# Harnessing Contact-Quenched, Profluorescent Chemical Probes for Sensitive Determination and High-Throughput Measurements of Enzyme Activity

## TL;DR

This paper introduces new profluorescent chemical probes that use contact quenching to measure enzyme activity in biological samples with high sensitivity and throughput.

## Contribution

The novel use of a nonsymmetrical mono-exo-BCN-cystamine backbone to create sensitive, contact-quenched, dual-labeled chemical probes for enzyme activity assays.

## Key findings

- The bis-FAM chemical probe with low background fluorescence showed effective fluorescence turn-on properties.
- The probes enabled sensitive assays for butyrylcholinesterase and paraoxonase 1 lactonase activities in human serum.
- A high-throughput assay for butyrylcholinesterase activity was successfully developed using the chemical probe.

## Abstract

Dual-labeled, profluorescent
chemical probes have been
developed
to quantify and visualize a specific enzyme’s activity in complex
biological media. The intact chemical probes often exhibit minimal
fluorescence due to fluorescence quenching, but their intrinsic fluorescence
can be released by a favorable enzyme-catalyzed reaction. Contact
quenching represents one of several fluorescence quenching mechanisms.
However, it is seldom intentionally implemented in the synthesis of
dual-labeled, profluorescent chemical probes, because the structure
of a contact quenching construct requires the formation of a ground-state
fluorophore-quencher/-fluorophore complex. The ability of such dual-labeled
molecular probes to act as intramolecular dimers cannot be predicted.
We previously revealed that a mono exo-bicyclo­[6.1.0]­nonyne
(exo-BCN)-derivatized cystamine framework was critical
to synthesizing sensitive, dual-labeled, profluorescent chemical probes
capable of contact quenching. Here, we exploited the nonsymmetrical mono-exo-BCN-cystamine backbone by sequentially
coupling it with two different carboxyfluorescein (FAM) derivatives
and subsequently synthesizing four nonsymmetrical bis-FAM chemical probes. The fluorescence turn-on properties of the bis-FAM chemical probe with the lowest background FAM fluorescence
were characterized by kinetic studies. Moreover, the release of two
FAM equivalents from the fluorescence turn-on chemical probe during
reactions was utilized to develop sensitive assays for measuring the
activity and inhibition of two serum biomarkers, butyrylcholinesterase
(BChE) and paraoxonase 1 (PON1) lactonase. We also developed an efficient,
high-throughput assay for detecting BChE activity based on the chemical
probe. Finally, the fluorescence assays successfully quantified the
activities of BChE and PON1 lactonase in human serum.