Polyamidoamine (PAMAM) dendrimer conjugates of clickable agonists of the A3 adenosine receptor and coactivation of the P2Y14 receptor by a tethered nucleotide

Article abstract of DOI:10.1021/bc900473v

We previously synthesized a series of potent and selective A₃ adenosine receptor (AR) agonists (North-methanocarba nucleoside 5′-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed click chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A₃AR activation was preserved in these multivalent conjugates, which bound with apparent Ki of 0.1-0.3 nM. They were substituted with nucleoside moieties solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amidelinked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A₃AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A₃ and P2Y₁₄ receptors (via amide-linked uridine- 5′-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.

Full text of DOI:10.1021/bc900473v

372
Bioconjugate Chem. 2010, 21, 372–384
Polyamidoamine (PAMAM) Dendrimer Conjugates of “Clickable” Agonists
of the A3 Adenosine Receptor and Coactivation of the P2Y14 Receptor by a
Tethered Nucleotide
Dilip K. Tosh,† Lena S. Yoo,† Moshe Chinn,† Kunlun Hong,‡ S. Michael Kilbey II,‡ Matthew O. Barrett,§
Ingrid P. Fricks,§ T. Kendall Harden,§ Zhan-Guo Gao,† and Kenneth A. Jacobson*,†
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland 20892, Center for Nanophase Materials Sciences, Oak Ridge
National Laboratory, Oak Ridge, Tennessee 37831, and Department of Pharmacology, University of North Carolina School of
Medicine, Chapel Hill, North Carolina 27599. Received October 29, 2009; Revised Manuscript Received December 9, 2009
We previously synthesized a series of potent and selective A3 adenosine receptor (AR) agonists (North-methanocarba
nucleoside 5′-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal
alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed “click” chemistry to azide-derivatized G4 (fourth-generation)
PAMAM dendrimers to form triazoles. A3AR activation was preserved in these multivalent conjugates, which
bound with apparent Ki of 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination
with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-
linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A3AR affinity. We
prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation
of multiple ligands. A bifunctional conjugate activated both A3 and P2Y14 receptors (via amide-linked uridine-
5′-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first
example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation
of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate
might be useful in disease treatment.
monomer agonist derivatives, and these multivalent conju-
gates are intended to bridge multiple binding sites present
in GPCR dimers and other higher-order receptor aggregates.
In general, we might expect the stability of the ligand in
biological systems and its pharmacokinetic and pharmaco-
dynamic properties to be enhanced in the conjugates in
comparison to the monomeric ligands.
INTRODUCTION
Poly(amidoamine) (PAMAM)1 dendrimers are treelike poly-
mers that have wide application in drug delivery in vivo (1, 2).
We recently reported the first example of using a PAMAM
dendrimeric scaffold for the polyvalent, nanoscale presenta-
tion of nucleosides and nucleotides to selectively activate
adenosine receptors (ARs) and P2Y receptors to induce
intracellular signal transduction (3-5). Both of these receptor
families are cell-surface G protein-coupled receptors (GPCRs).
The dendrimer carriers greatly multiply the size of the
The A3AR is found at various levels of expression in
neutrophils, eosinophils, cardiac myocytes, skeletal muscle,
astrocytes, neurons, and other cell types (6) and has distinct
effects on cell survival and response to stress. The A3AR
agonist N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine
(IB-MECA, CF101) 1 (Chart 1) and its 2-chloro derivative
deregulate the NFκB pathway and are currently in clinical
trials for the treatment of autoimmune inflammatory diseases
and cancer, respectively (7-11). Anti-ischemic effects of
A3AR agonists also suggest their use in protection of the
heart, lung, nervous system, and skeletal muscle (6, 12, 13).
We previously synthesized a series of North (N)-metha-
nocarba nucleoside 5′-uronamides as A3AR agonists of high
affinity and selectivity (14). These selective agonists, includ-
ing the anti-ischemic nucleoside MRS3558 2, contained a
rigid bicyclo[3.1.0]hexane (methanocarba) ring system of the
(N) conformation in place of the freely twisting ribose
moiety (9, 11, 12). These agonists also contained an
optimized N6-(3-chlorobenzyl) group, which enhanced A3AR
selectivity in humans, mice, rats, and other species (14).
Recently, we extended this series to include dialkyne groups
on an extended adenine C2 substituent, e.g., 3a (21). We
expected the proximal alkyne to promote receptor recognition
and the distal alkyne to serve as a site for coupling to reporter
groups while maintaining receptor recognition. Thus, we have
selected a region on the nucleoside for attachment that is
* Corresponding author. kajacobs@helix.nih.gov, Bldg. 8A, Rm.
B1A-19, NIH, National Institute of Diabetes and Digestive and Kidney
Diseases, Bethesda, MD 20892-0810, Tel. 301-496-9024, Fax 301-
480-8422.
† National Institutes of Health.
‡ Oak Ridge National Laboratory.
§ University of North Carolina School of Medicine.
1Abbreviations: AR, adenosine receptor; DMSO, dimethylsulfoxide;
EDC, N-ethyl-N′-dimethylaminopropylcarbodiimide; GPCR, G protein-
coupled receptor; IBMX, 3-isobutyl-1-methylxanthine; AR, adenosine
receptor; cAMP, adenosine 3′,5′-cyclic phosphate; CHO, Chinese
hamster ovary; IB-MECA, N6-(3-iodobenzyl)-5′-N-methylcarboxami-
doadenosine; DMEM, Dulbecco’s modified Eagle’s medium; EDTA,
ethylenediaminetetraacetic acid; GPCR, G protein-coupled receptor;
HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate methanaminium; HEK, human embryonic kidney;
I-AB-MECA, 2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcar-
boxamidoadenosine; MS, mass spectrometry; NECA, 5′-N-ethylcar-
boxamidoadenosine; NMR, nuclear magnetic resonance; DIPEA,
diisopropylethylamine; DMF, N,N-dimethylformamide; HEPES, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; PAMAM, polyami-
doamine; PLC, phospholipase C; UDPG, uridine-5′-diphosphoglucose;
UDPGA, uridine-5′-diphosphoglucuronic acid.
10.1021/bc900473v  2010 American Chemical Society
Published on Web 02/01/2010

