Gábor Benkovics

Single-isomer carboxymethyl-γ-cyclodextrin as chiral resolving agent for capillary electrophoresis

Herein we report on the synthesis, characterization and the novel capillary electrophoretic use of octakis-(2,3-di-O-methyl-6-O-carboxymethyl)-γ-cyclodextrin sodium salt (ODMCM). ODMCM is the first single-isomer carboxymethyl-γ-cyclodextrin that is fully methylated on its secondary side and carries ionizable carboxymethyl functions on its primary side. ODMCM was prepared with high isomeric purity through a four-step synthetic procedure. The purity of each intermediate was characterized by appropriate chromatographic methods, while the isomeric purity of the carboxymethylated product was determined by an HPLC method using a CD-Screen-IEC column and by a capillary electrophoretic method using indirect UV detection, as well. The structural identification of the ODMCM was carried out by 1D, 2D NMR spectroscopy and ESI-MS. The acid-base characterization of the chiral selector was carried out by 1H NMR-pH titration. The chiral separation ability of the synthesized selector was studied by chiral capillary electrophoresis. ODMCM was used as a background electrolyte additive to separate enantiomers of representative pharmacologically significant model molecules such as propranolol, citalopram, ketamine, tapentadol and dapoxetine. The effects of the selector concentration and the pH of the background electrolyte on the enantiorecognition properties were investigated. 1H NMR spectroscopy was further applied to get deeper insight of the host-guest inclusion complex formation. The pH-dependent enantioselectivity of this new single-isomer chiral selector was demonstrated by chiral capillary electrophoresis and 1H NMR spectroscopy.

Comparative evaluation of the chiral recognition potential of single-isomer sulfated beta-cyclodextrin synthesis intermediates in non-aqueous capillary electrophoresis

The enantioselectivity of neutral single-isomer synthetic precursors of sulfated-β-cyclodextrins was studied. Four neutral single-isomer cyclodextrins substituted on the secondary side with acetyl and/or methyl functional groups, heptakis(2-O-methyl-3,6-dihydroxy)-β-cyclodextrin (HM-β-CD), heptakis(2,3-di-O-acetyl-6-hydroxy)-β-cyclodextrin (HDA-β-CD), heptakis(2,3-di-O-methyl-6-hydroxy)-β-cyclodextrin (HDM-β-CD), heptakis(2-O-methyl-3-O-acetyl-6-hydroxy)-β-cyclodextrin (HMA-β-CD), and their sulfated analogs the negatively charged heptakis(2,3-di-O-methyl-6-sulfato)-β-cyclodextrin (HDMS-β-CD) and heptakis(2,3-di-O-acetyl-6-sulfato)-β-cyclodextrin (HDAS-β-CD) were investigated by non-aqueous capillary electrophoresis in the view of enantiodiscrimination for various drugs and related pharmaceutical compounds. The focus of the present work was on the chiral selectivity studies of the neutral derivatives, which are the synthesis intermediates of the sulfated products. The chiral recognition experiments proved that among the neutral compounds the HMA-β-CD shows remarkable enantioselectivity towards chiral guests in non-aqueous capillary electrophoresis, while HM-β-CD, HDA-β-CD and HDM-β-CD failed to resolve any of the 25 studied racemates under the applied experimental conditions. In order to get deeper insight into the molecular interactions between the studied single-isomer cyclodextrin and chiral fluoroquinolones (ofloxacin, gatifloxacin and lomefloxacin) and β-blockers (propranolol), 1H and ROESY NMR experiments were performed. The 2-O-methylation in combination with the 3-O-acetylation of the host was evidenced to exclusively carry the essential spatial arrangement for chiral recognition.

A multifunctional β-cyclodextrin-conjugate photodelivering nitric oxide with fluorescence reporting

This contribution reports the design, synthesis and photochemical properties of a novel cationic, water soluble, β-cyclodextrin (βCD) conjugate integrating an anthracene moiety and a nitroaniline derivative within the primary side of the βCD scaffold. Photoinduced energy transfer between the anthracene and the nitroaniline chromophores effectively suppresses the fluorescence of the anthracene unit. Excitation with visible light triggers the release of nitric oxide (NO) from the nitroaniline chromophore, accompanied to the concomitant revival of the anthracene fluorescence, which acts as an optical reporter for detecting the amount of the NO released. Furthermore, the anthracene moiety photogenerates singlet oxygen (1O2) sequentially to NO release. The conjugate is also able to accommodate hydrophobic guests within the βCD cavity, as proven by using naphthalene as a model compound. In view of the key role NO and 1O2 play as anticancer and antibacterial species, the present βCD derivative represents an intriguing candidate for further studies in biopharmaceutical research addressed to multimodal therapeutic applications.

