Unprecedented Monofunctional Metalation of Adenine Nucleobase in Guanine- and Thymine-Containing Dinucleotide Sequences by a Cytotoxic Platinum−Acridine Hybrid Agent

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Abstract
We have investigated the reactions of [PtCl(en)(ACRAMTU-S)](NO3)2 (2) (en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation, 1), the prototype of a new class of cytotoxic DNA-targeted agents, with 2‘-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy (1H, 195Pt) to identify the covalent adducts formed by platinum. In the mononucleoside model system, two adducts are observed, [Pt(en)(ACRAMTU)(dGuo)]3+ (P1, major) and [Pt(en)(dGuo)2]2+ (P2, minor). The reaction, which proceeds significantly slower (half-life 11−12 h at 37 °C, pH 6.5) than analogous reactions with cisplatin and reactions of 2 with double-stranded DNA, results in the unexpected displacement of the sulfur-bound acridine ligand in ∼15% of the adducts. This reactivity is not observed in double-stranded DNA, rendering 1 a typical nonleaving group in reactions with this potential biological target. In enzymatic digests of calf thymus DNA treated with 2, three adducts were identified: [Pt(en)(ACRAMTU)(dGuo)]3+ (A1, ∼80%), [Pt(en)(ACRAMTU){d(GpA)}]2+ (A2, ∼12%), and [Pt(en)(ACRAMTU){d(TpA)}]2+ (A3, ∼8%). A1 and P1 proved to be identical species. In the dinucleotide adducts A2 and A3, complex 2 covalently modifies adenine at GA and TA base steps, which are high-affinity intercalation sites of the acridine derivative 1. A2 and A3, which may be formed in the minor groove of DNA, are the first examples of monofunctional adenine adducts of divalent platinum formed in double-stranded DNA. The analysis of the adduct profile indicates that the sequence specificity of 1 plays an important role in the molecular recognition between DNA and the corresponding conjugate, 2. Possible biological consequences of the unusual adduct profile are discussed.