Quantitative analysis of DNA-porphyrin interactions

The binding of manganese(III)-tetra(4-N-methylpyridyl)porphyrin (MnTMpyP) with synthetic poly(dA-dT)2, poly(dI-dC)2, and poly(dG-dC)2 DNAs as well as calf thymus (CT) DNA has been quantitatively studied in detail using induced CD (circular dichroism) spectroscopy in the Soret absorption band. The CD spectra, which changed greatly depending on the porphyrin to DNA base-pair molar ratio (r), were normalized with respect to DNA concentration and deconvoluted. Three independent component binding modes (named mode 1, 2, and 3 in the order of increasing r values) were identified, which successfully simulated the observed CD spectra with negligibly small residuals for a wide range of r values. In the case of poly(dA-dT)2, poly (dI-dC)2, and CT DNA, all the three modes appeared, whereas in the case of poly(dG-dC)2 DNA, only modes 1 and 3 appeared in the r range studied. The r dependence of each binding mode, i.e., its relative affinity toward DNA, has been revealed by this analysis. Mode 1, which appeared as a single binding mode at very low r values (r < or = ca. 0.05), was inhibited by the addition of methyl green, a drug that preferentially binds to the major groove of poly (dA-dT)2 DNA. Berenil, a known minor groove binder to poly(dA-dT)2 or poly(dI-dC)2 DNA, inhibited modes 2 and 3. From these inhibition experiments as well as comparison of the component spectra for DNAs of different sequence, a binding site on DNA was proposed for each component binding mode. The number of DNA base pairs covered by a single molecule of porphyrin was estimated.     

Binding and photocleavage of cationic porphyrin-phenylpiperazine hybrids to DNA

The binding properties of cationic porphyrin-phenylpiperazine hybrids to calf thymus (CT) DNA were investigated by using absorption, fluorescence and circular dichroism (CD) spectra, and the apparent affinity binding constants (K(app)) of the porphyrins for CT DNA were determined by using a competition method with ethidium bromide (EB). Intercalation of porphyrin into CT DNA occurred when two phenylpiperazines were introduced at cis position onto the periphery of cationic porphyrin. The photocleavages of pBR322 plasmid DNA by the porphyrins were consistent with the values of K(app). With [porphyrin]/[DNA base pairs] ratio increased, the binding mode tended to be outside binding, and the cleavage abilities of the porphyrins varied. In the presence of sodium azide, a quencher of 1O2, the cleavage of DNA by the porphyrin of intercalation was less inhibited.     

Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-Tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine)phenyl]porphyrin

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine)phenyl]porphyrin (TθF₄TAP) has been synthesized. The binding interactions of TθF₄TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TθF₄TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TθF₄TAP binds to calf thymus DNA, [poly(dA-dT)]₂, and [poly(dG-dC)]₂ in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TθF₄TAP/CT DNA resembled that of TθF₄TAP/[poly(dA-dT)]₂, suggesting that TθF₄TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TθF₄TAP binds preferentially to [poly(dA-dT)]₂ over [poly(dG-dC)]₂. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TθF₄TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TθF₄TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation. © 1997 John Wiley & Sons, Inc. Biopoly 42: 203–217, 1997     

DNA-binding affinities and sequence specificities of protoberberine alkaloids and their demethylated derivatives: a comparative study

Berberrubine (1a), jatrorubine (2a), and palmatrubine (3a) have been chemically prepared by partial demethylation of berberine (1), jatrorrhizine (2), and palmatine (3), respectively. Their interactions with calf thymus (CT) DNA, poly(dA-dT)poly(dA-dT), poly(dG-dC)poly(dG-dC), and eight AT-rich 12-mer double-stranded DNAs have been investigated by means of competitive ethidium bromide (EB) displacement experiments. The results showed that DNA-binding affinities of these protoberberine alkaloids have been significantly improved by partial demethylation, and that all of these alkaloids have the preferable binding affinities with AT-rich DNA. Especially, the sequence specificities of DNA-binding of demethylated derivatives 1a, 2a, and 3a had changed to a certain extent when compared with the parent alkaloids 1, 2, and 3, respectively. The binding mode of these alkaloids was further confirmed by UV spectroscopic titration experiments. All the compounds bind to double-stranded DNA most probably via an intercalating mode.     

