Study of the interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)Pt(II) complex with poly(I), poly(C) and poly(I) x poly(C)

The interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)platinum(II) (Ptpyrr) with the polynucleotides poly(I), poly(C) and poly(I) x poly(C) acids was studied by circular dichroism, molecular fluorescence and (1)H NMR spectroscopies. Multivariate Curve Resolution, a factor analysis method, was applied for the analysis and interpretation of spectroscopic data obtained in mole ratio and kinetics studies. This procedure allows the determination of the number of different interaction complexes present during the experiments and the resolution of both concentration profiles and pure spectra for all of them. Two different interaction complexes were observed at the experimental conditions studied. The first one, at low Ptpyrr:polynucleotide ratio (r(Ptpyrr:poly)) values, corresponds to the interaction of Ptpyrr with hypoxanthine bases in the poly(I) moiety. This interaction leads to the destabilization and dissociation of the double-stranded conformation. The second complex was observed at higher r(Ptpyrr:poly) values and corresponds to the interaction of Ptpyrr to cytosine bases in poly(C) moiety. The formation of both complexes showed that the interaction of Ptpyrr with hypoxanthine bases occurred at the first stages of the reaction and with cytosine bases at longer reaction times. The results obtained show the utility of the Multivariate Curve Resolution approach for the analysis of data obtained by monitoring spectroscopically the interaction equilibria of platinum compounds with nucleic acids.     

Interactions of cis- and trans-platinum(II) complexes with dehydrogenase enzymes in the presence of different mono- and polynucleotides: evidence for a ternary complex

The inhibition of several dehydrogenase enzymes by cis- and trans-Pt(NH3)2Cl2 have been measured in the presence of baker yeast ribonucleic acid (RNA), calf thymus and salmon sperm deoxyribonuclic acid (DNA) and several mononucleotides (AMP and ATP). The binding constants for the interaction of the platinum complexes to the nucleotides have been calculated and a comparison of those values to the previously calculated platinum complex-enzyme binding constants strongly suggest that platinum compounds are more tightly bound to the enzymes. The binding of the platinum complexes to most of the enzymes was decreased in the presence of any nucleotide, yet it was observed that when using rabbit muscle (M4) lactate dehydrogenase the mononucleotides reduced the binding to a lesser degree while the polynucleotides actually enhanced the platinum-enzyme interaction. The implications of these interactions are discussed.     

Optically detected magnetic resonance of tryptophan residues in Escherichia coli ssb gene product and E. coli plasmid-encoded single-stranded DNA-binding proteins and their complexes with poly(deoxythymidylic) acid

Optically detected magnetic resonance (ODMR) spectroscopy has been applied to several single-stranded DNA-binding (SSB) proteins encoded by conjugative plasmids of enteric bacteria. Fluorimetric equilibrium binding isotherms confirm their preferential binding to single-stranded DNA and polynucleotides and reveal a limited protein solubility at low ionic strength. The plasmid SSB-like proteins show the highest affinity for polydeoxythymidylic acid; these complexes are the least sensitive to disruption by salt. ODMR data on these complexes suggest the existence of stacking interactions between tryptophan residue(s) and thymine bases, as evidenced by spectral red shifts of the tryptophan phosphorescence 0,0 band, reduction of the magnitude of D zero field splitting parameter, and a dramatic reversal of the polarity of the ODMR signals. Wavelength-selected ODMR results point to the existence of two distinct tryptophan sites in these complexes. The triplet state properties of the red-shifted site are drastically altered by its interaction with the thymine bases. The chromosomal Escherichia coli SSB protein-poly(dT) complex shows an additional tryptophan site with zero field splitting parameters similar to those of the free protein. This site can be attributed to Trp-135, which is missing in each of the other plasmid SSB proteins, suggesting that this particular residue is not involved in the interaction with polynucleotides.     

Interaction of cisplatin and DNA-targeted 9-aminoacridine platinum complexes with DNA

Interaction of acridine- and 9-aminoacridinecarboxamide platinum complexes with DNA was investigated with respect to their DNA sequence specificity and kinetics of binding. The DNA sequence specificity of the compounds was quantitatively analyzed using a polymerase stop assay with the plasmid pUC19. The 9-aminoacridinecarboxamide platinum complexes exhibited a different sequence specificity to that of cisplatin, shifted away from runs of consecutive guanines (the main binding site for cisplatin). This alteration was dependent on chain length. Shorter chain length compounds (n = 2, 3) showed a greater difference in sequence specificity, while longer chain length compounds (n = 4, 5) more closely resembled cisplatin. An acridinecarboxamide platinum complex showed a similar sequence specificity to cisplatin, revealing that the major change of sequence specificity was due to the presence of the 9-amino substituent. A linear amplification system was used to investigate the time course of the reaction. The presence of an intercalating group (acridinecarboxamide or 9-aminoacridinecarboxamide) greatly increased the rate of reaction with DNA; this is proposed to be due to a different reaction mechanism with DNA (direct displacement by the N-7 of guanine).     

