Platination of A GG Site on Single‐Stranded and Double‐Stranded forms of A 14‐Base Oligonucleotide with Diaqua Cisplatin followed by NMR and HPLC

Abstract

Detailed studies of the kinetics of platination of the single‐stranded 14‐base DNA oligonucleotide d(ATACATGGTACATA) and the corresponding duplex by cis‐[Pt(NH3)2(H2O)2]2+ show that HPLC and NMR are complementary methods which provide similar results. The 5'‐G and 3'‐G monofunctional intermediates were trapped, separated and characterized by NMR (via 15NH3 labeling) and enzymatic digestion followed by mass spectrometry. The kinetic data are compared with those for the corresponding reactions of cis‐[PtCl2(NH3)2] (cisplatin) and its monohydrolysed analogue. For both single and double strands of the oligonucleotide, the aqua complex shows little selectivity for the 5'‐G or the 3'‐G in the initial platination step, whereas the chloro‐complex preferentially platinates the 3'‐G. The base on the 3' side of the GG sequence appears to play an important role in controlling this selectivity; replacement of T by C increases the selectivity of duplex platination by the diaqua complex by a factor of about 6, and the selectivity of chelation of the 3'‐G monofunctional adduct by a factor of about 3. In general the reactivity of the 5'‐G in a GG sequence appears to be enhanced in a duplex compared with a single‐strand. For both the aqua‐monoadduct and chloro‐monoadduct, cis‐[Pt(NH3)2(NG)(H2O or Cl)], the 5'‐G monoadduct is much longer lived (t½≈ 4 h at 288 K for aqua, 80 h at 298 K for chloro) than the 3'‐G monoadduct (t½≤ 45 min at 288 K for aqua, 6 h at 298 K for chloro). Inspection of molecular mechanics models of the end states of various monofunctional adducts provided insight into H‐bonding and destacking interactions in these adducts and the sequence selectivity observed in their formation. Such adducts may play an important role in the mechanism of action of platinum anticancer drugs.

Metals in Medicine

“…Unexpected was the finding that the 5'-G monofunctional adduct formed during the reaction of cisplatin with the 14-mer DNA duplex d(ATACATGGTACATA)´d(TATGTAC-CATGTAT) is very long lived with a half-life of 80 h at 298 K. [96,97] The lifetimes of the two monofunctional G adducts of the GG single strand are similar, suggesting that the three-dimensional structure of DNA plays a role in stabilizing the 5'-G adduct either by shielding the Cl ligand from hydrolysis, or by constraining the position of the incoming 3'-G N7 ligand. Molecular modeling studies demonstrate that hydrogen-bonding between the NH 3 ligands and carbonyl groups on DNA plays a major role in determining the orientation of the PtÀCl bonds and their accessibility.…”

New clues for platinum antitumor chemistry: Kinetically controlled metal binding to DNA

“…This field has been reviewed by others (15,35,52,53) and myself (13,20). Guanine sites have strong affinity for Pt amine compounds and two adjacent guanines are preferred, with the first binding occurring most rapidly at the 3Ј G. Monofunctional adducts at 5Ј G may persist and play a role in protein crosslinking (54). The two most frequent binding sites, GG and AG (55), appear to be valid for several sources of DNA (56), and they are schematically depicted in Fig.…”

[1H, 15N] NMR Studies of the Platination of Phosphorothioate Nucleotides − Monofunctional Sulfur Adducts versus Macrochelation

“…This is consistent with reported observations for cis-{Pt( 15 NH 3 ) 2 } 2ϩ binding to similar oligonucleotides. [15,16] cis-[PtI 2 ( 15 NH 3 ) 2 ] reacts relatively rapidly with nucleotide phosphorothioates, initially at sulfur. In the case of the mononucleotide GDP-β-S, further reaction to form the S,N7 macrochelate was relatively rapid, whereas for the oligonucleotide Is, the S-bound monofunctional adducts were very long lived.…”