Biomedical subjects

M J Waring

Publications and source records attributed to M J Waring.

At least 73 records · Page 4Linked to original sources

PubMed1994

Localized chemical reactivity in DNA associated with the sequence-specific bisintercalation of echinomycin.

Four complementary footprinting and probing techniques utilizing DNAse I, methidiumpropyl EDTA (MPE).FeII, diethyl pyrocarbonate (DEPC) and KMnO4 as DNA-cleaving or DNA-modifying agents have been applied to investigate the sequence-specific binding to DNA of the antitumour antibiotic echinomycin. A 265 bp EcoRI-PvuII DNA restriction fragment excised from plasmid pBS was used as a substrate. Six regions of protection against DNAase I cleavage were located on the 265-mer: three sites encompass the sequences 5'-TCGA or 5'-GCGT and the three others contain 5'-GpG (CpC) dinucleotide sequences where the inhibition of DNAase I cutting by echinomycin is less pronounced. In contrast, MPE.FeII cleavage allows identification of only three echinomycin-binding sites on the 265-mer: two sites contain the sequence 5'-TCGA and one encompasses the sequence 5'-ACCA. Cleavage of DNA by MPE.FeII in the presence of echinomycin remains practically unaffected at the sequence 5'-GCGT, despite its identification by DNAase I as a strong site for binding the antibiotic, as well as at the two other sequences containing GpG steps. With both DNAase I and MPE.FeII, enhanced DNA cleavage is evident at AT-rich sequences in the presence of echinomycin. Enhanced reactivity towards KMnO4 and DEPC provides clear evidence for sequence-dependent conformational changes in DNA induced by the antibiotic. The experiments reveal that KMnO4 reacts most strongly with thymines located around, but not necessarily adjacent to, an echinomycin-binding site, whereas the carbethoxylation reactions caused by DEPC occur primarily at the adenine residues lying immediately 5' or 3' to the dinucleotide that denotes an echinomycin-binding site. The results reported here demonstrate that DEPC and KMnO4 serve as sensitive probes for different states of the DNA helix. It seems that the reaction with KMnO4 involves transient unstacking events, whereas the carbethoxylation reaction of DEPC requires larger-scale helix opening.

Base Sequence↗

PubMed1994

DNA recognition by intercalators and hybrid molecules.

Experiments are described which probe the role of the 2-amino group of guanine as a critical determinant of the recognition of nucleotide sequences in DNA by specific ligands. Homologous samples of tyrT DNA substituted with inosine or 2,6-diaminopurine residues in place of guanosine or adenine respectively yield characteristically modified footprinting patterns when challenged with sequence-selective antibiotics such as echinomycin, actinomycin or netropsin. The capacity of small molecules to recognise particular DNA sequences is exploited in the 'combilexin' strategy to target small molecules to defined sites in DNA. A composite molecule containing a distamycin moiety linked to an intercalating ellipticine derivative has been synthesised and shown to bind tightly to DNA but without much sequence-selectivity. Refinement of this molecule based on predictions from molecular modelling has led to the synthesis of a second generation derivative bearing an additional positive charge: this new hybrid molecule is strongly selective for binding to AT-rich tracts in DNA.

Antineoplastic Agents↗

PubMed1994

Interaction of two peptide-acridine conjugates containing the SPKK peptide motif with DNA and chromatin.