Chemical reactivity of monofunctional platinum-DNA adducts

Complexes formed in vitro between cis- or trans-PtCl2(NH3)2 (DDP) and DNA were found to contain monofunctional adducts that reacted with exogenous guanosine. [14C]Guo bound irreversibly to cis- and trans-DDP-DNA complexes to form bis-Gua adducts. The reaction was first order with respect to the concentration of both [14C]Guo and platinum-DNA complex, but the rate of the reaction varied nonlinearly as a function of the level of platinum binding on DNA. The reaction between [14C]Guo and these platinum-DNA complexes was used to probe the concentration and stability of the monofunctional adducts and to investigate their chemistry in situ. The concentration of monofunctional adducts was highest immediately after reaction of DDP with DNA for 2 h at 37 degrees C, at which time they represented greater than 15% of the cis-DDP-DNA lesions and on the order of 80% of the trans-DDP-DNA lesions. The cis-DDP-DNA complex reacted with [14C]Guo by two kinetically distinct processes, indicating two types of reactive adducts. The most reactive adduct represented 5% of the platinum lesions. These monofunctional adducts disappeared during the incubation of the platinum-DNA complexes in the absence of drug, probably as a result of chelation to DNA. The half-lives of this chelation at 37 degrees C, 10 mM NaClO4, were 15 and 30 h for the cis and trans complexes, respectively. Monofunctional adducts were formed on Gua bases in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of gene 5 protein of phage f1 with single and double-stranded DNA and polynucleotides

The fluorescence method was used to reveal some differences in the interaction of gene 5 protein of phage f1 with single- and double-stranded polynucleotides (DNA). The binding with the duplexes is non-cooperative and the Kapp is twice lower than that for the cooperative formation of the complex with single-stranded structures. In the complex with a double-stranded polynucleotide (DNA) the protein cover 3 nucleotide pairs. The complex dissociates with a lower concentration of salt and the contribution of the energy of nonelectrostatic interactions to the total energy of complex formation for it is lower than for the complex with single-stranded DNA. In the complex of protein with single-stranded structure the fluorescence of the tyrosine (Tyr) residues is quenched to a greater degree and their accessibility to the external quencher is lower than that of the complex with double-stranded polynucleotides (DNA). The suggestion is made that in destabilization of nucleic double helices by gene 5 protein of phage f1, a great role belongs to Tyr residues because of their high affinity to single-stranded structures and because of their different localization in the complexes with single- and double-stranded polynucleotides.     

Cis-platinum(II) complexes of carboxymethyl-dextran as potential antitumor agents. I. Preparation and characterization

Cis-diamminedichloro platinum (II) (cis-DDP) and cis-diamminediaquo platinum (II) nitrate (cis-aq) were demonstrated to form complexes with dextran (dex) substituted with carboxymethyl (CM) groups at an average substitution ratio of 1 mole CM per 2 mole glucose units of dextran. The complexes were formed by reacting each of the two platinum (II) derivatives with carboxymethyl-dextran (CM-dex) at room temperature (RT) or at 37 degrees C in an aqueous solution. The complexing rate depended on temperature, ratio of platinum (II) compounds to CM-dex in the reaction mixture, and time of reaction. Experiments were performed with two CM-dex preparations, derived from dex T-10 (Mr-10,000) and from dex T-40 (Mr-40,000). Soluble cis-DDP and cis-aq complexes formed with CM-dex T-10 and CM-dex T-40 could carry up to 15 mole or 60 mole of the platinum (II) compounds per 1 mole CM-dex, respectively but higher complexing ratios resulted in complex precipitation. Reactivity of cis-aq with CM-dex was higher than that of cis-DDP. NaCl interfered with complex formation, but did not cause dissociation of already formed complexes. The binding of cis-DDP and cis-aq to CM-dex is, however, reversible since the drugs could be exchanged by other acceptors of higher affinity to platinum (II) such as O-phyenlenediamine, or DNA.     

