Targeting the DNA cleavage activity of copper phenanthroline and clip-phen to A.T tracts via linkage to a poly-N-methylpyrrole

To target DNA A.T tracts, a three-ring polyamide containing an N-methylpyrrole amino acid has been linked, on solid support, to carboxylic derivatives of phenanthroline and dimers of phenanthroline: 2-Clip-Phen, 3-Clip-Phen, or 2-Clip-Phen containing a long tether. After metalation by CuCl(2), the DNA cleavage activities of the different conjugates were compared on a restriction fragment. Cleavage patterns showed that the polyamide moiety of conjugates directs the cleavage activity in the vicinity of A.T tracts but the precise cleavage selectivity of these conjugates was dependent on the type of phenanthroline residue linked to the poly-N-methylpyrrole entity.     

Conjugation of various acridines to DNA for site-selective RNA scission by lanthanide ion

Three types of DNA conjugates having 9-acridinecarboxamide, 9-aminoacridine, and 9-amino-6-chloro-2-methoxyacridine at the 5'-ends were synthesized and used for site-selective RNA scission together with another unmodified DNA and Lu(III) ion. The target phosphodiester linkages in the substrate RNA were selectively and efficiently activated and were hydrolyzed by free Lu(III) ion. The conjugate bearing 9-amino-6-chloro-2-methoxyacridine was the most active. However, its duplex with the substrate RNA was almost as stable as that of the 9-aminoacridine-bearing conjugate, which was much less active for the RNA activation. The 9-acridinecarboxamide-bearing conjugate was only marginally active. The substituents on the acridine groups in these conjugates positively participate in the present RNA activation, probably by fixing the orientation of the acridine rings.     

Design of phosphoramidite monomer for optimal incorporation of functional intercalator to main chain of oligonucleotide

Chirally pure phosphoramidite monomers bearing 9-amino-6-chloro-2-methoxyacridine were synthesized from D- or L-threoninol and omega-aminocarboxylic acid, and incorporated into oligonucleotides. These acridine-DNA conjugates formed stable duplexes with complementary RNA because of intercalation of the acridine to DNA/RNA heteroduplexes. The stability of duplexes was not very dependent on either the chirality of the central carbon bearing the acridine or the length of the side chain. However, the ability for site-selective activation of the phosphodiester linkage in front of the acridine, which induced Lu(III)-promoted RNA scission, was strongly dependent on these two factors. The largest activation was achieved when the monomer unit was prepared from L-threoninol and 4-aminobutyric acid and the acridine was bound to the amino group. By attaching the more acidic 9-amino-2-methoxy-6-nitroacridine to this optimized scaffold, a quite effective acridine-DNA conjugate for site-selective RNA scission was obtained.     

Oligonucleotide derivatives in nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insertion

Non-nucleotide phosporamidites were synthetized, having a branching backbone with different positions for functional groups. Phosphoramidite monomers obtained contain intercalator moiety, 6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with the tert-butyldimethylsilyl group for post-synthetic modification. Oligothymidilates containing one or more modified units in different positions of the sequence were synthesized. The melting point and thermodynamic parameters of the formation of complementary duplexes formed by modified oligonucleotides were defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.     

Deoxyribonucleic acid cleavage specificity of a series of acridine- and acodazole-iron porphyrins as functional bleomycin models

A series of metalloporphyrins linked through basic chains to certain DNA interactive groups has been synthesized. Several of these agents reproduce the characteristic properties of the antitumor glycopeptide bleomycin, including the oxygen-mediated scission of DNA in the presence of thiols, antibiobic activity under aerobic conditions, and activity against human and animal tumor models. Initial screening by scission of PM2-CCC-DNA identified six of the compounds, including those bearing acridine and acodazole intercalating groups, as the most active. The specificity of the oxygen-mediated scission of a 139 base pair HindIII/NciI restriction fragment of pBR322 by these six selected agents was then determined and compared with the action of pancreatic DNase by densitometric scans. All six of these compounds produce uniform base and sequence neutral cleavage of the restriction fragment at each base site. The six active compounds bear either of two types of intercalators, 6-chloro-2-methoxyacridine or acodazole, and with linkages to the ferric binding domain of -NH(CH2)2-, -NH(CH2)3-, -NH(CH2)4-, or -NH(CH2)3NH(CH2)3- and either porphyrin or deuteroporphyrin moieties. Comparison of the Kassoc values for binding to calf thymus DNA suggests that the enhanced binding observed with the linker -NH(CH2)3NH(CH2)3- contributes to the efficiency of sequence neutral DNA scission and may be a factor in the relative anticancer activities of these agents. The iron porphyrins give no evidence of the production of base propenals in DNA degradation, and the autoradiograms clearly indicate that a phosphate group is attached to the 5' end of the oligomer. The scission is partially suppressible by catalase and superoxide dismutase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterodimeric molecules including nucleic acid bases and 9-aminoacridine. Spectroscopic studies, conformations, and interactions with DNA