The interaction between DNA and two peptide-acridine conjugates containing one (1) or two (2) moieties of the Ser-Pro-Lys-Lys (SPKK) minor groove-binding peptide motif has been studied by a combination of hydrodynamic, biochemical and spectroscopic methods including diffusion-enhanced luminescence energy transfer (DELET) measurements with a Tb(III) lanthanide chelate as donor. Viscometric titrations do not reveal any significant difference between the two hybrid molecules which both unwind (by about 15 degrees) and extend the DNA similarly. DELET measurements show that the acridinyl chromophore of compounds 1 and 2 is much more accessible than that of a simple monointercalating drug such as acridine orange or ethidium. The accessibility factor increases proportionally with the peptide length, reflecting the extent of perturbation imposed upon the intercalating chromophore by the binding to DNA of the peptide moiety of the hybrids. Experiments with the osmium tetroxide-bispyridine reagent indicate that the two hybrid compounds both affect the local conformation of DNA rendering certain thymine residues conspicuously accessible to the probe. The drug-induced sites of hyperreactivity towards OsO4 in DNA are very similar with the exception of a short run of three T residues which is attacked more strongly in the presence of tetrapeptide-acridine conjugate 1 than with the octapeptide-acridine conjugate 2. These results are fully in agreement with previous footprinting studies and support the view that a minimum of two SPKK motifs is required to mimic the AT-specific minor groove binding antibiotic netropsin. On the basis of the DNA-binding properties of these two peptide-acridine hybrids, we present DNA-binding models in which the acridinyl moiety of compound 1 protrudes slightly outside the double helix but remains more or less parallel to the plane of the base-pairs. In contrast, with compound 2, where the octapeptide SPKKSPKK is bound to the minor groove, we postulate that the chromophore lies only partially overlapped with the base pairs in the intercalation site and, in addition, the heterocyclic chromophore is significantly tilted with respect to the double helix axis. Electric linear dichroism and DELET measurements with chromatin reveal that the presence of histone proteins affects the intercalative binding of compound 2 while it has practically no effect on the binding of compound 1.

Acridines↗

PubMed1994

Sequence-selective binding to DNA of cis- and trans- butamidine analogues of the anti-Pneumocystis carinii pneumonia drug pentamidine.

Footprinting experiments using both DNase I and methidium propyl-EDTA.Fe(II) have been used to investigate the sequence selectivity in binding to DNA of pentamidine and four butamidine analogues active against the Pneumocystis carinii pathogen, which afflicts patients with acquired immunodeficiency syndrome. In common with pentamidine, the butamidine drugs, which contain cis- or trans-1,4-but-2-ene linkers and either bis(amidine) or bis(imidazolidine) terminal groups, bind selectively to DNA sequences composed of at least 4 consecutive A.T base pairs. None of the drugs tolerates the presence of a G.C base pair within the binding site. Consistently in the DNase I and methidium propyl-EDTA.Fe(II) footprinting experiments, the cis-isomers produce stronger footprints than do the trans-isomers, despite their similar hydrogen-bonding potentialities. The present experimental data support the view that the conformation of the drug plays a determining role in the binding reaction. Starting from the known structure of a pentamidine-oligonucleotide complex, it is possible to rationalize the different capacities of the cis- and trans-butamidine analogues to recognize defined DNA sequences in terms of the radius of curvature of the molecule and the distance between the positively charged terminal groups. Together, these features constitute critical factors favoring (cis-conformation) or hampering (trans-conformation) the fitting of the drugs into the minor groove of DNA. In terms of structure-activity relationships, the AT-specific recognition of DNA by this series of butamidine derivatives cannot be directly correlated with their potencies against Pneumocystis carinii pneumonia.

Base Sequence↗

PubMed1993

Footprinting titration studies on the binding of echinomycin to DNA incapable of forming Hoogsteen base pairs.