Importance of DNA conformation in the reaction with cis-dichlorodiammine platinum (II)

Cis-dichlorodiammine platinum (II) has been reacted with synthetic polynucleotides either in B or in Z conformation. The binding of cis-dichlorodiammine platinum (II) stabilizes the Z conformation when reacted with poly (dG-m⁵dC) ·poly (dG-m⁵dC) in the Z conformation as shown by circular dichroism and by the antibodies to Z-DNA. On the other hand, the binding of cis-dichlorodiammine platinum (II) stabilizes a new conformation when reacted with poly(dG-dC)·poly(dG-dC) or poly (dG-m⁵dC)·poly(dG-m5dC) in the B conformation. The antibodies to Z-DNA bind to these platinated polynucleotides. In rabbits, the injection of platinated poly (dG-dC) poly (dG-dC) induces the synthesis of antibodies which recognize Z-DNA. In low salt conditions, the circular dichroism spectra of these platinated polynucleotides differ from those of B-DNA or Z-DNA. The characteristic³¹P nuclear magnetic resonance spectrum of Z-DNA is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Thermodynamic Characteristics of Plasminogen Activation by Indirect Activators

Several indirect plasminogen (Pg) activators are known including streptokinase and the monoclonal antibody IV-Ic, whose mechanism of activation is well studied. To characterize thermodynamically the activation of Pg by streptokinase (SK) and the monoclonal antibody (mAB) IV-Ic, the activation energies were calculated for various reaction stages. Activation energy of 7.4 kcal/mol was determined for the interaction of the chromogenic substrate S-2251 with plasmin (Pm) and activated equimolar complexes Pm-SK and Pg*SK at the steady-state reaction stage, and 18.7 kcal/mol with the complexes Pg*IV-Ic. A 2.5-fold increase in the energy of activation for the Pg*IV-Ic complex suggests a more intricate mechanism of its interaction with the substrate. At the stage of increasing active center concentrations and the formation of activated complexes Pg*SK and Pg*mAB IV-Ic, the activation energy was found to be 10.5 and 38 kcal/mol, respectively. At this reaction stage the conformational rearrangement of Pg molecule with the formation of active center is the limiting stage determining the reaction rate. Unexpectedly high energy of activation at the second stage of interaction between mAB IV-Ic and Pg suggests several simultaneous reactions and complexity of conformation rearrangement in the Pg molecule in activated complexes, thus requiring large energy expense. Formation of the active center is probably accompanied by its transition within a narrow temperature range into another conformation state with the change in activation parameters of the reaction. Quantitative evaluation of the studied reactions from the perspective of thermodynamics of the enzymatic reactions gives more comprehensive characteristics of the activation mechanism.     

Methyl iodide reactions on the surface of mechanically pre-activated platinum(II) salt in the heterogeneous system: K2PtCl4 powder–MeI vapor

Mechanically pre-activated K₂PtCl₄ salt consumes methyl iodide producing methyl chloride at room temperature. The reaction mechanism includes the following steps sequence: oxidative addition of methyl iodide to platinum(II) complexes with intermediate formation of methyl platinum(IV) complexes and further decomposition of the latter in the course of innersphere reductive elimination yielding methyl chloride. The first step of the reaction proceeds owing to the assistance of active centers regenerated in the course of each event of MeI into MeCl transformation taking part in the chain halogen substitution process. It could be assumed that the role of active centers is played by coordinatively unsaturated platinum(II) complexes located on the surface. These species bearing a positive efficient charge can render electrophilic assistance for the nucleophilic substitution. The chain termination can be caused by recombination of coordinatively unsaturated platinum(II) complexes and interstitial chloride ions forming an inactive K₂PtCl₄ complex.     

Complex between single-stranded DNA and gene 5 protein of bacteriophage M13 studied with linear dichroism and ultraviolet absorption

We have studied complexes between the gene 5 protein (gp5) of bacteriophage M13 and various polynucleotides, including single-stranded DNA, using ultraviolet absorption and linear dichroism. Upon complex formation the absorption spectra of both the protein and the polynucleotides change. The protein absorption changes indicate that for at least two of the five tyrosine residues per protein monomer the environment becomes less polar upon binding to the polynucleotides but also to the oligonucleotide p(dT)8. All gp5-polynucleotide complexes give rise to intense linear dichroism spectra. These spectra are dominated by negative contributions from the bases, but also a small positive dichroism of the protein can be discerned. The spectra can be explained by polynucleotide structures, which are the same in all complexes. The base orientations are characterized by a substantial inclination and propellor twist. The number of possible combinations of inclination and propeller twist values, which are in agreement with the linear dichroism results, is rather limited. The base orientations with respect to the complex axis are essentially different from those in the complex with the single-stranded DNA-binding protein gp32 of bacteriophage T4.     

