Solid-phase organic tagging resins for labeling biomolecules by 1,3-dipolar cycloaddition: application to the synthesis of a fluorescent non-peptidic vasopressin receptor ligand

Two novel solid-phase organic tagging (SPOrT) resins were synthesized to facilitate the labeling of peptides and small organic compounds with a fluorescent probe. Both resins were obtained from the commercially available backbone amide linker (BAL) resin. Following the solid-phase synthesis of model compounds, a tripeptide and benzazepine, the fluorescent probe derived from Lissamine Rhodamine B was incorporated through CuI-catalyzed 1,3-dipolar cycloaddition. Final cleavage in acidic media enabled access to both types of molecules in good yield with high purity. The SPOrT resin was successfully applied to the preparation of the first non-peptidic fluorescent compound with a nanomolar affinity for the human vasopressin V2 receptor (V2R) subtype. This molecule will find application in binding assays that use polarization or fluorescence resonance energy-transfer (FRET) techniques. The SPOrT resins are also well suited for other tags and the parallel synthesis of a fluorescently tagged library for protein screening.     

Studies of the carbon dioxide and epoxide coupling reaction in the presence of fluorinated manganese(III) acacen complexes: kinetics of epoxide ring-opening

Five-coordinate manganese(III) complexes of N, N'-bis(trifluoroacetylacetone)-1,2-ethylenediimine (tfacacen) have been synthesized and structurally characterized by X-ray crystallography. The presence of the electron-withdrawing -CF3 substituents enhances the electrophilicity of the metal center in these (tfacacen)MnX (X=Cl, N3, NCO, NCS) derivatives when compared with their (acacen)MnX (acacen=N, N'-bis(acetylacetone)-1,2-ethylenediimine) analogs. This is demonstrated by the increased propensity of the Mn(III) center in the tfacacen complexes to bind a sixth ligand. Binding studies were performed utilizing the upsilonN3 stretching frequency in (tfacacen)MnN3, which is sensitive to the coordination of a ligand at the vacant axial site. Of importance, cyclohexene oxide was shown to readily bind to (tfacacen)MnN3, thereby providing an opportunity for directly monitoring the dependence of the epoxide ring-opening process on the metal complex concentration. In this instance, as has been amply demonstrated in the (salen)CrX case, the ring opening of cyclohexene oxide was found to be second-order in [(tfacacen)MnN3], with an activation energy of 71.0+/-6.0 kJ/mol. In the presence of strongly coordinating anions or amine bases, the rate of epoxide ring opening by (tfacacen)MnN3 was greatly retarded. The manganese cyanate and thiocyanate complexes were examined in an effort to develop other initiators for epoxide ring opening which provide readily accessible infrared spectroscopic probes. Indeed, the thiocyanate ligand was found to be well-suited for monitoring the epoxide ring-opening reaction by infrared spectroscopy.     

Enantiodifferentiation of chiral cationic cages using trapped achiral BF4- anions as chirotopic probes

The addition of enantiopure TRISPHAT anions to chiral cationic cages of type [Co(4)(L)(6)(BF(4))](7+) leads to the enantiodifferentiation of the ligands of the racemic salts and, even more effectively, of the achiral tetrafluoroborate anion trapped inside.     

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination.     

Hydrophilic properties as a new contribution for computer-aided identification of short peptides in complex mixtures

A new method to predict elementary amino acid (AA) composition of peptides (molar mass <1,000 g/mol) is described. This procedure is based on a computer-aided method using three combined analyses-reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and capillary electrophoresis coupled with mass spectrometry-and using a software calculating all possible amino acid combinations from the mass of any given peptide. The complementarity between HILIC and RPLC was demonstrated. Peptide retention prediction in HILIC was successfully modelled, and the achieved prediction accuracy was as high as r2=0.97. This mathematical model, based on amino acid retention contributions and peptide length, provided the information about peptide hydrophilicity that was not redundant with its hydrophobicity. Correlations between respectively the hydrophobicity coefficients and RPLC retention time, hydrophilicity and HILIC retention time, and electrophoretic mobility and migration time were used for ranking all potential AA combinations corresponding to the given mass. The essential contribution of HILIC in this identification strategy and the need to combine the three models to significantly increase identification capabilities were both shown. Applied to an 18-standard peptide mixture, the identification procedure enabled the actual AA combination determination of the 14 di- to pentapeptides, in addition to an over 98 % reduction of possible combination numbers for the four hexapeptides. This procedure was then applied to the identification of 24 unknown peptides in a rapeseed protein hydrolysate. The effective AA composition was found for ten peptides, whereas for the 14 other peptides, the number of possible combinations was reduced by over 95 % thanks to the association of the three analyses. Finally, as a result of the information provided by the analytical techniques about peptides present in the mixture, the proposed method could become a highly valuable tool to recover bioactive peptides from undefined protein hydrolysates.     

Effective chiral recognition among ions in polar media

Effective homochiral recognition occurs between the cationic [Fe(eilatin)3]2+ complex and TRISPHAT anions even in polar media such as 90% acetone-CHCl3 (de > or = 89%).     

Molecular spur gears comprising triptycene rotators and bibenzimidazole-based stators

Dynamic gearing of molecular spur gears, the most common type of mechanical gear, is elucidated. Molecular design and conformational analysis show that derivatives of 4,4-bis(triptycen-9-ylethynyl)bibenzimidazole represent suitable constructs to investigate gearing behavior of collateral triptycene (Tp) groups. To test this design, DFT calculations (B97-D/Def2-TZVP) were employed and the results suggest that these molecules undergo geared rotation preferentially to gear slippage. Synthesis of derivatives was carried out, providing a series of molecular spur gears, including the first desymmetrized spur gear molecules, which were subsequently subjected to stereochemical analysis.     

Glycolipid class profiling by packed-column subcritical fluid chromatography

The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.