In order to investigate the possible importance of Hoogsteen base pairing to the DNA-binding ability of echinomycin, quantitative DNase I footprinting has been performed. The substrate was the tyrT DNA restriction fragment, either "native" or substituted with one of the purine analogs 2'-deoxy-7-deazaadenosine and 2'-deoxy-7-deazaguanosine in both strands. The modified DNA species were prepared by PCR and selectively labeled at the 5' terminus of one strand (usually the upper "Watson" strand) with [32P]ATP and polynucleotide kinase. Proper incorporation of the analog nucleotides was verified by Maxam-Gilbert G- and C-sequencing reactions as well as exposure to osmium tetroxide and diethyl pyrocarbonate. OsO4 was found to react strongly with the 7-deaza nucleotides, providing a good check of faithful incorporation. The previously observed echinomycin-induced hyperreactivity of purines toward diethyl pyrocarbonate was eliminated by incorporating the appropriate 7-deazapurine. The DNase I footprinting titration studies greatly refined the existing knowledge of the DNA-binding characteristics of echinomycin, as they revealed five general types of concentration-dependent behavior at single-bond resolution. Estimates of microscopic binding constants at individual DNA binding sites were obtained by measuring the antibiotic concentration which produced a half-maximal effect on the concentration of a given DNase I cleavage product. All binding sites contained one or more CpG steps, and all CpG steps analyzed formed part of a binding site for echinomycin. No consistent differences in the estimated binding constants for these sites were observed by comparing normal and modified DNAs, indicating that the abolition of formal Hoogsteen pairs did not significantly alter the thermodynamics of echinomycin-DNA interaction. The lack of any detectable decrease in binding constants for critical sites in the 7-deazapurine-substituted DNAs argues against any anti-syn conformational transition of purine nucleosides occurring in association with the bis-intercalative complex formation.

Amino Acid Sequence↗

PubMed1993

Characterization of a triple helix-specific ligand. BePI (3-methoxy-7H-8-methyl-11- [(3'-amino)propylamino]-benzo[e]pyrido[4,3-b]indole) intercalates into both double-helical and triple-helical DNA.

A benzo[e]pyridoindole derivative, 3-methoxy-7H-8-methyl-11-[(3'-amino)propylamino] -benzo[e]pyrido[4,3-b]indole (BePI), and its interactions with double and triple-helical DNA have been investigated by a variety of fluorescence, spectrophotometric, hydrodynamic and molecular modeling techniques. Binding to DNA stabilizes the doubly charged (+2) form of BePI, increasing the apparent pKa of the 10-NH proton by approximately 1 pH unit. Binding to DNA also quenches the fluorescence of BePI, with a greater extent of quenching upon binding triplex relative to duplex DNA. BePI preferentially binds (and stabilizes) triple-helical relative to double-helical DNA. This preferential binding is not restricted to triplexes containing solely T x A.T base triplets. In addition, BePI preferentially stabilizes the poly(dA).poly(dT) relative to the poly[d(A-T)].poly[d(A-T)] duplex. Viscosity studies demonstrate that, upon binding, BePI induces the unwinding of negative supercoils in the pBR322 plasmid, and increases the relative contour lengths of double and triple-helical polydeoxynucleotides. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to bound BePI molecules in both BePI/duplex and BePI/triplex complexes. In a BePI/triplex complex, an average of 4.8 bases appear to transfer excitation energy totally to a bound BePI molecule, while in various BePI/duplex complexes an average of only 2.5 bases appear to do so, indicating that energy transfer is more efficient in the former complex. Measurements of fluorescence quenching indicate that BePI is protected from quenching by acrylamide when bound to either double or triple-helical polynucleotides. The viscosity and fluorescence behavior of BePI are fully consistent with the conclusion that BePI intercalates into both double and triple-helical DNA. Molecular modeling studies suggest that stronger stacking interactions between intercalated BePI and adjacent bases in BePI/triplex relative to BePI/duplex complexes may account for the enhanced thermal stability of the former complex.

Base Sequence↗

PubMed1993

Preferential intercalation at AT sequences in DNA by lucanthone, hycanthone, and indazole analogs. A footprinting study.

DNAase I footprinting has been used to probe the DNA sequence selectivity of the antitumor intercalating agents lucanthone (1), hycanthone (2), 6-chlorolucanthone (7), and four indazole analogs (IA-3-IA-6). The latter have a benzothiopyranoindazole chromophore substituted with a diethylaminoethyl side chain identical to that attached to the thioxanthenone chromophore of compounds 1, 2, and 7. IA-3 and IA-5 are lucanthone analogs bearing a methyl group at position 4, whereas IA-4 and IA-6 are hycanthone analogs bearing a hydroxymethyl group. IA-3 and IA-4 have an additional chloro group at position 6. Studies employing the 160-bp tyrT DNA fragment as substrate to assay inhibition of DNAase I-mediated cleavage show that both lucanthone and hycanthone bind preferentially to AT sites. They discriminate against GC-rich sequences as well as short runs of a single base, which are often cut more readily in the presence of the drugs compared to the control. The indazole analogs exhibit more pronounced selectivity of binding to AT sequences and promote enhanced DNAase I cleavage both at GC-rich sequences and at homooligomeric runs of adenines or thymines. The results of further DNAase I cleavage inhibition assays, performed with three more restriction fragments having different base pair arrangements, are fully consistent with those obtained with the tyrT fragment. They reveal that the preferred binding sequences for lucanthone, hycanthone, and the indazole analogs are predominantly composed of alternating A and T residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