Concentration and temperature effects in interactions of the dye pyronine G with polynucleotides

The interaction of poly(A) and poly(A).poly(U) with pyronine G dye depending on the concentration of components and temperature was studied spectrophotometrically in the visible and UV ranges at pH (6.86). It was found that the interaction of pyronine G with poly(A) and poly(A).poly(U) results in the formation of two types of complexes. The relation of the equilibrium concentrations of these complexes depends on the initial concentrations of the components in solution. The formation of complex I results in shifting the spectrum towards the short wave range with regard to the monomer band and reflects the aggregation of the dye cations. Complex II is characterized by the shift towards the long wave range. Complex II is formed in considerable amounts for poly(A).pyronine G system at large P/D and for poly(A).poly(U).pyronine G system at P/D = 5-6 and is probably due to the interaction between the dye and polynucleotides of the intercalation type or reflects the interaction between the dye and two negatively charged phosphate groups. Analysis of temperature measurements of spectra confirms the formation of various types of complexes in the system studied.     

The effect of mRNA on the stability of the ribosome--aminoacyl-tRNA complex]

The effect of some olygo- and polynucleotides on the dissociation rate of the 14C-aminoacyl-tRNA - ribosome complex was investigated. Polyuridylic and polycytidylic acids were shown to accelerate significantly dissociation of the complex of lysyl- and phenylalanyl-tRNA with native ribosomes, but not to affect the complexes of these aminoacyl-tRNA's with 50S subunits. It is proposed that the template polynucleotides decrease the affinity of ribosomes to tRNA by association with the mRNA-binding site on 30S subunits.     

Anticancer activity of new imidazole derivative of 1R,2R-diaminocyclohexane palladium and platinum complexes as DNA fluorescent probes

The aim of this study was synthesis of two new water-soluble fluorescent palladium and platinum complexes with formulas of [Pt(DACH)(FIP)](NO₃)₂ and [Pd(DACH)(FIP)](NO₃)₂, respectively, where FIP is 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10] phenanthroline and DACH is 1R,2R-diaminocyclohexane. Fluorescence spectroscopy, circular dichroism (CD), thermal denaturation measurement, ionic strength, and kinetic study displayed groove binding of Pt complex on DNA, while due to binding of Pd complex, B form of DNA convert to Z form. Due to electrostatic interaction of Pd complex with DNA, the DNA form is converted and it provides enough space for Pd complex to insert between base stacking of DNA. UV–vis study shows two complexes could denature the DNA at low concentrations in exothermic process and Pt complex is more active than Pd complex. Finally, the anticancer and growth inhibitory activities of synthesized complexes were investigated against human colon cancer cell line HCT116 after incubation time of 24 h using MTT assay and higher activity was observed for the platinum complex. Interaction of the two metal derivative complexes was studied by molecular docking and molecular dynamics simulation. The results showed that Pt complexes have higher negative docking energy and higher tendency for interaction with DNA, and exert more structural change on DNA.     

Synthesis,characterization and DNA binding studies of platinum(II) complexes with benzimidazole derivative ligands

The aim of this study was to synthesize and evaluate plasmid DNA interaction of new platinum(II) complexes with some 2-substituted benzimidazole derivatives as carrier ligands which may have potent anticancer activity and low toxicity. Twelve benzimidazole derivatives carrying indole, 2-/or 3-/or 4-methoxyphenyl, 4-methylphenyl, 3,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 4-methoxybenzyl, 3,4,5-trimethoxybenzyl, 3,4,5-trimethoxystyryl, 3,4,5-trimethoxybenzylthio or dimethylamino ethyl groups in their position 2 and twelve platinum(II) complexes with these carrier ligands were synthesized. The chemical structure of the platinum complexes have been characterized by their elemental analysis and FIR, ¹H NMR and mass spectra and their ¹H NMR and FIR spectra were interpreted by comparison with those of the ligands. The interaction of all the ligands and their complexes with plasmid DNA and their restriction endonuclease reactions by BamHI and HindIII enzymes were studied by agarose gel electrophoresis. It was determined that complex 1 [dichloro-di(2-(1H-indole-3-yl)benzimidazole)platinum(II)·2H₂O] has stronger interaction than carboplatin and complex 10 [dichloro-di(2-(3,4,5-trimethoxystyryl)benzimidazole)platinum(II)·2H₂O] has stronger interaction than both carboplatin and cisplatin with plasmid DNA.     

Interaction of Hoechst 33258 with repeating synthetic DNA polymers and natural DNA

Fluorescence, circular dichroism and sedimentation through cesium chloride gradient techniques were performed to study the physical properties of the binding of the bisbenzimidazole dye Hoechst 33258 (H33258) to natural DNAs and synthetic polynucleotides of defined repeating units. These studies show that Hoechst 33258 exhibits at least two modes of interaction with duplex DNA: (1) a strong base pair specific mode which requires at least 4 consecutive AT base pairs and (2) a weaker mode of binding which is significantly reduced in the presence of high salt (0.4 M NaCl) and exhibits no apparent base specificity. The H33258 binding was found to be sensitive to the substitutions in the minor groove elements of a series of synthetic polynucleotides supporting the model of H33258 binding in the minor groove of the DNA with AT rich sequences. Similar mode of binding was predicted in natural DNAs by methylation of dye-DNA complexes. Footprint analysis of the complex of dye to a pBR322 fragment also supports that a minimum of 4 consecutive AT base pairs are required for H33258 binding to DNA.     

Reaction of [Pt(Gly-Gly-N,N',O)I]- with the N-acetylated dipeptide L-methionyl-L-histidine: selective platination of the histidine side chain by intramolecular migration of the platinum(II) complex

The reaction of the monofunctional [Pt(Gly-Gly-N,N′,O)I]⁻ complex, in which Gly-Gly is the dipeptide glycyl-glycine coordinated through two nitrogen and oxygen atoms, with the N-acetylated dipeptide l-methionyl-l-histidine (MeCOMet-His) studied by ¹H NMR spectroscopy. All reactions were carried out in 50 mM phosphate buffer at pD 7.4 and at 25 °C. In the initial stage of the reaction, the platinum(II) complex forms the kinetically favored [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-S)]⁻ complex, with unidentate coordination of the MeCOMet-His dipeptide through the sulfur atom of the methionine residue. In the second stage of the reaction, complete intramolecular migration of the [Pt(Gly-Gly-N,N′,O)] unit from the sulfur to the N3 nitrogen atom of imidazole was observed and a new platinum(II)-peptide complex, [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-N3)]⁻ was formed. In comparison with previous results obtained for the reaction of [Pt(dien)Cl]⁺ with different methionine- and histidine-containing peptides, this migration reaction was sufficiently fast and strongly selective to the N3 atom of the imidazole ring of the histidine side chain. This study is an important step in the development of new platinum(II) complexes for selective covalent modification of peptides and proteins.     

DNA binding of a proflavine derivative bearing a platinum hanging residue

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I = 0.1 M and 25 °C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides.     

Interaction of novel bis(platinum) complexes with DNA.

Bis(platinum) complexes [[cis-PtCl2(NH3)]2H2N(CH2)nNH2] are a novel series of potential anticancer agents in which two cis-diamine(platinum) groups are linked by an alkyldiamine of variable length. These complexes are potentially tetrafunctional, a unique feature in comparison with known anticancer agents. Studies of DNA interactions of bis(platinum) complexes in comparison with cisplatin demonstrate significant differences. Investigations of interstrand crosslink formation in which crosslinking of a short DNA fragment is detected by gel electrophoresis under denaturing conditions demonstrate that interstrand crosslinks are 250 fold more frequent among bis(platinum) adducts than among cisplatin-derived adducts under the conditions examined. These investigations indicate that bis(platinum) adducts contain a high frequency of structurally novel interstrand crosslinks formed through binding of the two platinum centers to opposite DNA strands. Unlike cisplatin, bis(platinum) complex binding does not unwind supercoiled DNA. Studies with the E. coli UvrABC nuclease complex demonstrate that both linear and supercoiled DNA containing bis(platinum) adducts are subject to incision by the repair enzyme complex. Initial studies using UvrABC nuclease as a probe to define the base and sequence specificity for bis(platinum) complex binding suggest that the specificity of the bis(platinum)s is similar, but not identical, to that of cisplatin.     

Formylation of porphyrin platinum complexes

The formylation reaction of platinum complexes of beta-unsubstituted porphyrins was studied. The interaction of deuteroporphyrin IX derivatives with the Vilsmeyer reagent led to the selective formylation of their macrocycles in the beta position. The resulting formyl derivatives of the porphyrins are of interest for fluorescent immunoassay.