Structural and thermodynamic studies of the interaction of distamycin A with the parallel quadruplex structure [d(TGGGGT)]4

The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by 1H NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)]4, we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGGMeGGT)]4, and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand-DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)]4 complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)]4 in a 4:1 binding mode, with two antiparallel distamycin dimers that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)]4 enhances ( approximately 10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groove-binder molecule complexed to a DNA quadruplex structure.     

Sequence‐Specific DNA Alkylation by Hybrid Molecules between Segment A of Duocarmycin A and Pyrrole/Imidazole Diamide

In the absence of distamycin A (Dist), hybrids 1 (X=N, CH) selectively alkylate the 3′ end of adenine in AT‐rich DNA sequences. However, these hybrids can form a heterodimer with Dist to alkylate G residues of predetermined DNA sequences efficiently and with high selectivity.     

Programmable oligomers for minor groove DNA recognition

The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im), pyrrole (Py), and hydroxypyrrole (Hp), four different ways. On the basis of the paradigm of unsymmetrical paired edges of aromatic rings for minor groove recognition, a second generation set of heterocycle pairs, imidazopyridine/pyrrole (Ip/Py) and hydroxybenzimidazole/pyrrole (Hz/Py), revealed that recognition elements not based on analogues of distamycin could be realized. A new set of end-cap heterocycle dimers, oxazole-hydroxybenzimidazole (No-Hz) and chlorothiophene-hydroxybenzimidazole (Ct-Hz), paired with Py-Py are shown to bind contiguous base pairs of DNA in the minor groove, specifically 5'-GT-3' and 5'-TT-3', with high affinity and selectivity. Utilizing this technology, we have developed a new class of oligomers for sequence-specific DNA minor groove recognition no longer based on the N-methyl pyrrole carboxamides of distamycin.     

Design and synthesis of novel thiazole-containing cross-linked polyamides related to the antiviral antibiotic distamycin

A family of naturally occurring oligopeptides includes netropsin, distamycin, anthelvencin, kikumycin B, amidinomycin, and norformycin. Netropsin (I) and distamycin (II) express their biological activities by targeting specific sequences of chemical functionalities in the minor groove of DNA. Both netropsin and distamycin can be regarded as polyamide chains in which each alpha-carbon has been replaced by a five-membered pyrrole ring. The repeat distance in such an augmented polyamide chain is almost the same as the distance from one base pair to the next along the floor of a minor groove within beta-DNA. In this paper we report the synthesis of 16-21 cross-linked polyamides containing a thiazole heterocyclic ring bearing the active functionalites NH(2), NHCHO, or H. 16 and 17 were synthesized by DCC and HOBt catalyzed reaction of 5 with 14 and 15, while the formylation products 18 and 19 were obtained by coupling the formylated 4-methyl-thiazolated acid 6 with 14 and 15. The deaminated compounds 20 and 21 were obtained by the coupling of 5-trichloroacetyl-4-methylthiazole 7 synthesized from 4-methylthiazole. All the six cross-linked polyamides 16-21 were tested for their DNA gyrase inhibition. The studies have shown these polyamides have better sequence recognition and a greater percentage of inhibition than the corresponding monomers. The compound 17 shows complete inhibition of gyrase at 0.5 microM concentration as compared to the naturally occurring distamycin at 1.0 microM.     

Positive ion electrospray ionization mass spectrometry of double-stranded DNA/drug complexes.

Positive ion electrospray ionization mass spectra of 16 base-pair double-stranded (ds)DNA have been obtained with essentially no ions from single-stranded DNA present. Single-stranded DNA was minimized by: (1) careful choice of DNA sequences; (2) the use of a relatively high salt concentration (0.1 M ammonium acetate, pH 8.5), and, (3) a low desolvation temperature (40 degrees C). Similarly, ESI-MS complexes of dsDNA with cisplatin, daunomycin and distamycin were obtained that contained only negligible amounts of single-stranded DNA. The complexes with daunomycin and distamycin were more stable to strand separation in the gas phase than dsDNA alone. This is in agreement with solution studies and with other recent gas phase results. These data contrast with many earlier ESI-MS studies of dsDNA and DNA/drug complexes in which ions from ssDNA are also normally observed.     

Synthesis of distamycin A polyamides targeting G-quadruplex DNA

A number of amide-linked oligopyrroles based on distamycin molecules have been synthesized by solid-state methods, and their interactions with a human intramolecular G-quadruplex have been measured by a melting procedure. Several of these molecules show an enhanced ratio of quadruplex vs. duplex DNA binding compared to distamycin itself, including one with a 2,5-disubstituted pyrrole group. Quadruplex affinity increases with the number of pyrrole groups, and it is suggested that this is consistent with a mixed groove/G-quartet stacking binding mode.     