PAMAM Dendrimer Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 373
Chart 1. Structures of Adenosine A3AR Agonist Monomers (1-3a) and the Design of Multivalent Dendrimer Conjugates in the (N)-
Methanocarba Series, Represented by the Triazole Derivative of General Formula 3ba
a Compound 3a was reported in ref 21.
distal from the pharmacophore and is predicted by modeling
to extend into the extracellular region of the A3AR (15).
Here, we coupled the distal alkyne of an A3AR agonist
containing a 2-octadiyne group to azido-derivatized G4 (fourth-
generation) PAMAM dendrimer carriers with Cu(I)-catalyzed
alkyne-azide cycloaddition, a type of click chemistry (16-18),
to form triazole derivatives, as in conjugate 3b, and determined
the receptor-binding affinity and selectivity. This variation of
click chemistry was used previously for coupling biologically
active molecules to polymers (19). In our previous studies of
dendrimer-bound ligands for GPCRs, the small molecular
ligands were amide-linked to the carrier (3-5). For this study,
we compared the amide linkages to PAMAM dendrimers with
the use of azido groups for conjugation of alkyne derivatives.
We also included PAMAM dendrimers with mixed peripheral
azido and amino groups in this study. The incorporation of
azides for click chemistry along with the conventional terminal
amino/carboxylic groups of the PAMAM dendrimers for amide
coupling allowed the facile conjugation of ligands for two
different GPCRs (Figure 1). We present the first example of
coactivation of two GPCRs by a hybrid PAMAM dendrimer
conjugate. The anti-inflammatory A3AR and the P2Y14 receptor,
which is activated by endogenous uridine-5′-diphosphoglucose
(UDPG) in the neuroimmune system (20), serve to illustrate
this approach.
3500, flat width 18 mm) from Spectrum Laboratories (Rancho
Dominguez, CA). Synthesis of compound 3a is described
elsewhere (21).
Nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker DRX-600 spectrometer with D2O as a solvent. The
chemical shifts are expressed as relative ppm from HOD (4.80).
We performed electrospray ionization mass spectrometry (MS)
and matrix-assisted laser desorption ionization (MALDI) time-
of-flight MS experiments on a Waters LCT Premier mass
spectrometer at the Mass Spectrometry Facility, National
Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK), NIH. We analyzed the azido dendrimer precursors
on a Varian 500 MHz NMR and on a Bruker Vertex 70 FT-IR.
Galbraith Laboratories (Knoxville, TN) performed the elemental
analysis.
G4 PAMAM, Conjugated with 6-Heptynoic acid (15). We
added freshly prepared aqueous sodium ascorbate (1 M, 42 µL,
42.1 µmol) to a mixture of G4 PAMAM dendrimer 13 (6.74
mg, 0.42 µmol) and 6-heptynoic acid (1.33 mg, 10.5 µmol) in
a mixture of t-butanol (0.3 mL) and water (0.3 mL), then added
7.5% aqueous cupric sulfate (70 µL, 20.8 µmol). The reaction
mixture was stirred at room temperature overnight, and the
product was purified by dialysis in water. The mixture was
lyophilized to yield compound 15 (5.45 mg, 68%) as a white,
foamy solid. MALDI-MS: calcd 19 029; found 18 928.
EXPERIMENTAL PROCEDURES
G4 PAMAM, Conjugated with A3AR Agonist 3a and
6-Heptynoic Acid (7). We added freshly prepared aqueous
sodium ascorbate (1 M, 31 µL, 31 µmol) to a mixture of
compound 15 (5.45 mg, 0.28 µmol) and compound 3a (4.13
mg, 7.74 µmol) in a mixture of t-butanol (0.3 mL) and water
(0.3 mL), then added 7.5% aqueous cupric sulfate (51 µL, 14.3
µmol). The reaction mixture was stirred at room temperature
overnight, and the product was purified by dialysis in water.
The mixture was lyophilized to yield compound 7 (5.8 mg, 60%)
as a white, foamy solid. 1H NMR (DMSO-d6, 600 MHz) δ 8.51
(s), 8.14 (s), 7.61 (s), 7.31-7.41 (m), 5.46 (s), 5.68-5.95 (m),
Chemical Synthesis. Materials and Methods. All reactions
were carried out under a nitrogen atmosphere. We purchased
N-ethyl-N′-dimethylaminopropylcarbodiimide hydrochloride
(EDC.HCl), UDPG acid trisodium salt (UDPGA), UDPG, G2.5
PAMAM (10 wt % solution in methanol), G3 PAMAM (20 wt
% solution in methanol), PAMAM dendrimers (G4, G5.5, and
G6, 5 wt % solution in methanol) with an ethylenediamine core
from Sigma-Aldrich (St. Louis, MO). All other reagents and
solvents, except those indicated, came from Sigma-Aldrich. We
purchased dialysis membranes (Spectra/Pore Membrane, MWCO

