title: An improved synthesis of a fluorophosphonate-polyethylene glycol-biotin probe and its use against competitive substrates.
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.
authors: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, USA.|Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109-1065, USA.
authors: Hao
authors: H
authors: Xu
authors: Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109-1065, USA.
authors: Hairat
authors: H
authors: Sabit
authors: Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109-1065, USA.
authors: Gordon L
authors: GL
authors: Amidon
authors: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, USA.|Vahlteich Medicinal Chemistry Core, University of Michigan, Ann Arbor, MI 48109-1065, USA.
authors: H D Hollis
authors: HDH
authors: Showalter
keywords: biotin; fluorophosphonate; high turnover rate; reversible substrate
references: Bioorg Med Chem. 2012 Jan 15;20(2):601-6
references: 21763150
references: Nat Biotechnol. 2003 Jun;21(6):687-91
references: 12740587
references: Toxicol In Vitro. 2009 Aug;23(5):887-96
references: 19470405
references: Arch Biochem Biophys. 2012 Jun 1;522(1):44-56
references: 22525521
references: Chembiochem. 2011 Nov 25;12(17):2587-98
references: 22025425
references: Proteomics. 2001 Sep;1(9):1067-71
references: 11990500
references: Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10335-40
references: 12149457
references: J Pharmacol Exp Ther. 2006 Dec;319(3):1477-84
references: 16966469
references: Toxicol Sci. 2005 Aug;86(2):291-9
references: 15888665
references: Biochemistry. 2001 Apr 3;40(13):4005-15
references: 11300781
references: Mol Cell Proteomics. 2008 Jul;7(7):1241-53
references: 18364346
references: Carbohydr Polym. 2014 Jan 16;100:126-34
references: 24188846
references: Am J Pharmacogenomics. 2004;4(6):371-81
references: 15651898
references: Synthesis (Stuttg). 2008 Jan 1;2008(2):215-220
references: 20585414
references: Proc Natl Acad Sci U S A. 2010 Dec 7;107(49):20941-6
references: 21084632
references: Nat Biotechnol. 2009 Apr;27(4):387-94
references: 19329999
references: Br J Clin Pharmacol. 1985 May;19(5):701-4
references: 2988590
references: Mol Pharm. 2008 Mar-Apr;5(2):294-315
references: 18254597
references: Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):14694-9
references: 10611275
references: Nat Chem Biol. 2005 Aug;1(3):130-42
references: 16408016