DNA-binding studies of mixed-ligand (ethylenediamine)ruthenium(II) complexes

A series of mixed-ligand ruthenium(II) complexes of the type [Ru(en)(2)bpy](2+) (bpy=2,2-bipyridine; 1), [Ru(en)(2)phen](2+) (phen=1,10-phenantroline; 2), [Ru(en)(2)IP](2+) (IP=imidazo[4,5-f][1,10]phenanthroline; 3), and [Ru(en)(2)PIP](2+) (PIP=2-phenylimidazo[4,5-f][1,10]phenanthroline; 4) have been isolated and characterized by UV/VIS, IR, and (1)H-NMR spectral methods. The binding of the complexes with calf thymus DNA has been investigated by absorption, emission spectroscopy, viscosity measurements, DNA melting, and DNA photo-cleavage. The spectroscopic studies together with viscosity measurements and DNA melting studies support that complexes 1 and 2 bind to CT DNA (=calf thymus DNA) by groove mode. Complex 2 binds more avidly to CT DNA than complex 1, complexes 3 and 4 bind to CT DNA by intercalation mode, 4 binds more avidly to CT DNA than 3. Noticeably, the four complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA.     

Binding of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin to an AT-rich region of a duplex DNA

Characterization of the interaction between DNA and small organic compounds is of considerable importance for gaining insights into the mechanism underlying molecular recognition, which could be highly relevant to drug design. In the present study, the interaction of a water-soluble cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin, with a self-complementary duplex DNA, d(GCTTAAGC)2, has been investigated by means of absorption, circular dichroism, and NMR spectroscopies. The optical studies indicated that TMPyP binds to the TTAA region of d(GCTTAAGC)2 with a binding constant of 2.5 x 10(6) M(-1) and a stoichiometric ratio of 1:1. The observation of intermolecular nuclear Overhauser effect connectivities demonstrated that TMPyP binds in the major groove of d(GCTTAAGC)2. A model for the binding of TMPyP in the major groove of the AT-rich region of d(GCTTAAGC)2 is proposed.     

Synthesis and DNA-recognition and -cleavage properties of multiply charged porphyrin esters

A series of systematically modified porphyrin esters, compounds 1-6, with multiple, permanent positive charges introduced at the meso-positions via N-methylated 4-, 3-, or 2-pyridyl moieties, were prepared and characterized. Their singlet-oxygen production, CT-DNA-binding properties, and plasmid-DNA photocleavage propensities were determined spectroscopically and by gel electrophoresis, and compared to those of the known, fourfold-charged parent porphyrin 4,4',4',4'-porphyrin-5,10,15,20-tetrayltetrakis(1-methylpyridinium) (TMPyP4). Some interesting structure-activity relationships could be established to rationalize effects affecting DNA binding mode and cleavage ability.     

Tricationic pyridium porphyrins appending different peripheral substituents: experimental and DFT studies on their interactions with DNA

Four tricationic pyridium porphyrins appending hydroxyphenyl, methoxyphenyl, propionoxyphenyl or carboxyphenyl group at meso-20-position of porphyrin core have been synthesized and their abilities to bind and cleave DNA have been investigated. Using a combination of absorption, fluorescence, circular dichroism (CD) spectra, thermal DNA denaturation as well as viscosity measurements, their binding modes and intrinsic binding constants (K_b) to calf DNA (CT DNA) were comparatively studied and also compared with those of 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP). The results suggest that the K_b values of these porphyrins are greatly influenced by the number of positive charges and steric hindrance. Theoretical calculations applying the density functional theory (DFT) have been carried out and explain their DNA-binding properties reasonably. The efficiency of DNA photocleavage by these porphyrins shows high dependence on the values of K_b.     