374 Bioconjugate Chem., Vol. 21, No. 2, 2010
Tosh et al.
Figure 1. Design of a bifunctional PAMAM dendrimer conjugate for the coactivation of two different GPCRs; shown here are agonists of the
A3AR and the P2Y14 receptor. Both receptors are distributed in the immune system and may occur on the same cell (27). The stoichiometry of
substitution of the dendrimer is not indicated.
3.86 (s), 2.66 (s), 2.21 (s), 1.8 (s), 1.61 (bs), 1.11-1.29 (m).
MALDI-MS: calcd 33 852; found 34 088.
NMR (DMSO-d6, 600 MHz) δ 8.11 (s), 7.85-8.01 (m), 7.58
(s), 7.36 (s), 7.18-7.32 (m), 5.06-5.21 (m), 4.88-4.93 (m),
4.65 (s), 4.59 (s), 3.86-3.91 (m), 3.07 (s), 2.65 (s), 2.39 (s),
2.21 (s), 1.85 (s), 1.74 (br s), 1.59 (br s), 1.21-1.33 (m), 1.03
(br s). MALDI-MS: calcd 8370; found 8369.
G3 PAMAM, Conjugated with A3AR Agonist 22 (11). We
followed the procedure used for compound 17 to synthesize
compound 11 (71%) from dendrimer 21 and compound 22.1H
NMR (DMSO-d6, 600 MHz) δ 8.88 (s), 8.11 (s), 7.73-8.05
(m), 7.58 (s), 7.32 (s), 7.13-7.28 (m), 5.43 (s), 5.03-5.21 (br
s), 4.87-4.96 (s), 4.85 (br s), 4.67 (s), 4.61 (s), 3.89 (s), 3.06
(s), 2.82 (s), 2.65 (s), 2.34-2.46 (m), 2.19 (s), 1.83 (s),
1.69-1.78 (m), 1.61 (s), 1.29 (s). MALDI-MS: calcd 13 694;
found 13 304.
G4 PAMAM, Conjugated with A3AR Agonist 24 (12). We
added HATU (4.67 mg, 12.28 µmol) to a mixture of compound
24 (6.72 mg, 12.35 µmol) and dendrimer 23 (2.73 mg, 12.28
µmol) in dry DMF (0.5 mL), then added DIPEA (2.15 µL, 12.22
µmol). The reaction mixture was stirred at room temperature
overnight, extensively dialyzed with water, then lyophilized to
yield compound 12 (5.5 mg, 61%).1H NMR (DMSO-d6, 400
MHz) δ 8.46 (br s), 8.10 (s), 7.79-7.93 (m), 7.60 (s), 7.27-7.36
(m), 5.45 (s), 4.67-4.93 (m), 3.87 (s), 3.08 (s), 2.86 (s), 2.66
(s), 2.41 (s), 2.19 (s), 1.78 (s), 1.61 (s), 1.23 (t, J ) 24 Hz),
0.80-0.89 (m). MALDI-MS: calcd 47 495; found 47 985.
Imidazole-1-Sulfonyl Azide Hydrochloride (26). A modifica-
tion of a published procedure was used (22). A suspension of
6.53 g NaN3 (25, 100 mmol, Aldrich, >99.5%) and 100 mL
acetonitrile (Aldrich, 99.8% anhydrous) in a 500 mL round-
bottom flask was cooled in an ice water bath under a flow of
nitrogen. We then added 8.15 mL sulfuryl chloride (100 mmol,
TCI American, >98.0%) in 20 mL anhydrous acetonitrile
dropwise to the ice-cooled suspension. The mixture was stirred
overnight at room temperature, after which we cooled the white
suspension to ∼0 °C with ice water and added 13.03 g imidazole
(191 mmol, TCI America, >98.0%). We warmed the resulting
G4 PAMAM, Conjugated with A3AR Agonist 3a (6). We
followed the procedure used for compound 7 to synthesize
compound 6 (69%) from 3a.1H NMR (DMSO-d6, 400 MHz) δ
8.50 (bs), 8.15 (bs), 7.60 (s), 7.29-7.59 (m), 5.44 (s), 4.67-5.05
(m), 4.16 (s), 3.86 (s), 2.78 (s), 2.67 (s), 2.08-2.23 (m), 1.82
(s), 1.61 (s), 0.98-1.30 (m). MALDI-MS: calcd 49 991; found
50 844.
G4 PAMAM, Conjugated with UDPGA (17). We added
EDC.HCl (12.5 mg, 65.2 µmol) to a solution of compound 5
(15.3 mg, 1.01 µmol) and UDPGA trisodium salt 16 (22.9 mg,
35.43 µmol) in dimethylsulfoxide (DMSO), then added 0.1 M
2-(N-morpholino)ethanesulfonic acid buffer (140 µL). We used
0.1 M HCl to adjust the pH of the reaction mixture to 4.5-5.0.
The reaction mixture was stirred at room temperature overnight,
and the product was purified by extensive dialysis followed by
1
lyophilization to yield compound 17 (22 mg, 64%). H NMR
(DMSO-d6, 400 MHz) δ 8.19 (s), 7.86-8.14 (m), 6.55 (m),
5.80 (s), 5.67 (s), 5.38-5.47 (m), 4.07 (s), 3.97 (s), 3.63 (br s),
3.25 (s), 3.12 (s), 2.91 (s), 2.69 (s), 2.24 (s). MALDI-MS: calcd
33 060; found 33 036.
G4 PAMAM, Conjugated with UDPGA and A3AR Agonist
3a (8). We followed the procedure used for compound 7 to
synthesize compound 8 (66%) from compound 17. MALDI-
MS: calcd 50 092; found 50 610.
G2.5 PAMAM, Conjugated with A3AR Agonist 19 (9). We
followed the procedure used for compound 17 to synthesize
compound 9 (54%) from dendrimer 18 and compound 19.1H
NMR (D2O, 600 MHz) δ 7.98 (br s), 6.88 (br s), 4.56 (br s),
3.85 (br s), 3.55 (br s), 3.45 (br s), 3.16-3.36 (m), 3.16 (br s),
3.08 (br s), 2.56-2.98 (m), 2.54 (s), 2.24-2.48 (m), 1.92 (br
s), 1.68 (br s). MALDI-MS: calcd. 10 598; found 10 783.
G2.5 PAMAM, Conjugated with A3AR Agonist 20 (10). We
followed the procedure used for compound 17 to synthesize
compound 10 (67%) from dendrimer 18 and compound 20.1H

PAMAM Dendrimer Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 375
0.43 g imidazole-1-sulfonyl azide hydrochloride (26, 2.05 mmol)
to the reaction mixture, which was stirred at room temperature
overnight. We then dialyzed the mixture against deionized water
for 7 d, changing the water every 2 h during the first 2 d and
every 4 h thereafter. After lyophilization, we collected 0.45 g
partially azide-derivatized G4 dendrimer. Elemental analysis of
the partially azide-derivatized G4 PAMAM found C, 49.91; H,
7.55; N, 28.07; O, 13.12. We compared results of the element
analysis with the composition of the G4 PAMAM dendrimer
and found that 47.2% of the primary amines were replaced with
azides, corresponding to ∼31 amines per dendrimer in 64
terminal positions.
Biological Methods: P2Y14 Receptor. Materials. We pur-
chased 3-isobutyl-1-methyl-xanthine from Sigma-Aldrich and
[3H]adenine from American Radiolabeled Chemicals (St. Louis,
MO). We obtained all cell culture medium and serum from
Gibco (Invitrogen, Carlsbad, CA).
Cell Culture. We generated C6 glioma cells stably express-
ing the human P2Y14-R (P2Y14-C6 cells) as previously
described by Fricks et al. (23). We cultured P2Y14-C6 cells
in Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 5% fetal bovine serum, 1% Geneticin (Gibco),
and 1% antibiotic-antimocotic (Gibco) at 37 °C in a 5%
CO2 environment.
Figure 2. 1H NMR spectra of G4-N3 (5, top), G4-NH2 from D2O (23,
middle). Schematic indication of assignments of the methylene groups
(bottom).
Accumulation of Adenosine 3′,5′-Cyclic Phosphate (cAMP). We
grew P2Y14-C6 cells in 24-well plates and incubated them with
1 µCi [3H]adenine/well in serum-free DMEM for at least 2 h
prior to assay. We initiated the assays by adding HEPES-
buffered, serum-free DMEM containing 200 µM 3-isobutyl-1-
methyl-xanthine and 30 µM forskolin, with or without drugs,
and incubation continued for 15 min at 37 °C. We terminated
incubations by aspirating the medium and adding 450 µL ice-
cold 5% trichloroacetic acid. We isolated [3H]cAMP by
sequential Dowex and alumina chromatography and quantified
it by liquid scintillation counting as previously described by
Harden et al. (24).
Data Analysis. We measured the concentrations of the
dendrimer-ligand complexes by the concentration of the
dendrimer, not the attached ligand. We determined EC50 and
apparent Ki values with Prism software (GraphPad, San Diego,
CA); they are presented as mean ( SE. The number of
nucleoside moieties on a given dendrimer that are accessible to
receptors is uncertain; the data are therefore shown as apparent
Ki values. We repeated all experiments at least three times.
Biological Methods: ARs. Receptor-Binding and Functional
Assays. We obtained [3H]adenosine-5′-N-methyluronamide ([3H]-
NECA, 42.6 Ci/mmol) from PerkinElmer Life and Analytical
Science (Boston, MA). We purchased [3H](2-[p-(2-carboxyeth-
yl)phenyl-ethylamino]-5′-N-ethylcarboxamido-adenosine) ([3H]-
CGS21680, 40.5 Ci/mmol) and [125I]N6-(4-amino-3-iodobenzyl)-
adenosine-5′-N-methyluronamide ([125I]I-AB-MECA, 2200 Ci/
mmol) from PerkinElmer. We prepared test compounds as 5 mM
stock solutions in DMSO and stored them frozen at -20 °C.
Cell Culture and Membrane Preparation. We cultured
Chinese hamster ovary (CHO) cells stably expressing the
recombinant hA1 and hA3 and human embryonic kidney (HEK)
293 cells stably expressing the hA2AAR in DMEM and F12
(1:1) supplemented with 10% fetal bovine serum, 100 units/
mL penicillin, 100 µg/mL streptomycin, and 2 µmol/mL
glutamine. In addition, we added 800 µg/mL Geneticin to the
A2A media and 500 µg/mL hygromycin to the A1 and A3 media.
After harvesting the cells, we homogenized them with an electric
homogenizer for 10 s, pipetted them into 1 mL vials, and then
stored them at -80 °C until we conducted the binding
experiments. We measured the protein concentration with a BCA
Protein Assay Kit from Pierce Biotechnology (Rockford, IL)
(25).
white slurry to room temperature, then stirred it for 3 h. We
then diluted it with ethyl acetate (200 mL) and separated and
discarded the aqueous layer. We washed the organic layer twice
with deionized water (2 × 100 mL), then twice with saturated
aqueous NaHCO3 (2 × 100 mL). We then dried it over
anhydrous MgSO4 overnight and filtered it. We added an
ethanolic HCl solution (196 mmol) dropwise to the filtrate while
stirring at ice-water temperature. After we added ∼3 mL, a
large amount of white solid appeared. We kept the mixture in
an ice bath for 15 min, then at room temperature for 30 min,
after which we filtered it. We washed the filter cake with ethyl
acetate (3 × 100 mL) to give 26 as a colorless solid (8.65 g,
49.9%), mp 99-101 °C. IR.(KBr) 2173, 1383, and 1160 cm-1
.
1H NMR (D2O, 500 MHz) δ 7.61 (dd), 8.02 (dd), 9.39 (dd);
13C NMR (D2O, 125 MHz) δ ) 120.0, 123.5, 137.6. Element
Analysis: calcd C, 17.19; H, 1.92; N, 33.41. Found: C, 17.38;
H, 1.98; N, 32.97.
Synthesis of Fully Azido-DeriVatized PAMAM (G4) (13). We
added anhydrous methanol (10 mL, Aldrich, >99.8%) and 18.15
g G4 PAMAM dendrimer (23, Dendritech, Midland, MI, 14.93
w/w % in methanol, [NH2] ) 12.2 mmol) to a 250 mL round-
bottom flask. We then added 3.12 g K2CO3 (Aldrich, >99.0%,
22.6 mmol) and 31.4 mg CuSO4 ·5H2O (Aldrich, 98.0%, 0.126
mmol), causing the solution to take on a pale bluish color. We
next added 2.73 g imidazole-1-sulfonyl azide hydrochloride (26,
13.1 mmol) to the reaction mixture, and it was stirred at room
temperature overnight. The mixture was then dialyzed against
deionized water for 7 d, during which we changed the water
every 2 h for the first 2 d and thereafter every 4 h. After
lyophilization, we collected 2.51 g azide-derivatized G4 den-
drimer. The proton NMR is shown in Figure 2. Element analysis:
PAMAM-G4: calcd C, 52.81; H, 8.48; N, 24.70, O, 14.01. Fully
azide-derivatized G4 PAMAM: calcd C, 47.03; H, 7.15; N,
33.31, O, 12.49; found C, 49.91; H, 7.55; N, 28.07; O, 13.12.
The element analyses indicated that 98.0% of primary amines
were replaced with azides.
Synthesis of Partially Azido-DeriVatized PAMAM (G4) (5).
We added anhydrous methanol (5 mL, Aldrich, >99.8%) and
5.13 g G4 PAMAM dendrimer (23, Dendritech, 14.93 w/w %
in methanol, [NH2] ) 3.45 mmol) to a 100 mL round-bottom
flask. We then added 0.803 g K2CO3 (Aldrich, >99.0%, 5.81
mmol) and 17.2 mg CuSO4 ·5H2O (Aldrich, 98.0%, 0.069
mmol); the solution took on a pale bluish color. Next, we added

376 Bioconjugate Chem., Vol. 21, No. 2, 2010
Tosh et al.
Table 1. Potency of a Series of Nucleoside-Dendrimer Conjugates at Three Subtypes of Human ARs

