Polyphenols emit light in the presence of hydrogen peroxide in an alkaline medium, and are utilized in detecting cobalt and formaldehyde using flow injection.1,2 However, polyphenol chemiluminescence (CL) is very weak compared to representative CL compounds, such as luminol and the acridium ester; therefore, their application for the assay of bioactive compounds was not reported.Recently, new enhancers, trans-4-(3-propionic acid)phenylboronic acid and 4-biphenylboronic acid, have been developed for the horseradish peroxidase (HRP) catalyzed CL of pyrogallol and purpurogallin. Especially, 4-biphenylboronic acid enhanced the peak light emission 314-fold during CL of the HRP-purpurogallin reaction. We found a strong enhancement effect during polyphenol CL development with the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). To evaluate the enhancement effect of EDC during polyphenol CL development, gallic acid (GA), gallic acid methyl ester (GM) and pyrogallol were used as models for the polyphenols.
ExperimentalApparatus CL reactions were carried out in a 75´12 mm roundbottom glass tube. CL measurements were performed using a photon-counting luminometer, Lumat LB 9501 (Berthold, Wildbad, Germany); its operation and data processing were performed using a personal computer (PC 9801 ES2, NEC Corp., Tokyo, Japan) with a luminometer program, LB 9501/9801 Ver.1.41 (Japan Berthold, Tokyo, Japan). The proton nuclear magnetic resonance ( 1 H-NMR) spectrum was taken in methanold 4 (CD 3 OD) with a Varian UNITY plus (USA) spectrometer at 500 MHz.
Reagents and solutionsDeionized and distilled water purified by a Mili-Q II (Japan Milipore, Tokyo, Japan) was used. GA was purchased from Aldrich (Milwaukee, WI, USA). GM was purchased from Tokyo Kasei Organic Chemicals (Tokyo, Japan). Pyrogallol and EDC were purchased from Nacalai Tesque (Kyoto, Japan). GA, GM, pyrogallol and EDC were dissolved in water just before use. Hydrogen peroxide (31%, v/v) was purchased from Mitsubishi Gas Kagaku (Tokyo, Japan). All other chemicals and solvents were of reagent grade.
Procedure for the estimation of enhancement effect of EDCTo 200 ml of 10 mM polyphenols (GA, GM or pyrogallol) was added 100 ml of 0 -1.0 M EDC. To the mixture was added 100 ml of 100 mM sodium hydroxide for GA and GM, or 10 mM for pyrogallol or water. After standing for 25 s, the CL reaction was initiated by the addition of 100 ml of 1.0 M hydrogen peroxide using an automatic injection system in the luminometer. The CL emission was measured for 2 min and the integral photon counts were used.
For confirmation of the structure of the EDC adduct with pyrogallolTo a stirred aqueous pyrogallol (0.63 g in 10 ml water, 5 mmol) solution was added EDC (0.19 g, 1 mmol) at room temperature, and the mixture was stirred for 2 h. The mixture was poured into 20 ml of water and the pyrogallol extracted with diethylether. The aqueous layer containing Ia (Fig. 2) was treated with active charcoal. The filtrate was lyophilized and 0.12 g of compound Ia was obtained as a brown so...