Synthesis and characteristics of a new fluorogenic substrate for horseradish peroxidase

N,N'-Dicyanomethyl-o-phenylenediamine was synthesized with a 90% yield by a reaction of o-phenylenediamine with chloroacetonitrite in triethylamine. Our experimental results showed that it was the effective fluorogenic substrate for horseradish peroxidase (HRP) and hemin. The K-m for the HRP system was 48 mu M, and that for hemin was 1.3 mu M Properties of the substrate were evaluated from the detection limits of enzymes and H2O2. The linear ranges for the determination of HRP and hemin were 21-150 pM and 2-20 nM, respectively. The linear range for the determination of H2O2 using HRP or hemin was 18-140 and 60-1000 nM, respectively. The structural elucidation of the fluorescent product using NMR and mass spectral techniques was proposed to be 1,2-dihydro-2-imido-imidazo[1,2-a] quinoxaline. Based on the product structure and earlier reports, the possible reaction mechanism of HRP and the substrate was also proposed, i.e., two steps for ring closures, one step of isomerization, and a final step of oxidative dehydrogenation. (C) 1996 Academic Press, Inc.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:A1996UT14500006&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Chemistry, AnalyticalSCI(E)12ARTICLE4428-4365