Molecular Recognition Section and Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, 20892-0810, USA.
Bioconjug Chem. 2010 Jul 21;21(7):1190-205. doi: 10.1021/bc900569u.
The P2Y(1) 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 P2Y(1) 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 N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y(1) 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 (K(i) 23 nM) and extended amine congener 15 (K(i) 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended epsilon-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 P2Y(1) receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y(1) receptor modeling and ligand docking. Attempted P2Y(1) 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.
P2Y(1) 受体是一种促血栓形成的 G 蛋白偶联受体 (GPCR)，可被 ADP 激活。通过使用环状受限的甲叉碳 (双环[3.1.0]己烷) 环作为核糖取代物，确定了嘌呤核苷酸 3'，5'-双磷酸 P2Y(1) 受体拮抗剂的核糖部分的 N 环构象偏好。设计了一系列共价连接的 N(6)-甲基-(N)-甲叉碳-2'-脱氧腺苷-3'，5'-双磷酸，其中包含扩展的 2-炔基链，并确定了它们与人 (h) P2Y(1) 受体的结合亲和力。这些功能化同系物的链含有亲水部分、反应性取代基或生物素，通过酰胺连接。酰胺中间基团的链长和位置的变化揭示了羧酸同系物 8（K(i) 23 nM）和扩展胺同系物 15（K(i) 132 nM）的高亲和力，它们都具有 2-(1-戊炔酰基)基团。含有扩展的 ε-氨基己酰间隔基链的生物素缀合物 18 比具有较短生物素化类似物的生物素缀合物具有更高的亲和力。或者，通过点击反应将同源 2-二炔基核苷酸衍生物的末端炔烃与烷基叠氮基团偶联，在保护双磷酸基团后，生成与 P2Y(1) 受体结合的三唑衍生物。功能化同系物保留受体亲和力与新的 P2Y(1) 受体建模和配体对接一致。通过酰胺形成或钯催化反应将炔烃与树枝状大分子上的炔烃偶联，试图将 P2Y(1) 拮抗剂缀合到 PAMAM 树枝状大分子载体上，或用 2-碘嘌呤衍生的核苷酸修饰，均未成功。含有在受体结合中优先选择的链长的二炔基中间体与叠氮化物衍生的树枝状大分子缀合，缀合物抑制 ADP 促进的人血小板聚集。这是首例将具有战略功能的 P2Y 受体拮抗剂连接到 PAMAM 树枝状大分子上，产生具有所需生物学效应（即抗血栓作用）的多价缀合物。
