A novel fluorescence assay for measuring phosphatidylserine decarboxylase catalysis.

Phosphatidylserine decarboxylases (PSDs) are central enzymes in phospholipid metabolism that produce phosphatidylethanolamine (PE) in bacteria, protists, plants, and animals. We developed a fluorescence-based assay for selectively monitoring production of PE in reactions using a maltose-binding protein fusion with Plasmodium knowlesi PSD (MBP-His6-Δ34PkPSD) as the enzyme. The PE detection by fluorescence (λex = 403 nm, λem = 508 nm) occurred after the lipid reacted with a water-soluble distyrylbenzene-bis-aldehyde (DSB-3), and provided strong discrimination against the phosphatidylserine substrate. The reaction conditions were optimized for enzyme, substrate, product, and DSB-3 concentrations with the purified enzyme and also tested with crude extracts and membrane fractions from bacteria and yeast. The assay is readily amenable to application in 96- and 384-well microtiter plates and should prove useful for high-throughput screening for inhibitors of PSD enzymes across diverse phyla.

Amine detection with distyrylbenzenedialdehyde-based Knoevenagel adducts.

Eight acceptor-substituted distyrylbenzene (DSB) derivatives were obtained by postfunctionalization of dialdehyde precursor 1 using Knoevenagel condensation. Solubility in a water/THF 9:1 mixture was achieved through the addition of branched oligoethylene glycol side chains. The acceptor compounds discriminate primary and secondary amines in aqueous solution. The fluorescence responses were analyzed by the multivariate analysis of variance (MANOVA) protocol, a statistical tool. In contrast to 1, the adducts show reactivity toward secondary and aromatic amines. Nitroolefin 2f is the most active dosimeter molecule. Reaction with amines is completed after less than 3 min, and the limit of detection (LOD) is improved by a factor of 10. Propylenediamine can be detected at 75 µM. This is a 10-fold improvement for the detection limit when compared to the detection limit of the starting dialdehyde.

Distyrylbenzene-aldehydes: identification of proteins in water.

Three different, water soluble, aldehyde-appended distyrylbenzene (DSB) derivatives were prepared. Their interaction with different albumin variants (human, porcine, bovine, lactalbumin, ovalbumin) was investigated (pH 11). All three fluorophores exhibit graded, protein-dependent fluorescence turn-on at slightly differing wavelengths. Linear discriminant analysis (LDA) differentiated all of the investigated albumins and was used to discern commercially available protein shakes. The three DSB derivatives barely react with the constituting amino acids but cysteine. In the proteins significant fluorescence signals are generated, probably due to a combination of imine/N,S-aminal formation and hydrophobic interactions between the DSBs and the proteins.

Detection of amines with extended distyrylbenzenes by strip assays.

We herein describe the synthesis and property evaluation of three novel aldehyde-substituted pentameric phenylenevinylenes carrying branched oligo(ethylene glycol) (swallowtail, Sw) substituents. The targets were synthesized by a combination of Heck coupling and Wittig or Horner reactions of suitable precursor modules. If the pentameric phenylenevinylene carries only two of these Sw substituents, it is no longer water-soluble. When six of the Sw substituents are attached, regardless of their position, the pentameric phenylenevinylenes are well water-soluble. The dialdehydes were investigated with respect to their amine-sensing capabilities both in water as well as in the solid state, sprayed onto thin layer chromatography (TLC) plates (alox, silica gel, reversed phase silica gel). The recognition of amine vapors using the sprayed-on phenylenevinylene dialdehydes is superb and allows the identification of different amines on regular silica TLC plates via color changes, analyzed by a statistical tool, the multivariate analysis of variance (MANOVA) protocol.

Water-soluble cruciforms and distyrylbenzenes: synthesis, characterization, and pH-dependent amine-sensing properties.

Three water-soluble fluorescent aldehyde-substituted distyrylbenzene derivatives were prepared using Heck or Horner methodologies. Water solubility was achieved through the addition of branched oligoethylene glycol side chains; these are attached via an ether bridge to the aromatic nucleus. The aldehydes are almost nonfluorescent in water, but addition of primary amines turns the fluorescence on; formation of imines results. Control of the basicity of the media allows further discrimination of the analytes employed. 1,3-Diaminopropane reacts with these aldehydes. Instead of an imine, a brightly fluorescent aminal forms. Amino acids are almost always nonreactive toward these aldehydes. Exceptions are lysine and cysteine, which form an imine and a thioaminal, respectively, discreating the aldehyde unit under fluorescence turn-on in water. The detection limit and time of completion of the sensing event were evaluated. Dialdehydes 3 and 16 were comparable on both counts. The cross-shaped 16 did react approximately twice as quickly with 1,3-diaminopropane.

Aldehyde-appended distyrylbenzenes: amine recognition in water.

Change in water: Aqueous solutions of aldehyde-substituted, water-soluble distyrylbenzenes reacted with amines to give imines or aminals with dramatically changed fluorescence. This approach allowed the detection and recognition of amines in water (see figure).