Sequence-selective binding of amiloride to DNA.

Nuclease footprinting techniques have been employed to investigate the interaction between the diuretic drug amiloride, a sodium channel blocker with potential therapeutic use in the treatment of cystic fibrosis, and three DNA fragments of defined sequence. Using either DNAse I or micrococcal nuclease as probes, an unusual pattern of sequence-selective recognition of DNA has been detected. Amiloride binds selectively to sites rich in adenine and thymine residues, frequently with an apparent preference for 5'-TpX-3' steps, and discriminates strongly against GC-rich sequences which are sometimes cut more readily in the presence of the drug compared to the control. A detailed comparison with the actions of known selective DNA-binding antibiotics and drugs reveals a unique pattern of binding sites, different from those of typical intercalators on the one hand and those of minor groove-binders on the other. Amiloride is believed to adopt a pH-dependent tricyclic hydrogen-bonded conformation in solution which allows it to intercalate into DNA; consistent with this belief, we find that the footprinting pattern largely disappears at pH values above the pKa. Preliminary studies with three amiloride analogues have indicated the importance of two functional groups in the recognition of DNA. The possible relevance of selective DNA binding to activity in vivo is considered.

Amiloride↗

PubMed1992

Binding of a distamycin-ellipticine hybrid molecule to DNA and chromatin: spectroscopic, biochemical, and molecular modeling investigations.

A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.

Autoradiography↗

PubMed1992

The 2-amino group of guanine is absolutely required for specific binding of the anti-cancer antibiotic echinomycin to DNA.

The 2-amino group of guanine is believed to be a critical determinant of potential DNA binding sites for echinomycin and related quinoxaline antibiotics. In order to probe its importance directly we have studied the interaction between echinomycin and DNA species in which guanine N(2) is deleted by virtue of substitution of inosine for guanosine residues. The polymerase chain reaction was used to prepare inosine-substituted DNA. Binding of echinomycin, assessed by DNAse I footprinting, was practically abolished by incorporation of inosine into one or both strands of DNA. We conclude that both the purines in the preferred CpG binding site need to bear a 2-amino group to interact with echinomycin.

Base Sequence↗

PubMed1992

Binding properties and DNA sequence-specific recognition of two bithiazole-linked netropsin hybrid molecules.

We report the DNA binding properties of two hybrid molecules which result from the combination of the DNA sequence-specific minor groove ligand netropsin with the bithiazole moiety of the antitumor drug bleomycin. The drug-DNA interaction has been investigated by means of electric linear dichroism (ELD) spectroscopy and DNase I footprinting. In compound 1 the two moieties are linked by a flexible aliphatic tether while in compound 2 the two aromatic ring systems are directly coupled by a rigid peptide bond. The results are consistent with a model in which the netropsin moiety of compound 1 resides in the minor groove of DNA and where the appended bithiazole moiety is projected away from the DNA groove. This monocationic hybrid compound has a weak affinity for DNA and shows a strict preference for A and T stretches. ELD measurements indicate that in the presence of DNA compound 2 has an orientation typical of a minor groove binder. Similar orientation angles were measured for netropsin and compound 2. This ligand which has a biscationic nature tightly binds to DNA (Ka = 6.3 x 10(5) M-1) and is mainly an AT-specific groove binder. But, depending on the nature of the sequence flanking the AT site first targeted by its netropsin moiety, the bithiazole moiety of 2 can accommodate various types of nucleotide motifs with the exception of homooligomeric sequences. As evidenced by footprinting data, the bithiazole group of bleomycin acts as a DNA recognition element, offering opportunities to recognize GC bp-containing DNA sequences with apparently a preference (although not absolute) for a pyrimidine-G-pyrimidine motif. Thus, the bithiazole unit of bleomycin provides an additional anchor for DNA binding and is also capable of specifically recognizing particular DNA sequences when it is appended to a strongly sequence selective groove binding entity. Finally, a model which schematizes the binding of compound 2 to the sequence 5'-TATGC is proposed. This model readily explains the experimentally observed specificity of this netropsin-bithiazole conjugate.

