of Cyclodextrins Bearing Guanidinoalkylamino and Aminoalkylamino Groups on Their Primary Side

The introduction of aminoalkylamino and guanidinoalkylamino substituents on the primary side of beta- and gamma-cyclodextrin (CDs) resulted in a series of novel compounds that were extensively characterized by NMR spectroscopy and mass spectrometry. Bromination of the primary side of beta- and gamma-CD, and reaction with neat alkylene diamines at a pressure of 7 atm afforded aminoalkylamino derivatives that were then guanylated at the primary amino group to give the corresponding guanidinoalkylamino-CDs. These compounds are water soluble and display pK(a) values that allow them to be mostly protonated at neutral pH; for example, pK(a(1)) approximately 6.4 and pK(a(2)) approximately 9.5 for the aminoethylamino-beta-CD and pK(a(1)) approximately 7.8 and pK(a(2)) approximately 11.0 for the guanidinoethylamino-beta-CD. The title CDs are rigid, cyclic alpha-D-glucopyranose oligomers (heptamers or octamers) with branches that resemble lysine and arginine side chains that enable multiple interactions with suitable substrates. Thus, they bear similarities to known cell-penetrating peptides. Indeed, the compounds were found to cross the membranes of HeLa cells and penetrate inside the cytoplasm quickly, the guadinylated ones within 15 min, as shown by fluorescence microscopy using fluorescein-labeled derivatives. The toxicity of the compounds, measured by performing MTT tests, ranged from 50 to 300 microM. Furthermore, some of the aminated CDs could facilitate the transfection of DNA expressing the green fluorescent protein (GFP) in HEK 293T cells, with effectiveness comparable to the commercial agent Lipofectamine 2000. Circular dichroism, atomic force microscopy and electrophoresis experiments confirmed the strong interaction of the compounds with DNA. Because of their carbohydrate, non-peptide nature the title compounds are not anticipated to be enzymatically labile or immunogenic, and thus they fulfill many of the criteria for non-hazardous transport vectors in biological and pharmaceutical applications.

Per(6-guanidino-6-deoxy)cyclodextrins: synthesis, characterisation and binding behaviour toward selected small molecules and DNA.

Per(6-guanidino-6-deoxy)-cyclodextrins , and are novel derivatives, resulting from homogeneous introduction of the guanidino group at the primary side of alpha-, beta- and gamma-cyclodextrins. The products were obtained from the corresponding amino derivatives, as direct guanidinylation of the known bromo-cyclodextrins provided mixtures. The new compounds were fully characterized by NMR spectroscopy and other analytical methods, and their interaction with guest molecules was studied. Strong complexation with 4-nitrophenyl phosphate () disodium salt was observed (K(binding) approximately 5 x 10(4) M(-1)), whereas the non-phosphorylated substrate nitrobenzene () formed a very weak complex. 2D ROESY spectra revealed cavity inclusion in both cases, however the orientation of was opposite to that of , such that the phosphate group is oriented toward the primary side facing the guanidine groups. The strong affinity of towards the phosphorylated guest suggested that interaction with DNA was possible. The new compounds were found to completely inhibit the migration of ultra pure calf thymus DNA during agarose gel electrophoresis, whereas no effects were observed with guanidine alone or with the plain cyclodextrins. Further, the condensation of DNA into nanoparticles in the presence of was demonstrated by atomic force microscopy, confirming strong electrostatic interaction between the biopolymer and the multicationic products . The strong guanidine-phosphate interactions between and DNA were therefore attributed to the clustering of the guanidine groups in the primary area of the cyclodextrin. Cavity effects could not be assessed.

Benzotriazol-1-ylmethyl isocyanide, a new synthon for CHNC transfer. Syntheses of α-hydroxyaldehydes, 4-ethoxy-2-oxazolines and oxazoles

Abstract Readily prepared benzotriazol-1-ylmethyl isocyanide (BetMIC) reacts under mild conditions with ketones and aldehydes, to afford oxazolines (convertible into α-hydroxyaldehydes) and oxazoles, respectively.

Positive effect of natural and negatively charged cyclodextrins on the stabilization of penicillins towards β-lactamase degradation due to inclusion and external guest–host association. An NMR and MS study

The complexation of penicillin (1a-c) and cephalosporin (2a,b) antibiotics with cyclodextrins (CDs), both natural [beta-CD (3b) and gamma-CD (3c)] and carboxylated [heptakis(6-oxycarbonylethylthio-6-deoxy)-beta-CD sodium salt (4b) and octakis(6-oxycarbonylethylthio-6-deoxy)-gamma-CD (4c) sodium salt], has been studied at neutral pH. Penicillins [ampicillin (1a), amoxicillin (1b) and dicloxacillin (1c) form inclusion complexes with the above CDs, as was shown by extensive NMR spectroscopic studies, whereas cephalosporins (cephalexin, cefadroxil) do not. Inclusion of the penicillins into either 3b or 4b was not accompanied by significant chemical shift changes in the 1H NMR spectra. On the contrary, with the wider 3c and its derivative 4c inclusion was evidenced by both chemical shift displacements of the cavity protons and intermolecular interactions, indicating the formation of primarily 1:1 guest-host inclusion complexes. The binding constants for 1a/3c, 1a/4c and 1c/3c were calculated as 19 +/- 4, 17 +/- 0.9 and 622 +/- 200 M(-1), respectively. With 4c, a 1:2 stoichiometry was also found. In addition, simultaneous formation of aggregates by external association takes place in solution, as shown by the ESI-mass spectrometric data. Studies on the hydrolysis of ampicillin under pseudo-first order conditions using an excess of 3c, 4c and of linear maltoheptaose at pH 7 showed that the drug hydrolysed at a similar rate in all cases. In the presence, however, of beta-lactamase enzyme and the carboxylated host 4c, ampicillin degraded twice as slowly (0.008 h(-1)) as in the presence of beta-lactamase alone (0.017 h(-1)). This was explained by the effective protection provided by both inclusion and external association of the host. The interaction, therefore, of penicillins with carboxylated CDs may present a means to lessen the chemical instability of these drugs in the presence of beta-lactamase enzymes.

The self-association of the drug acemetacin and its interactions and stabilization with β-cyclodextrin in aqueous solution as inferred from NMR spectroscopy and HPLC studies

Strongly concentration dependent, (1)H NMR chemical shifts of the non-steroidal anti-inflammatory drug acemetacin sodium salt (sodium [[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetoxy]acetate), were observed in aqueous solution. Self-titration and nOe experiments, point to a self-association model where stacking takes place via the indole portion of the drug. In addition, conformational isomerism (atropisomerism) of the anti to syn form was confirmed. Further increase of the concentration eventually led to stable chemical shifts and nearly simultaneous appearance of microcrystals. In the presence of betaCD, 1:1 inclusion complexation occurred through the p-chlorobenzoyl part of the drug, whereas with excess betaCD the indole part seemed to participate to a minor degree. The anti isomer is suggested to be involved in the inclusion process. In addition, aggregation of acemetacin was also evident, as competing with the conformational and inclusion equilibria. The present case demonstrates that many competitive processes are simultaneously active in a seemingly simple system. The measurements were strongly dependent upon the pH and use of buffered solutions was mandatory. Finally, for the quantitative analysis of acemetacin in the presence of betaCD, a special HPLC method was developed. The stability of the drug, studied by the identification of the degradation products and the pseudo-first order rate of hydrolysis, was found to be unaffected by the presence of betaCD.

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.

Anionic cyclodextrins as versatile hosts for pharmaceutical nanotechnology: Synthesis, drug delivery, enantioselectivity, contrast agents for MRI

The review presents a full library of single-isomer primary rim per-carboxylate- and per-sulfate-α-, -β- and -γ-cyclodextrin (CD) derivatives and their potential for pharmaceutical nanotechnology. Recent advances in cyclodextrin chemistry have enabled robust methods for the synthesis of single-isomer anionic CDs. Numerous nanobio-applications have been already reported for these negatively charged derivatives, which alone or in combination with other biodegradable molecular platforms can become important carriers for targeted drug delivery and release. Specialized applications are also discussed, such as chiral separations, as well as the ability of per-6-carboxylated-cyclodextrins to coordinate with metal cations and especially with lanthanide cations that makes them candidates as contrast agents for Magnetic Resonance Imaging.

