An improved synthesis of a fluorophosphonate-polyethylene glycol-biotin probe and its use against competitive substrates.

Hao Xu,Hairat Sabit,Gordon L Amidon,H D Hollis Showalter

doi:10.3762/bjoc.9.12

Hao Xu, Hairat Sabit + Show 2 more

Open Access

https://doi.org/10.3762/bjoc.9.12

Copy DOI

Abstract

The fluorophosphonate (FP) moiety attached to a biotin tag is a prototype chemical probe used to quantitatively analyze and enrich active serine hydrolases in complex proteomes in an approach called activity-based protein profiling (ABPP). In this study we have designed a novel synthetic route to a known FP probe linked by polyethylene glycol to a biotin tag (FP–PEG–biotin). Our route markedly increases the efficiency of the probe synthesis and overcomes several problems of a prior synthesis. As a proof of principle, FP–PEG–biotin was evaluated against isolated protein mixtures and different rat-tissue homogenates, showing its ability to specifically target serine hydrolases. We also assessed the ability of FP–PEG–biotin to compete with substrates that have high enzyme turnover rates. The reduced protein-band intensities resulting in these competition studies demonstrate a new application of FP-based probes seldom explored before.

Highlights

One of the goals of chemical biology is to develop small–molecule- and biomolecule-based probes to interrogate biological processes
Due to the absence of a UV chromophore, the purity of FP–PEG–biotin 1 could not be determined by HPLC
We have shown that for serine hydrolases with reversible substrates, FP probes can be useful tools if their concentration and incubation times are properly controlled

Summary

Introduction

One of the goals of chemical biology is to develop small–molecule- and biomolecule-based probes to interrogate biological processes. In this regard, fluorophosphonate (FP) probes have been extensively used in activity-based protein profiling (ABPP) in proteomic studies [1,2]. DFP is a serine hydrolase covalent inhibitor and from it have evolved analytical tools in which “handles“, such as biotin, rhodamine, and alkyne have been appended via a variety of linking chains [5,6,7,8] These FP analogues have proven to be powerful tools in the profiling of complex proteome samples [9] and in the identification of Beilstein J.

Results

Discussion

Conclusion