Base Sequence↗

PubMed1992

Selective binding to AT sequences in DNA by an acridine-linked peptide containing the SPKK motif.

The sequence selectivity of binding to DNA by an acridine-linked peptide ligand has been investigated by means of footprinting methodologies. The ligand conjugates an anilino-acridine intercalating chromophore with the potentially minor groove binder octapeptide SPKKSPKK. This basic peptide corresponds to a highly conserved DNA recognition motif found in histone H1 and several other nonhistone proteins. Three complementary techniques using DNase I, hydroxyl radicals and osmium tetroxide as sequencing probes have been employed to evaluate both the sequence specificity of binding and the drug-induced conformational changes in DNA. The results converge to demonstrate the AT-selectivity and support a model in which the peptide moiety lies in the minor groove. DNA-binding sites of the conjugate are restricted to a few alternating AT-sequences proximal to GC-rich regions. Binding to homooligomeric runs of A and T is clearly disfavoured by the hybrid whereas such sequences represent preferred binding sites for the unsubstituted basic peptide. These differences reflect the influence of the anilino-acridine chromophore, which evidently contributes to the DNA recognition process allowing the peptide only to contact defined DNA sequences.

Acridines↗

PubMed1992

Sequence specificity of the binding of 9-aminoacridine- and amsacrine-4-carboxamides to DNA studied by DNase I footprinting.

DNase I footprinting has been used to probe the sequence selectivity of binding of a series of intercalating amsacrine-4-carboxamides and a related 9-aminoacridine-4-carboxamide to three DNA restriction fragments. These ligands have good experimental antileukemic activity, and for those members of the series that gave evaluable footprints, our principal finding is that they bind preferentially to GC-rich regions in agreement with the conclusion of equilibrium and kinetic measurements. The highest affinity sites generally occur in clusters of GC base pairs with runs of AT pairs being excluded from binding. It is important to appreciate that the 9-aminoacridine- and amsacrine-4-carboxamides exhibit a very high degree of selectivity for GC sites which, to our knowledge, has not been previously matched by acridine derivatives in footprinting experiments. The principal determinant of specificity appears to be the 4-carboxamide group itself since neither variations in the terminal funtionality of the 4-carboxamide sidechain nor the presence of the 9-anilino substituent modifies sequence preferences. The molecular origins of selectivity may be discerned in terms of potential hydrogen bonding interactions between the 4-carboxamide moiety and carbonyl oxygen and amino groups of GC base pairs in the DNA minor groove at CG dinucleotide sites. The related therapeutic agent amsacrine failed to inhibit cleavage by DNase I, so no conclusion can be drawn concerning its binding selectivity, save to note that amsacrine does not possess the 4-carboxamide group which appears to be the crucial determinant of GC specificity. Whether selectivity for binding to GC-rich sequences is an important element in the antitumor activity of both the 9-aminoacridine- and amsacrine-4-carboxamides remains to be determined.

Aminacrine↗

PubMed1992

Echinomycin.

