Lown Jw

OPTIMIZATION OF CROSS-LINKED LEXITROPSINS

In attempts to optimize the cross-linked lexitropsin design, a number of cross-linked dimers composed of two tris(N-methylpyrrolecarboxamide) strands were synthesized and their binding interactions with poly d(A).poly d(T) and poly d(A-T).poly d(A-T) were characterized by circular dichroism and ethidium fluorometry. While all alkanediyl-linked dimers showed a similar binding behavior to the homo AT polymer, particularly at low ligand concentrations, the decanediyl linker was found to be the optimal linker permitting the bidentate antiparallel side-by-side binding of the corresponding dimer to the alternating AT polymer. Thus, in comparison with the monomer, the decanediyl-linked dimer has a binding strength enhancement of about 1400 times in the 1:1 binding mode. Moreover, the hydrophilicity of the linker dimer has a significant effect on the bidentate binding strength. The (3,6)-dioxaoctanediyl-linked dimer has a further binding strength enhancement of 10 times over the decanediyl-linked dimer. Overall, the best optimized dimer has a binding strength enhancement of over 14,000 times in comparison with the monomer in the 1:1 binding mode. This binding enhancement parallels that observed in the best optimized bisintercalators. Distance-restrained molecular modeling provides support for the experimental results. Dimers of longer linkers can readily accommodate a bidentate antiparallel side-by-side binding mode but those of shorter linkers necessitate marked structural distortions in the bound ligand molecules. It is further observed that the binding strength enhancement to the alternating AT polymer is not always accompanied by the binding specificity improvement. Our analysis suggests that the non-specific appendage-DNA backbone interaction is a key factor that controls the specificity improvement.

Structure and Dynamics of Ligand-Template Interactions of Topoisomerase Inhibitory Analogs of Hoechst 33258: High Field 1H-NMR and Restrained Molecular Mechanics Studies

The binding characteristics of Hoechst 33258 (1), a synthetic bis-benzimidazole, and its structural analog 2, with one of the benzimidazoles replaced by a pyridoimidazole, to the self-complementary decadeoxyribonucleotide sequences d(CGCAATTGCG)2 (A) and d-(CATGGCCATG)2 (B) respectively, were examined using high field 1H-NMR techniques. Selective complexation induced chemical shift changes, the presence of exchange signals and intermolecular NOE contacts between the ligands and the minor groove protons of the oligonucleotides suggest the preferred binding sites as the centrally located AATT segment for complex A1, and the CCAT segment for complex B2. The B-type conformations of the two DNA duplexes are preserved upon complexation, as confirmed by the 2D-NOESY based sequential connectivities involving DNA base and sugar protons. Close intermolecular NOE based contacts between the ligands and their respective DNA sequences were further refined to model the ligand-DNA complexes starting from the computer generated B-type structures for the oligonucleotides. Force field calculations of ligand-DNA interaction energies indicate a more favorable contribution from the van der Waals energy component in the case of complex A1 consistent with its stronger net binding compared with the complex B2. Overall, the incorporation of a pyridinic nitrogen in Hoechst 33258 structure alters its selectivity for base pair recognition from A.T to G.C, resulting largely from the formation of a hydrogen bond between the new basic center and the 2-NH2 group of a guanosine moiety. The rates for the exchange of ligands between the two equivalent binding sites (AATT for 1, and CCAT for 2) of the self-complementary DNA sequences, are estimated from analyses of coalescence of NMR signals to be 189s-1 at 301 K for A1 and 79s-1 at 297 K for B2; which correspond to delta G++ of 13.8 and 18.6 kcal.mol-1 respectively.

The Binding of Prototype Lexitropsins to the Minor Groove of DNA: Quantum Chemical Studies

Ab initio calculations (Hartree-Fock) using the 6-31 G basis set have been performed on two prototype lexitropsins or information-reading molecules. The latter are DNA minor groove binding agents related to the A.T recognizing netropsin in which each of the two N-methylpyrrole moieties is replaced in turn by 1-methylimidazole and which thereby confers the property of recognizing G.C sites.Ab initio treatment was possible by examining composities of separate non-conjugated segments of the molecules. Geometry optimized conformations, energies and distribution of electrostatic charges within the molecules were derived. The ab initio derived parameters of the geometry optimized conformations of these lexitropsins were used to interpret their interaction with different sequences within the minor groove of B-DNA.

DNA Binding Studies and Influence on the Activity of DNA Topoisomerases of Bis-Netropsins: Different Effects of Analogs Containing Cis and Trans Ethylene Linkers

Binding to DNA and synthetic duplex polymers of two bifunctional netropsins and effects on supercoiled plasmid DNA as well as their inhibitory potency on DNA topoisomerases have been investigated. Characteristic differences were found in the DNA binding properties of the two bis-netropsins containing a cis and trans tether as reflected by CD, thermal melting and sedimentation measurements. CD results indicate, that the bis-netropsins interact with DNA by a two-step binding mode depending on the ligand concentration. The trans bis-netropsin may form stable complexes with different DNAs at high salt concentration, whereas for cis bis-netropsin DNA complexes the second binding step is completely abolished. The variations in the DNA binding ability of trans and cis bis-netropsin show a close relationship to the differences observed in their inhibitory effects on DNA topoisomerases. It appeared that trans bis-netropsin more strongly blocks topoisomerase activity than the cis isomer and represents the most potent inhibitor of DNA gyrase. Differences in the DNA. binding ability of the bis-netropsins and their inhibitory potency on topoisomerase activity are explained in terms of bidentate and monodentate binding mode of the trans and cis isomer, respectively.

Quantum Chemical and Molecular Mechanics Studies on the Binding of Stereoisomers of the Oligopeptide Antibiotics Amidinomycin and Noformycin to the Minor Groove of B-DNA

Ab initio calculations (Hartree-Fock) using the 6-31G basis set have been performed on two chiral oligopeptide antitumor antibiotics amidinomycin 5 and noformycin 6. The latter are DNA minor groove binding agents related to the A.T recognizing netropsin 4 and distamycin 3 but, unlike the latter, bear stereocenters (two for 5 and one for 6) that may be expected to affect binding to the B-DNA receptor. Geometry optimized conformations, energies and distribution of electrostatic charges within the molecules were derived. The rotational barrier for bond C3-C6 in 6 was calculated to be ca. 6 kcal.mole-1 and the dipole moment for 6 was 7.69D and for 5 was 5.58D. The ab initio derived parameters of the geometry optimized conformations of the different possible stereoisomeric forms of 5 and 6 were used to interpret their different interactions with the minor groove of DNA at both A.T and G.C sequences and the results were compared with molecular mechanics calculations. The order of binding of the four stereoisomers of 5 at the preferred (A.T)n sequences by both ab initio and molecular mechanics calculations is 1S,3R > RR > RS > SS. The predicted energy differences for complexation with DNA of the other stereoisomers from that of 1S,3R are: RR (4.2%); RS (6.7%) and SS (21.5%). In the case of noformycin the 4R structure binds more effectively than the enantiomer. Considerations of phasing in the computed distances between hydrogen bond donating sites in the DNA-bound antibiotics provide further insight into the binding processes. In the complexes of noformycin 6 the N-N1-N4 and N1-N5 distances (9.05 and 9.15 A respectively for 4R-6 and 9.23 and 9.26 A respectively for 4S-6) are close to the optimum value of 9.1 A for effective binding. In the case of amidinomycin 5 the best agreement with the optimum value occurs with the strongest binding diastereomer 1S,3R (N1-N3 = 8.91, N1-N4 = 9.41 A). The unexpected result, consistent in both ab initio and molecular mechanics treatments, is that, in contrast to the cases of kikumycin 1 and anthelvencin 2, the natural 3S configuration of 5 and 4S of 6 do not confer maximal binding efficiency. This suggests that biogenetic factors in the generation of the oligopeptide antibiotics lead to maximum DNA binding in the cases of kikumycin and anthelvencin but not in the cases of amidinomycin and noformycin.

