Duplex-Promoted Platination of Adenine-N3 in the Minor Groove of DNA:  Challenging a Longstanding Bioinorganic Paradigm

Abstract

The interactions of [Pt(en)Cl(ACRAMTU-S)](NO3)2 (PT-ACRAMTU, en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) with adenine in DNA have been studied using a combination of analytical and high-resolution structural methods. For the first time, a cytotoxic platinum(II) complex has been demonstrated to form adducts in the minor groove of DNA through platination of the adenine-N3 endocyclic nitrogen. An acidic depurination assay was developed that allowed the controlled and selective (pH 2, 60 degrees C, 12 h) release of platinum-modified adenine from drug-treated nucleic acid samples. From the digested mixtures, three adducts were isolated by semipreparative reverse phase high-performance liquid chromatography and studied by electrospray ionization mass spectrometry (in-line LC-MS), variable-pH 1H NMR spectroscopy, and, where applicable, X-ray crystallography. The three species were identified as the N7 (A-I), N3 (A-II), and N1 (A-III) linkage isomers of [Pt(en)(ACRAMTU-S)(adenine)]3+ (A). Incubations carried out with the single- and double-stranded model sequences, d(TA)5 and d(TA)15, as well as native DNA indicate that the adduct profiles (A-I:A-II:A-IIIratios) are sensitive to the nature of the nucleic acid template. A-II was found to be a double-strand specific adduct. The crystal structure of this adduct has been determined, providing ultimate evidence for the N3 connectivity of platinum. A-II crystallizes in the triclinic space group P in the form of centrosymmetric dimers, {[Pt(en)(ACRAMTU-S)(adenine-N3)]2}6+. The cations are stabilized by a combination of adenine-adenine base pairing (N6...N1 2.945(5) A) and mutual acridine-adenine base stacking. Tandem mass spectra and 1H chemical shift anomalies indicate that this type of self-association is not merely a crystal packing effect but persists in solution. The monofunctional platination of adenine at its N7, N3, and N1 positions in a significant fraction of adducts breaks a longstanding paradigm in platinum-DNA chemistry, the requirement for nucleophilic attack of guanine-N7 as the principal step in cross-link formation. The biological consequences and potential therapeutic applications of the unique base and groove recognition of PT-ACRAMTU are discussed.

Understanding the Acid–Base Properties of Adenosine: The Intrinsic Basicities of N1, N3 and N7

“…[47] In contrast, divalent transition metal ions are expected [36,37,46] to coordinate not only to N7 but also to N3 in an innersphere manner. Indeed, there are several examples where a kinetically inert Pt II ion coordinates to an adenine N3, [48][49][50] but the corresponding observation has also been made with labile metal ions like Cu II , [51] and if a further suitable guiding binding site, like in several antivirally active nucleotide analogues is available, N3 coordination may become common, [42,52,53] which, knowing the intrinsic basicity of N3, is no real surprise anymore. Inner-sphere binding to N7 is well known [45,47] and has often been observed for adenine nucleotides which undergo macrochelate formation.…”

Characterization of the bisintercalative DNA binding mode of a bifunctional platinum-acridine agent

“…Our results suggest that bisintercalative binding, a potentially cytotoxic lesion in genomic DNA of this agent, is feasible and preferentially occurs in alternating TA sequences. Unlike the parent compound PT-ACRAMTU (20–22), PT-BIS(ACRAMTU) does not induce adducts with nucleobase nitrogen but binds to DNA in a reversible manner. (The inability of sulfur-bound ACRAMTU to act as a leaving group in reactions with double-stranded DNA has been amply demonstrated (21). )…”

Photoactivation of trans diamine platinum complexes in aqueous solution and effect on reactivity towards nucleotides

“…In order to complete the assignment of the peaks observed by NMR, the magnitudes of chemical shift differences for H8C8 and H2C2 between the free base and the metallated base in the 2D [ 1 H, 13 C] NMR spectra were analysed, since this appears to be a useful diagnostic tool [53,54] for assignment of the type of binding in metal-nucleotide adducts. The shift changes for the 13 C resonances were similar and small (up to ca.…”