RESUMO
Sensing of pyrophosphate anion (PPi) in the presence of nucleotide triphosphates allows the real time monitoring of the polymerase chain reaction. To get a deeper understanding of the factors involved in PPi/nucleotide triphosphate discrimination, a detailed study on the performance of a dimethyltin (IV)-catecholate complex capable of both separate fluorimetric or electrochemical detection of PPi in the presence of adenosine triphosphate (ATP) has been undertaken. Dimethyltin (IV) tightly binds PPi or ATP, and forms a stable 1:1 complex with tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid) in water. The complexation equilibria with all components are characterized quantitatively by potentiometric and spectroscopic titrations. Pyrophosphate anion can be detected owing to its ability to release free tiron from the complex by measuring either a fluorimetric or an electrochemical signal. On the contrary, ATP does not displace tiron but causes an interference with PPi in the fluorimetric detection method due to the formation of a ternary Me2Sn(IV)-tiron-ATP complex with optical properties intermediate between those of free and bound tiron. In the electrochemical (square wave voltammetry) method, the ternary ATP complex shows a separate peak which does not coincide with the peaks of neither free nor bound tiron, thus making possible the simultaneous detection of ATP in addition to PPi.
Assuntos
Difosfato de Adenosina/análise , Difosfatos/análise , Eletroquímica/métodos , Fluorometria/métodos , Compostos Orgânicos de Estanho/química , Espectrofotometria/métodos , Difosfato de Adenosina/química , Difosfatos/química , Limite de DetecçãoRESUMO
Stability constants of boronic acid diol esters in aqueous solution have been determined potentiometrically for a series of meta-, para-substituted phenylboronic acids and diols of variable acidity. The constants ß(11-1) for reactions between neutral forms of reactants producing the anionic ester plus proton follow the Hammett equation with ρ depending on pKa of diol and varying from 2.0 for glucose to 1.29 for 4-nitrocatechol. Observed stability constants (K(obs)) measured by UV-vis and fluorometric titrations at variable pH for esters of 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron) generally agree with those expected on the basis of ß(11-1) values, but the direct fitting of K(obs) vs pH profiles gives shifted pKa values both for boronic acids and diol as a result of significant interdependence of fitting parameters. The subsituent effects on absorption and fluorescence spectra of Tiron arylboronate esters are characterized. The K(obs) for Tiron determined by (11)B NMR titrations are approximately 1 order of magnitude smaller than those determined by UV-vis titrations under identical conditions. A general equation, which makes possible an estimate of ß(11-1) for any pair of boronic acid and diol from their pKa values, is proposed on the basis of established Brönsted-type correlation of Hammett parameters for ß(11-1) with acidity of diols. The equation allows one to calculate stability constants expected only on basis of acid-base properties of the components, thus permitting more strict evaluation of contributions of additional factors such as steric or charge effects to the ester stability.
Assuntos
Ácidos Borônicos/química , Ésteres/química , Concentração de Íons de Hidrogênio , Estrutura MolecularRESUMO
Highly selective detection of pyrophosphate in the presence of inorganic phosphate, halides, acetate, ATP, other nucleotides and phosphate esters in neutral aqueous solutions is achieved by using fluorescent dimethyltin(iv)-alizarin red S complex.