The story of echinomycin is of an antibiotic whose anti-cancer activity was rediscovered thanks to scientific investigation of its mode of action at the molecular level. It was the first DNA bis-intercalator identified (in 1974). Molecular models for echinomycin and its congeners are now well-founded on crystallographic data. These are beginning to throw light on significant variations in conformation which affect the ability of the antibiotics to recognise specific nucleotide sequences in DNA. Kinetic and other physical experiments have revealed much about the stability and selectivity of antibiotic-DNA complexes: stacking forces are important, as are hydrogen bonding interactions. Echinomycin preferentially recognises sites in DNA containing the CpG step, but other sites of lower occupancy exist and the antibiotic can migrate between them. Currently a good deal of attention is being paid to the suggestion that echinomycin might change Watson-Crick base pairs to a Hoogsteen form.

Anti-Bacterial Agents↗

PubMed1992

DNA-binding properties of a distamycin-ellipticine hybrid molecule.

We have synthesized a distamycin-ellipticine hybrid compound and investigated its interaction with DNA, using various optical and gel electrophoresis techniques. Binding of the hybrid to DNA is evidenced by spectral shifts, fluorescence quenching, and induced linear dichroism. Absorbance measurements have been used to generate Scatchard plots, which reveal that the interaction cannot be described adequately in terms of a single binding mode, probably because of simultaneous intercalation and minor groove binding of the ligand. Competition with added distamycin has been used to verify involvement of the N-methyl-pyrrole portion of the hybrid molecule in the binding reaction. From electric linear dichroism experiments, it is estimated that the orientation of the DNA-bound ellipticine chromophore in the hybrid differs by about 10 degrees from the orientation of the equivalent chromophore lacking a distamycin tail. Topoisomerase assays establish that binding of the hybrid unwinds the DNA helix by a minimum of 11 degrees, which is consistent with intercalation but notably smaller than the unwinding angle of ellipticine. In footprinting experiments, it is found that the AT- and GC-specificity of distamycin and ellipticine, respectively, appear to be merged in the binding of the hybrid, which produces a pattern of protection distinct from the characteristic patterns for either of the parent compounds. The hybrid is an extremely effective inhibitor of cutting by DNase I.

Chelating Agents↗

PubMed1991

DNA-sequence specific recognition by a thiazole analogue of netropsin: a comparative footprinting study.

Four different footprinting techniques have been used to probe the DNA sequence selectivity of Thia-Net, a bis-cationic analogue of the minor groove binder netropsin in which the N-methylpyrrole moieties are replaced by thiazole groups. In Thia-Net the ring nitrogen atoms are directed into the minor groove where they could accept hydrogen bonds from the exocyclic 2-amino group of guanine. Three nucleases (DNAase I, DNAase II, and micrococcal nuclease) were employed to detect binding sites on the 160bp tyr T fragment obtained from plasmid pKM delta-98, and further experiments were performed with 117mer and 253mer fragments cut out of the plasmid pBS. MPE.Fe(II) was used to footprint binding sites on an EcoRI/HindIII fragment from pBR322. Thia-Net binds to sites in the minor groove containing 4 or 5 base pairs which are predominantly composed of alternating A and T residues, but with significant acceptance of intrusive GC base pairs. Unlike the parent antibiotic netropsin, Thia-Net discriminates against homooligomeric runs of A and T. The evident preference of Thia-Net for AT-rich sites, despite its containing thiazole nitrogens capable of accepting GC sites by hydrogen bonding, supports the view that the biscationic nature of the ligand imposes a bias due to the electrostatic potential differences in the receptor which favour the ligand reading alternating AT sequences.

Base Sequence↗

PubMed1991

Optically detected triplet-state magnetic resonance studies of the DNA complexes of the bisquinoline analogue of echinomycin.