DNA-binding and photocleavage properties of cationic porphyrin-anthraquinone hybrids with different lengths of links

Four cationic porphyrin–anthraquinone (Por–AQ) hybrids differing in lengths of flexible alkyl linkage, 5-[4-(1-N-anthraquinonon-yl)-l-oxophenyl]-10,15,20-tris(N-methylpyridinium-4-yl)porphyrin triiodide, (l = acetyl, pentanoyl, octanoyl, undecanoyl, designed as [AQATMPyP]I₃, [AQPTMPyP]I₃, [AQOTMPyP]I₃ and [AQUTMPyP]I₃, respectively, see Fig. 1), were synthesized and their interactions with DNA were investigated. The results of spectroscopic, denaturation and viscosity measurements suggest that [AQATMPyP]I₃ binds to DNA through non-intercalative mode while the other three hybrids with longer links bind via bis-intercalative mode. Ethidium bromide (EB) competition experiment was carried out to determine the binding constants (K_b) of these compounds for CT DNA, and [AQPTMPyP]I₃ shows the largest K_b among these hybrids. The photocleavage mechanism and wavelength-dependent cleaving abilities of these hybrids to pBR322 plasmid DNA were also comparably investigated.     

Cationic porphyrins bearing diazolium rings: synthesis and their interaction with calf thymus DNA

Two novel cationic porphyrins bearing five-membered rings at the meso-positions, meso-tetrakis(1,3-dimethylimidazolium-2-yl)porphyrin (H2TDMImP) and meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (H2TDMPzP), have been synthesized. These two compounds interact with calf thymus DNA (CTDNA) in different binding modes from that of mesotetrakis(N-methylpyridinium-4-yl)porphyrin (H2TMPyP). H2TDMImP outside binds to the minor groove of CTDNA while H2TDMPzP intercalates into CTDNA. These two novel cationic porphyrins strongly bind to CTDNA even at high ionic strength and the binding constant of H2TDMPzP to CTDNA is comparable to that of H2TMPyP. The binding of H2TDMImP to CTDNA is enthalpically driven. The favorable free energy changes in binding of H2TDMPzP to CTDNA come from the large negative enthalpy changes accompanied by small positive entropy changes.     

DNA binding studies of a new dicationic porphyrin. Insights into interligand interactions

Cationic porphyrins have an affinity for DNA and potential for applications in the fields of photodynamic therapy and cellular imaging. This report describes a new dicationic porphyrin, 5,15-dimethyl-10,20-di(N-methylpyridinium-4-yl)porphyrin, abbreviated H2tMe2D4. Although tetrasubstituted, H2tMe2D4 presents modest steric requirements and forms in reasonable yield by a "2+2" synthetic method. Accordingly, studies of the zinc(II)- and copper(II)-containing derivatives, Zn(tMe2D4) and Cu(tMe2D4), have also been possible. Methods used to characterize DNA-binding motifs include absorption, emission, linear, and circular dichroism spectroscopies, as well as viscometry. An unusually detailed picture of porphyrin uptake emerges. As the ratio of DNA to porphyrin increases during a typical titration, H2tMe2D4 or Cu(tMe2D4) initially aggregates on the host and then shifts to intercalative binding at close quarters before finally dispersing into non-interacting intercalation sites of the host. Emission studies of the copper(II) porphyrin have been very valuable. The existence of a measurable signal is diagnostic of intercalative binding, and the saturation behavior establishes that internalization typically monopolizes approximately three base pairs. In the moderate loading regime, emission data are most telling because dipole-dipole interactions between near-neighbor porphyrins tend to confuse other spectroscopic assays. The third ligand, Zn(tMe2D4), behaves differently in that the uptake is a strictly cooperative process. The mode of binding also varies with the base content of the DNA host. When the DNA is rich in A=T base pairs, the porphyrin remains five-coordinate and binds externally; however, Zn(tMe2D4) loses its axial ligand and binds by intercalation if the host contains only G[triple bond]C base pairs.     

