ResearchNucleoside conjugates of quantum dots for characterization of G protein-coupled receptors : strategies for immobilizing A 2 A adenosine receptor agonists

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-(2carboxyethyl)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 μM). 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. Background Quantum dots (QDs) are crystalline semiconducting nanoparticles that, when properly derivatized, are compatible with biological systems to provide a chemically and photochemically stable fluorescent label [1]. The spectral characteristics are dependent on the particle size, which typically ranges from 2 10 nm, resulting in emission wavelengths in the 500 800 nm range. QDs have been chemically functionalized, leading to specfic interactions with cellular components for the purposes of biological imaging and therapeutics [2]. For example, antibodies have been covalently coupled to QDs for detection of tumors by confocal microscopy or whole body imaging using a near-infrared label [3-7]. In some cases, small molecular fluorescent prosthetic groups were superior to QDs as a mean of labeling cancer-related receptor sites to follow their regulation [8]. G protein-coupled receptors (GPCRs) are important pharmaceutical targets on the cell surface. We have developed a general approach toward functionalization of small molecular ligands of GPCRs that allow them to be conjugated to carriers, coupled to other pharmacophores, or immoblized on polymers without losing the ability to bind to the receptor with high affinity [9]. In fact, the attachment of functionalized congeners to carriers has * Correspondence: kajacobs@helix.nih. gov 1 Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,