New synthetic strategies for xanthene-dye-appended cyclodextrins

Summary Xanthene dyes can be appended to cyclodextrins via an ester or amide bridge in order to switch the fluorescence on or off. This is made possible through the formation of nonfluorescent lactones or lactams as the fluorophore can reversibly cyclize. In this context we report a green approach for the synthesis of switchable xanthene-dye-appended cyclodextrins based on the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). By using 6-monoamino-β-cyclodextrin and commercially available inexpensive dyes, we prepared rhodamine- and fluorescein-appended cyclodextrins. The compounds were characterized by NMR and IR spectroscopy and MS spectrometry, their UV–vis spectra were recorded at various pH, and their purity was determined by capillary electrophoresis. Two potential models for the supramolecular assembly of the xanthene-dye-appended cyclodextrins were developed based on the set of data collected by the extensive NMR characterization.

Synthesis, analytical characterization and capillary electrophoretic use of the single-isomer heptakis-(6-O-sulfobutyl)-beta-cyclodextrin

This contribution reports the synthesis, characterization and capillary electrophoretic application of heptakis-(6-O-sulfobutyl-ether)-β-cyclodextrin sodium salt, (6-(SB)7-β-CD). The compound was obtained through a five-steps synthesis and it represents the first example of single-isomer sulfobutylated cyclodextrin that carries the negatively charged functions exclusively on its primary side and it is unmodified on the lower rim. The purity of each intermediate was determined by appropriate liquid chromatographic methods, while the isomeric purity of the final product was established by an ad-hoc developed HPLC method based on a CD-Screen-IEC column. The structural identification of 6-(SB)7-β-CD was carried out by 1D, 2D NMR spectroscopy and ESI-MS. The chiral separation ability of 6-(SB)7-β-CD was studied by chiral capillary electrophoresis using the single-isomer host as a background electrolyte additive to separate the enantiomers of a representative set of pharmacologically significant model compounds such as verapamil, dapoxetine, ondansetron, propranolol, atenolol, metoprolol, carvedilol, terbutaline, amlodipine and tadalafil. The enantiomer migration order and the effects of the selector concentration on the enantiorecognition properties were investigated. NMR spectroscopy was applied to deepen and further confirm the host-guest interactions and in the case of the model compound dapoxetine a potential representation for the supramolecular assembly was developed based on the dataset collected by the extensive 2D NMR analysis. This single-isomer chiral selector offers a new alternative to the widely applied randomly sulfobutylated- and sulfated-beta-cylodextrins as well as to the single-isomer sulfated and carboxymethylated derivatives in chiral separations.

Designed positively charged cyclodextrin hosts with enhanced binding of penicillins as carriers for the delivery of antibiotics: The case of oxacillin

In an effort to identify the optimal cyclodextrin (CD) host for delivery of penicillins to mammalian cells that will also offer protection against β-lactamase-induced hydrolysis, nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) have been employed to study the inclusion complexes formed in aqueous solution between designed CD derivatives and two aminopenicillins, ampicillin and amoxicillin, and two antistaphylococcal penicillins, methicillin and oxacillin. Anionic and cationic thioether-substituted-β- and -γCD derivatives were thus synthesized and compared with the neutral, parent CDs for complexation with the penicillins. The synthesized derivatives were shown to present ∼20% elongated cavity space in solution. Moreover, the cationic ones are >98% protonated at physiological pH. The most efficient host was the positively charged octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) that formed the strongest complex with oxacillin (Kb ∼1700M-1) in an enthalpically and entropically favorable process (ΔHb=-10.5kJ/mol,TΔSb=8.0kJ/mol). In vitro biological tests demonstrated that γCys reduces 2.3-fold the rate of hydrolysis of oxacillin in the presence of oxa-1 β-lactamase while displaying cell crossing capability and efficient internalization into macrophages as well as a sufficiently safe cytotoxicity profile. Overall, γCys could be considered as a promising vehicle for protection and delivery of oxacillin.