Different Binding Mode in AT and GC Sequences for Unfused-Aromatic Dications

Abstract

We have previously synthesized a 2,5-diphenylfuranamidine dication (4) and presented evidence that this compound binds to AT sequences in DNA by a minor-groove interaction mode but binds to GC sequences by intercalation (1,2). To probe these sequence-dependent binding modes in more detail, and particularly to obtain additional evidence for the binding mode in GC rich sequences, we have synthesized and studied the DNA complexes of 1–3 which have the furan ring of 4 replaced by 2,6-substituted pyridine (1), pyrimidine (2), or triazine (3) ring systems. The three compounds with a six-membered central ring system bind to AT DNA sequences more weakly than the furan compound, but retain the minor-groove binding mode. The pyridine and pyrimidine derivatives bind to GC sequences of DNA more strongly than the furan, but the triazine derivative binds more weakly. The aromatic proton signals of 1–3, as previously observed with 4 shift upfield by approximately 0.5 ppm or greater on complex formation with polyd(G-C)₂. This and other spectroscopic as well as viscosity and kinetics results indicate that 1–4 bind to GC sites in DNA by intercalation. A nonclassical intercalation model, with the twisted-unfused, aromatic ring system intercalated into an intercalation site of matching structure can explain all of our and the literature results for the GC binding mode of these unfused, aromatic compounds.     

Synthesis and characterization of water-insoluble and water-soluble dibutyltin(IV) porphinate complexes based on the tris(pyridinyl)porphyrin moiety,their anti-tumor activity in vitro and interaction with DNA

The water-insoluble and water-soluble organotin(IV)porphinate complexes based on the tris-(4-pyridinyl)porphyrin and tris(N-methyl-4-pyridiniumyl)porphyrin moieties were synthesized and characterized by elemental analysis, (1)H NMR, IR and electrospray ionization mass spectra. The in vitro activity of the compounds against P388 leukemia and A-549 was determined. The results show that the anti-tumor activities of organotin(IV)porphinate is related to the water solubility of the compounds and the central ion in the porphyrin ring. The interaction between the water-soluble dibutyltin(IV) porphinate (7 and 10) complexes and DNA has been investigated. The result shows that compounds 7 and 10 cause DNA hypochromism measured by A(260), a slight increase in the viscosity of the DNA, and an increase in the melting point of DNA by 2.9 and 1.6 degrees C, respectively at DNA(base)/Drug(Por) ratios of 60. The binding constants to DNA were 1.35+/-0.16 x 10(7) M(-1) (7) and 1.45+/-0.12 x 10(6) M(-1) (10) determined using EB competition method based on the porphyrin concentration, which is 20 and five times greater than that of precursor porphyrins [5-p,o-(carboxy)methoxyphenyl-10,15,20-tris(N-methyl-4-pyridiniumyl)] porphyrin (p,o-tMPyPac) to DNA. Electrophoresis test shows that the compounds cannot cleave the DNA. According to the electrophoresis test result and all the above results, the cytotoxic activity against P388 and A-549 tumor cells appears not to come from the cleavage of DNA caused by the compounds but from the high affinity of compounds to DNA.     

Design,synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIα-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIα decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA–topoisomerase IIα covalent complex indicating that they acted as topoisomerase IIα poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.     

Porphyrin intercalation and non-specific 'edge on' outside binding to natural DNA

A theoretical two-mode binding model for porphyrin binding to natural DNA is presented. One of the binding modes is assumed to be base sequence specific with binding sites n base-pairs long. The other binding mode has binding sites which consist of only one base-pair and can involve cooperativity. The model fits satisfactorily to data for H2TMPyP-4, Cu(II)TMPyP-3 and Cu(II)TMPyP-4 binding to calf thymus DNA in both a high (mu congruent to 1.0 M) and a low (mu congruent to 0.2 M) ionic strength buffer. The results show that the fraction of porphyrin bound in the non-specific mode reaches a maximum at certain input DNA to porphyrin concentrations ratios. The value of this maximum decreased, and its position shifted to higher DNA to porphyrin concentration ratios for binding in the high ionic strength buffer. The value of the cooperativity parameter obtained through the fitting process suggests that the non-specific binding is positively cooperative. The results are compared with the data analysed using other techniques.     

