title: Nucleoside conjugates of quantum dots for characterization of G protein-coupled receptors: strategies for immobilizing A2A adenosine receptor agonists.
abstract: BACKGROUND
  Quantum dots (QDs) are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand probes with fluorescent reporter groups are needed for detection and characterization of G protein-coupled receptors (GPCRs).
  
  
  RESULTS
  Synthetic strategies for coupling the A2A adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine) to functionalized QDs were explored. Conjugates tethered through amide-linked chains and poly(ethyleneglycol) (PEG) displayed low solubility and lacked receptor affinity. The anchor to the dendron was either through two thiol groups of (R)-thioctic acid or through amide formation to a commercial carboxy-derivatized QD. The most effective approach was to use polyamidoamine (PAMAM) D5 dendrons as multivalent spacer groups, grafted on the QD surface through a thioctic acid moiety. In radioligand binding assays, dendron nucleoside conjugate 11 displayed a moderate affinity at the human A2AAR (Kiapp 1.02 +/- 0.15 muM). The QD conjugate of increased water solubility 13, resulting from the anchoring of this dendron derivative, interacted with the receptor with Kiapp of 118 +/- 54 nM. The fluorescence emission of 13 occurred at 565 nm, and the presence of the pendant nucleoside did not appreciably quench the fluorescence.
  
  
  CONCLUSIONS
  This is a feasibility study to demonstrate a means of conjugating to a QD a small molecular pharmacophore of a GPCR that is relatively hydrophobic. Further enhancement of affinity by altering the pharmacophore or the linking structures will be needed to make useful affinity probes.
authors.affiliation: Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. kajacobs@helix.nih.gov.
authors.forename: Arijit
authors.initials: A
authors.lastname: Das
authors.forename: Gangadhar J
authors.initials: GJ
authors.lastname: Sanjayan
authors.forename: Miklós
authors.initials: M
authors.lastname: Kecskés
authors.forename: Lena
authors.initials: L
authors.lastname: Yoo
authors.forename: Zhan-Guo
authors.initials: ZG
authors.lastname: Gao
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson