Peptide bis-intercalator binds DNA via threading mode with sequence specific contacts in the major groove

BACKGROUND: We previously described a general class of DNA polyintercalators in which 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) intercalating units are connected via peptide linkers, resulting in the first known tetrakis- and octakis-intercalators. We showed further that changes in the composition of the peptide tether result in novel DNA binding site specificities. We now examine in detail the DNA binding mode and sequence specific recognition of Compound 1, an NDI bis-intercalator containing the peptide linker gly-gly-gly-lys. RESULTS: 1H-NMR structural studies of Compound 1 bound to d(CGGTACCG)(2) confirmed a threading mode of intercalation, with four base pairs between the diimide units. The NMR data, combined with DNAse I footprinting of several analogs, suggest that specificity depends on a combination of steric and electrostatic contacts by the peptide linker in the floor of the major groove. CONCLUSIONS: In view of the modular nature and facile synthesis of our NDI-based polyintercalators, such structural knowledge can be used to improve or alter the specificity of the compounds and design longer polyintercalators that recognize correspondingly longer DNA sequences with alternating access to both DNA grooves.     

Intramolecular charge separation in a hydrogen bonded tyrosine-ruthenium(II)-naphthalene diimide triad

Long-lived charge-separated states in the ns to [micro sign]s range were observed upon laser flash excitation of a donor-chromophore-acceptor triad based on tris(bipyridine) ruthenium(ii) as photo-sensitizer, naphthalene diimide as acceptor, and a hydrogen bonded phenol as donor.     

N-methylpiperazinocarbonyl-2',3'-BcNA and 4'-C-(N-methylpiperazino)methyl-DNA: introduction of basic functionalities facing the major groove and the minor groove of a DNA:DNA duplex

Piperazino-functionalized 2',3'-BcNA and 4'-C-hydroxymethyl-DNA are appropriate molecular architectures for the introduction of basic functionalities facing the major groove and the minor groove of nucleic acid duplexes, respectively. 4'-C-(N-Methylpiperazino)methyl-DNA monomers induce significantly increased thermal stability of a DNA:DNA duplex.     

Alteration in the choice of DNA repair pathway with increasing sequence selective DNA alkylation in the minor groove

BACKGROUND: Many conventional DNA alkylating anticancer drugs form adducts in the major groove of DNA. These are known to be chiefly repaired by both nucleotide (NER) and base (BER) excision repair in eukaryotic cells. Much less is known about the repair pathways acting on sequence specific minor groove purine adducts, which result from a promising new class of anti-tumour agents. RESULTS: Benzoic acid mustards (BAMs) tethering 1-3 pyrrole units (compounds 1, 2 and 3) show increasing DNA sequence selectivity for alkylation from BAM and 1, alkylating primarily at guanine-N7 in the major groove, to 3 which is selective for alkylation in the minor groove at purine-N3 in the sequence 5'-TTTTGPu (Pu=guanine or adenine). This increasing sequence selectivity is reflected in increased toxicity in human cells. In the yeast Saccharomyces cerevisiae, the repair of untargeted DNA adducts produced by BAM, 1 and 2 depends upon both the NER and BER pathways. In contrast, the repair of the sequence specific minor groove adducts of 3 does not involve known BER or NER activities. In addition, neither recombination nor mismatch repair are involved. Two disruptants from the RAD6 mutagenesis defective epistasis group (rad6 and rad18), however, showed increased sensitivity to 3. In particular, the rad18 mutant was over three orders of magnitude more sensitive to 3 compared to its isogenic parent, and 3 was highly mutagenic in the absence of RAD18. Elimination of the sequence specific DNA adducts formed by 3 was observed in the wild type strain, but these lesions persisted in the rad18 mutant. CONCLUSIONS: We have demonstrated that the repair of DNA adducts produced by the highly sequence specific minor groove alkylating agent 3 involves an error free adduct elimination pathway dependent on the Rad18 protein. This represents the first systematic analysis of the cellular pathways which modulate sensitivity to this new class of DNA sequence specific drugs, and indicates that the enhanced cytotoxicity of certain sequence specific minor groove adducts in DNA is the result of evasion of the common excision repair pathways.     

Dual mechanisms of DNA damage by MoCH(3)(eta(3)-allyl)(CO)(2)(phen) complexes

DNA damage by MoCH3(eta3-allyl)(CO)2(phen) complexes has been shown to occur by two mechanisms: by backbone cleavage via the abstraction of H1' and/or H5' from the deoxyribose moiety and by base modification, resulting in G-specific cleavage via the formation of base-labile residues methylguanine, methoxyguanine, and 8-oxo-G.     

Cysteine cyclic pyrrole-imidazole polyamide for sequence-specific recognition in the DNA minor groove

Pyrrole-imidazole (PI) polyamides are small DNA-binding molecules that can recognize predetermined DNA sequences with high affinity and specificity. Hairpin PI polyamides have been studied intensively; however, cyclic PI polyamides have received less attention, mainly because of difficulties with their synthesis. Here, we describe a novel cyclization method for producing PI polyamides using cysteine and a chloroacetyl residue. The cyclization reaction is complete within 1 h and has a high conversion efficiency. The method can be used to produce long cyclic PI polyamides that can recognize 7 bp DNA sequences. A cyclic PI polyamide containing two β-alanine molecules had higher affinity and specificity than the corresponding hairpin PI polyamide, demonstrating that the cyclic PI polyamides can be used as a new type of DNA-binding molecule.     

Duplex-promoted platination of adenine-N3 in the minor groove of DNA: challenging a longstanding bioinorganic paradigm

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.     

Identification of oligo(p-phenylene vinylene)-naphthalene diimide heterocomplexes by scanning tunneling microscopy and spectroscopy at the liquid-solid interface

Co-assembly of a melamine derivative covalently equipped with two oligo(p-phenylene vinylene) chromophores and a naphthalene diimide dye results in the formation of heterocomplexes at the liquid-solid interface which was shown using bias dependent imaging and scanning tunneling spectroscopy, despite the disordered nature of the assemblies.     

Strong binding in the DNA minor groove by an aromatic diamidine with a shape that does not match the curvature of the groove

A combination of biophysical techniques has been used to characterize the interaction of an antitrypanosomal agent, CGP 40215A, with DNA. The results from a broad array of methods (DNase I footprinting, surface plasmon resonance, X-ray crystallography, and molecular dynamics) indicate that this compound binds to the minor groove of AT DNA sequences. Despite its unusual linear shape that is not complementary to that of the DNA groove, a high binding affinity was observed in comparison with other similar but more curved diamidine compounds. The amidine groups at both ends of the ligand and the -NH groups on the linker are involved in extensive and dynamic H-bonds to the DNA bases. Complementary and consistent results were obtained from both the X-ray and molecular dynamics studies; both of these methods reveal direct and water-mediated H-bonds between the ligand and the DNA.     

Competitive Na(+) and Rb(+) binding in the minor groove of DNA

Sequence-dependent coordination of alkali ions to the nucleotide bases in the minor groove of AT-tract B-DNA has recently been inferred from X-ray crystallography, solution NMR and computer simulations. Here, we present new (23)Na and (87)Rb magnetic relaxation dispersion (MRD) data that demonstrate competitive and long-lived binding of Na(+) and Rb(+) ions in the minor groove of the B-DNA duplex [d(CGCGAATTCGCG)](2). The Na(+)/Rb(+) selectivity of the minor groove is found to be weak, consistent with local structural flexibility. The ion occupancies derived from the MRD data are substantially higher than previously reported, suggesting that groove-bound ions significantly influence the energetics and structural polymorphism of DNA in vivo. For example, in the presence of 0.20 M Na(+) and 0.56 M Rb(+) at 4 degrees C, the ApT site in the minor groove is occupied by a Rb(+) ion, a Na(+) ion, or a water molecule 40, 10, and 50% of the time, respectively. In the absence of Rb(+), the Na(+) occupancy increases to 50%. At 4 degrees C, the mean residence time of groove-bound ions is 0.2 +/- 0.1 micros for Rb(+) and 10 ns to 100 micros for Na(+). A shorter correlation time of 2 ns is attributed to counterions bridging cross-strand phosphate groups.     