Heterodimeric molecules have been examined in which the 9-amino-6-chloro-2-methoxyacridine ring is linked to the nucleic acid bases adenine, thymine, and guanine by polymethylenic chains (CH2)n of varying length (n = 3, 5, 6). A detailed analysis has been performed, including hypochromism measurement in the UV, chemical shift variations in Fourier transform proton magnetic resonance, and fluorescence emission. All these techniques show that all molecules exist mainly under folded conformations in water in the temperature range 0-90 degrees C, with the acridine and the base rings being stacked one on top of the other. The thermodynamic parameters for the folded in equilibrium unfolded conformational equilibrium were estimated. The geometry of the intramolecular complexes could be determined. (1) All these data give information on the strength and nature of the base-acridine interactions as a function of different parameters such as solvent, temperature, etc. (2) The molecules under study constitute "spectroscopic models" for the drug-base complexes as they occur in DNA. In particular, we show the dramatic influence of the relative orientations of the two stacked chromophores in the complex upon the magnitude of % H, the percent hypochromism. The quenching and enhancement of acridine fluorescence emission induced respectively by guanine and adenine is evidenced and quantitatively estimated. (3) These base-acridine heterodimers bind to DNA to an extent that is inversely proportional to their degree of intramolecular stacking.     

4-Aminoquinoline quinolizidinyl- and quinolizidinylalkyl-derivatives with antimalarial activity

A set of quinolizidinyl and quinolizidinylalkyl derivatives of 4-amino-7-chloroquinoline and of 9-amino-6-chloro-2-methoxyacridine were prepared and tested in vitro against CQ-sensitive (D-10) and CQ-resistant (W-2) strains of Plasmodium falciparum. All compounds but one exerted significant antimalarial activity. Some of the quinolizidine derivatives were from 5 to 10 times more active than chloroquine on the CQ-resistant strain. No toxicity against mammalian cells was observed.     

RNA-ligand interactions: affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element

Semisynthetic aminoglycoside derivatives may provide a means to selectively target viral RNA sites, including the HIV-1 Rev response element (RRE). The design, synthesis, and evaluation of derivatives based upon neomycin B, kanamycin A, and tobramycin conjugates of 9-aminoacridine are presented. To evaluate the importance of the acridine moiety, a series of dimeric aminoglycosides as well as unmodified "monomeric" aminoglycosides have also been evaluated for their nucleic acid affinity and specificity. Fluorescence-based binding assays that use ethidium bromide or Rev peptide displacement are used to quantify the affinities of these compounds to various nucleic acids, including the RRE, tRNA, and duplex DNA. All the modified aminoglycosides exhibit a high affinity for the Rev binding site on the RRE (K(d) 相似文献   

Adenosine-guanosine preferential photocleavage of DNA by azido-benzoyl- and diazocyclopenta-dienylcarbonyloxy derivatives of 9-aminoacridine.

The photoreactions of 9-[6-(4-azidobenzamido)hexylamino]acridine (AHA) and 9-[6-(2-diazocyclopentadienylcarbonyloxy)hexylamino]acridine (DHA) with double stranded DNA result in formation of single strand nicks and alkali labile sites (adducts) with an efficiency of 6 x 10(-3) nicks per AHA and 3 x 10(-2) nicks per DHA molecule. The alkali dependent DNA cleavage by AHA shows a pronounced A+G preference whereas that by DHA is practically sequence independent. In the presence of diacridines, however, DHA exhibits a preference for cleavage at guanosines. These DNA photocleaving reagents could be useful for DNA photofootprinting and photosequencing.     

Mechanisms of DNA cleavage by copper complexes of 3-clip-phen and of its conjugate with a distamycin analogue

Mechanisms of DNA oxidation by copper complexes of 3-Clip-Phen and its conjugate with a distamycin analogue, in the presence of a reductant and air, were studied. Characterisation of the production of 5-methylenefuranone (5-MF) and furfural, associated with the release of nucleobases, indicated that these copper complexes oxidised the C1′ and C5′ positions of 2-deoxyribose, respectively, which are accessible from the DNA minor groove. Oxidation at C1′ was the major degradation route. Digestion of DNA oxidation products by P1 nuclease and bacterial alkaline phosphatase allowed characterisation of glycolic acid residues, indicating that these copper complexes also induced C4′ oxidation. However, this pathway was not associated with base propenal release. The ability of the copper complex of the 3-Clip-Phen conjugate with the distamycin analogue to produce sequence-selective DNA cleavage allowed confirmation of these mechanisms of DNA oxidation by PAGE. Comparison of DNA cleavage activity showed that conjugation of 3-Clip-Phen with a DNA minor groove binder, like the distamycin analogue, decreased both its ability to perform C1′ oxidation as well as the initial rate of the reaction, but this conjugate is still active after 5 h at 37°C, making it an efficient DNA cleaver.     