PAMAM Dendrimer Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 377
Table 1. Continued
a NA: not active in inhibition of radioligand binding (<10%) at 1 µM. All experiments were done on CHO or HEK293 (A2A only) cells stably
expressing one of four subtypes of human ARs. The binding affinity for A1, A2A, and A3ARs was expressed as Ki values (n ) 3-5) and was determined
by using agonist radioligands ([3H]NECA, [3H]CGS21680, or [125I]I-AB-MECA, respectively), unless noted. A percentage in parentheses refers to
inhibition of radioligand binding at the indicated concentration. The concentrations of the dendrimer-ligand complexes were measured by the
concentration of the dendrimer, not the ligand. Therefore, all binding Ki values of dendrimers are expressed as apparent Ki values. b Affinity values from
Tosh et al. (15, 21) and Melman et al. (14). c 2, MRS3558; 3a, MRS5221; 4a, MRS5233; 6, MRS5246; 7, MRS5247; 8, MRS5259; 11, MRS5216.
d Coumpound 5 is the dendrimer precursor of 8 and is similar to the dendrimer precursor of 6 and 7.
Binding Assays. Each tube in the binding assay contained 50
µL of increasing concentrations of the test ligand in Tris-HCl
buffer (50 mM, pH 7.5) containing 10 mM MgCl2, 50 µL of
the appropriate agonist radioligand, and 100 µL membrane
suspension, added sequentially. For the A1AR (20 µg protein/
tube), we used the radioligand [3H]NECA (∼3.5 nM, precise
final concentration is calculated for each experiment). For the
A2AAR (20 µg/tube), we used the radioligand [3H]CGS21680
(∼10 nM). For the A3AR (21 µg/tube), we used the radioligand
(Scheme 2), or the conventional all amino/carboxylate groups
(Scheme 3). The synthetic methods used to partially or fully
substitute terminal amino groups of a G4 PAMAM dendrimer
with azides are shown in Scheme 4 (22). Schemes 1-4 and
Table 1 indicate the approximate, average number of attached
moieties on each dendrimer, which is actually a statistical
distribution of substitution.
We recently introduced terminal alkynyl groups at the C2
position of (N)-methanocarba nucleoside derivatives to serve
as sites on the A3AR agonists for cross-linking by click
chemistry (21). Thus, a dialkyne was introduced at the C2
position in 2, and further reaction with azides generally occurred
exclusively at the distal alkyne. Prior to coupling the nucleosides
to dendrimer carriers, we explored in detail the structure-activity
relationship at the distal region of an alkyne-functionalized chain
at the 2 position and the resultant click products (21).
The synthetic route to the multivalent dendrimer conjugates
of nucleosides for activation of the A3AR that contain 5′-N-
methyluronamide (N)-methanocarba 2-alkynyl triazoles 6 and
7 is shown in Scheme 1. Click chemistry efficiently coupled
the A3AR agonist 3a to a PAMAM dendrimer fully substituted
with peripheral azido groups. We used two versions of this
azido-functionalized dendrimeric scaffold: 15, containing extra
appended carboxylic acid groups for increasing water solubility,
and 13, lacking these groups. We added the carboxylic acid
moieties to the azido dendrimer 13 by partial reaction of the
peripheral azido groups with hexynoic acid 14. Thus, we
prepared two nucleoside-containing dendrimer derivatives 6 and
7. Derivative 6 was fully substituted with the adenosine
analogue, with 64 nucleoside moieties per dendrimer molecule.
Derivative 7 was partially (28 out of 64) substituted with the
adenosine analogue and contained 24 carboxylic acid moieties.
Twelve azido groups per molecule of 7 remained unreacted.
[
125I]I-AB-MECA (∼0.3 nM). We determined nonspecific
binding with a final concentration of 10 µM unlabeled NECA
diluted with the buffer. We incubated the mixtures at 25 °C for
60 min in a shaking water bath. We terminated binding reactions
by filtration through Brandel GF/B filters under a reduced
pressure with a M-24 cell harvester (Brandel, Gaithersburg,
MD). We washed filters three times with 3 mL of 50 mM ice-
cold Tris-HCl buffer (pH 7.5). We then placed filters for A1
and A2AAR binding in scintillation vials containing 5 mL
Hydrofluor scintillation buffer and counted with a PerkinElmer
Liquid Scintillation Analyzer (Tri-Carb 2810TR). We counted
filters for A3AR binding with a Packard Cobra II γ-counter
(PerkinElmer). We determined the Ki values with GraphPad
Prism for all assays.
Accumulation of cAMP. To determine cAMP production (26),
we incubated protein kinase A with [3H]cAMP (2 nM) in
K2HPO4/EDTA buffer (K2HPO4, 150 mM; EDTA, 10 mM), 20
µL cell lysate, and 30 µL 0.1 M HCl or 50 µL cAMP solution
(0-16 pmol/200 µL for standard curve). We separated bound
radioactivity by rapid filtration through Whatman GF/C filters
and washed the filters once with cold buffer. We measured
bound radioactivity with liquid scintillation spectrometry.
RESULTS
In the next phase of our study, we combined different GPCR
ligands, i.e., the A3AR and another receptor, on the same
dendrimer carrier. We selected the P2Y14 receptor, which is
activated by naturally occurring uridine-5′-diphosphoglucose
(UDPG), for this purpose, because, like the A3AR, it occurs on
Chemical Synthesis. We prepared various PAMAM den-
drimer derivatives of nucleosides and nucleotides (Table 1,
6-12) to act as multivalent ligands of GPCRs. The peripheral
groups of the precursor dendrimers contained either all azido
groups (Scheme 1), mixtures of azido and amino groups

378 Bioconjugate Chem., Vol. 21, No. 2, 2010
Tosh et al.
Scheme 1. Use of Click Chemisry to Synthesize PAMAM Dendrimer Derivatives 6 and 7, Containing a Functionalized 2-Alkynyl A3AR
Agonista
a An optional water-solubilizing group was added in 7, in the form of an aliphatic carboxylic acid.
immune cells and because we recently introduced high-potency
dendrimer conjugates for this subtype (5, 27). Each ligand would
provide selectivity for a particular GPCR subtype, and the
overall selectivity of the conjugate would be a function of the
union of the sets of selectivity of the attached nucleosides. We
sequentially coupled alkyne and carboxylic acid derivatized
ligands for the two receptors to a novel multifunctional PAMAM
dendrimer. Thus, alkynes reacted exclusively with azido terminal
groups and carboxylic acids with terminal amino groups on the
same dendrimer to randomly (but in a defined ratio) produce a
multivalent, binary conjugate.
We proceeded to synthesize a multivalent, binary conjugate
of a mixed azido/amino dendrimer 5, as shown in Scheme 2.
In the dendrimer precursor 5, we determined, through NMR
(28) (Figure 2) and elemental analysis, that the ratio of amines
to azides was ∼31:33. Therefore, of the 64 terminal groups on
each G4 dendrimer, ∼33 would be available for cycloaddition,
and the remaining terminal groups would be available for amide
formation. We first coupled the amino groups of 5 to the
functionalized, P2Y14-selective nucleotide UDPGA 16, which
we previously showed to be a suitable functionalized congener
for chain attachment without loss of receptor activation (5). We
then coupled the azido groups of 17 to the functionalized
nucleoside 3a, which is a suitable functionalized congener for
chain attachment without loss of binding affinity at the A3AR
(21). The product of this second coupling reaction was the mixed
dendrimer 8, in which the average ratio of UDGPA and
nucleoside moieties was ∼31:33 per G4 dendrimer. Thus,
conjugate 8 would be expected to potently activate both A3 and
P2Y14 receptors and perhaps maintain selectivity over other ARs
and P2Y receptors.
To compare alkyne-azide click chemistry with the more
standard amide formation as a means of linking A3AR-selective
agonists to PAMAM dendrimers, we prepared various dendrimer
derivatives starting with our previously reported amino and
carboxylic-functionalized congeners 19, 20, 22, and 24 (14, 15).
The reaction sequences are shown in Scheme 3. We included a
variety of dendrimer sizes and sites of conjugation on the
nucleoside.
The synthesis of the PAMAM dendrimer intermediate 5
containing equiproportioned peripheral azido and amino groups
for conjugation of multiple ligands is shown in Scheme 4. We
used the reagent imidazole-1-sulfonyl azide hydrochloride 26
for the conversion of peripheral amino groups to azido groups
(22). We prepared a fully azido-derivatized G4 dendrimer 13
similarly and used it to synthesize conjugates 6 and 7.
Quantification of Pharmacological Activity. We tested the
dendrimer nucleoside conjugates similarly in binding assays at
three hAR subtypes; we used standard radioligands (29-31)
and membrane preparations from CHO cells (A1 and A3) or
HEK293 cells (A2A) stably expressing a hAR subtype (Table
1) (32, 33). We used the previously reported agonist molecular
probes of the A3AR (1, 2, 3a, and 4) for comparison in the
biological assays (15, 21).
We included the parent dendrimer 5, which contained both
amino and azido groups, as a control; it was inactive in AR

PAMAM Dendrimer Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 379
Scheme 2. Synthesis of Two PAMAM Dendrimer Derivatives Containing a Functionalized Agonist of the P2Y14 Receptor Derived by
Amide Linkage of UDPGA 16a
a Conjugate 17 contains only the P2Y14 receptor agonist; conjugate 8 contains an A3AR agonist linked through a click reaction of its 2-alkynyl
functionalized chain. The dendrimer precursor 5 contained a random distribution of peripheral amino and azido groups in a ratio of ∼33:31.
binding at 1 µM. We considered compound 4a to be a model
compound for the attachment of the 2-octadiyne derivative 3a
to azido-bearing PAMAM dendrimers by click chemistry. As
reported previously, the triazole-containing monomer 4a main-
tained both high potency and selectivity (at least 100-fold) for
the A3AR (21).
Both 6 and 7 were readily soluble in DMSO at a concentration
of 1 mM, but when we diluted the DMSO stock solutions with
phosphate-buffered saline for a final calculated concentration
of 30 µM, we observed some cloudiness in 6 but not in 7.
Dendrimer derivative 8 contained an additional covalently
bound GPCR ligand, a nucleotide designed to activate the P2Y14
receptor. We found that combining strategically functionalized
nucleosides and nucleotides on the same dendrimer carriersfor
example, to activate both adenosine and P2Y receptorssprovided
high-affinity recognition at both GPCRs. However, the Ki value
of 8 in binding to the A3AR (39.5 nM) indicated that it was
considerably less potent than conjugates 6 and 7, which
contained only the A3AR agonist ligand. The A3AR selectivity
of 8 was only 2-fold in comparison to the A1AR and 15-fold in
comparison to the A2AAR. Thus, it was much less A3AR
selective than were 6 and 7.
The multivalent dendrimer derivatives 6 and 7 obtained from
3a through click conjugation were considerably more potent
than 4a in binding assays at the A1, A2A, and A3 ARs. The
affinity of 6 and 7 in binding to the A3AR was particularly high,
with Ki values of 0.1-0.3 nM (Figure 3). The fully substituted
dendrimer 6 was only 2.3-fold more potent at the A3AR than
the less heavily loaded 7, which was substituted with nucleosides
at 44% of the theoretically available terminal positions. The
selectivity of these dendrimer conjugates for the A3AR in
comparison to the A1AR was still present (161- and 110-fold
for 6 and 7, respectively) but diminished in comparison to the
monomer 4a (>500-fold). However, the A3AR selectivity of 6
(230-fold) and 7 (180-fold) in comparison to the A2AAR was
similar to that of 4a. Thus, the presence of the carboxylic acid
chain in 7 had only minor effects on the observed AR affinity
and selectivity, but it increased the observed aqueous solubility.
A functional assay of adenylate cyclase inhibition carried out
in C6 cells expressing the human P2Y14 receptor demonstrated
that potency at this receptor was enhanced in the dendrimer
conjugate 8 over that of the monomer, with an EC50 value of
2.24 ( 0.80 nM (Figure 4). Both UDPG and UDPGA displayed
potencies in the same assay of 200-300 nM (5). This degree

