Fluorescent pH Indicators for Neutral to Near-Alkaline Conditions Based on 9-Iminopyronin Derivatives

Abstract

Monitoring enzymatic activities at pH ranges compatible with their physiological optimum using fluorescent assays is important for high-throughput screening and engineering of novel biocatalysts as well as applications in biosensing and diagnostics. However, the number of fluorescent dyes exhibiting necessary optical properties at alkaline pHs is limited. Here, we report the design, synthesis, and biochemical application of fluorescent 9-iminopyronin derivatives possessing unique and distinct emission properties to provide optimal sensitivity at neutral to near-alkaline conditions. They exist in two acid−base forms, allowing for a dual-emission ratiometric sensing with a linear response of pH between 6.0 and 8.5 and detection sensitivity of a 0.06 pH unit. Selected fluorophores were used as the sensors for monitoring the enzymatic hydrolytic activity of a model enzyme haloalkane dehalogenase.

Ketone Incorporation Extends the Emission Properties of the Xanthene Scaffold Beyond 1000 nm†

“…An additional feature of certain xanthene derivatives is an exocyclic double bond (69,70). This was recently investigated by Johnsson and coworkers, where photoactivatable silicon rhodamines possessing an exocyclic alkene were generated through bisaryl lithium addition to glutaric anhydride (69).…”

Synthesis of Catechol Derived Rosamine Dyes and Their Reactivity toward Biogenic Amines

“…In fact, 9-aminopyronin dyes are described in the literature as chemically stable dyes having high molar absorption coefficients and high fluorescence quantum yields. Being initially introduced by K. Burgess et al [ 30 ], their synthesis typically involves the reaction of xanthenone derivatives with amines in the presence of trifluoromethanesulfonic anhydride or oxalyl chloride [ 31 , 32 , 33 , 34 ]. Alternatively, 9-aminopyronins can be prepared by reacting 9H-xanthene-9-thione with electron-withdrawing groups such as 4-aminopyridine and 2-aminoquinoline [ 35 ].…”

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation

“…The two distinct topics discussed in this paper, haloalkane dehalogenases and halide assays, are united by the unmet demand for more powerful dehalogenase assays. [10] We have developed a novel halide assay based on the biocatalytic oxidation of halides under mild conditions. The HOX assay is up to three orders of magnitude more sensitive and much safer than the currently most widely used methods employing hazardous mercury compounds.…”

“…[7a] This creates an unmet demand for sensitive and high-throughput assays to screen for haloalkane dehalogenase activity. [10] Using gas chromatography and mass spectrometry (GC-MS) to identify the alcohol products of dehalogenation reactions remains the analytical gold standard but these methods lack the throughput required for screening large libraries, especially since often several substrates need to be tested. [8] Therefore, preliminary characterisation of haloalkane dehalogenases often relies on detection of either the protons or halide ions produced.…”

“…[11,13] More sensitive assays employing fluorescent pH indicators have recently been developed, enabling the detection of 40 to 400 μM protons. [10] The use of very low buffer concentrations (< 2 mM) means that pH is easily changed, also by factors other than enzymatic dehalogenation, reducing the reliability of pH assays. For this reason, the Iwasaki assay for halide ions has remained popular in dehalogenase assays, despite the involvement of extremely toxic mercury derivatives.…”