The polymeric DNA and model duplex oligonucleotide complexes of the bisquinoline analogue of echinomycin (2QN) have been studied by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoline chromophores of the drug used as intrinsic probes. Plots of ODMR transition frequencies versus monitored wavelength revealed heterogeneity in the phosphorescence emission of 2QN which was ascribed to the presence of a major and minor conformation of the drug in aqueous solutions (referred to as the red and blue forms of 2QN, respectively, in this report). ODMR results, in conjunction with findings from low-temperature phosphorescence investigations, indicate that the quinoline chromophores of the major (red) form of 2QN are involved in aromatic stacking interactions in complexes with the natural DNAs from Escherichia coli, Micrococcus lysodeikticus, Clostridium perfringens, and calf thymus as evidenced by red shifts in the phosphorescence 0,0-band of the drug, reductions in the phosphorescence lifetime and zero-field splitting (zfs) D and E parameters, and polarity reversals of the ODMR slow passage signals upon complex formation between the analogue and DNA. The polarity reversals, which reflect shifts in the triplet-state sublevel populations induced by complex formation, apparently result from changes in the triplet sublevel decay constants upon binding to the natural DNAs. The 2QN complexes of the double-stranded alternating copolymers poly(dG-dC).poly(dG-dC) [abbreviated as poly[d(G-C)2]] and poly(dA-dT).poly(dA-dT) [abbreviated as poly(dA-dT).poly(dA-dT) [abbreviated as poly[d(A-T)2], the homopolymer duplexes poly(dG).poly(dC) [abbreviated as poly(dG.dC)] and poly(dA).poly(dT) [abbreviated as poly(dA.dT)], and the self-complementary oligonucleotides d(ACGT)2, d(TCGA)2, and d(ACGTACGT)2 were also investigated. The extent of reduction of the zfs D parameter (delta D) for the major form of 2QN upon complex formation with the polymeric DNAs was found to scale linearly with the standard free energy of the drug-DNA interaction (delta G degrees) calculated from previously reported binding studies for these targets [Fox, K. R., et al. (1980) Biochem. J. 191, 729-740]. This relationship between spectroscopic and thermodynamic properties of the 2QN-polynucleotide complexes is a consequence of the effects of base stacking interactions on the electronic states of the intercalator, which were postulated to arise from second-order shifts of the ground-state and the triplet-state energies of the complex on the basis of a modification of the solvent effect theory of van Egmond et al. [(1975) Chem. Phys. Lett. 34, 423-426].

Animals↗

PubMed1991

Conformational heterogeneity of quinoxaline peptides in solution.

The conformational heterogeneity of several quinoxaline antibiotics, a class of naturally occurring quinoxaline peptides with antitumor properties, and their synthetic analogues was investigated in polar and nonpolar solvents by high performance liquid chromatography (HPLC) with uv photodiode array detection, uv-absorbance, low-temperature phosphorescence, and nmr techniques. Multiple peak formation and interconversion in the HPLC and 1H-nmr analysis of triostin A, its under-N-methylated synthetic analogues (des-N-tetramethyltriostin A [TANDEM] and [N-MeCys3, N-MeCys7]-TANDEM [MCTAN-DEM]), and echinomycin were examined as a function of temperature, solvent polarity, and residence time in solution prior to analysis. Slow interconversion between HPLC peaks, ascribed to the presence of multiple solution conformers, was exhibited by these peptides although at very different interconversion rates. Among the triostins, the rate of interconversion appeared to vary with the degree of N-methylation of the residues in the cyclic depsipeptide chain. Interconversion of the n and p conformers of triostin A in chloroform occurred on a chromatographic timescale (a few minutes with kn----p calculated to be 0.02 s-1 at 25 degrees C) while the solution conformers of TANDEM in methanol equilibrated very slowly to one preferred conformer over a period of several weeks at ambient temperature. MCTANDEM, a synthetic analogue of triostin A with an intermediate degree of N-methylation of the residues in the peptide ring, consisted of an equilibrium mixture of n and p conformers in methanol that interconverted on a chromatographic time scale. Two additional conformers of MCTANDEM developed within a few weeks' residence time in methanol at ambient temperature. Echinomycin was found to exist in methanol as an interconverting mixture of at least four minor conformers in addition to the major isoform (95% by peak area) of the peptide. The solution conformers of the quinoxaline peptides investigated in this report are most likely a consequence of hindered rotation about the N-methylated peptide bonds in the depsipeptide ring and/or intramolecular hydrogen bonding.

4-Quinolones↗