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Long Range 1,4 and 1,6-Interstrand Cross-Links Formed by a Trinuclear Platinum Complex. Minor Groove Preassociation Affects Kinetics and Mechanism of Cross-Link Formation as Well as Adduct Structure
journal contribution
posted on 2004-02-25, 00:00 authored by Alexander Hegmans, Susan J. Berners-Price, Murray S. Davies, Donald S. Thomas, Anthony S. Humphreys, Nicholas FarrellReported here is a comparison of the kinetics of the stepwise formation of 1,4- and 1,6-GG
interstrand cross-links by the trinuclear platinum anticancer compound 15N-[{trans-PtCl(NH3)2}2{μ-trans-Pt(NH3)2(H2N(CH2)6NH2)2}]4+, (1,0,1/t,t,t (1) or BBR3464). The reactions of 15N-1 with the self-complementary
12-mer duplexes 5‘-{d(ATATGTACATAT)2} (I) and 5‘-{d(TATGTATACATA)2} (II) have been studied at
298 K, pH 5.3 by [1H,15N] HSQC 2D NMR spectroscopy. The kinetic profiles for the two reactions are
similar. For both sequences initial electrostatic interactions with the DNA are observed for 1 and the
monoaqua monochloro species (2) and changes in the chemical shifts of certain DNA 1H resonances are
consistent with binding of the central charged {PtN4} linker unit in the minor groove. The pseudo first-order rate constants for the aquation of 1 to 2 in the presence of duplex I (3.94 ± 0.03 × 10-5 s-1), or II
(4.17 ± 0.03 × 10-5 s-1) are ca. 40% of the value obtained for aquation of 1 under similar conditions in the
absence of DNA. Monofunctional binding to the guanine N7 of the duplex occurs with rate constants of
0.25 ± 0.02 M-1 s-1 (I) and 0.34 ± 0.02 M-1 s-1 (II), respectively. Closure to form the 1,4- or 1,6-interstrand
cross-links (5) was treated as direct from 3 with similar rate constants of 4.21 ± 0.06 × 10-5 s-1 (I) and
4.32 ± 0.04 × 10-5 s-1 (II), respectively. Whereas there is only one predominant conformer of the 1,6
cross-link, evidence from both the 1H and [1H,15N] NMR spectra show formation of two distinct conformers
of the 1,4 cross-link, which are not interconvertible. Closure to give the major conformer occurs 2.5-fold
faster than for the minor conformer. The differences are attributed to the initial preassociation of the central
linker of 1 in the minor groove and subsequently during formation of both the monofunctional and bifunctional
adducts. For duplex I, molecular models indicate two distinct pathways for the terminal {PtN3Cl} groups to
approach and bind the guanine N7 in the major groove with the central linker anchored in the minor groove.
To achieve platination of the guanine residues in duplex II the central linker remains in the minor groove
but 1 must diffuse off the DNA for covalent binding to occur. Clear evidence for movement of the linker
group is seen at the monofunctional binding step from changes of chemical shifts of certain CH2 linker
protons as well as the Pt−NH3 and Pt−NH2 groups. Consideration of the 1H and 15N shifts of peaks in the
Pt−NH2 region show that for both the 1,4 and 1,6 interstrand cross-links there is a gradual and irreversible
transformation from an initially formed conformer(s) to product conformer(s) in which the amine protons of
the two bound {PtN3} groups exist in a number of different environments. The behavior is similar to that
observed for the 1,4-interstrand cross-link of the dinuclear 1,1/t,t compound. The potential significance of
preassociation in determining kinetics of formation and structure of the adducts is discussed. The
conformational flexibility of the cross-links is discussed in relation to their biological processing, especially
protein recognition and repair, which are critical determinants of the cytotoxicity of these unique DNA-binding agents.
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PtN 3 Clchemical shiftsduplexrate constants15 N shiftsTATACATAmonofunctional binding stepTACATATmonoaqua monochloro speciesGGDNA 1 H resonancesguanine N 7CH 2 linker protonsgrooveTrinuclear Platinum ComplexMinor Groove Preassociation Affects Kinetics6 NH 21 HBBR15 NHSQC 2 D NMR spectroscopyconformerinterstrandAdduct Structure ReportedIINMR spectra show formation
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