Importance of <i>ortho</i> Proton Donors in Catalysis of Hydrazone Formation

Anthranilic acids were recently reported as superior catalysts for hydrazone and oxime formation compared to aniline, the classic catalyst for these reactions. Here, alternative proton donors were examined with varied p<i>K</i>_a in an effort to enhance activity at biological pH. The experiments show that 2-aminobenzenephosphonic acids are superior to anthranilic acids in catalyzing hydrazone formation with common aldehyde substrates

Water-Soluble Organocatalysts for Hydrazone and Oxime Formation

The formation of oximes and hydrazones is widely used in chemistry and biology as a molecular conjugation strategy for achieving ligation, attachment, and bioconjugation. However, the relatively slow rate of reaction has hindered its utility. Here, we report that simple, commercially available anthranilic acids and aminobenzoic acids act as superior catalysts for hydrazone and oxime formation, speeding the reaction considerably over the traditional aniline-catalyzed reaction at neutral pH. This efficient nucleophilic catalysis, involving catalyst–imine intermediates, allows rapid hydrazone/oxime formation even with relatively low concentrations of the two reactants. The most efficient catalysts are found to be 5-methoxyanthranilic acid and 3,5-diaminobenzoic acid; we find that they can enhance rates by factors of as much as 1–2 orders of magnitude over the aniline-catalyzed reaction. Evidence based on a range of differently substituted arylamines suggests that the <i>ortho</i>-carboxylate group in the anthranilate catalysts serves to aid in intramolecular proton transfer during imine and hydrazone formation

Fluorescence Quenchers for Hydrazone and Oxime Orthogonal Bioconjugation

We describe the synthesis and properties of new fluorescence quenchers containing aldehyde, hydrazine, and aminooxy groups, allowing convenient bioconjugation as oximes or hydrazones. Conjugation to oligonucleotides proceeded in high yield with aniline as catalyst. Kinetics studies of conjugation show that, under optimal conditions, a hydrazine or aminooxy quencher can react with aldehyde-modified DNA to form a stable hydrazone or oxime adduct in as little as five minutes. The resulting quencher-containing DNAs were assessed for their ability to quench the emission of fluorescein in labeled complements and compared to the commercially available dabcyl and Black Hole Quencher 2 (BHQ2), which were conjugated as phosphoramidites. Results show that the new quenchers possess slightly different absorbance properties compared to dabcyl and are as efficient as the commercial quenchers in quenching fluorescein emission. Hydrazone-based quenchers were further successfully incorporated into molecular beacons and shown to give high signal to background ratios in single nucleotide polymorphism detection <i>in vitro.</i> Finally, aminooxy and hydrazine quenchers were applied to quenching of an aldehyde-containing fluorophore associated with living cells, demonstrating cellular quenching within one hour

Fast Alpha Nucleophiles: Structures that Undergo Rapid Hydrazone/Oxime Formation at Neutral pH

Hydrazones and oximes are widely useful structures for conjugate formation in chemistry and biology, but their formation can be slow at neutral pH. Kinetics studies were performed for a range of structurally varied hydrazines, and a surprisingly large variation in reaction rate was observed. Structures that undergo especially rapid reactions were identified, enabling reaction rates that rival orthogonal cycloaddition-based conjugation chemistries

Fast Hydrazone Reactants: Electronic and Acid/Base Effects Strongly Influence Rate at Biological pH

Kinetics studies with structurally varied aldehydes and ketones in aqueous buffer at pH 7.4 reveal that carbonyl compounds with neighboring acid/base groups form hydrazones at accelerated rates. Similarly, tests of a hydrazine with a neighboring carboxylic acid group show that it also reacts at an accelerated rate. Rate constants for the fastest carbonyl/hydrazine combinations are 2–20 M^–1 s^–1, which is faster than recent strain-promoted cycloaddition reactions

Hybridization Thermodynamics of DNA Oligonucleotides during Microchip Capillary Electrophoresis

Capillary electrophoresis (CE) is a powerful analytical tool for performing separations and characterizing properties of charged species. For reacting species during a CE separation, local concentrations change leading to nonequilibrium conditions. Interpreting experimental data with such nonequilibrium reactive species is nontrivial due to the large number of variables involved in the system. In this work we develop a COMSOL multiphysics-based numerical model to simulate the electrokinetic mass transport of short interacting ssDNAs in microchip capillary electrophoresis. We probe the importance of the dissociation constant, <i>K</i>_D, and the concentration of DNA on the resulting observed mobility of the dsDNA peak, μ_w, by using a full sweep of parametric simulations. We find that the observed mobility is strongly dependent on the DNA concentration and <i>K</i>_D, as well as ssDNA concentration, and develop a relation with which to understand this dependence. Furthermore, we present experimental microchip capillary electrophoresis measurements of interacting 10 base ssDNA and its complement with changes in buffer ionic strength, DNA concentration, and DNA sequence to vary the system equilibria. We then compare our results to thermodynamically calculated <i>K</i>_D values