Synthesis, DNA binding, and cleavage studies of novel PNA binding cyclen complexes

A novel coumarin‐appended PNA binding cyclen derivative ligand, C1 , and its copper(II) complex, C2 , have been synthesized and characterized. The interaction of these compounds with DNA was systematically investigated by absorption, fluorescence, and viscometric titration, and DNA‐melting and gel‐electrophoresis experiments. DNA Melting and viscometric titration experiments indicate that the binding mode of C1 is a groove binding, and C2 is a multiple binding mode that involves groove binding and electrostatic binding. From the absorption‐titration data, we can state that the primary interaction between CT DNA and the two compounds may be H‐bonds between nucleobases. Fluorescence studies indicate that the binding ability of C1 to d(A)₉ is as twice or thrice as that of other oligodeoxynucleotides. Agarose gel‐electrophoresis experiments demonstrate that C2 is an excellent chemical nuclease, which can cleave plasmid DNA completely within 24 h.     

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.     

Binding, electrochemical activation, and cleavage of DNA by cobalt(II) tetrakis-N-methylpyridyl porphyrin and its beta-pyrrole brominated derivative

The binding of nucleic acids by water-soluble cobalt(II) tetrakis-N-methylpyridyl porphyrin, (TMPyP)Co, and its highly electron-deficient derivative cobalt(II) tetrakis-N-methyl pyridyl-beta-octabromoporphyrin, (Br(8)TMPyP)Co, was investigated by UV-visible absorption, circular dichroism (CD), and electrochemical and gel electrophoresis methods. The changes of the absorption spectra during the titration of these complexes with polynucleotides revealed a shift in the absorption maxima and a hypochromicity of the porphyrin Soret bands. The intrinsic binding constants were found to be in the range of 10(5)-10(6) M(-1). These values were higher for the more electron-deficient (Br(8)TMPyP)Co. Induced CD bands were noticed in the Soret region of the complexes due to the interaction of these complexes with different polynucleotides, and an analysis of the CD spectra supported a mainly external mode of binding. Electrochemical studies revealed the cleavage of polynucleotides by (TMPyP)Co and (Br(8)TMPyP)Co in the presence of oxygen preferentially at the A-T base pair region. Gel electrophoresis experiments further supported the cleavage of nucleic acids. The results indicate that the beta-pyrrole brominated porphyrin, (Br(8)TMPyP)Co, binds strongly and cleaves nucleic acids efficiently as compared with (TMPyP)Co. This electrolytic procedure offers a unique tool in biotechnology for cleaving double-stranded DNA with specificity at the A-T regions.     

Quantitative footprinting analysis using a DNA-cleaving metalloporphyrin complex

The results of quantitative footprinting studies involving the antiviral agent netropsin and a DNA-cleaving cationic metalloporphyrin complex are presented. An analysis of the footprinting autoradiographic spot intensities using a model previously applied to footprinting studies involving the enzyme DNase I [Ward, B., Rehfuss, R., Goodisman, J., & Dabrowiak, J. C. (1988) Biochemistry 27, 1198-1205] led to very low values for netropsin binding constants on a restriction fragment from pBR-322 DNA. In this work, we show that, because the porphyrin binds with high specificity to DNA, it does not report site loading information in the same manner as does DNase I. We elucidate a model involving binding equilibria for individual sites and include competitive binding of drug and porphyrin for the same site. The free porphyrin and free drug concentrations are determined by binding equilibria with the carrier (calf thymus DNA) which is present in excess and acts as a buffer for both. Given free porphyrin and free netropsin concentrations for each total drug concentration in a series of footprinting experiments, one can calculate autoradiographic spot intensities in terms of the binding constants of netropsin to the various sites on the 139 base pair restriction fragment. The best values of these binding constants are determined by minimizing the sum of the squared differences between calculated and experimental footprinting autoradiographic spot intensities. Although the determined netropsin binding constants are insensitive to the value assumed for the porphyrin binding constant toward its highest affinity sites, the best mean-square deviation between observed and calculated values, D, depends on the choice of (average) drug binding constant to carrier DNA, Kd.(ABSTRACT TRUNCATED AT 250 WORDS)