Synthesis and characterization of trans-[Pt(NH3)2Cl2] adducts of d(CCTCGAGTCTCC).d(GGAGACTCGAGG).
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.
Lepre
C A
CA
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Chassot
L
L
Costello
C E
CE
Lippard
S J
SJ
D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D002945:Cisplatin / Q000378:metabolism*; D004355:Drug Stability; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010758:Phosphorus; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011522:Protons; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013696:Temperature
D011089:Polydeoxyribonucleotides; D011522:Protons; C034697:transplatin; D010758:Phosphorus; D002945:Cisplatin