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Issue 8, 2013
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4-Alkyloxyimino-cytosine nucleotides: tethering approaches to molecular probes for the P2Y6 receptor

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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 (N4 and 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 conjugate 16 activated the human P2Y6R expressed in 1321N1 human astrocytoma cells with an EC50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 (t1/2 of 18 min) and surface-bound fluorescence. Known P2Y6R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R.

Graphical abstract: 4-Alkyloxyimino-cytosine nucleotides: tethering approaches to molecular probes for the P2Y6 receptor

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The article was received on 10 May 2013, accepted on 30 May 2013 and first published on 17 Jun 2013


Article type: Concise Article
DOI: 10.1039/C3MD00132F
Med. Chem. Commun., 2013,4, 1156-1165

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    4-Alkyloxyimino-cytosine nucleotides: tethering approaches to molecular probes for the P2Y6 receptor

    P. S. Jayasekara, M. O. Barrett, C. B. Ball, K. A. Brown, E. Kozma, S. Costanzi, L. Squarcialupi, R. Balasubramanian, H. Maruoka and K. A. Jacobson, Med. Chem. Commun., 2013, 4, 1156
    DOI: 10.1039/C3MD00132F

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