Discrimination of hairpin polyamides with an alpha-substituted-gamma-aminobutyric acid as a 5'-TG-3' reader in DNA minor groove

Pyrrole-imidazole (Py-Im) polyamides containing stereospecifically alpha-amino- or alpha-hydroxyl-substituted gamma-aminobutyric acid as a 5'-TG-3' recognition element were synthesized by machine-assisted Fmoc solid-phase synthesis. Their binding properties to predetermined DNA sequences containing a core binding site of 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T, T.A, G.C, and C.G) were then systematically studied by surface plasmon resonance (SPR). SPR results revealed that the pairing of stereospecifically alpha-amino-/alpha-hydroxyl-substituted gamma-aminobutyric acids, (R or S)-alpha,gamma-diaminobutyric acid (gammaRN or gammaSN) and (R or S)-alpha-hydroxyl-gamma-aminobutyric acid (gammaRO or gammaSO), side-by-side with beta-alanine (beta) in such polyamides significantly influenced the DNA binding affinity and recognition specificity of hairpin polyamides in the DNA minor groove compared with beta/beta, beta/gamma, and gamma/beta pairings. More importantly, the polyamide Ac-Im-gammaSO-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSO) favorably binds to a hairpin DNA containing a core binding site of 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T) with dissociation equilibrium constant (K(D)) of 1.9 x 10(-)(7) M over N.N' = T.A with K(D) = 3.7 x 10(-)(6) M, with a 19-fold specificity. By contrast, Ac-Im-gammaSN-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSN) binds to the above sequence with N.N' = A.T with K(D) = 8.7 x 10(-)(7) M over N.N' = T.A with K(D) = 8.4 x 10(-)(6) M, with a 9.6-fold specificity. The results also show that the stereochemistry of the alpha-substituent, as well as the alpha-substituent itself may greatly alter binding affinity and recognition selectivity of hairpin polyamides to different DNA sequences. Further, we carried out molecular modeling studies on the binding by an energy minimization method, suggesting that alpha-hydroxyl is very close to N3 of the 3'-terminal G to induce the formation of hydrogen bonding between hydroxyl and N3 in the recognition event of the polyamide Ac-Im-gammaSO-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSO) to 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T). Therefore, SPR assays and molecular modeling studies collectively suggest that the (S)-alpha-hydroxyl-gamma-aminobutyric acid (gammaSO) may act as a 5'-TG-3' recognition unit.     

Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,characterization, and interaction with plasmid DNA

A new family of conjugates between a Zn(II)-tach complex and (indole)₂ or benzofuran-indole amide minor groove binders connected through alkyl or oxyethyl linkers of different lengths has been prepared. The conjugates bind strongly to DNA. However, the complexation to DNA to promote the Zn(II) catalyzed hydrolytic cleavage of the DNA results instead in its inhibition. This inhibition effect has been confirmed also using Cu(II). Modeling studies suggest that in the most stable complex conformation, the minor groove binder and the linker lie in the minor groove hampering the interaction between the metal complex and the phosphate backbone of DNA. Therefore, the linear arrangement of minor groove binder-linker-metal complex appears to be effective to ensure tight binding but unproductive from a hydrolytic point of view.     

