Sensitive fluorogenic substrate for alkaline phosphatase

Abstract

Alkaline phosphatase serves as both a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered upon P–O bond cleavage in a process mediated by a “trimethyl lock”. Although this substrate requires a second, non-enzymatic step to manifest fluorescence, we demonstrated that the first, enzymatic step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.

A single diamagnetic catalyCEST MRI contrast agent that detects cathepsin B enzyme activity by using a ratio of two CEST signals

“…Other peptide sequences can be incorporated into the synthesis scheme to create catalyCEST MRI contrast agents that sense other proteases (26). Other classes of enzymes that modify an aryl functional group may also be detected using this platform technology, such as esterases (7), phosphatases (27), and sulfatases (28). The salicylic acid moiety may be replaced by other molecules that have intramolecular hydrogen bonding that causes highly shifted CEST signals and are less likely to undergo slow degradation (11,17,29).…”

Bright Building Blocks for Chemical Biology

“…Thus, N -acyl rhodamine 110 derivatives are valuable fluorogenic compounds for measuring enzyme activity or serving as activatable labels for advanced fluorescence microscopy experiments. 72 , 82 , 84 , 85 …”

Trimethyl Lock: A Multifunctional Molecular Tool for Drug Delivery, Cellular Imaging, and Stimuli‐Responsive Materials

“…[67] Although the cytotoxic activity of compound 53 is roughly two times lower than that of 52,i ts bioavailability and effectiveness in an in vivo murine tumor model have been shown to be superior to those of its parent drug. Thus, phosphatase-sensitive TML-luciferin derivative 54 [78] and TML-morpholino urea rhodamine 55 [79] provide more stable alternative substrates for highly sensitivea ssay development. [68][69][70][71][72][73][74][75] Beyond these applicationsi np rodrug design, phosphatase-sensitiveT ML fragments have been used for the design of compounds 54 and 55 (Scheme 11)a sf luorogenic substrates for enzyme-linked immunosorbent assays (ELISAs) by utilizing the alkaline phosphataseo fEscherichia coli.…”

“…[77] In addition, common substrates for sensitivea ssays based on fluorescence( e.g.,3 ,6-fluorescein diphosphate and 4-methylumbelliferyl phosphate) or on luminescence( e.g.,l uciferin 6'-O-phosphate) show limited stability. Thus, phosphatase-sensitive TML-luciferin derivative 54 [78] and TML-morpholino urea rhodamine 55 [79] provide more stable alternative substrates for highly sensitivea ssay development.…”