Recognition and cleavage of DNA by rebeccamycin- or benzopyridoquinoxaline conjugated of triple helix-forming oligonucleotides

Indolocarbazole and benzopyridoquinoxaline derivatives have been shown to have anti-tumor activity and to stimulate DNA topoisomerase I-mediated cleavage. Two indolocarbazole compounds (R-6 and R-95) and one benzopyridoquinoxaline derivative (BPQ(1256)) were covalently attached to the 3'-end of a 16mer triple helix-forming oligonucleotide (TFO). These conjugates bind to DNA with a higher affinity than the unsubstituted oligonucleotides. Furthermore, they induce topoisomerase I-mediated and triplex-directed DNA cleavage in a sequence-specific manner.     

DNA cleavage and binding selectivity of a heterodinuclear Pt–Cu(3-Clip-Phen) complex

The synthesis and nuclease activity of a new bifunctional heterodinuclear platinum–copper complex are reported. The design of this ditopic coordination compound is based on the specific mode of action of each component, namely, cisplatin and Cu(3-Clip-Phen), where 3-Clip-Phen is 1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine. Cisplatin is not only able to direct the Cu(3-Clip-Phen) part to the GG or AG site, but also acts as a kinetically inert DNA anchor. The nuclease activity of this complex has been investigated on supercoiled DNA. The dinuclear compound is not only more active than Cu(3-Clip-Phen), but is also capable of inducing direct double-strand breaks. The sequence selectivity of the mononuclear platinum complex has been investigated by primer extension experiments, which reveal that its interaction with DNA occurs at the same sites as for cisplatin. The Taq polymerase recognizes the resulting DNA damage as different from that for unmodified cisplatin. The sequence-selective cleavage has been investigated by high-resolution gel electrophoresis on a 36-bp DNA fragment. Sequence-selective cleavages are observed in the close proximity of the platinum sites for the strand exhibiting the preferential platinum binding sites. The platinum moiety also coordinates to the other DNA strand, most likely leading only to mono guanine or adenine adducts. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and structural properties of oligonucleotides covalently linked to acridine and quindoline derivatives through a threoninol linker

Oligonucleotide conjugates containing acridine and quindoline derivatives linked through a threoninol molecule were synthesized. We showed that these conjugates formed duplexes and quadruplexes with higher thermal stability than the corresponding unmodified oligonucleotides. When acridine is located in the middle of the sequence, DNA duplexes have a similar stability independently of the natural base present in front of acridine. Self-complementary oligonucleotides and thrombin binding aptamers (TBA) carrying the acridine and quindoline molecules are studied by NMR.     

Substituted benz[a]acridines and benz[c]acridines as mammalian topoisomerase poisons

Coralyne and several other synthetic benzo[a,g]quinolizium derivatives related to protoberberine alkaloids have exhibited activity as topoisomerase poisons. These compounds are characterized by the presence of a positively charged iminium group, which has been postulated to be associated with their pharmacological properties. The objective of the present study was to devise stable noncharged bioisosteres of these compounds. Several similarly substituted benz[a]acridine and benz[c]acridine derivatives were synthesized and their relative activity as topoisomerase poisons was determined. While the benz[c]acridine derivatives evaluated as part of this study were devoid of topoisomerase poisoning activity, several dihydrobenz[a]acridines were able to enhance DNA cleavage in the presence of topo I. In contrast to certain protoberberine derivatives that did exhibit activity as topo II poisons, none of the benz[a]acridines derivatives enhanced DNA cleavage in the presence of topo II. Among the benz[a]acridines studied, 5,6-dihydro-3,4-methylenedioxy-9,10-dimethoxybenz[a]acridine, 13e, was the most potent topo I poison, with comparable potency to coralyne. These data suggest that heterocyclic compounds structurally related to coralyne can exhibit potent topo I poisoning activity despite the absence of an iminium cation within their structure. In comparison to coralyne or other protoberberine derivatives, these benz[a]acridine derivatives possess distinctly different physicochemical properties and represent a novel series of topo I poisons.     

Structural investigations of DNA-histone complexes. A spin label study.