Systematical Synthesis of Distamycin Analogues and Their Interaction with Herring Sperm DNA

A simple procedure for the systematical synthesis of eight distamycin analogues containing N‐methylpyrrole (Py) and N‐methylimidazole (Im) has been developed by a chloroform reaction and DCC coupling reaction without amino protection and deprotection, and the interaction of the analogues with Herring Sperm DNA was investigated by fluorescence spectroscopy.     

Molecular dynamics simulation of the sliding of distamycin anticancer drug along DNA: interactions and sequence selectivity

Molecular dynamics simulations and umbrella sampling have been used to investigate the sliding of distamycin anticancer drug along the DNA minor groove. The potential energy surface calculated for the sliding of drug shows three minima. The global minimum corresponds to the binding of drug to the AT-rich region, which is the origin of sequence selectivity of distamycin. This selectivity originates from both structural factors and energy contributions. The analysis of energy contributions of binding was performed by the MM–PBSA method. The analysis of hydrogen bonds and van der Waals, electrostatic, and solvation interactions show that structural or steric factors are more important in the selectivity of distamycin than energetic factors. The results of this study can be applied in the design of new derivatives of distamycin anticancer drug with improved properties.     

A total synthesis of distamycin a,an antiviral antibiotic

A new total synthesis of distamycin A (1) is described. The route followed passes through an intermediate acid (2a) which can serve as a convenient starting material for the synthesis of distamycin analogs.     

Synthesis of Distamycin Analogs and Their Interactions with Calf Thymus DNA

Two distamycin analogs (PyPyPy‐γ‐Dp and PyPyPyPy‐γ‐Dp) were synthesized by a haloform reaction and the DCC/HOBT coupling reaction in a ample and fast way without amino protection. By using calf thymus DNA, the interaction between the analogs and DNA duplex was studied by CD, and ITC.     

Study on the Interaction between Distamycin Analogs and DNA from Herring Sperm by Fluorimetry

Distamycin is a naturally occurring antibiotic which binds to the AT rich regions in minor groove of DNA1. We have reported the interaction between the analogs and calf thymus DNA by circular dichroism spectropolarimetry (CD) and isothermal titration calorimetry (ITC)2. Due to the greater sensitivity of fluorescence-based techniques in comparison with CD, we employ the fluorimety to explore the interaction between four new distamycin analogs and DNA from herring sperm. O2NNNOHNNO…     

Synthesis of Nitrated Analogs of Distamycin and their DNA Cleaving and Protecting Activities under UV Irradiation

Two nitrated analogs of distamycin were synthesized by a novel haloform reaction and the DCC/HOBT coupling reaction in a convenient and effective way with out amino group protection. Our results have demonstrated that these polyamides show DNA cleaving activity under long UV (LUV 365 nm) irradiation. Furthermore, it is an unexpected discovery that these analogs can prevent DNA dam age under short UV (SUV 302 nm).     

Interaction between antitumor drugs and a double-stranded oligonucleotide studied by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry was used to investigate the complex formation between a double-stranded oligonucleotide and various antitumor drugs belonging to two categories: intercalators (ethidium bromide, amsacrine and ascididemin) and minor groove binders (Hoechst 33258, netropsin, distamycin A, berenil and DAPI). The goal of this study was to determine whether the relative intensities in the mass spectra reflect the relative abundances of the species in the solution phase. The full-scan mass spectra suggest non-specific binding for the intercalators and specific binding for the minor groove binders. The preferential stoichiometries adopted by each minor groove binder were determined by studying the influence of the drug concentration on the spectra. We obtained 2:1 > 1:1 for distamycin, 1:1 > 2:1 for Hoechst 33258 and DAPI and only the 1 : 1 complex for netropsin and berenil. These features reflect their known behavior in solution. The compared tandem mass spectra of the 1 : 1 complexes with Hoechst 33258 and netropsin, when correlated with published crystallographic data, suggest the possibility of inferring some structural information. The relative binding affinities of the drug for the considered duplex were deduced with two by two competition experiments, assuming that the relative intensities reflect the composition of the solution phase. The obtained affinity scale is netropsin > distamycin A > DAPI > Hoechst 33258 > berenil. These examples show some of the potential uses of mass spectrometry as a useful tool for the characterization of specific drug binding to DNA, and possibly a rapid drug screening method requiring small amounts of materials.     

