Oxygenolysis of flavonoid compounds: DFT description of the mechanism for quercetin.

Flavonoids are naturally occurring phenol derivatives present in substantial amounts in a large variety of plants, fruits and vegetables daily eaten by humans. Most of these compounds exhibit several interesting biological activities, such as antiradical and antioxidant actions. Indeed, by complexation with specific enzymes, flavonoids are notably liable to metabolize molecular dioxygen. On the basis of experimental results describing oxygenolysis of the flavonoid quercetin, activated by the enzyme quercetin 2,3-dioxygenase (2,3-QD),ur attention has focused on the role of metal center in the activation of the substrate quercetin. Thus, in the present study, by means of DFT calculations at the B3LYP/ 6-31(+)G* level on model molecular systems, we describe different mechanisms for dioxygen metabolization by quercetin. Stationary points are described, and energetic and structural analyses along the reaction paths are reported. Our calculations show that the copper cation must act as an oxidant towards the substrate and that the reaction proceeds through a 1,3-cycloaddition.     

High-throughput purification of combinatorial libraries I: a high-throughput purification system using an accelerated retention window approach

We have developed a high-throughput purification system to purify combinatorial libraries at a 50-100-mg scale with a throughput of 250 samples/instrument/day. We applied an accelerated retention window method to shorten the purification time and targeted one fraction per injection to simplify data tracking, lower QC workload, and simplify the postpurification processing. First, we determined the accurate retention time and peak height for all compounds using an eight-channel parallel LC/UV/MS system, and calculated the specific preparative HPLC conditions for individual compounds. The preparative HPLC conditions include the compound-specific gradient segment for individual compounds with a fixed gradient slope and the compound-specific UV or ELSD threshold for triggering a fraction collection device. A unique solvent composition or solvent strength was programmed for each compound in the preparative HPLC in order to elute all compounds at the same target time. Considering the possible deviation of the predicted retention time, a 1-min window around the target time was set to collect peaks above a threshold based on UV or ELSD detection. Dual column preparative instruments were used to maximize throughput. We have purified more than 500 000 druglike compounds using this system in the past 3 years. We report various components of this high-throughput purification system and some of our purification results.     

Trace analysis of rapamycin in human blood by micellar electrokinetic chromatography

A capillary electrophoretic method with UV detection at 278 nm has been developed for analysis of the immunosuppressant rapamycin (sirolimus) in human blood at low microgram per liter levels. Separation has been achieved in an acidic carrier electrolyte containing sodium dodecylsulfate and 30% (v/v) acetonitrile. For sample clean-up and preconcentration, an off-line solid-phase extraction step using a silica-based reversed-phase material and an on-capillary focussing technique were employed. The latter allows the injection of increased sample volumes without excessive band broadening. Although this new method is less sensitive than existing liquid chromatographic procedures combined with mass spectrometry, it is fully suited to routine analysis of rapamycin in blood from patients treated with this drug. Last but not least the low costs make it an attractive alternative to established methods.     

Dendritic molecular capsules for hydrophobic compounds

Reichardt's dye, a highly solvatochromic dye, was encapsulated within poly (glycerol succinic acid) ([Gn]-PGLSA-OH) dendrimers to investigate the interior environment of these dendritic macromolecules. The absorption maximum for the encapsulated Reichardt's dye in water was indicative of a relatively high dielectric constant present within the dye/dendrimer complex. (1)H NMR of the encapsulated complex showed the presence of aromatic protons from Reichardt's dye along with the aliphatic protons of the dendrimer. Additionally, there were substantial changes in T(1) and T(2) times of the encapsulated dye when compared with the free dye, and (1)H NOESY spectra for the complex showed a significant number of intermolecular NOE cross-peaks. These data reveal the close through-space proximity of the dye to the dendrimer and the restricted motion of the encapsulated dye. To demonstrate the potential use of these macromolecules as drug delivery vehicles, the poorly water-soluble anticancer drug 10-hydroxycamptothecin (10HCPT) was encapsulated within a carboxylated PGLSA dendrimer ([G4]-PGLSA-COONa). Cytotoxicity assays with human breast cancer cells showed a significant reduction of cell viability, demonstrating that 10HCPT retains activity upon encapsulation.     

Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis

Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mM ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 microM and a limit of quantitation of 12 microM for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing with control cell samples.     

Advances in Catalysis. Volume 25; edited by D.D. Eley, H. Pines, and P.B. Weisz, Academic Press, New York, San Francisco and London, 1976, xii + 457 pages, $42.50

Photolysis of 1,2-divinyl- and 1-phenyl-2-vinyltetramethyldisilane and 1-phenylethenylpentamethyldisilane has been investigated. All three compounds afforded a new type of silicon—carbon double-bonded intermediate via photorearrangement of a silyl group to the terminal carbon of a vinyl group.     

Free energy perturbation approach to the critical assessment of selective cyclooxygenase-2 inhibitors

The discovery of selective cyclooxygenase-2 (COX-2) inhibitors represents a major achievement of the efforts over the past few decades to develop therapeutic treatments for inflammation. To gain insights into designing new COX-2-selective inhibitors, we address the energetic and structural basis for the selective inhibition of COX isozymes by means of a combined computational protocol involving docking experiment, force field design for the heme prothetic group, and free energy perturbation (FEP) simulation. We consider both COX-2- and COX-1-selective inhibitors taking the V523I mutant of COX-2 to be a relevant structural model for COX-1 as confirmed by a variety of experimental and theoretical evidences. For all COX-2-selective inhibitors under consideration, we find that free energies of binding become less favorable as the receptor changes from COX-2 to COX-1, due to the weakening and/or loss of hydrogen bond and hydrophobic interactions that stabilize the inhibitors in the COX-2 active site. On the other hand, COX-1-selective oxicam inhibitors gain extra stabilization energy with the change of residue 523 from valine to isoleucine because of the formations of new hydrogen bonds in the enzyme-inhibitor complexes. The utility of the combined computational approach, as a valuable tool for in silico screening of COX-2-selective inhibitors, is further exemplified by identifying the physicochemical origins of the enantiospecific selective inhibition of COX-2 by -substituted indomethacin ethanolamide inhibitors.