Lepre C A, Chassot L, Costello C E, Lippard S J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1990 Jan 23;29(3):811-23. doi: 10.1021/bi00455a031.
The reaction of trans-diamminedichloroplatinum(II) (trans-DDP), the inactive isomer of the anticancer drug cisplatin, with the single-stranded deoxydodecanucleotide d(CCTCGAGTCTCC) in aqueous solution at 37 degrees C was monitored by reversed-phase HPLC. Consumption of the dodecamer follows pseudo-first-order reaction kinetics with a rate constant of 1.25 (4) x 10(-4) s-1. Two intermediates, shown to be monofunctional adducts in which Pt is coordinated to the guanine N7 positions, were trapped with NH4(HCO3) and identified by enzymatic degradation analysis. These monofunctional adducts and a third, less abundant, one are rapidly removed from the DNA by thiourea under mild conditions. When allowed to react further, the monofunctional intermediates formed a single main product that was characterized by 1H NMR spectroscopy and enzymatic digestion as the bifunctional 1,3-intrastrand cross-link trans-[Pt(NH3)2[d(CCTCGAGTCTCC)-N7-G(5),N7-G(7]]). Binding of the trans-[Pt(NH3)2]2+ moiety to the guanosine N7 positions decreases the pKa at N1 and leads to destacking of the intervening A(6) base. The double-stranded trans-DDP-modified and unmodified DNAs were obtained by annealing the complementary strand to the corresponding single strands and then studied by 31P and 1H NMR and UV spectroscopy. trans-DDP binding does not induce large changes in the O-P-O bond or torsional angles of the phosphodiester linkages in the duplex, nor does it significantly alter the UV melting temperature. trans-DDP binding does, however, cause the imino protons of the platinated duplex to exchange rapidly with solvent by 50 degrees C, a phenomenon that occurs at 65 degrees C for the unmodified duplex. A structural model for the platinated double-stranded oligonucleotide was generated through molecular dynamics calculations. This model reveals that the trans-DDP bifunctional adduct can be accommodated within the double helix with minimal distortion of the O-P-O angles and only local disruption of base pairing and destacking of the platinated bases. The model also predicts hydrogen bond formation involving coordinated ammine ligands that bridge the two strands.
通过反相高效液相色谱法监测了抗癌药物顺铂的无活性异构体反式二氨二氯铂（II）（trans-DDP）与单链脱氧十二聚核苷酸d（CCTCGAGTCTCC）在37℃水溶液中的反应。十二聚体的消耗遵循准一级反应动力学，速率常数为1.25（4）×10⁻⁴ s⁻¹。用NH₄（HCO₃）捕获了两种中间体，经证明它们是单功能加合物，其中Pt与鸟嘌呤N7位配位，并通过酶促降解分析进行了鉴定。在温和条件下，这些单功能加合物和第三种含量较少的加合物可被硫脲从DNA中快速去除。当进一步反应时，单功能中间体形成了一种单一的主要产物，通过¹H NMR光谱和酶促消化鉴定为双功能1,3-链内交联反式-[Pt（NH₃）₂[d（CCTCGAGTCTCC）-N7-G（5），N7-G（7]]）。反式-[Pt（NH₃）₂]²⁺部分与鸟苷N7位的结合降低了N1处的pKa，并导致中间A（6）碱基的解堆积。通过将互补链与相应的单链退火获得了双链反式-DDP修饰和未修饰的DNA，然后通过³¹P和¹H NMR以及紫外光谱进行了研究。反式-DDP的结合不会在双链体中引起O-P-O键或磷酸二酯键扭转角的大变化，也不会显著改变紫外解链温度。然而，反式-DDP的结合确实会导致铂化双链体的亚氨基质子在50℃时与溶剂快速交换，而未修饰的双链体在65℃时会出现这种现象。通过分子动力学计算生成了铂化双链寡核苷酸的结构模型。该模型表明，反式-DDP双功能加合物可以容纳在双螺旋中，O-P-O角的扭曲最小，仅局部破坏碱基配对和铂化碱基的解堆积。该模型还预测了涉及桥接两条链的配位氨配体的氢键形成。
