The development of new catalytic methods, in particular enzymatic processes, is increasingly being followed with combinatorial and evolutionary methods, whereby sensitive assays for catalysis play an essential role. [1±3] Most enzyme assays are based on chromogenic or fluorogenic substrates. [1,4, 5] In many cases, however, it is desirable to measure the reaction of a well-defined substrate of interest, and not that of a different fluorogenic or chromogenic derivative. Such direct detection can be achieved with instrumental methods, such as HPLC and mass spectrometry, [6] by using product-selective fluorescent sensors, [7] or with indirect colorimetric assays based on susbstrate-or product-binding proteins, such as cat-ELISAs (catalytic enzyme-linked immunosorbent assays) [8] or the QUEST method (ªquerying for enzymes using the three-hybrid systemº). [9] More simply, one can also measure spectrophotometrically a physico-chemical parameter that responds to the progress of the reaction, such as measuring the temperature by IR thermography, [10] or, for reactions releasing acids or bases, the pH value of the reaction medium with pH indicators. [11] Herein we report a new enzyme assay based on following the evolution of pM (À lg[M], where M is free metal ions) as a function of reaction time. The change in pM is recorded using an orange fluorescent metal-sensor derived from quinacridone.
The orange fluorescent sensor 3, which is obtained from quinacridone (1) via dibromide 2, responds to the presence of small concentrations of Cu 2 by an almost quantitative quenching of its fluorescence (l em(max) 558 nm). The effect can be explained by formation of a macrocyclic chelate M ´3, in which energy transfer quenching occurs due to the proximity of the quenching metal ion to the fluorophore (Scheme 1). [12] The complex M ´3 contains a 17-membered ring. The chelate effect induced by such a large ring is expected to be weak. Indeed the macrocycle is readily broken by addition of excess metal ion, leading to the formation of an M 2 ´3 complex. This suggests that other weak metal chelators, Scheme 1. Principle of the fluorescence enzyme assay using pM.