DNA Interaction of the Imidazole-containing Lexitropsin ImPy: Titration Viscometric Study in Comparison to Netropsin

The imidazole (Im) containing lexitropsin ImPy related to netropsin (Nt) is a sequence reading DNA ligand which, in contrast to Nt, permits binding to a GC base pair. The ImPy induced DNA conformational changes differ significantly from those induced by Nt as monitored by titration viscometry, although interaction modes have also been resolved with boundaries at the same ligand to DNA phosphate ratio, r. Evidently ImPy covers similar binding sites (in the same sequence) as Nt for natural calf thymus DNA at r < 0.023. This result suggests that the preferred binding sites of ImPy are A tracts (cf. K.E.R. JBSD 9(1993) 973), in agreement with previous data. The respective DNA coil expansion, most probably caused by unbending (l.c.), is similar but smaller compared to the Nt-DNA interaction. These results again suggest that, at low r values, the van der Waals interaction in the narrowed minor groove of AT clusters provides a dominating energy contribution to ImPy binding. At r > 0.03 the DNA coil expansion increases to extremely high values in that r range where Nt binding (to mixed AT/GC sequences) induces no effect at all owing to steric hindrance with the amino group of guanine. On the basis of many quantitative results for the Nt-DNA systems these effects can be understood in terms of an unbending of intrinsic helix bends (l.c.). They are of considerable interest in connection with the ability of such compounds to influence the direction of the local regulatory relevant DNA curvature.

α-DNA. Synthesis, Characterization and Base-Pairing Properties of Unnatural α-Oligodeoxyribonucleotides

Abstract The novel hexadeoxyribonucleotides α-d(CpCpTpTpCpC) and α-d(CpApTpGpCpG), in which each glycosidic linkage exhibit the anomeric α-configuration, were synthesized by the phosphotriester method. 1H-NMR and thermal denaturation studies provided evidence for these a-oligonucleotides to exhibit a secondary structure similar to that of the natural nucleic acids.

Site selective alkoxymethylation of imidazo[4,5-b]pyridines: structural analysis by high field NMR methods

The alkylation reactions of 2-aryl- 1(3)H-imidazo[4,5-b]pyridines (equivalent to 1-deazapurines) with alkoxymethyl chlorides and bromoacetonitrile are described. The structural assignments of the products were made by the use of two-dimensional 1 H- 1 H NOE (NOESY) and selective INEPT (INAPT) 13 C nmr experiments utilizing polarization transfer from carbon-bound hydrogens in the alkyl side chains to selected 13 C resonances via long-range 3 J CH couplings. Although three isomeric N-alkyl derivatives could arise from a single heterocycle based on considerations of tautomeric equillibria, however, the reactions exhibit marked site selectivity even under quite different reaction conditions. Thus, N-3 alkyl derivatives are produced exclusively in basic (Et 3 N/NaH) nonpolar media following an S E 2cB mechanism

Quantum Chemical Studies Employing an Ab Initio Combination Approach on the Binding of the Bis-Benzimidazole Hoechst 33258 to the Minor Groove of DNA

Ab initio calculations (Hartree-Fock) using the 3-21G and the STO-3G Gaussian basis sets were performed on the sequence selective minor groove binding bis-benzimidazole Hoechst 33258. Geometry optimized conformations, energies and distribution of electrostatic charges within the molecule were derived. The binding of the optimized conformations of the drug to both alternating and non-alternating (AT)n sequences was studied.

1H and 31P-NMR Assignments of the Non-exchangeable Protons of the Consensus Acceptor Exon: Intron Junction d(CpTpApCpApGpGpT)

The consensus acceptor exon:intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique 1H-1H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of 31P-1H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon:intron junction adopts a random coil conformation in solution under the experimental conditions employed.