380 Bioconjugate Chem., Vol. 21, No. 2, 2010
Tosh et al.
Scheme 3. Preparation of Amide-Linked Nucleoside Conjugates of PAMAM Dendrimers
Scheme 4. Synthesis of Azido-Derivatized PAMAM Dendrimers,
Which Served As Precursors for the GPCR Ligand Conjugates
by Reacting with Imidazole-1-Sulfonyl Azide Hydrochloride 26
with selectivity for the P2Y14 receptor. Dendrimers 8 and 17
displayed EC50 values in activation of phospholipase C (PLC)
by the human P2Y2 receptor of 1.39 ( 0.54 µM and 733 (
223 nM (n ) 3), respectively, assayed as described in stably
transfected 1321N1 astrocytoma cells (34). The native agonist
of the P2Y14 receptor, UDPG, also activated the human P2Y2
receptor, with an EC50 value of 10 µM (34).
PAMAM dendrimer conjugates (9-12) of carboxylic- and
amino-functionalized congeners of A3AR-selective agonists
consistently displayed lower affinity in A3AR binding than
did the click-linked dendrimers 6 and 7. The amide-linked
conjugates included derivatives of both terminal carboxylic
acid-functionalized dendrimers 9 and 10 and terminal amine-
functionalized dendrimers 11 and 12, ranging from generation
2.5 to 4. The affinities of these dendrimers at the hA3AR
were in the range 12-42 nM, and the nature of the residual
terminal groups of the dendrimer carrier had little effect. We
did not observe the subnanomolar affinity seen in click-linked
nucleoside-dendrimer conjugates. Thus, the high affinity is
partially the result of the linkers and the coupling chemistry
used, which in 6 and 7 produce a triazole rather than an amide
linkage.
The selectivity of the amide-linked dendrimers in comparison
to the A2AAR was high (x-fold, for compound): >300, 9; .24,
10; 327, 11; 62, 12. However, there was a substantial gain in
affinity at the A1AR over the corresponding monomeric nucleo-
sides. Thus, 10 and 11 lost selectivity for the A3AR in
comparison to the A1AR. Compound 12 was reversed in its
selectivity (6-fold more potent at the A1AR than at the A3AR).
Conversely, we considered compound 4b to be a model
nucleoside derivative for the amide-linked dendrimer derivatives
of potency enhancement at the P2Y14 receptor was consistent
with previous results of conjugation of UDPGA derivatives to
PAMAM carriers (5). Dendrimer 17, which contained only the
ligand of the P2Y14 receptor (i.e., 31 nucleotides bound per G4
dendrimer) and not the A3AR-activating moiety, displayed an
EC50 value at the P2Y14 receptor of 2.41 ( 0.77 nM. Curiously,
nucleotide-containing dendrimers 8 and 17 also interacted with
at least one other subtype of P2Y receptor, but nevertheless

PAMAM Dendrimer Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 381
Figure 4. Activation of the Gi-coupled P2Y14 receptor stably expressed
in C6 cells by the mixed nucleoside/nucleotide G4 dendrimer conjugate
8 (MRS5259) and the nucleotide G4 dendrimer conjugate 17 (MRS2949).
Inhibition of forskolin (FSK)-stimulated cAMP is shown in comparison
to the effects of UDPG (n ) 3).
Table 2. Maximal Efficacy of (N)-Methanocarba Adenosine
Derivatives in a Functional Assay at the A3AR
Figure 3. Inhibition of radioligand binding by dendrimer nucleoside
conjugates in membranes of CHO cells expressing the human A1 (A)
and A3 (C) ARs and HEK-293 cells expressing the human A2AAR (B).
Inhibition curves at three ARs are shown for the dendrimer-nucleoside
conjugates 6 and 7, in which the linkage to an azide-derivatized
dendrimer was formed by click cycloaddition, for the mixed nucleoside/
nucleotide conjugate 8 and for the amide-linked nucleoside conjugate
12. Both 6 and 7 were highly selective for the hA3AR in comparison
to the hA1 and hA2AARs.
% inhibition of
concentration
cAMP accumulationa
at the hA3AR
compound
(µM)
IB-MECA
Cl-IB-MECA
NECA
3a
4ab
5
1.0
1.0
1.0
10
99 ( 6
97
100
60.2 ( 17.0
93.6 ( 17.7
1.7 ( 1.4
98.7 ( 14.3
91.4 ( 20.2
111 ( 20
88.2 ( 15.1
91.3 ( 12.3
101 ( 10
92.1 ( 21.2
10
1.0
0.1
0.1
0.1
1.0
1.0
1.0
0.1
6
7
8
9 and 12, and it was clearly A3AR selective. Compound 4c (63-
fold selective for the A3AR versus the A1AR) was a model for
N6-functionalized dendrimer conjugates 10 and 11, although it
was a higher homologue with 4 methylenes. Thus, both model
compounds 4b and 4c were more potent and selective in binding
to the A3AR than were the corresponding amide-linked
dendrimers.
We determined binding at the rat A3AR expressed in CHO
cells for selected compounds. The Ki values of the N6-chain
derivatized, amide-linked dendrimers 10 and 11 were 62.0 (
36.5 and 4.94 ( 1.96 nM (n ) 3), respectively. The triazole-
linked adenosine conjugate 7 with water-solubilizing groups
displayed a Ki value of 0.08 ( 0.02 nM in binding to the rat
A3AR. Thus, there was a relatively low degree of species-
dependent variation in the observed A3AR affinity (4-fold greater
in rat than human).
We tested the nucleoside conjugates in a functional assay at
the A3AR (inhibition of forskolin-stimulated cAMP production
in A3AR-expressing CHO cells) as shown in Table 2. These
multivalent derivatives displayed A3AR agonist properties with
varying degrees of maximal inhibition of forskolin-stimulated
cAMP production at 0.1-10 µM (concentration selected to be
in >100-fold excess of the Ki value for each). In general, the
relative efficacy was similar to that of NECA (1 µM), taken as
a reference standard. The dendrimer control was inactive. The
EC50 value of the amide-linked adenosine conjugate 11 was 2.93
( 0.37 nM, determined with a full curve in CHO cells stably
9c
10
11
12
a The efficacy at the human A3AR was determined by inhibition of
forskolin-stimulated cyclic AMP production in AR-transfected CHO
cells, as described in the text. Percent inhibition is shown at the
indicated concentration, in comparison to the maximal effect of a full
agonist NECA at 1 µM. Data are expressed as mean ( standard error (n
) 3-5). ND, not determined. b Values from Tosh et al. (21). c EC50
values were determined with full curves for compounds 7 (0.12 ( 0.03
nM), 8 (1.36 nM), and 11 (2.93 ( 0.37 nM).
expressing the human A3AR (Figure 5). Conjugate 11 was a
full agonist in the A3AR-mediated inhibition of cAMP produc-
tion. We also determined EC50 values for cAMP inhibition with
full curves for the triazole-linked adenosine conjugate with
water-solubilizing groups 7 (0.12 nM) and 8 (1.36 nM).
DISCUSSION
We used the Cu(I)-catalyzed alkyne-azide click reaction
for linking functionalized congeners of AR ligands (21, 35)
to other moieties, specifically PAMAM dendrimer derivatives.
In this study, we compared different chemical strategies for
tethering GPCR ligands to macromolecular carriers. Den-
drimer derivatives prepared by click chemistry were particu-

