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

• Main Authors: Hao Xu, Hairat Sabit, Gordon L. Amidon, H. D. Hollis Showalter
• Format: Article
• Language: English
• Published: Beilstein-Institut 2013-01-01
• Series: Beilstein Journal of Organic Chemistry
• Subjects: biotin; fluorophosphonate; high turnover rate; reversible substrate
• Online Access: https://doi.org/10.3762/bjoc.9.12
• ISSN: 1860-5397

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.