Supramolecular structures based on regioisomers of cinnamyl-α-cyclodextrins – new media for capillary separation techniques

Summary This work focuses on the preparation and application of supramolecular structures based on mono-cinnamyl-α-cyclodextrins (Cin-α-CD). Pure regioisomers of Cin-α-CD having the cinnamyl moiety at the 2-O- or at the 3-O-position, respectively, were prepared, characterized and applied in capillary electrophoresis as additives to the background electrolyte. These new monomer units with a potential to self-organize into supramolecular structures were synthesized via a straightforward one-step synthetic procedure and purified using preparative reversed-phase chromatography allowing a large scale separation of the regioisomers. The ability of the monomers to self-assemble was proved by various methods including NMR spectroscopy and dynamic light scattering (DLS). The light scattering experiments showed that the monomer units have distinguishable ability to form supramolecular structures in different solvents and the size distribution of the aggregates in water can be easily modulated using different external stimuli, such as temperature or competitive guest molecules. The obtained results indicated that the two regioisomers of Cin-α-CD formed different supramolecular assemblies highlighting the fact that the position of the cinnamyl group plays an important role in the intermolecular complex formation.

Novel β-cyclodextrin–eosin conjugates

Eosin B (EoB) and eosin Y (EoY), two xanthene dye derivatives with photosensitizing ability were prepared in high purity through an improved synthetic route. The dyes were grafted to a 6-monoamino-β-cyclodextrin scaffold under mild reaction conditions through a stable amide linkage using the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The molecular conjugates, well soluble in aqueous medium, were extensively characterized by 1D and 2D NMR spectroscopy and mass spectrometry. Preliminary spectroscopic investigations showed that the β-cyclodextrin–EoY conjugate retains both the fluorescence properties and the capability to photogenerate singlet oxygen of the unbound chromophore. In contrast, the corresponding β-cyclodextrin–EoB conjugate did not show either relevant emission or photosensitizing activity probably due to aggregation in aqueous medium, which precludes any response to light excitation.

Delivery of cyclodextrin polymers to bacterial biofilms — An exploratory study using rhodamine labelled cyclodextrins and multiphoton microscopy

Cyclodextrin (CD) polymers are interesting nanoparticulate systems for pharmaceutical delivery; however, knowledge regarding their applications towards delivery into complex microbial biofilm structures is so far limited. The challenge is to demonstrate penetration and transport through the biofilm and its exopolysaccharide matrix. The ideal functionalization for penetration into mature biofilms is unexplored. In this paper, we present a novel set of rhodamine labelled βCD-polymers, with different charge moieties, i.e., neutral, anionic, and cationic, and explore their potential delivery into mature Staphylococcus epidermidis biofilms using multiphoton laser scanning microscopy (MPM). The S. epidermidis biofilms, being a medically relevant model organism, were stained with SYTO9. By using MPM, three-dimensional imaging and spectral investigation of the distribution of the βCD-polymers could be obtained. It was found that the cationic βCD-polymers showed significantly higher integration into the biofilms, compared to neutral and anionic functionalized βCDs. None of the carriers presented any inherent toxicity to the biofilms, meaning that the addition of rhodamine moiety does not affect the inertness of the delivery system. Taken together, this study demonstrates a novel approach by which delivery of fluorescently labelled CD nanoparticles to bacterial biofilms can be explored using MPM. Future studies should be undertaken investigating the potential in using cationic functionalization of CD based delivery systems for targeting anti-microbial effects in biofilms.

Characterization of a single-isomer carboxymethyl-beta-cyclodextrin in chiral capillary electrophoresis

In this work, the synthesis, characterization, and chiral capillary electrophoretic study of heptakis‐(2,3‐di‐O‐methyl‐6‐O‐carboxymethyl)‐β‐CD (HDMCM), a single‐isomer carboxymethylated CD, are presented. The pH‐dependent and selector concentration‐dependent enantiorecognition properties of HDMCM were investigated and discussed herein. The enantioseparation was assessed applying a structurally diverse set of noncharged, basic, and zwitterionic racemates. The increase in the selector concentration and gross negative charge of HDMCM improved the enantioseparation that could be observed in the majority of the cases. HDMCM was also successfully applied as BGE additive in NACE using a methanol‐based system in order to prove the separation selectivity features and to highlight the broad applicability of HDMCM. Over 25 racemates showed partial or baseline separation with HDMCM under the conditions investigated, among which optimal enantiomer migration order was found for the four stereoisomers of tadalafil, tapentadol, and dapoxetine, offering the possibility of a chiral CE method development for chiral purity profiling of these drugs.