382 Bioconjugate Chem., Vol. 21, No. 2, 2010
Tosh et al.
activated both A3 and P2Y14 receptors, although not to the
exclusion of other ARs.
The bifunctional dendrimer 8 and its nucleotide-dendrimer
precursor 17 both activated one other P2Y receptor examined,
the hP2Y2 receptor, with intermediate potency. UDPG itself
activates the hP2Y2 receptor with an EC50 of 10 µM for
stimulation of PLC in stably transfected 1321N1 cells (34).
Therefore, the potency at both subtypes of P2Y receptors was
much greater in the dendrimer conjugates than in the monomer,
but the desired selectivity for the P2Y14 receptor was maintained.
Thus, both covalently tethered GPCR ligands, which were
previously shown to be suitable functionalized congeners for
coupling to carriers, retained their activity as the mixed
dendrimer conjugate 8. This conjugate had reduced A3AR
affinity in the presence of tethered nucleotide moieties but not
reduced P2Y14 potency due to the tethered nucleoside moieties.
This is the first example of targeting two different GPCRs with
the same dendrimer conjugate, which is intended for activation
of heteromeric aggregates of GPCRs.
Many examples exist of two small molecules linked with a
spacer group to make binary conjugates for activation of two
different GPCRs (35, 38, 39). Some researchers have systemati-
cally varied the length of the spacer to achieve the correct fit to
the two adjacent binding sites. This multivalent approach was
taken in part because of uncertainty about the geometric
orientation of protomer binding sites in a GPCR dimer. Flexible
dendritic carriers facilitate empirical testing of a variety of spatial
orientations of the same conjugate. This obviates the need to
probe the optimal geometry a priori of a single spacer chain to
achieve simultaneous binding to two sites.
Both A3 and P2Y14 receptors are distributed in the immune
system and appear to occur on the same cell, as was shown for
RBL-2H3 mast cells (27). Further experiments will be needed
to determine whether heteromers composed of A3 and P2Y14
receptors exist and whether this conjugate is capable of bridging
the binding sites on both protomers. This approach of mixing
receptor ligands on the same dendrimer carrier can be extended
to many other combinations of GPCRs.
We know that GPCRs tend not to be evenly or randomly
distributed over the surface of a given cell; we demonstrated
this for the A3AR expressed heterologously in CHO cells (4).
In general, GPCRs may exist as complexes with other receptors
and associated proteins. The receptors to which a given GPCR
pairs or forms a higher-order aggregate can dramatically affect
its pharmacology, and the binding of agonist to both protomers
does not have equivalent pharmacological effects (40). We lack
effective ligand tools for separating the effects of one receptor
dimeric combination from another. This approach of mixing
ligands for two different receptors (35, 38, 39, 41) promises to
be a means of achieving selectivity for receptors in a given
tissue, based on association or aggregation of GPCRs, in
comparison to another tissue in which the same receptor may
be alternatively paired. At this early stage, we do not know the
topological requirements for selectively targeting these combi-
nations of receptors, because we are just beginning to detect
their existence. The multivalent binding to other types of cell
surface receptors has been explored through structural modifica-
tion of the carrier polymer (42).
Figure 5. Activation of the Gi-coupled A3AR stably expressed in CHO
cells by 11. The agonist effect measured is inhibition of forskolin-
stimulated cAMP production, in comparison to the maximal effect of
a full agonist NECA at 1 µM. Curve is representative of three
determinations.
larly potent and selective in binding to the A3AR. Multivalent
nucleoside-dendrimer conjugates 6 and 7 showed much
greater A3AR affinity than the corresponding monomer, the
model nucleoside 4a. Furthermore, amide-linked nucleoside
conjugates were considerably weaker than click-linked
conjugates in binding to the A3AR. This illustrates the
dramatic effect that both the multivalency and the intermedi-
ate structures contained within the functionalized chain can
have on interaction with the target receptor (35). A com-
parison with various amide-linked dendrimers showed that
the triazole linkage had a general A3AR affinity-enhancing
effect. Click chemistry was previously used to link small
molecules to dendrimeric structures, but ours is the first
application for studying PAMAM dendrimer derivatives of
small-molecular GPCR ligands. Recently, peptide ligands
were conjugated to dendrimers by click chemistry for
activation of a GPCR (18).
Other studies have focused on PAMAM dendrimers as
reversible drug carriers, for example, to deliver anticancer drugs
that dissociated at the site of action (36). Our approach
derivatizes a small molecule but allows it to remain active while
covalently attached to the carrier (35). The binding sites of the
receptors are accessible from the extracellular medium. Thus,
activation of the desired receptor would not benefit from either
drug release or cellular internalization of the dendrimer. The
flexible dendrimer conjugate is available to spread over the cell
surface and to adopt a conformation that can bridge multiple
binding sites (37).
The efficiency of the click reaction readily allowed complete
substitution in the case of one of the dendrimer derivatives, i.e.,
6. In previous experiences with amide coupling of functionalized
nucleosides, we encountered difficulty in the complete substitu-
tion of amine-terminated dendrimers (3, 4). Click chemistry
proved to be a more efficient coupling technique and allowed
orthogonal reaction control over the coupling chemistry when
two distinct receptor ligands were bound to the same dendrimer
carrier (as in 8). This conjugate was most readily synthesized
by amide coupling of the carboxy-functionalized nucleotide
followed by click coupling of the alkyne-functionalized ad-
enosine derivative.
This study presents the first example of targeting two different
GPCRs with the same dendrimer conjugate, intended for
activation of heteromeric aggregates of GPCRs. Each ligand
provided selectivity for a particular GPCR subtype, and the
overall selectivity of the conjugate was a function of the union
of the sets of selectivity of the attached nucleosides. Specifically,
the dendrimer conjugate 8 contained ligands for both the A3AR
and the P2Y14 receptor. The principle of attachment of UDPG
derivatives to PAMAM dendrimers to enhance potency was
introduced previously (5). This bifunctional dendrimer potently
Multivalent conjugates of GPCR ligands provide quantita-
tively and qualitatively different pharmacological properties than
do monomers. Prosthetic groups for targeting or therapeutic
purposes or reporter groups for diagnostic purposes might be
introduced. Specific combinations of receptor ligands on the
same dendrimer carrier might achieve synergistic effects and
prove useful for disease treatment, such as inflammatory
conditions, to which the current example of adenosine and P2Y
receptor ligands apply. Various combinations may be proposed

PAMAM Dendrimer Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 383
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could be applied to other combinations of dendrimer-bound
GPCR ligands.
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supported by the Intramural Research Program of the NIH,
NIDDK. Research conducted at the Center for Nanophase
Materials Sciences at Oak Ridge National Laboratory is
sponsored by the Scientific User Facilities Division, Office of
Basic Energy Sciences, U.S. Department of Energy.
Supporting Information Available: 1H NMR and mass
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