On the gene delivery efficacies of pH-sensitive cationic lipids via endosomal protonation: a chemical biology investigation

In an effort to probe the importance of endosomal protonation in pH-sensitive, cationic, lipid-mediated, non-viral gene delivery, we have designed and synthesized a novel cholesterol-based, endosomal pH-sensitive, histidylated, cationic amphiphile (lipid 1), its less pH-sensitive counterpart with an electron-deficient, tosylated histidine head group (lipid 2) as well as a third new cholesterol-based, cationic lipid containing no histidine head group (lipid 3). For all the novel liposomes and lipoplexes, we evaluated hysicochemical characteristics, including lipid:DNA interactions, global surface charge, and sizes. As anticipated, lipid 2 showed lower efficacies than lipid 1 for the transfection of 293T7 cells with the cytoplasmic gene expression vector pT7Luc at lipid:DNA mole ratios of 3.6:1 and 1.8:1; both lipids were greatly inhibited in the presence of Bafilomycin A1. This demonstrates the involvement of imidazole ring protonation in the endosomal escape of DNA. Conversely, endosome escape of DNA with lipid 3 seemed to be independent of endosome acidification. However, with nuclear gene expression systems in 293T7, HepG2, and HeLa cells, the transfection efficacies of lipid 2 at a lipid:DNA mole ratio of 3.6:1 were found to be either equal to or somewhat lower than those of lipids 1 and 3. Interestingly, at a lipid:DNA mole ratio of 1.8:1, lipids 2 and 3 were remarkably more transfection efficient than lipid 1 in both HepG2 and HeLa cells. Mechanistic implications of such contrasting relative transfection profiles are delineated.     

Synthesis of a library of 3-oxopiperazinium and perhydro-3-oxo-1,4-diazepinium derivatives and identification of bioactive compounds

The design and synthesis of a library of novel families of 3-oxopiperazinium and perhydro-3-oxo-1,4-diazepinium derivatives is reported. The library was composed of 44 3-oxopiperazinium derivatives (11 of these compounds had a spiranic skeleton) and 22 perhydro-3-oxo-1,4-diazepinium compounds. The synthetic procedure involved a 6-step sequence carried out in solution, along with the use of solid-phase linked scavengers and microwave activation for the rapid removal of the excess of amine reagents. A final cyclization step performed under mild conditions led to the charged heterocyclic moiety. Screening of this library in two biological assays identified active compounds that inhibit the activity of the vanilloid receptor TRPV1 and modulators of the multidrug resistance phenomenon. Thus, this synthetic sequence represents a facile and convenient entry to unprecedented libraries of this sort of tetraalkylammonium derivatives that may be of use for identification of novel scaffolds of diverse biological activity.     

Selective immunoclean-up followed by liquid or gas chromatography for the monitoring of polycyclic aromatic hydrocarbons in urban waste water and sewage sludges used for soil amendment

A selective clean-up procedure using immunoaffinity solid-phase extraction was applied for the trace-level determination of polycyclic aromatic hydrocarbons (PAHs) in urban waste water and sewage sludges used for soil amendment. Anti-pyrene antibodies have been immobilized on a silica-based sorbent and the cross-reactivity of the antibodies towards structurally related compounds were allowed to extract the whole class of priority PAHs. The selectivity of the antibodies provided clean extracts from sludges and, therefore, the identification and quantification were shown to be easier using either liquid chromatography (LC) with UV diode array and fluorescence detection in series or gas chromatography-mass spectrometry (GC-MS), although some loss of up to 50% was observed for the clean-up. The identification of the PAHs by matching of UV and MS spectra was greatly improved. The procedure, including immunoclean-up and LC coupled to diode array and fluorescence detection, was validated using certified reference materials with native PAHs of concentrations in the range of 0.57-2.16 mg/kg (dry sludges).     

Relationships between Retention Factors and Analyte Hydrophobicity on Cyanopropyl and n‐Octadecyl Bonded Silicas,Cross‐Linked Polymers and Porous Graphitic Carbon Stationary Phases. Consequences for the Trace Analysis of Highly Polar Organic Compounds

LC retention data have been measured using various stationary phases with an emphasis on highly polar to moderately polar neutral organic compounds having octanol‐water partition coefficients (K_ow) in log units between 0 and 3. The relationships between the retention factor measured in water and the octanol‐water partition coefficient are linear but with different slopes for octadecyl (C₁₈) silicas, and two polystyrene divinylbenzene (PS‐DVB) phases with low and high surface areas. These relationships confirm that highly cross‐linked polymers can provide more than 1000‐times higher retention values than C₁₈ silicas for moderately polar analytes but close values for highly polar ones. They also explain why C₁₈ silicas and polymers are equivalent for the separation of very polar analytes. In contrast, due to a different retention mechanism, no relation exists between the retention shown by porous graphitic carbons (PGC) and analyte hydrophobicity, but highly polar analytes are in general much more strongly retained than by any other sorbent. The potential of PGC for both the extraction and the separation of analytes is shown. Due to the difference in separation mechanism, PGC is the analytical phase that should be used for confirmation of the identity of analytes instead of a cyanopropylsilica column as recommended in some environmental procedures. Applications are presented for the trace‐determination of triazines and polar degradation products in ground and surface water with detection limits below the 0.1 μg/L level.     

Selective on-line immunoextraction coupled to liquid chromatography for the trace determination of benzidine, congeners and related azo dyes in surface water and industrial effluents.

A new extraction immunosorbent involving antigen-antibody interactions was coupled on-line to liquid chromatography for the selective extraction in aqueous samples of benzidine and congeners, widely used as intermediate compounds in the manufacturing of dyes and pigments. Due to the cross-reactivity of the antibodies for analytes with chemical structures closely related to that of the analyte used for immunization, the immunoextraction sorbent was shown to be able to extract aminoazobenzene and related azo dyes with good recoveries. The on-line coupling was optimized for the trace determination of benzidine, dichlorobenzidine, aminoazobenzene and some azo dyes with detection limits in the range 0.1 to 1 microgram/l. The high selectivity of the immunoextraction was shown by comparing the analysis of an industrial textile effluent obtained using precolumns packed either with a non-selective polymeric sorbent or with the anti-benzidine immunosorbent. In such complex samples, extraction and clean-up are achieved in the same step.     

Superoxide dismutase-like activity of cobalt(II) complexes based on a sugar platform

A SOD-like activity evaluated by a modified McCord-Fridovich test was evidenced for two Co(II) complexes built from "glycoligands" using a sugar platform derived from d-galactose and D-galactal and functionalized by three 2-picolyl groups.     

A direct meta-lithiation route to 1,3-disubstituted ferrocenes

Ferrocenyl sulfides afford meta-lithiation products with up to 94% regioselectivity on reaction with s-BuLi; the resulting 1,3-disubstituted ferrocenes can then be reacted with a variety of electrophiles.