title: 4-Alkyloxyimino-cytosine nucleotides: tethering approaches to molecular probes for the P2Y6receptor.
abstract: 4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we probed two positions (N4and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y6R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate16activated the human P2Y6R expressed in 1321N1 human astrocytoma cells with an EC50of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with16to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized16(t1/2of 18 min) and surface-bound fluorescence. Known P2Y6R ligands inhibited labeling. Theoretical docking of16to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified theN4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R.
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA. ; Tel: +1 301-496-9024.
authors.forename: P Suresh
authors.initials: PS
authors.lastname: Jayasekara
authors.affiliation: Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7365, USA.
authors.forename: Matthew O
authors.initials: MO
authors.lastname: Barrett
authors.affiliation: Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7365, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Ball
authors.affiliation: Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7365, USA.
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Brown
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA. ; Tel: +1 301-496-9024.
authors.forename: Eszter
authors.initials: E
authors.lastname: Kozma
authors.affiliation: Department of Chemistry, American University, Washington, DC 20016, USA.
authors.forename: Stefano
authors.initials: S
authors.lastname: Costanzi
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA. ; Tel: +1 301-496-9024.
authors.forename: Lucia
authors.initials: L
authors.lastname: Squarcialupi
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA. ; Tel: +1 301-496-9024.
authors.forename: Ramachandran
authors.initials: R
authors.lastname: Balasubramanian
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA. ; Tel: +1 301-496-9024.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Maruoka
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA. ; Tel: +1 301-496-9024.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson