Flow‐Through Synthesis on Teflon‐Patterned Paper To Produce Peptide Arrays for Cell‐Based Assays

A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell‐based assays. Solvent‐repelling barriers made of Teflon‐impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow‐through syntheses on paper. The confinement and flow‐through mixing significantly improves the peptide yield and simplifies the automation of this synthesis. The synthesis of 100 peptides ranging from 7 to 14 amino acids in length gave over 60 % purity for the majority of the peptides (>95 % yield per coupling/deprotection cycle). The resulting peptide arrays were used in cell‐based screening to identify 14 potent bioactive peptides that support the adhesion or proliferation of breast cancer cells in a 3D environment. In the future, this technology could be used for the screening of more complex phenotypic responses, such as cell migration or differentiation.     

Synthesis and Structure–Activity Correlation Studies of Secondary‐ and Tertiary‐Amine‐Based Glutathione Peroxidase Mimics

In this study, a series of secondary‐ and tertiary‐amino‐substituted diaryl diselenides were synthesized and studied for their glutathione peroxidase (GPx) like antioxidant activities with H₂O₂, cumene hydroperoxide, or tBuOOH as substrates and with PhSH or glutathione (GSH) as thiol cosubstrates. This study reveals that replacement of the tert‐amino groups in benzylamine‐based diselenides by sec‐amino moieties drastically enhances the catalytic activities in both the aromatic thiol (PhSH) and GSH assay systems. Particularly, the N‐propyl‐ and N‐isopropylamino‐substituted diselenides are 8–18 times more active than the corresponding N,N‐dipropyl‐ and N,N‐diisopropylamine‐based compounds in all three peroxide systems when GSH is used as the thiol cosubstrate. Although the catalytic mechanism of sec‐amino‐substituted diselenides is similar to that of the tert‐amine‐based compounds, differences in the stability and reactivity of some of the key intermediates account for the differences in the GPx‐like activities. It is observed that the sec‐amino groups are better than the tert‐amino moieties for generating the catalytically active selenols. This is due to the absence of any significant thiol‐exchange reactions in the selenenyl sulfides derived from sec‐amine‐based diselenides. Furthermore, the seleninic acids (RSeO₂H) derived from the sec‐amine‐based compounds are more stable toward further reactions with peroxides than their tert‐amine‐based analogues.     

Probing the Formation of a Seleninic Acid in Living Cells by the Fluorescence Switching of a Glutathione Peroxidase Mimetic

Glutathione peroxidase (GPx) is a selenoenzyme that protects cells against oxidative damage. Although the formation of a seleninic acid (‐SeO₂H) by this enzyme during oxidative stress has been proposed, a selenic acid has not been identified in cells. Herein, we report that the formation of a seleninic acid can be monitored in living cells by using a redox‐active ebselen analogue with a naphthalimide fluorophore. The probe reacts with H₂O₂ to generate the highly fluorescent seleninic acid. The electron withdrawing nature of the ‐SeO₂H moiety and strong Se???O interactions, which prevent the photoinduced electron transfer, are responsible for the fluorescence.     

Reactivity of superoxide anion radical with a perchlorotriphenylmethyl (trityl) radical

The reaction of superoxide radical with a tricarboxylate derivative of perchlorotriphenylmethyl radical (PTM-TC) is studied. PTM-TC is a stable ("inert") free radical, which gives a single sharp electron paramagnetic resonance (EPR) peak in aqueous solutions. PTM-TC also gives a characteristic optical absorption at 380 nm. Superoxide, on reaction with PTM-TC, induced a decrease in the intensity of the EPR signal and optical absorption of PTM-TC at 380 nm. The signal loss was specific to superoxide and linearly dependent on the superoxide flux in the system. Competitive kinetics experiments revealed that PTM-TC reacts with superoxide with an apparent second-order rate constant of 8.3x10(8) M(-1) s(-1). Electrochemical and mass spectrometric analyses of the reaction suggested the formation of perchlorotriphenylmethane and molecular oxygen as products. The high sensitivity of detection of PTM-TC combined with the high rate constant of the reaction of superoxide with PTM-TC may offer a potential opportunity for measurement of superoxide in biological systems. In conclusion, the PTM-TC molecule has high sensitivity and specificity for superoxide radicals and thus may enable quantitative detection of superoxide generation in biological systems using EPR and/or spectrophotometric methods.     

Synthesis, characterization, and antioxidant activity of some ebselen analogues

Simple synthetic routes for several analogues of the anti-inflammatory organoselenium drug, ebselen, are described. The compounds are characterized by (1)H, (13)C, and (77)Se NMR spectroscopy and mass spectral techniques and, in some cases, by single-crystal X-ray diffraction studies. The glutathione peroxidase (GPx)-like antioxidant activity has been studied by using H(2)O(2), tBuOOH, and Cum-OOH as substrates, and thiophenol (PhSH, 4-Me-C(6)H(4)SH) and glutathione (GSH) as cosubstrates. Density functional theory (DFT) calculations have been performed on these systems to understand the effects of various substituents on the (77)Se NMR chemical shifts; these results have been compared with the experimental data. The experimental and theoretical results suggest that the presence of a phenyl substituent on the nitrogen atom is important for the antioxidant activity of ebselen. While ebselen and its analogues are poor catalysts in aromatic thiol assays, these compounds exhibit high GPx activity when GSH is used as the cosubstrate. The poor catalytic activity of ebselen analogues in the presence of aromatic thiols such as PhSH and 4-Me-C(6)H(4)SH can be ascribed to the undesired thiol exchange reaction that takes place at the selenium center due to SeO nonbonding interactions. To understand the effects of different peroxides on the catalytic activities, we have determined the initial rates at various concentrations of GSH and peroxides. These data suggest that the nature of peroxide has little effect on the catalytic efficiencies, although the initial reaction rates observed with hydrogen peroxide were found to be higher than that with tBuOOH and Cum-OOH. In contrast to the effect of peroxides, the nature of thiols appears to have a dramatic effect on the catalytic activity of ebselen and its related derivatives.     

An enantiopure galactose oxidase model: synthesis of chiral amino alcohols through oxidative kinetic resolution catalyzed by a chiral copper complex

An enantiopure galactose oxidase (GO) enzyme model has been synthesized from readily available (R)-BINAM and Cu(OTf)₂, and the enantiopure GO model has been effectively used in situ as an efficient chiral catalyst for the synthesis of chiral amino alcohols through oxidative kinetic resolution (OKR), where molecular oxygen is used as the sole oxidant. Under the proposed catalytic conditions, both ortho- and para-substituted amino alcohols were resolved with good to excellent enantiomeric excesses through oxidative kinetic resolution.