DOI: 10.1002/cbic.200700644 Self-Cleavable Bioluminogenic Luciferin Phosphates as Alkaline Phosphatase Reporters Wenhui Zhou,* [a] Christine Andrews,* [b] Jianquan Liu, [a] John W. Shultz, [b] MichaelP. Valley, [b] Jim J. Cali, [b] Erika M. Hawkins, [b] Dieter H. Klaubert, [a] Robert F. Bulleit, [b] and Keith V. Wood [b] 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 immuno- sorbent assays (ELISA) [1] and DNA hybridization assays. [2] It is also used as an in situ probe to monitor the expression and translocation of fusion proteins from the cytoplasm [3] and for vis- ualization of the spatial distribu- tion of target biomolecules, such as cognate ligands or receptors in cells, tissues, and embryos. [4] Among the many methods for detecting AP activity, there are various phosphate substrates, such as the colorimetric p-nitro- phenyl phosphate, [5] the fluores- cent AttoPhos$, [6] and the chemi- luminescent adamantyl 1,2-di- ACHTUNGTRENNUNGoxetane AMPPD derivatives [7] (Scheme 1). It is the ultrasensitiv- ity of chemiluminescence, specif- ically 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 chemi- luminescent 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 ultrasensitivi- ty is needed in order to promote the bioluminescent AP assay for practical applications. Chemical modification of the 6-hy- droxyl group of luciferin (or the 6-amino group of aminoluci- ferin) is an effective means to approach bioluminescent assays for enzymes of interest, [8] and 6-luciferin phosphate (Scheme 1) has been previously shown to detect AP activity. [9] However, the detection limit of 10 À19 mol of AP was 2–3 orders of magni- tude lower than that for the AMPPD assay. [7] Since the hydroly- sis of phosphate monoesters is highly dependent on the pK a of the leaving group [10] and the lower pK a 8.5 [11] of the luciferin phenol compared to a pK a ~ 9.0 [7] of the adamantyl dioxetane phenol favors both nucleophilic attack and P ÀO bond fission Scheme 1. Chemical structures of substrates for AP enzyme. A) Known chemiluminescent substrate AMPPD deriva- tives and bioluminescent substrate 6-luciferin phosphate; B) proposed self-cleavable luciferin phosphates, amino- luciferin trimethyl lock phosphate 1, and luciferin p-hydroxymethylphenyl phosphate 2. [a] Dr. W. Zhou, Dr. J. Liu, Dr. D. H. Klaubert Research and Development, Promega Biosciences, Inc. 277 Granada Drive, San Luis Obispo, CA 93401 (USA) Fax: (+ 1)805-5431531 E-mail: wenhui.zhou@promega.com [b] C. Andrews, Dr. J. W. Shultz, Dr. M. P. Valley, J. J. Cali, E. M. Hawkins, Dr. R. F. Bulleit, Dr. K. V. Wood Research and Development, Promega Corporation 2800 Woods Hollow Road, Madison, WI 53711-5399 (USA) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. 714 # 2008 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2008, 9, 714 – 718