Cu2+ -mediated assembly of the minor groove binders on the DNA template with sequence selectivity

The ligands (Bpy-H, 5) have been designed to connect the Hoechst33258 skeleton for DNA binding and 2,2'-bipyridine for Cu(2+) complexation. It has been revealed that the new Hoechst ligand long Bpy-H (5L) having a long linker exhibits Cu(2+)-mediated assembly on the DNA template having two A(3)T(3) sites in a selective manner depending on the length of the linker of the ligand as well as on the distance between the two A(3)T(3) sites of DNA. [reaction: see text]     

Synthesis and molecular modelling of double-functionalised nucleosides with aromatic moieties in the 5'-(S)-position and minor groove interactions in DNA zipper structures

A series of six double-functionalised nucleosides, in which aromatic moieties were inserted into the 5'-(S)-C-position, were synthesised and incorporated into DNA duplexes. The aromatic moieties were thymine-1-yl, phenyl, 1,2,3-triazol-1-yl, 1,2,3-triazol-4-yl, 4-(uracil-5-yl)-1,2,3-triazol-1-yl and 4-phenyl-1,2,3-triazol-1-yl. The DNA duplexes were studied with UV melting curves, CD spectroscopy and molecular modelling. The results showed that the aromatic moieties in some cases interact in the minor groove forming DNA zipper structures. The strongest specific interaction was found between two thymines or between a thymine and a phenyl group in a crossed (-3)-zipper motif (i.e., with two base pairs interspacing the modifications). Modelling revealed that the interaction is aromatic stacking across the minor groove. Also, the extended uracil-triazole moiety demonstrated zipper contacts in the minor groove as well as binding to the floor of the groove.     

Short lexitropsin that recognizes the DNA minor groove at 5'-ACTAGT-3': understanding the role of isopropyl-thiazole

Isopropyl-thiazole ((iPr)Th) represents a new addition to the building blocks of nucleic acid minor groove-binding molecules. The DNA decamer duplex d(CGACTAGTCG)(2) is bound by a short lexitropsin of sequence formyl-PyPy(iPr)Th-Dp (where Py represents N-methyl pyrrole, (iPr)Th represents thiazole with an isopropyl group attached, and Dp represents dimethylaminopropyl). NMR data indicate ligand binding in the minor groove of DNA to the sequence 5'-ACT(5)AG(7)T-3' at a 2:1 ratio of ligand to DNA duplex. Ligand binding, assisted by the enhanced hydrophobicity of the (iPr)Th group, occurs in a head-to-tail fashion, the formyl headgroups being located toward the 5'-ends of the DNA sequence. Sequence reading is augmented through hydrogen bond formation between the exocyclic amine protons of G(7) and the (iPr)Th nitrogen, which lies on the minor groove floor. The B(I)/B(II) DNA backbone equilibrium is altered at the T(5) 3'-phosphate position to accommodate a B(II) configuration. The ligands bind in a staggered mode with respect to one another creating a six base pair DNA reading frame. The introduction of a new DNA sequence-reading element into the recognition jigsaw, combined with an extended reading frame for a small lexitropsin with enhanced hydrophobicity, holds great promise in the development of new, potentially commercially viable drug lead candidates for gene targeting.     

In(2)(OH)(3)(BDC)(1.5) (BDC = 1,4-benzendicarboxylate): an In(III) supramolecular 3D framework with catalytic activity

The new hybrid inorganic-organic polymer In(2)(OH)(3)[O(4)C(8)H(4)](1.5) has been hydrothermally obtained. Conditions for the synthesis are reported. The crystal structure of this material has been established by single-crystal X-ray diffraction: it is monoclinic, with space group P2(1)/c (Nomicron. 14), a = 6.772(1) A, b = 10.329(2) A, c = 20.152(3) A, beta = 97.573(3) degrees. The In atoms are octahedrally coordinated by three hydroxide groups and three different molecules of carboxylate ligand. The resulting polymeric 3D structure can be envisaged as having been generated from a honeycomb (6,3) 2D that is cross-linked by the BDC organic anions. Data of IR and TGA-DTA studies, as well as the results of reduction of nitroaromatics and selective oxidation of organic sulfide reactions catalyzed by the new material, are reported.