Non-covalent interactions in the crystallization of the enantiomers of 1,7-dioxaspiro[5.5]undecane (olive fly sex pheromone) by enantiospecific cyclodextrin hosts, hexakis(2,3,6-tri-O-methyl)-α-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin

The enantiomers of racemic olive fly sex pheromone 1,7-dioxaspiro[5.5]undecane (1) have been isolated by crystallization with enantiospecific cyclodextrin hosts: (S)-(1) crystallizes with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) and (R)-(1) with hexakis(2,3,6-tri-O-methyl)-alpha-cyclodextrin (TMalphaCD). The crystal structure of TMbetaCD/(S)-(1) from synchrotron radiation data at 100 K, determined for the first time, proves that TMbetaCD crystallizes with only the (S)-enantiomer from the racemic mixture. Comparison with the 100 K structure of TMalphaCD/(R)-(1) redetermined with synchrotron data has provided insight into the interactions between each of the hosts with the corresponding enantiomeric guests. Owing to the high resolution of the data and the unusually high quality of the crystals, localization of the H atoms has been achieved, a rare accomplishment for cyclodextrin X-ray structures. This made possible, apart from the geometry of the complexes, the detailed description of a five-membered-ring water cluster with very well ordered hydrogen bonding. The enantiospecificity exhibited by the described systems reveals the subtle differences of the weak intermolecular forces involved in the selective binding of the two optical antipodes by the two hosts. The binding geometry in the two complexes is different, but it is effected in both by numerous host-guest C-H.O interactions, resulting from induced fit of the hosts toward each of the enantiomeric guests. In TMalphaCD/(R)-(1) two of these H.O host-guest distances, directed toward the acetal O atoms defining the chirality of the guest, are much shorter than the rest and also shorter than all the H.O distances in TMbetaCD/(S)-(1). Moreover, (R)-(1) interacts not only with the enclosing host, but with other hosts in the crystal lattice, in contrast to (S)-(1) in the TMbetaCD/(S)-(1) complex which is isolated inside channels formed by the host molecules. The above differences are reflected in the much higher binding constant of TMalphaCD/(R)-(1) compared with that of TMbetaCD/(S)-(1) ( approximately 6800 and approximately 935 M(-1), respectively), determined by NMR in aqueous solution, and the ability of TMalphaCD to selectively precipitate (R)-(1) from racemic (1) in much higher yield than TMbetaCD precipitates (S)-(1).

Isomerizations of N-(α-aminoalkyl)-1,2,4-triazoles and N-(α-aminoalkyl)tetrazoles

The title compounds exist as equilibrium mixtures of the N-1 and N-2 isomers in solution. However, the crystal structure of 3-(1-hydroxycyclohexyl)-1-(α-pyrrolidinomethyl)-1,2,4-triazole discloses only this isomer in the solid state. Evidence for the nature of the isomerization processes in solution is provided. The kinetic and thermodynamic parameters are measured and qualitatively assessed.

Synthesis of 3(5)-substituted 1,2,4-triazoles by lithiation of 1-(1-pyrrolidinomethyl)-1,2,4-triazole

1,2,4-Triazole 1 is readily converted into 3(5)-substituted 1,2,4-triazoles 4 by a three-step sequence: (i) Mannich reaction with formaldehyde and pyrrolidine; (ii) lithiation of 1-(1-pyrrolidinomethyl)-1, 2,4-triazole 2, followed by addition of an electrophile; (iii) deprotection of the N-aminal groups of the N-protected 3-substituted 1,2,4-triazoles 3 using sodium borohydride (NaBH4) in ethanol. 3(5)-Substituted 1,2,4-triazoles 4 result in good overall yields.