Artificial Membrane High-performance Liquid Research Articles

Development of Safirinium dyes for new applications: fluorescent staining of bacteria, human kidney cells, and the horny layer of the epidermis

Low-molecular synthetic fluorophores are convenient tools in bioimaging applications. Several derivatives of Safirinium dyes as well as their reactive N-hydroxysuccinimide (NHS) esters bearing diverse substituents were synthesized and evaluated experimentally in terms of their lipophilicity by means of reverse-phase and immobilized artificial membrane high-performance liquid chromatography. Subsequently, the selected compounds were employed as novel cellular imaging agents for staining Gram-positive and Gram-negative bacteria, human kidney cell line, as well as human skin tissue. The analyzed dyes allowed for visualization of cellular structures such as mitochondria, endoplasmic reticulum, and cellular nuclei. They proved to be useful in fluorescent staining of stratum corneum, especially in the aspect of xenobiotic exposure and its penetration into the skin. The best results were obtained with the use of moderately lipophilic NHS esters of Safirinium Q. The development of Safirinium dyes is a promising alternative for commercially available dyes since the reported molecules have low molecular masses and exhibit efficient staining and remarkable water solubility. Moreover, they are relatively simple and low-cost in synthesis.

Predicting the phospholipophilicity of monoprotic positively charged amines

The sorption affinity of eighty-six charged amine structures to phospholipid monolayers (log KIAM) was determined using immobilized artificial membrane high-performance liquid chromatography (IAM-HPLC). The amine compounds covered the most prevalent types of polar groups, widely ranged in structural complexity, and included forty-seven pharmaceuticals, as well as several narcotics and pesticides. Amine type specific corrective increments were used to align log KIAM data with bilayer membrane sorption coefficients (KMW(IAM)). Using predicted sorption affinities of neutral amines, we evaluated the difference (scaling factor ΔMW) with the measured log KMW(IAM) for cationic amines. The ΔMW values were highly variable, ranging from -2.37 to +2.3 log units. For each amine type, polar amines showed lower ΔMW values than hydrocarbon based amines (CxHyN+). COSMOmic software was used to directly calculate the partitioning coefficient of ionic structures into a phospholipid bilayer (KDMPC-W,cation), including quaternary ammonium compounds. The resulting root mean square error (RMSE) between log KDMPC-W,cation and log KMW(IAM) was 0.83 for all eighty-six polar amines, and 0.47 for sixty-eight CxHyN+ amines. The polar amines were then split into five groups depending on polarity and structural complexity, and corrective increments for each group were defined to improve COSMOmic predictions. Excluding only the group with sixteen complex amine structures (≥4 polar groups, Mw > 400, including several macrolide antibiotics), the resulting RMSE for corrected KDMPC-W,cation values improved to 0.45 log units for the remaining set of 138 polar and CxHyN+ amines.

Phospholipophilicity of CxHyN(+) amines: chromatographic descriptors and molecular simulations for understanding partitioning into membranes.

Using immobilized artificial membrane high-performance liquid chromatography (IAM-HPLC) the sorption affinity of 70 charged amine structures to phospholipids was determined. The amines contained only 1 charged moiety and no other polar groups, the rest of the molecule being aliphatic and/or aromatic hydrocarbon groups. We systematically evaluated the influence of the amine type (1°, 2°, 3° amines and quaternary ammonium), alkyl chain branching, phenyl ring positioning, charge positioning (terminal vs. central in the molecule) on the phospholipid-water partitioning coefficient (KPLIPW). These experimental results were compared with quantum-chemistry based three-dimensional (3D) molecular simulations of the partitioning of charged amines, including the most likely solute conformers, using a hydrated phospholipid bilayer in the COSMOmic module of COSMOtherm software. Both IAM-HPLC retention data and the simulations suggest that the molecular orientation of charged amines at the location in the bilayer with the lowest calculated Gibbs free energy exerts a strong influence over the partitioning within the membrane. The most favourable position of charged amines coincides with the region where the phosphate anions in the phospholipid bilayer are most abundant. Hydrocarbon units oriented in this layer are located more towards the aqueous phase and contribute less to the overall membrane affinity than hydrocarbon units extending into the more hydrophobic core of the bilayer. COSMOmic simulations explain most of the trends between the structural differences observed in IAM-HPLC based KPLIPW. For this set of cationic structures, the mean absolute difference between COSMOmic simulations and IAM-HPLC data, accounting only for amine type corrective increments, is 0.31 log units.

Robustness of an Immobilized Artificial Membrane High-Performance Liquid Chromatography Method for the Determination of Lipophilicity

Hydrophobicity is an intrinsic property that relates to a chemical’s tendency to partition between a polar and a nonpolar phase. Traditionally, hydrophobicity has been described by the logarithm of...

Estimation of phospholipophilicity of 1-[3-(arylpiperazin-1-yl)-propyl]-pyrrolidin-2-one derivatives on immobilized artificial membrane stationary phase and its correlation with biological data

A molecule library containing 42 1-[3-(arylpiperazin-1-yl)-propyl]-pyrrolidin-2-one derivatives has been designed and synthesized. The phospholipophilicity of the obtained compounds has been determined using immobilized artificial membrane high-performance liquid chromatography (IAM-HPLC). The performed analysis allowed the calculation of log k(we) values for each of the tested compounds. Experimental phospholipophilicity data (log k(we)) has been compared with the affinity of the tested compounds to alpha(2)-adrenoceptors. Performed quantitative structure-activity relationship studies indicated that, for the tested compounds, there are dependences between affinity for alpha(2)-adrenoceptors and their log k(we) values. The obtained results confirmed that the applied chromatographic IAM-HPLC method could be useful in fast characterization of the phosholipophilicity of structurally closely related compounds as well as for larger series of compounds, such as drug candidates. It could also be used as a tool for further research into this group of compounds.

Cytochrome p450 purification and immunological detection in an insecticide resistant strain of german cockroach ( Blattella germanica, l.)

A German cockroach strain, Munsyana (MA) had 80-fold resistance to the pyrethroid insecticide cypermethrin, 4.5-fold greater total cytochrome P450 content and 2.5-fold greater cytochrome P450-mediated N-demethylation of 4-chloro- N-methylaniline compared to the susceptible Johnson Wax (JWax) strain. Immobilized artificial membrane high performance liquid chromatography (IAM-HPLC) of microsomal proteins from the MA strain enriched cytochrome P450 greater than 70-fold. Following purification, a single protein band of M r=49 000 (P450 MA), was detected by silver-staining SDS PAGE gels. Antiserum to the purified protein from the MA strain (anti-P450 MA) was produced in mice. Anti-P450 MA inhibited cytochrome P450-mediated N-demethylation by 4-fold in both MA and JWax strains. In Western blots of microsomal proteins, anti-P450 MA differentiated single MA and JWax individuals by recognizing an M r 49 000 protein band in only the MA strain. In JWax cockroaches, the M r 49 000 band was only detectable in Western analysis following induction with pentamethylbenzene (PMB). PMB induction also increases N-demethylation 2.6 and 8.0-fold in the MA and JWax strains, respectively. These results are consistent with the hypothesis that insecticide resistance in the MA strain is due to over-expression of a cytochrome P450.

Enzyme-based high-performance liquid chromatography supports as probes of enzyme activity and inhibition: The immobilization of trypsin and α-chymotrypsin on an immobilized artificial membrane high-performance liquid chromatography support

Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes α-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of Nα-benzoyl- dl-arginine- p-nitroanilide (BAPNA), and the IAM-α-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized α-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.