We have prepared two acridine spin labels, 6-chloro-9-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)amino]-2-methoxyacridine (I) and 9-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)amino]-acridine (II) and have used them to study the binding of lysine-rich histone (H1) to DNA using electron spin resonance (ESR). ESR spectra of I in the presence of DNA, polydA-polydT and polydG-polydC were characteristic of highly immobilized radicals with maximum hyperfine splitting (2T11) of 59G, 62.5G and 59G respectively. However, the 2T11 values for II in the same systems were 55.5G, 55.5G and 62.5G respectively. Addition of H1 at a low P/D released ionically bound I and II from DNA. In the presence of 0.1 M NaCl, which prevents ionic binding, H1 still caused a significant release of bound II but not I from DNA. At a high P/D (with or without NaCl) H1 caused no displacement of either I or II. Our findings suggest that H1 does not affect the intercalating sites and probably binds to one of the grooves of DNA, most probably the major groove, and specifically in the A-T-rich regions.     

Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine

Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of twisted intercalating nucleic acids possessing acridine derivatives. Thermal stability studies

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been synthesized via the postsynthetic modifications of oligonucleotides possessing insertions of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) at the 5'-end or in the middle as a bulge. In the first postsynthetic step, oligonucleotides 8 and 9 on the CPG support were treated with a Sonogashira coupling reaction mixture containing 9-chloro-2-ethynylacridine or 9-chloro-2-iodoacridine, respectively. After the postsynthetic step, treatment of the oligonucleotides with 32% aq ammonia or 50% ethanolic solution of tris(2-aminoethyl)amine led to the substitution of chloride on acridine concurrent with deprotection of the bases and cleavage of the oligonucleotides from CPG. Molecular modeling of the parallel triplex with a bulged insertion of the monomer (R)-3-O-[4-(9-aminoacridin-2-ylethynyl)benzyl]glycerol in the triplex-forming oligonucleotide (TFO) showed that the acridine moiety was stacking between the bases of the duplex, while phenyl was placed between the bases of the TFO. Thermal denaturation studies and fluorescence properties of TINA-acridine oligonucleotide duplexes and triplexes are discussed.     

The absolute configuration of the enantiomers of 6-chloro-9-(4′-diethylamino-1′-methylbutyl)-amino-2-methoxyacridine (quinacrine)

Absolute configurations have been assigned to the enantiomers of the antimalarial drug quinacrine dihydrochloride. Condensation of (?)-(R)-4-amino-1-diethylaminopentane (from L -glutamic acid) with 6,9-dichloro-2-methoxyacridine gave (?)-(R)-6-chloro-9-(4′-diethylamino-1′-methylbutyl) amino-2-methoxyacridine [(?)-(R)-quinacrine] while (+)?(S)-quinacrine was obtained from the enantiomeric diamine.     

Targeted gene correction with 5' acridine-oligonucleotide conjugates

Single-stranded oligonucleotides (SSOs) mediate gene repair of punctual chromosomal mutations at a low frequency. We hypothesized that enhancement of DNA binding affinity of SSOs by intercalating agents may increase the number of corrected cells. Several biochemical modifications of SSOs were tested for their capability to correct a chromosomally integrated and mutated GFP reporter gene in human 293 cells. SSOs of 25 nucleotide length conjugated with acridine at their 5' end increased the efficiency of gene correction up to 10-fold compared to nonmodified SSOs. Acridine and psoralen conjugates were both evaluated, and acridine-modified SSOs were the most effective. Conjugation with acridine at the 3' end of the SSO inhibited gene correction, whereas flanking the SSO by acridine on both sides provided an intermediate level of correction. These results suggest that increasing the stability of hybridization between SSO and its target without hampering a 3' extension improves gene targeting, in agreement with the "annealing-integration" model of DNA repair.     

Antimalarial acridines: Synthesis,in vitro activity against P. falciparum and interaction with hematin

A series of acridine derivatives were synthesised and their in vitro antimalarial activity was evaluated against one chloroquine-susceptible strain (3D7) and three chloroquine-resistant strains (W2, Bre1 and FCR3) of Plasmodium falciparum. Structure–activity relationship showed that two positives charges as well as 6-chloro and 2-methoxy substituents on the acridine ring were required to exert a good antimalarial activity. The best compounds possessing these features inhibited the growth of the chloroquine-susceptible strain with an IC₅₀ ? 0.07 μM, close to that of chloroquine itself, and that of the three chloroquine-resistant strains better than chloroquine with IC₅₀ ? 0.3 μM. These acridine derivatives inhibited the formation of β-hematin, suggesting that, like CQ, they act on the haem crystallization process. Finally, in vitro cytotoxicity was also evaluated upon human KB cells, which showed that one of them 9-(6-ammonioethylamino)-6-chloro-2-methoxyacridinium dichloride 1 displayed a promising antimalarial activity in vitro with a quite good selectivity index versus mammalian cell on the CQ-susceptible strain and promising selectivity on other strains.