Functionalized Congeners of P2Y 1 Receptor Antagonists: 2-Alkynyl ( N )-Methanocarba 2′-Deoxyadenosine 3′,5′-Bisphosphate Analogues and Conjugation to a Polyamidoamine (PAMAM) Dendrimer Carrier

The P2Y1 receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of the P2Y1 receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphates containing extended 2-alkynyl chains was designed and binding affinity at the human (h) P2Y1 receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (Ki 23 nM) and extended amine congener 15 (Ki 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended ε-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y1 receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y1 receptor modeling and ligand docking. Attempted P2Y1 antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to produce a multivalent conjugate exhibiting a desired biological effect, i.e. antithrombotic action

Polyamidoamine (PAMAM) Dendrimer Conjugates of “Clickable” Agonists of the A 3 Adenosine Receptor and Coactivation of the P2Y 14 Receptor by a Tethered Nucleotide

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 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

Role of Branching on the Structure of Polymer Brushes Formed from Comb Copolymers

The distinguishing feature of a polymer brush at equilibrium is the stretched configuration of the chains that results from tethering the polymer chains by one end at the solid-fluid interface. The stretched configuration of the chains and the crowded nature of the interfacial layer is the origin of many of the useful properties of polymer brushes: these layers resist compression and aggregation, effectively dissipate shear stresses, and respond reversibly to changes in their solution environment

Synthesis and Characterization of an ABC Miktoarm Star Terpolymer of Cyclohexadiene, Styrene, and 2-Vinylpyridine

Several research groups have expended great effort toward the preparation of miktoarm (mixed-arm) star copolymers and terpolymers. We describe herein a novel synthesis, using anionicpolymerization,ofapoly(cyclohexadiene)[PCHD]-polystyrene [PS]-poly(2-vinylpyridine) [P2VP] miktoarm star terpolymer. Previously, miktoarm A2B and A3B star polydiene copolymers comprised partially of poly(cyclohexadiene) have been synthesized by Tsoukatos and Hadjichristidis by employing chlorosilane linking techniques. More recently, an ABC star terpolymer of PS, polyisoprene, and PCHD was prepared by Yang and co-workers by using meta-double-diphenylethylene-based linking chemistry

Looped Polymer Brushes Formed by Self-Assembly of Poly(2-vinylpyridine)-Polystyrene-Poly(2-vinylpyridine) Triblock Copolymers at the Solid-Fluid Interface. Kinetics of Preferential Adsorption

The kinetics of assembly of a series of poly(2-vinylpyridine)-polystyrene-poly(2-vinylpyridine) (PVP-b-PS-b-PVP) triblock copolymers from the selective solvent toluene onto a silicon surface has been studied using phase-modulated ellipsometry. The adsorbed amount and thickness have been determined independently as functions of time. Even though the adsorbed amount as a function of time follows the traditional two-step process that is typical of the self-assembly of diblock copolymerssthere is an initial fast adsorption followed by a slow buildup of the layer (brush regime) - the thickness shows an “overshoot” that corresponds to the brush regime. We attribute this phenomenon, not observed in the self-assembly of amphiphilic diblock copolymers, to having both ends of the chain tethered. The final ellipsometric thicknesses of the brush made from the triblocks are less than that expected for a single-end tethered brush made from a diblock copolymer with a buoy block of similar molecular weight. This result supports the conclusion that PVP-b-PS-b-PVP triblock copolymers adsorb mainly in a looplike conformation

MALDI-TOF MS Characterization of Carboxyl-End-Capped Polystyrenes Synthesized Using Anionic Polymerization

A comparative study of three strategies used to synthesize well-defined carboxyl-end-capped polystyrenes is reported. Structural characterization was carried out using a combination of thin-layer chromatography (TLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For the reaction of polystyryl anions with 4-bromo-1,1,1-trimethoxybutane, in addition to the major product, Bu-PS-CH2CH2CH2COOH, four byproducts were observed. Their mass values suggest the following structures: Bu-PS-H, Bu-PS-CH3, Bu-PS-CH2CH=CH-CHO, and Bu-PS-CH2CH2CH2- CO-PS-Bu. The reaction of polystyryl anions with succinic anhydride generated the targeted compound, Bu-PS-CO-CH2CH2COOH, but also yielded the byproducts Bu-PS-H and dimer Bu-PS-CO-CH2CH2-CO-PS-Bu. Under appropriate conditions, the reaction of carbon dioxide gas with polystyryl anions could yield quantitative carboxyl chain-end-functionalization

Impact of Solvent Quality on the Density Profiles of Looped Triblock Copolymer Brushes by Neutron Reflectivity Measurements

Preferential adsorption of poly(2-vinylpyridine)-deuterated polystyrene-poly(2-vinylpyridine) (PVP-dPS-PVP) triblock copolymers from toluene onto silicon leads to the formation of dPS loops tethered by the PVP end blocks. Using neutron reflectometry, we have determined the segment density profiles of these looped polymer brushes in toluene, a good solvent for the dPS block, and in cyclohexane at 20 °C (poor solvent), 32 °C, (near-Θ solvent), and 50 °C (marginal solvent). While the swelling behavior qualitatively agrees with that observed for singly grafted brushes, there are interesting differences in the local structural details: In a good solvent, the segment density profiles are composed of an inner parabolic region and a long, extended tail. In cyclohexane, the profiles are described by exponential decays. We ascribe these features to a novel polydispersity effect that arises due to tethering the PS loops by both ends. The results also show that the less dense layers undergo more significant changes in swollen height as solvent quality is changed and that the looped brushes of different molecular weight, asymmetry, and tethering density adhere to scaling relationships derived for lightly cross-linked polymer gels