Synthesis of pyrrole urea and pyrrole carbonylurea derivatives

A series of urea and carbonylurea distamycin analogs whereby the linker has two NH groups for hydrogen bonding with base pairs of DNA were synthesized. The urea and carbonylurea derivatives are prepared from the in situ generation of pyrrole isocyanate (prepared from compound 3) and acyl isocyanate (compound 9), followed by the reaction with an amine. The synthetic feasibility for the further transformations of the pyrrole urea and pyrrole carbonylurea derivatives was also addressed. The binding abilities of these molecules to calf thymus DNA were evaluated by DNA melting temperature (T_m) analysis.     

Synthesis of directly linked diazine isosteres of pyrrole-polyamide that photochemically cleave DNA

A distamycin model containing an isosteric diazine linked pyrrole has been designed and synthesized. The key steps of the synthesis involved the successful diazotization of the 4-amino-pyrrole derivatives to give the diazomium salts, which undergo coupling reactions with N-methylpyrrole to yield the directly linked diazine compounds. The amide isosteric-diazine pyrrole I demonstrated photo-induced DNA damage upon iradiation with UV light (365 nm). Spectrophotometric and mass spectrometric identification suggest that the azo-linkage in I did not dissociate during irradiation. Moreover, compound I produced DNase I footprints with the HexB DNA fragment at AT sites, as well as some other mixed sequences (5'-ATGTCG-3'), indicative of the additional role of the diazine-linkage for interaction at the duplex DNA.     

Spectroscopic binding studies of novel fluorescent distamycin derivatives

Novel distamycin-porphyrin conjugates were synthesized and their interaction with calf thymus DNA was studied. Minor groove binding of the distamycin part of the molecule was confirmed. The porphyrin part of the conjugates exhibited intercalation and the non-specific electrostatic interaction with the phosphate groups of DNA.     

Binding of distamycin A to UV-damaged DNA

We have found that distamycin A can bind to DNA duplexes containing the (6-4) photoproduct, one of the major UV lesions in DNA, despite the changes, caused by photoproduct formation, in both the chemical structure of the base moiety and the local tertiary structure of the helix. A 20-mer duplex containing the target site, AATT.AATT, was designed, and then one of the TT sequences was changed to the (6-4) photoproduct. Distamycin binding to the photoproduct-containing duplex was detected by CD spectroscopy, whereas specific binding did not occur when the TT site was changed to a cyclobutane pyrimidine dimer, another type of UV lesion. Distamycin binding was analyzed in detail using 14-mer duplexes. Curve fitting of the CD titration data and induced CD difference spectra revealed that the binding stoichiometry changed from 1:1 to 2:1 with photoproduct formation. Melting curves of the drug-DNA complexes also supported this stoichiometry.     

Photophysical and Duplex‐DNA‐Binding Properties of Distamycin Dimers Based on 4,4′‐ and 2,2′‐Dialkoxyazobenzenes as the Core

Distamycin‐based tetrapeptide ( 1 ) was covalently tethered to both ends of the central dihydroxyazobenzene moiety at either the 2,2′ or 4,4′ positions. This afforded two isomeric, distamycin–azobenzene–distamycin systems, 2 (para) and 3 (ortho), both of them being photoisomerizable. Illumination of these conjugates in solution at approximately 360 nm induced photoisomerization and the time course of the process was followed by UV/Vis and ¹H NMR spectroscopy. The kinetics of the thermal reversion at various temperatures of cis to trans isomers of the conjugates obtained after photoillumination were also examined. This afforded the respective thermal‐activation parameters. Both the molecular architecture and the location of the substituent around the core azobenzene determined the rate and activation‐energy barrier for the cis‐to‐trans back‐isomerization of these conjugates in solution. Duplex–DNA binding of the conjugates and the changes in DNA‐binding efficiency upon photoisomerization was also examined by CD spectroscopy, thermal denaturation studies, and a Hoechst displacement assay. The conjugate 2 showed higher DNA‐binding affinity and a greater change in the DNA‐binding efficiency upon photoisomerization compared with its 2,2′‐disubstituted counterpart. The experimental findings were substantiated by using molecular‐docking studies involving each conjugate with a model duplex d[(GC(AT)₁₀CG)]₂ DNA molecule.     

Electrospray ionization tandem mass spectrometric characteristics and fragmentation mechanisms of distamycin analogues

The electrospray ionization tandem mass spectrometric (ESI-MS/MS) characteristics and fragmentation mechanisms of eight distamycin analogues containing N-methylpyrrole and N-methylimidazole were investigated. The members of two isomeric groups of distamycin analogues with the same elemental composition can be distinguished by MS/MS spectra of protonated molecules and of significant fragment ions.