Serum paraoxonase (PON1) is an high-density lipoprotein (HDL)-associated enzyme with antiatherogenic and detoxification properties that hydrolyzes a wide range of substrates, such as esters, organophosphates (eg, paraoxon) and lactones. For a long time, PON1 was considered to be an aryl esterase and paraoxonase, and its activity was measured accordingly. However, it recently became apparent that PON1 can catalyze both the hydrolysis [1, 2] and formation [3] of a variety of lactones. Indeed, structure–reactivity studies [4] and laboratory evolution experiments [5] indicate that PON1’s native activity is as a lactonase, and that the paraoxonase and aryl esterase are promiscuous activities. Studies of PON1’s activation by binding to HDL particles carrying ApoA-I indicate high specificity towards lactone substrates, and lipophilic lactones in particular.[6] Finally, the lactonase activity is the only activity shared by all other members of the PON family, some of which exhibit no paraoxonase or aryl esterase activity.[2] The activity of PON1 in human sera has been the subject of numerous studies that address a possible linkage between polymorphisms of PON1, various environmental factors that modulate its activity and susceptibility to atherosclerosis and other disorders.[7] The assays, however, use phenyl acetate or paraoxon, which have no physiological relevance. A more relevant assay must address the lactonase activity. However, current methods for measuring lactonase activities with aliphatic lactones are based on pH indicators [1, 4] and HPLC.[2, 3] The latter is highly laborious, while the pH-indicator assay requires repetitive calibrations and gives accurate results only with pure enzyme samples in which the pH and buffer strength can be tightly controlled. We have therefore developed novel aliphatic chromogenic/fluorogenic lactone substrates that would be favoured by PONs, and could be used with biological samples, for example, serum, cells or cell lysates. The development of facile enzymatic assays that are suitable for high-throughput screens is of importance in many fields, including diagnostics, drug discovery, functional genomics and enzyme engineering and evolution.[8]

We adopted the general strategy of Reymond et al., by which, the primary enzymatic reaction produces an unstable intermediate that rapidly collapses to release a fluorescent/chromogenic moiety.[8] In fact, this group has recently developed a fluorescence-based lactonase assay using 6-and 7-member ring lactones substituted with umbelliferone.[9] However, these substrates significantly differ from the favourable substrates of PON1 that comprise 5-and 6-membered ring oxo-lactones with long alkyl side chains.[2, 4, 6] They also exhibit high background rates at the pH optimum for PON1 (∼ 8.0). We therefore designed 5-thioalkyl butyrolactones (TXBLs) that release a thiol moiety upon hydrolysis of the oxo-lactone ring (Scheme 1). The released thiol can be detected with chromogenic or fluorogenic probes such as Ellman’s reagent (DTNB)[10] or CPM.[11]