Self-Cleavable Bioluminogenic Luciferin Phosphates as Alkaline Phosphatase Reporters

Alkaline phosphatase (AP)—a stable enzyme with high specific activity for the hydrolysis of phosphate esters—is widely used as a conjugated enzyme label in enzyme-linked immunosorbent assays (ELISA) and DNA hybridization assays. It is also used as an in situ probe to monitor the expression and translocation of fusion proteins from the cytoplasm and for visualization of the spatial distribution of target biomolecules, such as cognate ligands or receptors in cells, tissues, and embryos. Among the many methods for detecting AP activity, there are various phosphate substrates, such as the colorimetric p-nitrophenyl phosphate, the fluorescent AttoPhos<, and the chemiluminescent adamantyl 1,2-diACHTUNGTRENNUNGoxetane AMPPD derivatives (Scheme 1). It is the ultrasensitivity of chemiluminescence, specifically with 1,2-dioxetane AMPPD derivatives, that has made this the overwhelming choice for monitoring AP activity. Although a luciferase-coupled bioluminescent assay is not only generically similar to the chemiluminescent assay and could show similar sensitivity, it also has the additional potential of creating recombinant luciferase to AP protein fusions, which might be preferable for the detection of AP activity in situ. The development of a suitable substrate to reach this ultrasensitivity is needed in order to promote the bioluminescent AP assay for practical applications. Chemical modification of the 6-hydroxyl group of luciferin (or the 6-amino group of aminoluciferin) is an effective means to approach bioluminescent assays for enzymes of interest, and 6-luciferin phosphate (Scheme 1) has been previously shown to detect AP activity. However, the detection limit of 10 19 mol of AP was 2–3 orders of magnitude lower than that for the AMPPD assay. Since the hydrolysis of phosphate monoesters is highly dependent on the pKa of the leaving group and the lower pKa 8.5 [11] of the luciferin phenol compared to a pKa ~9.0 of the adamantyl dioxetane phenol favors both nucleophilic attack and P O bond fission Scheme 1. Chemical structures of substrates for AP enzyme. A) Known chemiluminescent substrate AMPPD derivatives and bioluminescent substrate 6-luciferin phosphate; B) proposed self-cleavable luciferin phosphates, aminoluciferin trimethyl lock phosphate 1, and luciferin p-hydroxymethylphenyl phosphate 2.